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Lupine Publishers | The Prevention and Treatment of Malaria in Traditional Medicine of Tetun Ethnic People in West Timor Indonesia

Abstract

Native people in West Timor Indonesia have been exposed to malaria since long time ago. Because of this experience, it is believed that this community has developed their local concept about malaria, and how to manage it. This research was intended to document and analyze local knowledge and practices of malaria prevention and treatment developed by Tetun ethnic people in West Timor. The research was a field study, conducted through some interviews, discussions and observations. The results of this study showed that this community has long been developing various methods to prevent and threat malaria. The prevention and treatment of malaria in traditional medicine of Tetun ethnic people consists of both herbal and non-herbal methods and supported by some prohibitions and restrictions. The results also showed that the practice of traditional medicine for prevention and treatment of malaria by Tetun ethnic people can be explained scientifically. Medicinal plants that widely used like Strychnos ligustrina, Carica papaya, Momordica sp., Cleome rutidosperma, Physalis angulata, Alstonia spectabilis, Alstonia scholaris and Melia azedarach have been proven to have antimalarial activities as anti-plasmodial, antipyretic, analgesic, anti-inflammatory and immunostimulant.

Keywords: Local Knowledge; Traditional Medicine; Malaria Prevention and Treatment; Tetun Ethnic; West Timor

Introduction

Traditional communities in ancient times developed their local knowledge about the prevention and treatment of a disease based on their experience interacting with the disease for a long time. This local knowledge was then become a guidance for them to establish strategies to prevent and treat the disease, which were practiced widely in the community, and become their traditional medicine [1,2]. Traditional medicine is a term imposed on pre-scientific medical systems, and defines as a sum total of knowledge, skills and practices based on theories, beliefs and experiences of different cultural customs used in health care, disease prevention and increased physical and mental performance, which have been used for generations from one generation to the next [3,4]. Malaria is an ancient disease that has not been fully eradicated until this time [5]. Since long time ago, malaria was the main infectious disease that often attacks Timorese people, especially in Belu and Malaka Districts in West Timor (Indonesia). Several old manuscripts noted that Timorese people in early of 19th century were suffered from malaria which caused many deaths [6,7]. Until this time, Belu and Malaka Districts are still hyper-endemic areas of malaria. According to the Global Fund report, in 2014, Belu and Malaka Districts were classified as high malaria endemic areas, with the Annual Parasite Insidence (API) of 12.87o/oo and 11.58o/oo respectively, higher than Indonesian average API 1.38o/oo. Various programs for malaria prevention and eradication sponsored by the Indonesian Ministry of Health and World Health Organization such as insecticide-impregnated net, fogging, mass blood survey for early diagnosis and prompt treatment, and treat malaria patient using Artemisinin Combination Therapy (ACT) have been implemented, but decreasing of the API value is still not too convincing [8]. Cultural factors that influence public attitudes and acceptance on the programs of prevention and treatment of malaria are estimated to be one of the obstacles to the success of these programs. The implementation of various disease control programs and strategies often faces major challenges stemming from the social and cultural situation of the community. The social and cultural situation of a community in a particular place can negatively influence the choice, acceptance and use of interventions in disease control. Many programs of disease control and eradication are unsuccessful because of these social and cultural barriers. Therefore, it is very necessary to understand the local knowledge of the community, including an understanding of the health-illness concept that they believe in. An understanding of this can help policy makers in designing a sustainable and more effective disease control programs [9]. The Tetun ethnic is one of native communities that inhabit territories from the central part of Timor island (in Belu and Malaka districts, Indonesia) to the east (in Republic Democratic de Timor Lester, RDTL). Tetun people are still using traditional medicines to date, and often running various traditional medication rituals [10]. Because of their long-time interaction with malaria, it should be assumed that they have developed their own local knowledge about malaria and methods to prevent and treat it. Therefore, this research was intended to study the local knowledge of the Tetun ethnic people regarding malaria and the methods they have developed for the prevention and treatment of this disease.

Introduction

Study Design

This study is a kind of research in the field of medical anthropology. This study was conducted as a qualitative exploratory research, with a field study as main technique, supported by a literature study.

Profile of Study Site and People

This research was conducted in Belu and Malaka Districts located in the central part of Timor island. These areas are located at 9°15’ S-9°34’ S and 124°40’ E-124°54’ E. Belu and Malaka are two of Indonesian territories that border directly with the Republic Democratic Timor Leste (RDTL). The topography of Belu Districts is mainly hilly, while Malaka is generally a stretch of flat land. Some areas of Malaka at the south part meet the rainy season twice in a year, while the areas of north part and also Belu areas are only have one rainy season. The main rainy season takes place between November-March due to wind that brings rain from the Indonesian Ocean. This rain occurs evenly in Malaka and Belu regions. The additional rainy season in April-June, which is limited in some areas of Malaka, is affected by wind from Australia that carries moisture from the Timor Sea. Based on the ethnolinguistics, there are four indigenous ethnic groups that live in Belu and Malaka Districts, namely Tetun, Dawan, Kemak, and Bunaq (Marae). Tetun ethnic is the majority ethnic group in Belu and Malaka, consists of approximately 80% of the population. They scaterred in almost all sub-districts of Belu and Malaka [11].

The Informants

The informants of this study were people of Tetun ethnic who have lived for long time in Belu or Malaka Districts. They were people with good knowledge and experiences of traditional medicine practices. The informants were selected through the purpossive and snowball tehniques. A total of 94 informants (42 men and 52 women) with the age of 40-90 years old were involved in this study. They came from 15 vilages of five sub-districts in Malaka (Wewiku, Malaka Barat, Weliman, Malaka Tengah and Kobalima Timur Subdistricts), and 14 vilages of ten sub-districts in Belu (Raimanuk, Tasifeto Barat, Nanaet Duabesi, Tasifeto Timur, Lasiolat, Raihat, Lamaknen, Kakuluk Mesak, Atambua Barat and Atambua Selatan Sub-districts). These informants consist of traditional public healers, home healers, and traditional medicine users.

Data Collection

Data were collected through several interviews, discussions, and observation. Interviews were conducted with a semi-structured questionnaire. Interviews were intended to collect informations about local knowledge on health-illness concept, symptoms, signs and causes of malaria, traditional methods for the prevention and treatment of malaria, and medicinal plants used for the prevention and treatment of malaria. More deep questions were developed spontaneously based on the answers given by the informants to the previous questions. Interviews and discussions were conducted in Tetun (local language) and Indonesian. We recorded the contents of every interview by wrote a detailed essence of the conversation, but not fully word by word. Several interviews were recorded with audio and video recorder. In this field study, we were assisted by several local guides to search for informants, accompanied in the interviews, to interpreted specific local terms that strange for us, and help us to search, document and collect plant specimens. All plants mentioned by informants were collected in-situ and documented by making photographs and herbaria for taxonomic identification. This field study was conducted from April 2017 to December 2017.

Data Analysis

Data obtained from interviews, discussions and observations were analyzed qualitatively, and presented in narrative or qualitative descriptions [12]. The steps of qualitative analysis are as follows:

a) Transcription of data: first of all, the interview data, discussions and field observation records were well-transcribed in a neat text.

b) Data reduction: transcripts were analyzed to marked meaningful parts, and then grouped based on the same characteristics into certain categories, i.e. the local knowledge about health-illness, local concepts about malaria, methods for the prevention and treatment of malaria, and plants used for the prevention and treatment of malaria.

c) Presentation of data: data that has been grouped were arranged regularly according to each category to make them easy to understand. Data of plants used in malaria prevention and treatment were presented in a table.

d) Verification and conclusion: determined the meaning of the data presented.

Results and Discussion

Local Concepts about Health-Illness

The concept of health and illness in Tetun community is very simple. Tetun people define health as a condition of normal, good and not sick. Illness is interpreted as a condition in which someone feels unwell or sick or has a disease in the body. Tetun traditional people state a condition as health or ill by seeing physical signs. A person is said to be health if he/she looks physically strong, fresh, agile, has a bright face and good appetite; and vice versa, if the physical performance seems weak, lethargic, pale face, lack of appetite, then the person is said to be sick or has an illness in the body. Someone is said to have recovered from illness when showing physical signs such as being able to get up, not feel dizzy anymore, being able to walk quickly and to work again, and his/her appetite is back and improved. The concept of Tetun people about health and illness is also associated with the ability to carry out daily life activities. Someone who is still able to work or move without feeling bad or pain in his body, then that person is not said to be sick. People who are clinically suffering from a certain disease but not feel sick and still able to carry out daily activities without being disturbed by the disease, then that person is not considered sick. WHO and Indonesian Ministry of Health define health as a state of complete physical, mental and social well-being, and not merely the absence of diseases or infirmity [13]. Comparing the concept of health according to Tetun people’s understanding with this official definition, it can be concluded that the concept of health of Tetun ethnic people is inadequate to describes whole condition called health, because for this community, health and illness are more related to physical performance than psychological and social performance.

Local Concept about Symptoms and Signs, and Causes of Malaria

The indigenous people of Tetun know malaria as is in mana’s (hot body, fever) with primary signs and symptoms are high fever, shivering, intermittent fever, headache, muscle and joint pain, pale, yellow eyes, and abdominal pain and/or diarrhea. Many informants did not know that swollen spleen (splenomegaly) is also one of the signs of malaria that is already severe, but they assumed that the swollen spleen can cause fever (they say “malaria”). In general, almost all the informants assumed that malaria is a common, mild and not serious disease, only a sick of hot body or fever. This local concept seems to greatly influences people’s perceptions of the danger of malaria and result in reduction of their alertness on malaria and the seriousness of managing this disease. In the local knowledge of Tetun ethnic people, the causes of malaria are: sweet food and drink, chilled, sunburn, fatigue, presence of other disease in the body, magic, cold food and drink, lack of sleep, inadequate post-natal care, spicy food, alcohol, and oily or fatty food. Tetun ethnic people assumed that sweet food and drink, sunburn, magic, spicy food, alcohol, and oily or fatty food cause an excessive heat in the body, and as a result, someone will get high fever malaria. Chilled, cold food and drink, lack of sleep is assumed to cause cold entering the body, and as the result, someone will get shivering malaria. The fatigue, presence of other disease in the body and inadequate post-natal care for mother and infant are assumed to destroy the equilibrium of hot and cold in the body and result in malaria with high fever and/or shivering. According to some informants, mosquito as malaria transmitter was a new knowledge that coming from outside, introduced by the Catholic missionaries from Europe. According to Foster dichotomous on causes of disease [14], the causes of malaria in the local concept of Tetun people are naturalistic, not personalistic. Factors such as sweet foods or drinks, long time in rain, water or cold places, long working under the hot sun, fatigue and the presence of other diseases in the body are naturalistic properties that cause heat-cold balance in the human body to be disrupted, and then causes someone to get malaria. Many Tetun people do not consider mosquito as carrier of malaria, causing them to have low awareness of the threat of mosquitoes. This may be one of the causes of the still high endemic of malaria in Belu and Malaka until this time [15].

Methods for The Prevention and Treatment of Malaria

The Tetun ethnic people have their own patterns or habits of life that they do for generations to prevent malaria attacks. The methods that are considered effective in preventing attacks of malaria are: luli or hale’u, drink medicinal concoction of bitter herbs, eat bitter food, and drink tua moruk. Luli or hale’u means avoiding things that can cause malaria (according to their local concepts about the cause of malaria), which are: not eating sweets frequently, not working for long time under the rain or hot sun, and not too tired at work or physical activities. Eating bitter foods, especially papaya and bitter melon, and drinking bitter palm sap tua moruk are also considered effective to prevent someone from being attacked by malaria. Some informants who previously linked malaria with mosquitoes stated that repelling mosquitoes using smoke of burned aromatic plants and sleeping under mosquito nets are effective for malaria prevention. The treatment of malaria in traditional medicine of Tetun ethnic consists of herbal and non-herbal methods. Herbal method consists of drinking herbal concoction, inhaling the vapor of boiled medicinal plant, massage with paste of medicinal plant, bath with water of boiled medicinal plant, and attach the paste of medicinal plant as a cataplasm on the swollen spleen. A non-herbal method is sunu kok, that is burning the waist above the swollen spleen using a piece of coconut shell coal or a heated metal. The results of the interviews showed that most traditional medication for malarial patient usually combine two or more methods. It was found also that the role of traditional healer in the treatment of malaria patient is not so important. Tetun ethnic people assumed that malaria is a common and not a serious disease, thus the treatment of malaria does not require a high competency healer. Several informants stated that they usually conducted self- and home-medication for malaria complaint. In the traditional medicine of Tetun ethnic people, the treatment of malaria is a simple treatment for reducing heat or fever [15]. The assumption of malaria as a common, mild and not a serious disease results in lack of awareness about dangers of malaria. It was found that in many cases, health workers often complain of disobedience of patients who stop taking antimalarial drugs immediately after they feel cured (being able to get up, not feel dizzy anymore, being able to work again, and the appetite is improved), even though Plasmodium in their blood has not been completely eliminated. As the result, the success of the malaria eradication program in this area has increased very slowly [8].

Plants Used for The Prevention of Malaria

Tetun ethnic people believe that consumption of bitter food or drink can prevent someone from malaria attacks. Therefore, small children are often forced by their parents to eat stew and drink decoctions of flowers, leaves and young fruit of Carica papaya, or young fruit of Momordica sp. (M. charantia or M. balsamina). Some informants gave information that if they feel tired, achy and lack of appetite, they will drink decoction of Carica papaya leaves, fruit of Momordica charantia, Melia azadarach leaves, Alstonia scholaris, Alstonia spectabilis or Strychnos ligustrina stem bark. Consumption of these plants’ decoction is believed to restoring body freshness, increasing appetite, eliminating fatigue, and thus, preventing from malaria attack. Some informants also believed that drinking tua moruk is effective in malaria preventon. Tua moruk is a traditional drink made by fresh tapped palm sap soaked with the stem bark of Alstonia scholaris, Alstonia spectabilis or Strychnos ligustrina soaked in it. Several publications of other previous studies showed that the bitter plants used by Tetun people to prevent malaria has been shown to have pharmacological activities as antiplasmodium and immunostimulant [16-18].

Plants Used for The Treatment of Malaria

In this study, we recorded a total of 96 species from 39 families used by Tetun people in various formula for drink, massage, bath, inhalation and cataplasm (Table 1). Strychnos ligustrina, Carica papaya, Cleome rutidosperma, Physalis angulata, Alstonia spectabilis, Alstonia scholaris and Melia azedarach are some of the most widely plants used in various formula for drink. For massage, Garuga floribunda, Jatropha curcas, Acorus calamus, Allium cepa, Drynaria quercifolia, Ocimum sp. and Ruta graveolens are common. For bathing, people use Tamarindus indica, Psidium guajava, Melicope latifolia and Blumea balsamifera. Leaves of Brucea javanica, Annona muricata and Annona reticulata are used in inhalation method. Root of Moringa oleifera and leaves of Ficus hispida are used as cataplasm to reduce the swollen spleen [19]. Several plants were found in various formula for more than one mode of application. Several previous publications showed that most of these plants are also used in other traditional medicine for the same purpose in many areas of Indonesia and the world [16,18], and have been scientifically proven to have pharmacological activities as true antimalarial (antiplasmodial) and/or indirect antimalarial such as antipyretic, analgesic, anti-inflammatory and immunostimulant [20].

Table 1: Plants used by Tetum ethnic people for the treatment of malaria.

Conclusion

The practice of preventing and treating malaria in the traditional medicine of Tetun ethnic people is a direct implementation of their local knowledge about malaria. The local concept of signs and symptoms and the causes of malaria encourage traditional people to create methods to prevent and treat malaria. The local concept of the Tetun ethnic people about malaria is the main reference in the creation of rules regarding prohibitions and restrictions, and recommendations for preventing attacks of malaria. The local concept of the causes of malaria determines the choice of plants for the treatment of malaria. Scientifically, these plants have been proven to have activities as true antimalarial and indirect antimalarial. The local concept of malaria as a common, mild and harmless disease causes that the role of traditional healer is not always needed in the treatment of malaria. Methods for the prevention and treatment of malaria developed by Tetun ethnic people consist of both herbal and non-herbal methods and supported by the implementation of several prohibitions and restrictions to provide healing for the sufferers of malaria.

Acknowledgement

We thank to Indonesian Ministry of Research, Technology and Higher Education, for financial support (Research Contract No. 0668/K8/KM/2018).

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The Optimal Pain Management Methods Post Thoracic Surgery: A Literature Review| Lupine Publishers

Journal of Surgery|Lupine Publishers

Abstract

Post-operative pain control is one of the key factors that can aid in fast and safe recovery after any surgical interventions. Thoracic surgery can cause significant postoperative pain which can lead to delayed recovery, delayed hospital discharge and possibly increased risk of chest complications in the form of atelectasis and even lower respiratory infections. Therefore, appropriate pain management following thoracic surgery is mandatory to prevent development of such morbidities including chronic pain.

Keywords:

Thoracic Surgery, Analgesia, VATS, Robotics, Thoracotomy

Introduction

Thoracic surgical procedures can result in severe pain which can present as a challenge to be appropriately managed postoperatively. In particular, thoracotomies are well known for their severity of pain due to the incision, manipulation of muscles and ligaments, retraction of the ribs with compression, stretching of the intercostal nerves, possible rib fractures, pleural irritation, and postoperative tube thoracotomy [1]. Recognition of this has contributed to the development of minimally invasive techniques such as video assisted thoracoscopic surgeries (VATS) and lately robotic surgery [1]. These techniques not only aim to produce better aesthetic results, but also reduce post-operative pain and enhance recovery without compromising the quality of treatment offered. Poor pain management can lead to several and serious complications such as lung atelectasis, hypostatic pneumonia due to avoidance of deep breathing in these patients as a result of pain and superimposed infection [1]. Pain management as a result, does not only lead to greater patient satisfaction, but it also reduces morbidity and mortality in patients undergoing thoracic surgery [2]. Historically, post-operative pain management for thoracic surgery involved the use of narcotics alongside parenteral or oral anti-inflammatory agents [2]. Post chest tube removal patients typically are transitioned to oral analgesia. Multiple additional pain control adjuncts were also implemented with differing levels of success [1]. Over time, intra-operative techniques have been developed which aims to target pain reduction postoperatively [2]. As our understanding of both pain management and the factors that play a role in the development of pain has increased, we have been able to target these and improve postoperative pulmonary morbidity and pain scores [1,2]. We aim to review different means of pain control in this paper in order to assess their effectiveness in achieving optimum results.

Thoracotomy

The mechanism of pain in thoracotomy involves the innervation of the intercostal, sympathetic, vagus and phrenic nerves [3]. Additionally, shoulder pain may result from stretching of the joints during the operation.

After a thoracotomy, pain can persist for two months or more, and in certain incidences it recurs after a period of cessation. The incidence of chronic pain post thoracotomy is reported to be 22-67% in the population [4]. Good surgical technique and effective acute post-operative pain treatment are evident means of preventing post-thoracotomy pain and consequent pulmonary complications [4]. Due to the multifactorial character of the pain, a multimodal approach to target pain is advised. Typically, both regional and systemic anaesthesia are administered. A combination of opioids such as fentanyl or morphine are typically used [5]. A variety of techniques for the administration of local anaesthetics are available at present, and the effectiveness of each is assessed in this paper.

a) Thoracic Epidural Analgesia (TEA)

TEA was the most widely used method of means of analgesia. It was the gold standard means of pain relief [6,7]. It is typically inserted prior to general anaesthesia, at the level of T5-T6, midway along the dermatomal distribution of the thoracotomy incision. A study by Tiippana et al. [8] measured the visual analogue scale (VAS) in order to assess the presence of pain during rest and at the time at which they coughed in 114 patients of whom 89 had TEA and 22 who had other methods of pain control. TEA was effective in alleviating pain at rest and during coughing. In TEA patients, the incidence of chronic pain of at least moderate severity was 11% and 12% at 3 and 6 months, respectively. The study found that at one week after discharge, 92% of all patients needed daily pain medication. The study advised for extended postoperative analgesia for up to the week post-discharge to be administered in order to manage this. The study however concluded overall, that TEA was effective in controlling evoked post-operative pain. However, the study did encounter problems of technical form in 24% of the epidural catheters. The incidence of chronic pain, however, was lower compared with previous studies where TEA was not used. Several other studies support that TEA is superior to less invasive methods. According to Shelley B. et al. [9] TEA was preferred by 62% of the respondents over paravertebral block (PVB) with 30% and other analgesic techniques with 8%. Limitations of this technique included hypotension and urinary retention. Certain patients with active infection and on anticoagulation are excluded from epidural placement.

b) Paravertebral Block (PVB)

PVB is considered an effective method for pain management and its use has been increased in the recent years. This technique involves injecting local anaesthetic into the paravertebral space and it is able to block unilateral multi-segmental spinal and sympathetic nerves. Previous studies have shown that it is effective in achieving analgesia and is associated with a lower incidence of side effects such as nausea, vomiting, hypotension and urinary retention [10,11]. As the lungs are collapsed, it is associated with a lower risk of pneumothorax.

In a study by Davies R.G. et al. [10] there was no significant difference in pain scores, morphine consumption and supplementary use of analgesia between TEA and PVB. The rate of failed technique was lower in PVB (OR =0.28, p=0.007). Respiratory function was improved at both 24 and 48 hours with PVB but only significantly improved at 24 hours.

c) Intercostal Nerve Block (ICNB)

ICNBs are generally administered as single injections at least two dermatomes above and below the thoracotomy incision [12]. It is performed percutaneously or under direct vision, using single injections or through placement of an intercostal catheter. It can also be formed using cryotherapy. It is associated with reduced post-operative pain scores; however, it is less effective than TEA in controlling chronic pain [12]. This was illustrated by a study by Sanjay et al. [12] which found that patients that underwent ICNB had higher pain scores 4 hours post-operatively, than those who received epidural anaesthesia using 0.25% bupivacaine (p<0.05). The study concluded that in the early post-operative period there was significant impact in pain relief for both techniques, but thereafter, epidural anaesthesia was proven to significantly reduce post thoracotomy pain over ICNB. Due to the multifactorial nature of post-thoracotomy pain, various approaches are required in order to target pain. ICNBs are useful in the blockade of intercostal nerves, whilst PVB and TEA appear to block the intercostal and sympathetic nerves. Due to the inability of regional anaesthesia to block the vagus and phrenic nerves which are implicated in the pathophysiology of pain, NSAIDs and opioids are required as adjuncts. TEA is proven to be the most effective means of treating pain alongside PVB; however, it is associated with more side effects than PVB. At present, there are a limited number of studies directly comparing pain control and post-operative outcomes between PVB and TEA. There is no conclusive evidence that either method is superior to the other regarding pain control.

Video-Assisted Thoracoscopic Surgery (VATS)

Existing evidence supports the noninferiority of thoracic PVB when compared to TEA for postoperative analgesia [13]. PVB is versatile and may be applied both unilaterally or bilaterally. It can be used to avoid contralateral sympathectomy, consequently minimising hypotension. This is an apparent advantage it has over thoracic epidural. Furthermore, it offers a more favourable side effect profile when compared to epidural anaesthesia. At present, the factors taken into consideration when selecting a regional technique include tolerance of side effects associated with TEA, consensus on best practice/technique, and operator experience [13]. A randomised controlled trial by Kosiński et al. [14] compared the analgesic efficacy of continuous thoracic epidural block and percutaneous continuous PVB in 51 patients undergoing VATS lobectomy. The primary outcome measures were postoperative static (at rest) and dynamic (coughing) visual analogue pain scores (VAS), patient-controlled morphine use and side-effect profile. The study found that pain control (VAS) was superior in the PVB group at 24 hours, both at rest (1.7 vs3.3, p=0.01) and on coughing (5.8 vs 6.6, p=0.023), and control of pain at rest was also superior in the PVB group at 36 hours (3.0 vs 3.7 (p=0.025) and at 48 hours (1.2 vs 2.0, p=0.026). There were no significant differences in the postoperative morphine requirements. In regard to side-effect profile, the study showed that the incidence of postoperative urinary retention (defined as no spontaneous micturition for 8 hours or ultrasound-assessed volume of the urinary bladder >500ml) was greater in the epidural group (64.0% vs 34.6%, p=0.0036), as was the incidence of hypotension (32.0% vs 7.7%, p=0.0031). There was no significant difference in the incidence of atelectasis (4.0% vs 7.7%, p=0.0542). However, the incidence of pneumonia was significantly more frequent in the PVB group (3.8% vs 0%, p=0/0331). Kosiński et al. concluded that PVB is as effective as thoracic epidural block in regard to pain management as it offers a superior safety profile with minimal postoperative complications. A further randomised controlled trial by Okajima et al. [15] compared the requirements for postoperative supplemental analgesia in 90 patients who received wither a PVB or thoracic epidural infusion for VATS lobectomy, segmentectomy or wedge resection. The main outcome measures were pain scores at rest (verbal rating scale 0= none and 10=maximum pain), blood pressure, side effects and overall satisfaction scores relating to pain control (1=dissatisfied and 5=satisfied). The study found a similar frequency of supplemental analgesia (50mg diclofenac sodium suppository or 15mg pentazocine intramuscularly) for moderate pain in both groups, with 56% of those in the PVB group requiring ≥2 doses, compared to 48% in the epidural group (p=0.26). Hypotension, defined as a systolic blood pressure <90mmHg, occurred more frequently in the epidural group (21.2% vs 2.8%, p=0.02). There was no difference in the incidence of pruritus (3.0% vs 0%, p=0.29) and post-operative nausea and vomiting (30.3% vs 25.0%, p=0.62) between both groups. The study found no statistical difference between patient-reported satisfaction in pain control between epidural and PVB using the verbal rating scale (5.0 vs 4.5, p=0.36). The study concluded that PVB offered additional to equivalent analgesia to epidural, a lower incidence of haemodynamic instability postoperatively. A further study by Khoshbin et al. [16] performed an analysis on 81 patients undergoing VATS for pleural aspiration +/- pleurodesis, lung biopsies or bullectomy. The main outcome was postoperative pain levels, documented every 6 hours and scored against the Visual analogue Scale (0= no pain, 10= worst possible pain). In both PVB and epidural groups, bupivacaine 0.125% was the local anaesthetic of choice, with clonidine added to the epidural infusion at 300μg in 500ml. The study showed that there was no significant difference in mean pain scores between PVB or EP (2.1 vs 2.9, p=0.899), therefore concluding that PVB is as effective as epidural in controlling pain post-VATS.

Robotic Lung Surgery

Minimally invasive techniques are considered advantageous over open surgical approaches due to their shorter recovery times, reduced perceived levels of pain post-operatively and shorter postoperative length of stay in hospital [17-19]. Robotic surgery has become a popular method in recent years. Debate remains regarding whether robotic surgery is superior to VATS in regard with pain reduction. A case control study by Louie et al. [19] compared 45 robotic assisted lobectomies (RAL) to 34 VATS lobectomies. The study showed that both groups had a similar mean ICU stay (0.9 vs 0.6 days) and a mean total length of stay (4.0 vs 4.5 days). The study showed that patients that underwent robotic lobectomies had a shorter duration of analgesic use post-operatively (p=0.039) and a shorter time resuming to normal everyday activities (p=0.001). A limitation in this study was an inaccurate record of the amount of pain relief used by the patients, ultimately working as a confounding factor when interpreting the results. In a separate study by Jang et al. [18] 40 patients undergoing RAL were compared retrospectively to 80 VATS patients (40 initial patients and 40 most recent patients), all with resectable non-small cell lung cancer. The study showed that the post-operative median length of stay was significantly shorter in RAL patients compared to the initial VATS patients. The rate of post-operative complications was significantly lower in the RAL group (10%) compared to the initial VATS group (32.5%) and similar to the recent VATS group (17.5%). Post-operative recovery was easier for patients in both the RAL and VATS group due to earlier mobilisation, allowing them to return to their everyday activities quicker. In a retrospective review by Kwon et al. [17] 74 patients undergoing robotic surgery, 227 patients undergoing VATS and 201 patients undergoing anatomical pulmonary resection were assessed and compared with regard to acute (visual pain score) and chronic pain (Pain DETECT questionnaire). The study showed that there was no significant difference in acute or chronic pain between patients undergoing robotic assisted surgery and VATS. Despite no significant difference in pain scores, 69.2% of patients who underwent robotic-assisted surgery felt the approach affected their pain versus 44.2% of the patients who underwent VATS (p=0.0330). These results all support the superiority of robotic surgery over VATS and open approaches with regard to pain, length of hospital stay and recovery times. Both robotic surgery and VATS have their benefits i.e. two-versus three-dimensional view, instrument manoeuvrability, and reduced post-operative pain.

Conclusion

Since post-thoracotomy pain is multifactorial, a multimodal approach is required. In particular, ICNB blocks the intercostal nerves, and PVB and TEA appear to block the intercostal and sympathetic nerves. NSAIDs and opioids are required as valgus and phrenic nerve cannot be blocked by regional anaesthesia. TEA is evident to be the most effective in treating pain alongside with PVB. It is however associated with more side effects than PVB.

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lupine-publishers-prjfgs · 5 years ago

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Lupine Publishers | Principles of the Military Consultations in Ancient Egypt

Lupine Publishers- Anthropological and Archaeological Sciences Journal Impact Factor

Introduction

A way from the regular theory, that the royal king always had the perfect and ideal order and decision [1], when he always is depicting as an intelligent and wise man, making the correct solution against the doubts and uncertainties of his councilors [2], The pattern is basically that the king makes and demonstrates his superior judgment by the ensuing success [3].

From the beginning of the ancient Egyptian history, the royal king must had his own courtiers and advisors among the great ones [3A,3B], to consult with them, concerning the vital matters, especially when are relating to the country fate [4], the literary texts depicted several kinds of councils, whether were holding at the royal palace or outside it.

The first witness of holding a royal council was belonging to the 4th dynasty, at the reign of the great king Khufu, this council documented in Westcar papyrus [5] (= Berlin Papyrus 3033), when the great king Khufu summoned his sons to tell him an exciting marvel event that had happened in the past, the texts contains a cycle of tales within a single theme related to the birth of the kings who will succeed his line family rule, the episode begins when the king one day felt with a boredom, so he went around every chamber of his palace, finding something fun to entertain [6]. The story refers indirectly, the king’s need to own his close and special courtiers [7], who were emerging obviously at the Middle Kingdome literary texts (Berlin leather Roll of Senusert 1) [8].

Methodology

The research is concerned with the texts which refer to the royal consultation dating since the second intermediate period (Hyksos era) till the reign of the king Thutmose III, it contains four councils, three of them were held at the 2nd intermediate period (Apophis, Seqenenre and Kamose), while the 4th one belonged to the king Thutmose III’ reign.

The research will intend to analyze those councils, the reasons of its holding, members of the council and their positions, the place of its holding, the full dialogue that happened between the king and his councilors, and however the role of both (the king and his councilors) at these councils, as well as the results and the decisions which had been adopted.

Principle of Military Consultation in the Second

During the end of the 12th dynasty of the Middle kingdom [9], considerable details are indicating an increasing in numbers and activities of the Asiatics who infiltrated and settled around the eastern borders of the Delta [10], that settlement facilitated the operation of Hyksos invasion [11], which happened in the second half of the 13th dynasty of the Middle kingdom [12]. So, the country was divided into individual parts, the Hyksos invaders established the 15th dynasty, that predominated the whole Delta region until the middle Egypt borders at south, while the fugitive king of the 13th dynasty had to leave and ruled his remaining days of his reign to govern from Thebes [13], when the local governors of Thebes province hosted him and his royal family after the fall of the political capital el-Lisht, a short time passed, the Theban governors had inherited the kingship and authority and were establishing the 16th dynasty, which began the first liberal operations against the Asiatics invaders.

This latter dynasty was followed by a strong family (the 17th dynasty) whose kings began the true struggle and liberation wars against Hyksos, the first engagement occurred during the reign of Seqenenre Taa in the time of Ippy (Apophis) king of Hyksos.

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Lupine Publishers | Historical Silahtaraga Power Plant-Black Sea Decovil Line Research, Double Military Decovil Photogrammetry Study

Lupine Publishers- Anthropological and Archaeological Sciences Journal Impact Factor

Abstract

In this article, which we prepared in addition to the works carried out by tracing a lost cultural heritage, In 1915 Turkish geography, investigation and photogrammetry study on the narrow-gauge railroad line built to transport coal from the Black Sea coast to the Golden Horn will be included. In the study, a CAD model created by measurements made from old photographs related to the subject will be used as data in the prototype to be produced by SLS (Selective Laser Sintering) method. Then, we believe that the miniature model and production adventure of the Decovil locomotive, which we have brought to the present with the noncommercial serial production of the model, will be the means of remembering at least a cultural heritage that has not reached today.

Introduction

In this part of the study, the research of the historical railway and its archaeological importance will be shared. In this review, news published on the internet pages and books and some collection materials prepared by the researchers were used. The narrow-gauge railroad line located in the boundaries of Istanbul, in Kagithane district was founded in 1915. In order to uncover the lost story of this railway which ended in 1950 with the dismantling of the rails, a book published named “100 years later on the trail of a lost railway”. In the study carried out by the Municipality of Kagithane as a multi-disciplinary team, the team of writers created an important task in bringing the cultural heritage to the present day by bringing together the written documents, photographs and pieces of the railway which have the chance to reach today.

In the studies, many details related to the narrow-gauge railroad line, which was built for the purpose of transporting coal from the lignite basin in Agacli (25 km area starting from Kilyos to the Terkos Lake on the Black Sea coast) to the power plants in the Golden Horn, have been delivered to our day[1]. If we need to share some valuable details about the railway: The Kagithane- Black Sea decovil line, which was effectively used to meet energy needs during the First World War, was built between 1914 -1916 and is 57 km long (Figure1). The distance between the rails of the railway is 60cm and this system is called as decovil [2]. The name dekovil comes from the company founded in 1875 of the surnames of the French engineer and businessman Paul Decauville who lived between 1846 -1922 [3].

The period when the line was established, World War I continues in the region. There is an energy problem in Istanbul due to the imports of coal stopped from the UK due to the war and the damage of ships bringing coal from Zonguldak to the region during the war. The fact that the Canakkale Strait was closed due to the war made it impossible to import coal through the Mediterranean. In the Ottoman geography of the period, coal was used as an energy source in ships and power plants rather than domestic fuel. Today it is a museum building; the building, known as Silahtaraga Power Plant of the period, meets the electricity needs of Istanbul (Figure2). With the planned decovil line, it is aimed to evaluate the coal reserve on the Black Sea coast and to transport it to the Silahtaraga Power Plant without the need for sea transportation. In this way, the solution to the energy problem of Istanbul will be produced. Although the existence of the coal reserves of Agacli, Ciftalan region on the Black Sea coast has been known since the Byzantine Period, no studies have been conducted to make the reserve available for use. After the preliminary investigation, it is determined that the desired yield can be obtained by mixing the lignite coal in the region with Zonguldak hard coal, and it is decided to use the coal in the region and construction of the decovil line is started. The entire installation works are photographed by Hasan Mukadder Dolen, the railway regiment officer of the period. Hasan Mukadder Dolen’s photo collection was left to his grandson Emre Dolen after his death in 1975. It is known that many photographs and information about the historical railway have survived through this channel. Following the first line completed in 1915, a second line was built in Ciftalan in 1916. Railway rails and locomotives produced by Germany’s decovil line, with many stations, vehicles and employees is important in terms of energy logistics of the period. It is mentioned in the historical documents that the rails and locomotives transported from Germany to the Ayestefanos Railway Regiment warehouses in Yesilkoy by the Danube River were later brought to Eyup, Silahtaraga by ships (Figure3).

The first line starts from Silahtaraga and reaches Agacli village after Kagithane stream; the other line runs through the Belgrade forests to the village of Ciftalan. The light rail line that reached the Black Sea coast from Kagithane, which is the famous promenade of the period, undertook an important duty in coal transportation during the years it was established, but was forgotten by being out of use in time. The line was transferred to the Ministry of Commerce in 1922 and to the Ministry of Economy after the proclamation of the Republic [4]. Although traces of the line disappeared in the region after 1956, the rails remained largely underground and in many regions the rails were removed. It is known that one of the locomotives is currently located in the Celtek coal mine depot of the Special Provincial Administration of Amasya. In the photogrammetry study for the protection of cultural heritage, CAD model will be created by using original photographs of locomotives known as Zwilling Heeres Feldbahn (Double Military Decovil) produced in Munich in 1890 by Krauss Werkshof [1] (Figures 4 & 5). The first prototype of the model produced with SLS (Selective Laser Sintering) one of today’s 3d print technologies, will be used for silicon mould technique in mass production.

Literature on Photogrammetry

In this part of the article, the photogrammetry study carried out with measurements taken from photographs of locomotives used in historical railway will be shared. In this context, sharing of literature knowledge about photogrammetry and its usage areas and then modelling study were included in the study. Visual analysis techniques are used in many scientific research areas. In the fields of anthropology and sociology, from the use of photographs of past periods [7] to airborne imaging technologies[8]; visual analysis techniques in different scientific fields from ecology, geography to medical science [9], are basically based on the use of photography as a source of information. Photographs used as data in the social field allow interpretations, social-cultural determinations and visual analysis of the time of the photograph [10]; in technical fields, it can also be used as a digital data source. The use of photography for numerical data acquisition will be explained within the framework of photogrammetry concept. The word, which consists of a combination of ancient Greek “photos” (light), “grama” (drawing) and “metron” (measurement), means measuring with the help of pictures. Photogrammetry, which is used only in mapping, has been used in different areas in the following years. Basically, the photographic analysis to determine the shape, size and position of an object is called photogrammetry [11]. Photogrammetry is divided into three main sections (topographic photogrammetry, interpretation photogrammetry, special purpose photogrammetry) according to the application areas. Photogrammetry used in the fields of architecture, dentistry and archaeology is included in this third group [12]. In recent years, many studies have been done to document the cultural heritage with photogrammetric methods [13], photogrammetry has been used extensively in histor ical works documentation and model formation processes [12]. In such studies, the measurements taken on the photos allow the creation of the 3-D model of the historical work on the computer with digital photogrammetric techniques [14]. While the measurement process is carried out with the points and lines determined by the software, different methods can be used. In our study, the CAD model, which is designed with the measurements with calliper and ruler from old photographs, will be discussed within the scope of special-purpose photogrammetry.

Modelling Process

In this part of our study, we will discuss the modelling process created by taking measurements from the historical photographs of the railway line locomotive of Kağithane. In the modelling study, the locomotive CAD model was created in CATIA V5. The modelling; cabin, nose, mechanical parts and rails, including a total of 4 body consists of (Figure 7). Part design tools are used in the modelling. The modelling of the locomotive as 4 bodies is taken into consideration for the production criteria for the silicone mould to be needed during mass production. In this sense, the model has been modelled and divided into pieces so as to enable post-production assembly. In the modelling study, first the technical drawings (Figure 6) made by Alan Prior were used for general information about the model; in the detail drawings, black and white photographs taken from different angles were used. After the results of modelling, some forms are very detailed for the casting process and line softening is performed according to the model casting process (Figure 7).

Data Transfer to 3d Printing System After Modelling

(STL File, Quality Problems) STL (Stereolithography) data is needed for additive manufacturing of the model. The CAD data generated for this reason is exported in the STL format in the CATIA software. In this process, STL mesh quality is important for the surface quality of the model to be produced. The quality of the prototype to be produced with SLS will affect the quality of the silicone mould from this prototype. The quality problems in the STL data are related to the number of mesh on the surface and the settings of the CATIA display and the necessary arrangement is made as follows: First, the screen settings are set in the ”tools“ - ”options” – “performance” section in the top menu of the CATIA Part Design module. In this section, the 3D Accuracy and 2D Accuracy “fixed” values are revised to “0.01”. The value 0.01 remains constant until changed again. The next editing is done in the CATIA STL Rapid Prototyping module. In the “tesselation” command, with “sag” value, 0.001mm and “grouped” option preference, the mesh quality of each part is determined (Figure 8: on the effect of mesh quality adjustment on surface quality in STL data).

Model Production Process

The technique used in the prototype production is SLS (Selective Laser Sintering) and the material used is PA 2200 (polyamide). If we need to give basic information about SLS production system: The SLS technique is made by sintering micron-size polymer powder in layers, using laser power. In 1986, Carl Deckard, a student at the University of Texas, developed this method of powdered material, which he called PGLSS (Part Generation by Layer wise Selective Sintering). Later on, this production technique called SLS, (with the description text: computer-aided laser apparatus which sequentially sinters a plurality of powder layers to build the desired part in a layer-by-layer fashion) is patented on October 1986 [15]. The method of SLS production is as follows: Firstly, files saved in STL format are opened in Netfabb software and settled in the production area. (Figure5). All parts are sliced at intervals of 0.1 mm (100 microns) after placement. Then the file sliced into 100 microns is saved in SLI format. Although there are 60 microns slicing options within the system, 100 microns will be sufficient for the desired quality. Then the process will continue in the EOS PSW software. After the material preference and parameter selection in EOS PSW software, the file will be transferred to the production bench. The material preference is selected as PA2200 and the layer thickness is 100 microns. The production parameter is then determined. After the prototype production to be performed in EOS P110 (Figure 9), silicone moulding will be carried out for mass production.

The first prototype produced with PA 2200 material, is used to form the silicone mould. After that, the model which is replicated in the manufacturer company by casting process from polyester material is painted with handwork and the final product is obtained (Figure 10). In classical applications, the silicone mould is taken from the prototype modelled by the sculptor and polyester casting process is performed. In the prototype production subject to our study, the process was completed by using digital technologies and methods. In the study, modelling was performed in parametric cad software, enabling the revisions needed in the process to be made quickly.

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Lupine Publishers | Peer Review of Statistics in Surgical Research: Identify The X-Factor or Toss a Coin!

Lupine Publishers | Journal of Cardiology & Clinical Research

Opinion

Professor Peter Bacchetti’s excellent article [1], highlighting “the other problem of peer review of finding flaws that are not really there based on unfounded statistical criticism, and its demoralizing effect on authors”. I wish to add some thoughts to the debated issues. Professor David Horrobin’s original classics on the subject [2,3]. have not yet been surpassed. It was updated recently [4] and prompted some contributory thoughts [5]. Having enough experience as author of reject articles and some as peer reviewer, I find the most devastating effect to author’s morale is making no comment, giving no reason for rejection or not replying all. The BMJ is guilty on this account as an article of mine was rejected that was accepted elsewhere after minor editing [6]. The BMJ, however, is in the good company of most biomedical journals who apply the COPE rules. The article lacked statistics of any kind that perhaps might be one of the reasons it was disliked at BMJ. To Editors’ credit, however, it took about a month to say ‘No’ that caused no momentum loss, unlike other Journals who reach the same verdict on other articles after 6 months or a year that drag another year or two before the author could recover and gather enough time, interest and energy to face the damn thing again. One subtle aim of that article [6], mentioned to BMJ Editors, was an attempt to say that “there is science and in particular evidence based medicine without statistics”.

It is a devil’s advocate to say statistics has not only been made into a “big lie” but also ‘false God’. It was invented elsewhere but currently worshiped only at most medical and surgical journals. A look at Science and Nature testifies such prestigious magazines have reduced statistics to real size and value as a “tool for testing a hypothesis”. It is not too basic a question for every biomedical peer reviewer to find out the exact role, aim and limitations of statistics. Some was mentioned in an article [7], nobody noticed save the late great Professor GD Chisholm editor of Br J Urology. It was based on a study that was rejected by a grant committee. It aimed at resolving 2 of the most serious puzzles of current clinical practice, postoperative hyponatraemia and the multiple vital organ dysfunction or failure syndrome [8]. However, giving data and statistics [7,8]. before clarifying the theories [9]. has proved as wrong as putting the cart in front of the horse. Einstein’s methods on proposing the special and general relativity theory is the correct way. When statistics was haled in the sixties everyone thought it was the only mean to discover “The Unifying Theory”.

This has proved both immensely costly and wrong. The basic fact is ‘statistics cannot, was not intended to and will never could, make a discovery’. Observation, mental experiments and the X factor are the only way to make a discovery long before it is verified and proved by practical studies and statistical tests. Before explaining the X-factor please allow me tell a relevant true story that symbolizes the current problem with statistics. Two friends of mine in UK had a disagreement, made a bit on a round of drinks and decided the first person to enter the hospital club will be the judge. Guess who did? I did but having no clue on how to resolve the conflict suggested that a flip of a coin might be the best way. They agreed also to my condition that while head or tail will determine the winner among them, if the coin stood on edge the judge should be the winner of all. It did and I won. Another conflict started on: Who should buy the 3rd round of drinks? Both agreed that it was my turn. I explained that buying the 3rd round will gain good company but lose all winnings, and my turn should be the 5th round! The point is statistics can tell the probability of head or tail and exclude the odd but when evaluating to either 0 or 100% and the truth is known, instead of expiring it generates residual arguments. Professor Richard Smith contributed to this debate by quoting Dr Hedge on Professor Robert Fox’s famous thought that “swabbing the rejects with the accepts does not make a difference.”

He added that perhaps it has already been done at BMJ” and asked “How can you know?” With due respect Sir, I frankly think nobody can. Despite a proven incremental value of an average article it does not make a noticeable difference or great loss to scientific advances. Statistically speaking that means a quality article submitted to BMJ has 50% chance of being accepted or rejected. So, why not save everybody the trouble and toss a coin? Here is where statistics has shot itself in the foot. It gives an average chance to the average and an odd chance to the odd but can’t tell which is important. The odd chance of a tossed coin to stand on edge matches that of a breakthrough scientific or medical article coming an editor or peer reviewer’s way but detecting such article makes all the difference. Some call it a hunch or gut feeling. Others qualify it by the three-pronged tests of quality, relevance and civility. Identifying the “X-factor” that makes such an article stand out is worth all the trouble. I honestly do not know but it is the arresting beauty found in Einstein’s famous papers, Newton’s laws, Mozart’s music and Shakespeare’s writing among many examples that include medicine [2-4]. I wrote 2 articles on such para-scientific para-medical stuff to identify the X-factor, “Rules and lures of the science game” and “The Mozarts of Science” sent to journals nearly two years ago and have not received a reply yet. I think a message of “Ignore the big headed bustard” arrived. Qualified people to find out the X-factor are COPE members. Another question that requires a ‘Yes’ or ‘No’ answer would be: if any of Einstein’s papers is evaluated using the current peer review standard and statistics adopted by most biomedical journals, would it be accepted?

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Lupine Publishers | Post Endodontic Pain Reduction using three Irrigants with Different Temperature

Lupine Publishers | Journal of Otolaryngology Research Impact Factor

Abstract

Objective: The purpose of this research was to evaluate whether meticulous irrigation with three different temperatures would help in a decrease dental pain.

Materials and Methods: All 120 patients had teeth chosen for conventional RCT for prosthetic reasons in teeth with vital pulps. All canals were cleaned and shaped with Reciprocal files. Final irrigation was done with cold saline solution (6 OC, 4 OC, and room temperature).

Results: A total of 120 of 135 patients (69 females and 51 male) were included whereas 15 were excluded as not achieving the necessities of the study. All patients presented with a vital upper or lower molar, premolar, or front teeth. No statistically major change (P>0.05) between the groups was found regarding the degree or duration of pain.

Conclusion: The approach in both selecting the patients participating in the research and analyzing the data in this research allows us to determine that cryotherapy is an aid of clinical procedures to clean and shape the canals to decrease the occurrence of post-endodontic pain and the need for medication in patients presenting with a diagnosis of vital pulp.

Keywords: Apical healing; Flare-ups; Pain; Post endodontic pain; Post-operative pain

Introduction

Post-endodontic pain is an undesirable sensation occurred in patients regardless of the preoperative periapical status of the tooth treated. Therefore, prevention and management of post endodontic pain are essential in endodontic practice [1]. Organic material, microorganisms, and irrigating solutions extruding beyond the apical constriction during root canal therapy (RCT) will originate inflammation and periodontal ligament complications, such as severe pain or flare-ups. It must be noticed that the amount of extruded material (debris and/or irrigate) varies widely in the reported studies which indicate problems and inconsistencies in treatment methodologies [2-4]. Recent literature has showed that keeping apical patency would not generate more postoperative difficulties [5-7]. A recently issued in vitro study showed that intracanal delivery of cold irrigating solution at 2.5 °C with negative pressure flushing reduced the external surface temperature to close 10 °C [8-10], would be enough to create a local anti-inflammatory beneficial consequence in peri radicular tissues. Cryotherapy proposes that using cold over some procedures may decrease the diffusion of nerve signs, bleeding, edema, and local inflammation and is therefore effective in the reducing of pain. Therefore, the purpose of this research was to evaluate whether meticulous irrigation with three irrigating practices with different temperature would help in a decrease of post-endodontic pain.

Three expert endodontists with a private practice of 17 years and skilled in the procedures and procedures studied were included in the research and performed 40 RCTs each (a total of 120) in upper/lower front or back teeth with irreversible pulpitis recognized by pulp sensitivity testing with hot and cold. Pulpal response tests were achieved by the main author, and a digital X ray diagnosis was documented by three certified clinicians. Additional clinical necessities for patients´ inclusion were as follows: Necessities of the research were agreed and spontaneously accepted, healthy patients were included, teeth with enough coronal structure and diagnosed with vital pulps, no previous RCT, and no analgesics or antibiotic consumption 7 days before the procedures. A total of 120 of 135 patients (69 females and 51 male) aged 18 – 60 years were referred and integrated in this research, whereas 15 were rejected as not accomplishing the necessities wanted. All participants showed with a vital upper or lower molar, premolar or front teeth designated for conventional RCT for dental rehabilitation reasons.

Methods

Dental procedures

Root canal treatment was done in one visit. Topical anesthetic (Anesthesia Topical, Astra, Mexico) was used. Patients received 2 carpules of articaine 2% with epinephrine 1:200,000 (Septodont, Saint-Maur des-Fosses, France). Situations in which supplementary anesthesia was needed, intra-ligamental anesthesia (2mL articaine 2%) was supplied. For the upper front teeth, the solution was administered by tender and slow local infiltration. For the lower teeth, one of the carpules was used for the lingual and alveolar nerve block, the other one for a moderate bucal infiltration nearby the tooth to be treated.

Irrigation protocols

Group 6 °C. The R25 (size 25/ .08) instrument was employed in tinny and curved canals, and R40 files (40/ .06) were used in broad root canals. Three in-and-out pecking series were employed with a fullness of not more than 3mm until getting the calculated WL. Patients allocated to this group receive a final irrigation with 5mL of cold (6 °C) 17% EDTA followed by 10mL of cold (6 °C) sterile saline solution dispensed to the WL using a cold (6 °C) metallic micro-cannula.

Group 4: Canals were instrumented as in group A. Patients allocated to this set received a final irrigation with 5mL of cold (4 °C) 17% EDTA followed by 10mL of cold (4°C) sterile saline solution dispensed to the WL using a cold (4 °C) metallic micro-cannula for 1 minute.

Group RT: The R25 (size 25/ .08) instrument was employed in tinny and curved root canals, and R40 files (40/ .06) were used in wide canals. Three in-and-out series were employed with a space of not more than 3mm until getting the calculated WL. Reciprocal instruments were used in one tooth only (single use). Participants allocated to this control group were treated similarly to the experimental groups, except that they received a final flush with 5mL (room temperature) of 17% EDTA followed by 10 mL (room temperature) of sterile saline solution delivered to the WL.

Statistical analysis

The related issues preoperatively recorded were integrated into the examination as follows: age and sex, occlusal contacts, and maxilla or mandibular teeth. Changes in the strength of pain among groups were studied using the ordinal (linear) X2 test. Variances in VAS-recorded standards after 24, 48, and 72 hours and in the quantity of analgesic intake among the two groups tested.

Results

Table 1: Distribution by group of teeth and location.

Table 2: Kruskal/Wallis test applied to the post-endodontic pain.

Table 1 displays the distribution of variables; a total of 120 participants took part in this study: 69 (57.5%) were women, and 51 (42.5%) were men. The ages fluctuated among 18 and 60 years; 87 (72.5%) were upper teeth, and 33 (27.5%) were lower teeth. The clinical management of the patients is showed in Table 1. No significant modification (P > 0.05) between the groups was encountered concerning the grade or period of pain. Rendering to the VAS examination, marks were seen 24 – 72 hours late in the 3 groups with a significant decline successively (Tables 2 & 3).

Table 3: Kruskal/Wallis test applied to the post-endodontic pain.

Discussion

Pain is tough to comprehend and calculate especially when it occurs unexpectedly in patients. The major trouble in knowledge painful and discomfort is the participant’s individual valuation and its dimension. For this objective, organization of the estimation form has to be entirely understood by participants. In our research, a simple spoken classification was followed in the feedback procedure with four classes: no pain, slight, modest, and intense pain. These classes were clearly comprehended by participants and were described by the occurrence or nonappearance of the necessity for pain-relieving treatment. Preoperative pain is one of the main predictors of post-endodontic pain [11-14]. Thus, only teeth with irreversible pulpitis indicated for RCT because of prosthodontic purposes were treated in this research. In our research, we reduced the variation in the procedures following protocols based on recommendations by authors and manufacturers. While successful endodontic treatment depends on various variables, an important point to consider in the shaping of the root canal system is the amount of the irrigating solution. Proper disinfecting and filling the root canal system is facilitated by the keeping of its original shape from the entrance to the apical third, without any iatrogenic event.

Conclusion

According to the conditions established for this study, there was no statistically significant difference between the instrumentation systems assessed.

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Mipo - A Biological Method for Fixation of Complex Fractures of Tibia- Lupine Publishers

Surgery Open access journal| Lupine Publishers

Abstract

Introduction: Management of proximal and distal tibial fractures is challenging because of the complexity of injury, limited muscle coverage and poor vacularity. Surgical management of tibial fractures includes multiple options: external fixation, IM nailing, ORIF and minimally invasive plate osteosynthesis (MIPO). Open reduction and internal fixation with a plate may end in wide dissection and tissue devascularization. Fixation of tibial fractures with MIPO allows protection of soft tissue and blood supply. This is a report of a series of prospectively studied closed proximal and distal tibial fractures treated with MIPO.

Materials and Methods: A total of 20 patients with closed proximal or distal tibial or fractures were enrolled in the study between 2016 and 2017 and completed follow-up. Demographic characteristics, mechanism of injury, time required for union, range of motion and complications were recorded. Eleven patients had proximal tibial fractures and nine had distal tibial fractures. Patients were followed up to 2 and 6 weeks and then at intervals of 4 to 6 weeks until 12 months.

MResults: Mean age of the patients was 39.5 years (range 18-65 years). Thirteen cases were the consequence of high-energy trauma and seven were the result of low-energy trauma. The clinical and radiological outcome were excellent in 13 patients, good in 6 patients and fair in 1 patient. The average time for fracture union was 16.4 weeks, ranging from 16 to 20 weeks. None of our patients developed any complications.

Conclusion: MIPO is a reliable method of management for tibial fractures; it provides a high union rate and good functional outcome with minimal soft tissue problems.

Keywords: Tibial fractures, MIPO, Soft tissue, Locking plate, Osteointegration, Functional outcome, Clinical outcome

Abbreviations: MIPO: Minimally Invasive Plate Osteosynthesis, LCP: Locking Compression Plate

Introduction

Proximal tibial fractures are serious injuries and pose a treatment challenge. They usually result from high energy injuries, damage is usually extensive and open fractures, compartment syndromes, and vessel injuries are frequently related [1-4]. On the other hand, distal tibial fractures are typically the result of combined compressive and shear forces, and may end in instability of the metaphysis, with or without articular depression, and injury to the soft tissue [5]. The aim of fracture treatment is to obtain early fracture union in the most acceptable anatomical position with early and maximum functional return of activity. Conservative management of closed comminuted fractures with cast are associated with complications like prolonged immobilization, deformity, shortening, angulations, joint stiffness etc. Precise reduction by open reduction and internal fixation with conventional plate, frequently lead to complications like non-union, delayed union, infection, implant failure and need for bone grafting [6]. In addition, it also results in significant soft tissue stripping [3,7] The Minimally Invasive Plate Osteosynthesis (MIPO) is a technique which enables indirect reduction and stable fixation with minimal biological footprint and preserving vascular supply at the fracture site [3,8]. The aim of this prospective study was to evaluate clinical results and complications of MIPO in complex tibial fractures.

Materials and Methods

Case 1

Between 2016 and 2017, 20 patients with acute, closed proximal and distal tibial fractures were selected. Among them 11 had proximal tibial fractures and 9 had distal tibial fractures. The exclusion criteria included open fractures, tibial shaft fractures in which intramedullary nailing was done, fractures with neurovascular injury, and pathological fractures. There were 15 males and 5 females who consented to be a part of this study. Fractures were analysed preoperatively using radiographs and CT scans if there was any articular involvement (Figure 1). We prospectively gathered their data. Demographic characteristics, mechanism of injury, time required for union, range of motion and complications were recorded The Locking compression plate which are anatomically pre-contoured were used. Preoperative calculations were done on radiographs to ascertain the size of the plate, accurate size of locking, cortical and cancellous screws after subtraction of the magnification factor. Patients were operated on as soon as they were medically fit. Surgeries were performed under Spinal Anaesthesia. Patients were positioned supine on a radiolucent table. The involved leg was draped and a pneumatic tourniquet applied.

Figure 1: X-Ray showing the fracture of tibia and fibula at junction of proximal two-third and distal one-third.

An incision of 2 to 3 cm over the proximal aspect of the tibia on the medial or lateral sides was made, according to the fracture site. For distal tibial fractures, a straight incision was made at the level of the medial malleolus or antero-laterally with preservation of the vessels and nerves depending on the fracture site. Fractures were reduced indirectly by a reduction forceps under fluoroscopic guidance and by ligamentotaxis using an external fixator. A sub muscular plane was made with a long hemostat and chisel without causing additional periosteal damage. The locking plate was applied sub muscularly to fix the fracture. The plate was fixed with screws, which were inserted percutaneously through the primary surgical incisions. Reduction and position of the plate was checked under a C-arm (Figure 2). Postoperatively, the limbs were protected using an above knee splint for 2 days. Active and passive range of motion exercises were permitted to avoid knee stiffness and to strengthen the quadriceps. Suture removal was done after 2 weeks and non-weight-bearing walking was allowed for 6 weeks. Partial weight bearing was allowed depending on fracture healing and patient compliance. At week 12, full weight bearing was permitted as tolerated. At each follow up patients was assessed for pain at the fracture site, tenderness, range of movement at knee and ankle, operative scar and radiological union at 6,10,14,18 and 22 weeks. Clinical and radiological assessments based on the criteria laid down by S.J.LAM9 were recorded using a proforma. The criteria are as follows:

Figure 2: Post-operative AP and Lateral view showing the fracture with implant in situ.

a) Excellent: Range of movement of adjacent joints 80-100 % of normal. No pain in performing daily activities.

b) Good: Range of movement of adjacent joints 60-80% normal. Pain not enough to cause any modification of patient daily routine.

c) Fair: Range of movement of adjacent joints 30-60% normal. Pain enough to cause restriction patients daily activities.

d) Poor: Range of movement of adjacent joints less than 30% of normal. Pain enough to cause severe disability or non union.

Result

A total of 20 enrolled patients completed follow-up for one year and were included in the study; there were 15 males and 5 females. Mean age of the patients was 39.5 years (range 18-65 years). Thirteen cases were the consequence of high-energy trauma and seven were the result of low-energy trauma. Proximal tibial fracture was in eleven cases and distal tibial fractures in nine cases. Majority of the patients were operated within 3 days of injury. On S.J.LAM criteria, 13 patients showed excellent results, 6 patients had good results and 1 patient had fair result. The average time for fracture union was 16.4 weeks, ranging from 16 to 20 weeks (Figure 3). None of our patients developed any complications.

Figure 3: X- Ray showing radiological union at the fracture site.

Discussion

MIPO allows indirect fracture reduction and percutaneous sub muscular implant placement [3]. The minimal-invasive method agrees in theory to combine the principles of a stable construct to a closed reduction with conservation of the haematoma [10]. Contrast to conventional plates, the locking compression plate (LCP) is anatomically pre-contoured; it does not depend on on friction and provides superior stability. Thus, it reduces the risk of loss of reduction through locking screws secured in the plate. The key aspect that led.com to use locking plates to treat fractures of the tibial shaft was the difficulty we would have faced in controlling the reduction of the proximal or distal fractures with intramedullary nailing. Indirect reduction and subcutaneously applied plates protects soft tissue and periosteal blood supply; hence, MIPO should provide undisturbed union and a low complication rate. In spite of the advantage of well-preserved blood supply, MIPO does not help in reduction. The fragments may not be anatomically reduced and interfragmentary compression may be inadequate, which consequences in delayed union. Barei DP et al. [11] demonstrated that after open reduction and internal fixation, 20% of the cases developed superficial or deep infections in spite of acceptable functional outcome. In this study, all patients had radiological union without any complications. In the present study, early surgical intervention was performed. Majority of patients were operated within 3 days because delay in intervention makes fracture reduction challenging. The time to consolidation reported in our study was to some extent less, when matched to the data known from the literature concerning plating osteosynthesis [4,5]. The average time for fracture union in our series was 16.4 weeks. MIPO decreases postoperative pain and aids early rehabilitation, which improves articular cartilage nutrition and healing. It is cosmetically more acceptable owing to less scar formation [3]. Uniqueness of this study was early mobilization, high mean time to fracture union, which was 16.4 weeks, no complications and majority of the patients having excellent outcome. The current study has some limitations. This is a non-randomized study with no control group. In addition, the sample size was small and the mean follow up was for 12 months. In conclusion, MIPO is a reliable method of treatment for distal tibial fractures; it provides a high union rate and good functional outcome with minimal soft tissue complications.

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lupine-publishers-prjfgs · 5 years ago

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Enforcement of Regulatory Bodies to Trade Indian Medicinal Plants and DNA Bar-coding In Wildlife Forensics

Lupine Publishers| Journal of Forensic

Abstract

India's diverse topology and habitat flourishes near about 45,000 species of plants. Certain species among these plants are medicinal plant species which are endemic to India. These plants are valuable for their medicinal or aromatic properties. Due to India's biodiversity abundance, plants from the wild are wildly exploited resulting in over exploitation of medicinal plants. The present review is carried out to ascertain the rules and their regulations in various states of India which are presently saving the national heritage and helping officials to apprehend herb smugglers. The review also presents the data suggesting the present scenario of advancements in the field of wild life forensic genetics to deal with such cases and technical advancements in the field of plant/part identification during the confiscations in various states.

Keywords: DNA bar-coding; Laws protecting plants; Indian Forest ACT; Wild Life Protection Act; CITES Appendix

Introduction

Wildlife forensics uses scientific tools to confiscate criminals dealing with restricted wildlife products. In recent times, very less literature could be found on crimes related to plants which are likewise smuggled for their socioeconomic importance. Many newspaper headlines mention such trade happenings around the world [1]. Various national and international regulatory bodies are also actively regulating such trade practices. But, due to lack of awareness and limited research work on these aspects of wildlife forensics, herbal medicine industry is growing. According to traffic.org India and china ranked 3rd and 2nd respectively to export plant material used in traditional healing systems. Plants with medicinal values are majorly collected from the wild [2]. To regulate herbal plants in trade, organizations like CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) plays a major role by providing guidelines to the signatory bodies. CITES convention has provided three appendixes i.e. Appendix I deals with species which are threatened or near to extinction, Appendix II deals with the species which might become threatened in the coming future, Appendix III of cites deals with plant requested to be included under regulation by member signatory countries. CITES is dedicated to provide fortification to plants as well as animals worldwide [3]. CBD is a different biodiversity convention which utilizes Nagoya protocol that gives the ability to access genetic resources of the native country and also ensure fair and unbiased sharing of remuneration arising from their use of these assets between it signatory countries of the convention [4]. Laws protecting Non wood forest produce in India. According to Indian Forest Act, 1927, thirteen Chapters have been solely dedicated to the protection of wildlife flora. The law stated to facilitate the lawful and unlawful activities of forests, duties and powers to forest officers and penalties and punishments as per the state Government rules. The act defines itself by stating the following definition.

"Consolidate the law related to forest, the transit of forest produce and the duty livable on timber and other forest produce". The word "forest produce" include timber, charcoal, caoutchouc, catechu, wood-oil, resin, natural varnish, barck, ac, mahua flower, mahua seeds and myrabolams. The chapter 1 clause2 (4b) in the Indian forest act also gives the liberty to add the trees, leaves, flowers, fruits, grass, creepers reeds, moss and all parts of such plant produce and other parts or produce that are not directly mentioned in the act to be protected and preserved by same regulations prescribed by the act [5]. By describing the forest produce broadly, the Indian forest act take the responsibility to even protect the non-economical plants which are mentioned or not mentioned in the act [6]. Basically the non-wood forest produce is economically important produce of the forest other than wood, which is being exploited by the law breakers to earn the instant money [7]. NTFPs are the active ingredient of budding small scale and fully developed large scale industries to commercialize the products to earn profit [8]. The produce (medicinal herbs) is profitable forest produce which is often used by pharmaceutical industries to launch health related products in the market [9]. These companies or organizations have permits and licenses to collect the permitted plant varieties from the locals [10]. The local herb dwellers have certain powers given by state and center governments through Indian biodiversity act etc. to earn their living by using their traditional knowledge of gathering wild flora [11]. This lawful business turns illegal when these dwellers and industrial set up without prior permissions gather and motivate forest dweller to supply the produce in bulk [12]. With limited knowledge of endangered and threatened species restricted from wild collection or deliberate collection to earn instant money is the main reason of utilization of forest produces [13].

According to the chapter 2 of INDIAN FOREST ACT which defines the reserve forest area under section 28 clause (f) which states "Any person who fells, girdles, lops, taps or burns any tree or strip off the bark leaves from, or otherwise damages the same and clause (g) removes any forest produce shall be punishable with imprisonment for a term which may extend to six months or with fine or both in comparison to the damage [14]. Similar State Provision according to section 26. In this Bihar Sec. (26) the Forest range Offence is said to be cognizable and punishable with sentence for a maximum term of 6 months which may extend up to 24 years or with fine up to 5000/- or both + compensation[15]. In Gujarat Sec (26) of Indian Forest Act, punishment up to 1 year or with fine up to 1000/. In Himachal Pradesh according to Sec (26) punishment for forest offence is two years along with the fine of 5000. In Madhya Pradesh & West Bengal I year punishment is decided for the offender and fine of 2000 is imposed on such illegal activities. In Uttar Pradesh the maximum imprisonment for violating the rules of Indian forest act maximum punishment of 2 years or a fine of Rs. 5000/- or both is imposed on the offender when proved guilty for violation of sect. (26) (b), (h) (i) (j) but if the equivalent crime is committed second time and subsequently, sentence up to 2years or fine up to Rs. 20,000 but not less than Rs. 5000/- or both are imposed [16]. For the abuse other clauses of Sec. (26) imprisonment up to six month or a fine up to Rs. 1000/- or both is provided and for second and subsequent violation of these subsections is punishable by imprisonment up to six month or a fine up to Rs. 2000/- or both. Similarly, in chapter 3 which defines the protected forest areas under section 33 clauses is punishable with same penalty as is defined for the activities in reserve forest declared by the officials [17].

State Provisions According to Section 33

In Bihar state according to the section (33) imprisonment minimum of six months, maximum 2 of years fine minimum Rs. 1000/- maximum Rs. 5000/- imprisonment + fine both is availed to punish the convicted criminal but in case of Haryana:-15Sec (33) Imprisonment up to 1 year or with fine up to Rs. 1000 is imposed for the conviction in similar crime [18]. In Madhya Pradesh punishment lesser than Bihar Punishment is decided upto1 year or with fine up to Rs. 1000/ [19]. In Maharashtra punishment for violating Indian forest act rules is only up to 1 year or with fine up to Rs. 2000. Similarly in West Bengal Sec. (33) up to 1 year or with fine up to Rs. 1000 only. Uttar Pradesh is strict in terms of punishment for forest offences as their Maximum imprisonment of two years or fine up to Rs. 5000/- or both and for second and subsequent violation maximum term of 2 years or fine up to Rs. 10,000/-[20]. Wildlife protection Act. 1972 and the listed species Wildlife protection Act. 1972, SCHEDULE VI six plants are protected strictly by this Act which offers severe penalties to the offenders [20].The penalty is minimum 3 years which may extend depending upon the severity of the illegal activity and with fine of rupees 25000. If the convict is license holder the license will be cancelled and upon repetitive offence the penalty may increase up to 7 years [21]. Chapter ii of WPA outlines the protection of definite plants by declaring two clauses which are as follows:

a) Willfully pick, uproot, damage, destroy, acquire or collect any specified plant from any forest land and any area specified, by notification, by the Central Government.

b) Posses, sell, offer for sale, or transfer by way of gift or otherwise, or transport any specified.

Plant, whether alive or dead, or part or derivative thereof: Provided that nothing in this section shall prevent a member of a scheduled tribe, subject to the Provisions of Chapter IV, from picking, collecting or possessing in the district he resides any Specified plant or part or derivative thereof for his bona fide personal use." Further in section 17B of chapter 2 the law provides relaxation to educational institutions, researchers, scientific institutions to collect plant samples by acquiring the permission from chief wildlife warden [22]. The plants mentioned in this section are also prohibited to be cultivated without the prior permission from chief wildlife warden. The license is necessary to commercially cultivate the plants and sell them to the wholesalers. These rules are followed more strictly in protected and preserved forest areas or any such areas which are being declared protected by the government officials [23].

First species listed is Beddomescycad (Cycas beddomei) is native to trimulla hills of Andhra Pradesh. The male cones incorporate bioflavonoid which is used in the management of rheumatoid arthritis and muscle pains. It is listed in cites Appendix1 and Wild Life Protection Act 1972 Schedule 6 and CITES appendixes [24]. Blue Vanda (Vanda soerulec) is another endangered native plant of India which is found at 2000m- 3000m altitude. The plant is traded for its ornamental properties [25]. Kuth (Saussurea lappa) is list in WPA is also listed in CITES appendix 1. It is found at 2500 to 3000m Himalayas, Jammu Kashmir forest areas, Western Ghats, and kishenganga valley. It is traded by the name kuth kauri, kuth mithi. Basically the root of the plant contains Sesquiterpene lactones which are used to treat serious ailments like asthma, inflammatory diseases, and ulcer and stomach problems [26]. In India, only two genera namely Cypripedium and Paphiopedilum listed in Cites 1 and WPA schedule 6 can be found in Himalayan and NorthEast Indian region except one species of Paphiopedilum which is endemic to Western Ghats. This orchid specie is specifically traded for its ornamental and aromatic properties [27]. Pitcher plant's natural habitat is India and mostly originates in Jarain area of the Jaintia Hills, the Baghmara area of the Garo Hills of Meghalaya or in Assam.

The plant is exploited from the wild for its ornamental and medicinal use. It is enumerated in cites Appendix1 and Wild Life Protection Act 1972 Schedule 6 [28]. Red Vanda is listed CITES appendix 1 and schedule 6 of WPA is found in Meghalaya. It is being traded internationally for its ornamental properties [29]. Customs Act. 1962, regulates the import and export of India. Under this act, section 112 defines the penalties for improper import and export of any good which is restricted by any law in action at that present time will be confiscated and fine of rupees 5000 or value of goods whichever is higher will be imposed. According to the section 118 and 119 deals with smuggling goods and goods used to conceal the smuggling goods. The section give the power to custom officers to seal any import and export goods and goods which are used to conceal the smuggled goods which are brought out of the limits of custom area and custom ports[30,31].

International treaties to protect plants

a) International Treaty on Plant Genetic Resources for Food and Agriculture (IT PGRFA)

Secretary-General of the United Nations [32].

b) The International Tropical Timber Agreement (ITTA, 1983), International Tropical Timber Agreement, 1994 (ITTA, 1994 or ITTA2) [33].

c) Convention on International Trade in Endangered Species of Wild Fauna and Flora, also known as the Washington Convention) [34].

d) Convention on the Conservation of European Wildlife and Natural Habitats, also known as the Bern Convention (or Berne Convention), council of Europe [35].

e) Convention on Biological Diversity (CBD) Secretary- General of the United Nations [36].

f) Conservation of Antarctic Fauna and Flora Secretary- General of the United Nations [37].

Trade of restricted medicinal herbs

Wildlife trafficking have emerged with its medium from physical markets to online e commerce sites. Numbers of species are being sold on the web portals with dedicated websites, WebPages through face book, Instagram YouTube and many more social networking sites illegally just because these platforms provide greater anonymity to the seller. The plants which are being sold on these platforms are basically threatened rare or endangered [38]. EVAN MATI 307 medicinal products corresponding to ca. 283 species. Ten species out of 35 were found to be locally endangered and listed in IUCN [39]. The plants are also advertised online on daily bases and delivered without being securitized by the authorities. This may be due to lack of awareness on this issue specifically in India. Figure number 1-4 clearly shows such online business [40].

DNA Bar-Coding to Identify Plants in Trade

DNA bar-coding is presently quick and precise tool to identify plant species. Chloroplast genome off plant sequencing can convey a consistent barcode to accurately identify plants. The development of specific barcodes provides a new approach to distinguish closely related Species to tackle illegal trade of medicinal plants in trade [41]. Table 1 shows the ingenuities of Investigation work in wildlife forensics with respect to flora. The scanty research work in this area provide ample scope to scientist and researcher to explore this budding field of wild life forensics and establish barcode libraries specific to plant families or location specific libraries showing mutations if any [42-46].

Table 1: Shows the research work in the field of wildlife forensic

Conclusion

Due to India's biodiversity abundance, plants from the wild are wildly exploited resulting in depletion of important medicinal herbs. The illegal trade practices often go unnoticed because lack of awareness and lesser punishments as compared to the reimbursements involved to such trades practices. The punishments mentioned as such in the paper are demonstrating the lack of momentousness of law enforcement toward this serious issue. The Scanty literature on DNA Bar-coding provide ample opportunities of research in this untouched area of wildlife genetics which will directly benefit the law enforcement agencies to convict forest offenders

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lupine-publishers-prjfgs · 5 years ago

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Lupine Publishers | Induction of Systemic Acquired Resistance in Papaya by Foliar Application of HrpN Recombinant Protein for Increased Resistance against Papaya Dieback Pathogen

Lupine Publishers | Agriculture Open Access Journal

Abstract

Plants are continuously exposed to undesirable pest and pathogen threats. In response, plants have developed numerous mechanisms to protect themselves against the pathogens. Systemic acquired resistance (SAR) is an inducible disease resistance response in plant species. It is found in a large range of plant species including papaya and characterized by broad spectrum disease control and an associated coordinated expression of a set of pathogenesis related (PR) genes and proteins which are also known as SAR markers. Expression and purification of HrpN from Erwinia mallotivora, the causal agent of papaya dieback, was carried out. In this report, HrpN recombinant protein was tested and characterized for its effect and potential as elicitor that can increase papaya defence against E. mallotivora through the activation of SAR mechanism. Based on disease severity analysis, control plants which were untreated, showed faster disease infection rate and severity when compared to the recombinant protein treated plants. Increased resistance towards the papaya dieback pathogen was shown to be associated with increased expression of selected plant defined genes using quantitative Real Time analysis which were observed after the papaya was sprayed with the recombinant HrpN protein. Based on physiological and molecular analysis, the selected protein has induced SAR; increased selected SAR associated defence gene expression and increased the papaya resistance against the papaya dieback pathogen.

Keywords: Systemic acquired resistance; Recombinant protein; Erwinia mallotivora

Abbreviations: SAR: Systemic Acquired Resistance; E. mallotivora: Erwinia mallotivora; SA: Salicylic Acid; Hrp: Hairpin Proteins; PR: Pathogenesis-Related; MTI: MAMP-triggered immunity; ETI: effector-triggered immunity; SA: salicylic acid; BTH: Benzo Thiodiazole

Core Ideas

a) HrpN recombinant protein was tested and characterized for its effect and potential as elicitor that can increase papaya defence against E. mallotivora through the activation of SAR mechanism.

b) Increased selected SAR associated defence gene expression and increased papaya resistance against the papaya dieback pathogen observed.

Introduction

Papaya is a popular and commercially available fruit in the tropical and subtropical regions. It is highly known not only for its nutritious quality but also for its medicinal functions [1]. During its prime time, Eksotika, Sekaki and Solo were the Malaysia’s flagship varieties for export with an export value of about RM100–120 million per year, a total volume of 58,149 mt which accounted for 21% of the global trade in 2004 [2,3]. Papaya dieback disease caused by Erwinia mallotivora is the main cause for the rapid decline of Malaysian papaya production, amounting to 60% decrease in papaya production [4,5]. When attacked by pathogen, plants defend themselves through activation of plant defence mechanism which includes oxidative burst of cells, alteration in cell wall composition and de-novo synthesis of compounds like phytoalexin and elevated expression of pathogenesis-related (PR) proteins. The plant defence mechanisms include MAMP-triggered immunity (MTI), effectortriggered immunity (ETI) and systemic acquired resistance (SAR) signify different layers of active plant defence strategy [6]. Plants also have the ability to activate quantitative protection against extensive spectrum of microorganisms upon inoculation with a pathogen, exogenous application of proteins from microorganism or through application of chemicals [7].

The elevated resistance of the whole plant is known as SAR which is an inducible defence response present in a wide range of plant species including papaya [8]. Systemic plant resistance or Systemic acquired resistance involves a salicylic acid (SA)-mediated pathway of defence reactions within the plant [9,10]. During activation of SAR, induced plants showed an earlier boost of exogenous salicylic acid and activation of pathogenesis- related (PR) protein genes [11,12]. Production of PR genes/proteins can lead to increased resistance against pathogen attack [13,14]. Initiation and activation of PR proteins cascade can be produced by exposing the plant to a virulent, avirulent, and nonpathogenic microbe, or molecules with low molecular weight and sometimes volatile molecules such as salicylic acid and jasmonate [15- 17]. The inductions of SAR by using external inducers have been investigated in the past in plants such as tobacco [18] and Arabidopsis thaliana [19]. Incitation of defence reaction occurs not just at the establishment of pathogen recognition but additionally in distal regions of the plant and can last for weeks upon induction [20]. SAR is an effective innate immune response that offers protection against certain infection of pathogens. SAR may also be introduced by treating plants with salicylic acid (SA) and SA analogues; 2,6- dichloroisonicotinic acidity (INA) and benzothiodiazole (BTH) [21-23].

Phytopathogens are known to secrete proteins and virulence factors collectively known as effectors that are essential for pathogenesis and colonization of their host plants [24]. Pathogenicity of E. mallotivora depends on these effectors, which control the pathogen ability to cause disease and to elicit specific defence responses in papaya plants [25]. Erwinia mallotivora genome was already sequenced and bioinformatics tools were utilised to predict genes that potentially encode virulence factors and toxins along with other molecules that promote pathogenesis [26]. Like many other plant pathogenic bacteria, E. mallotivora contains type III secretion system (T3SS) that delivers effectors proteins into host plant. The T3SS apparatus is a key virulence determinant in many Gram-negative plant bacteria. Due to its importance, a lot of studies have been conducted to disable or block the function of T3SS by targeting known T3SS processes [27]. This could serve as a method to control plant microbial-associated diseases.

Effector proteins as virulence factors are known to suppress diverse signalling pathways required for plant innate immunity [28]. Apart from being effectors, some effectors proteins, known as hairpins, have been revealed to elicit plant defence and SAR responses [29]. Type III secreted hairpins are glycine-rich and also heat-stable proteins that are secreted from Gram-negative plantpathogenic bacteria. The hairpin proteins have been proven to elicit defence response and activate SAR for increased disease tolerance against diverse plant pathogens [23]. In selected cases, during fungial, oomycetal or plant pathogen attack, increased defence responses without the symptom exhibited by hypersensitive response cell death were recorded in plants treated with foliar application of hairpins proteins or genetically modified plants that constitutively expressed hairpins genes. This was observed in Arabidopsis after spray treatment with E. amylovora HrpN [30]. Activation of activated SAR in the plant conferred disease resistance to Hyaloperonospora sp. and Pseudomonas syringae pv. Tomato and in addition stimulated the expression of the pathogenesis-related (PR) 1 genes [31].

Another successful research finding includes reduced diseases caused by Phytophthora infestans and Botrytis cinerea in tomato through application of HrpN hairpin proteins [32]. Rice sprayed with Hpa1, another type of Hrp protein also showed strong resistance to X. oryzae pv. oryzae and Magnaporthe grisea [33]. Past studies have implied SAR strategy as another useful approach for controlling plant diseases through the activation of host plant defences by application of various agents or external inducers. Thus, this research aims to assess application of selected recombinant hairpin protein from the papaya dieback pathogen for SAR activation as an alternative new strategy to control papaya dieback disease. In an effort to develop recombinant proteins as potential SAR inducer, cloning and expression of HrpN from E mallotivora was carried out in a bacterial system. Our study is carried out to evaluate the effectiveness of HrpN recombinant protein in inducing Systemic Acquired Resistance (SAR) in papaya for enhanced disease resistance to papaya dieback pathogen.

Materials and Methods

Bacterial strains and growth conditions

Escherichia coli strains, Top10 (Invitrogen,USA) and BL21, were cultivated and grown in LB medium at 37°C respectively. Antibiotic ampicillin (Amp) was used at the concentration of 50μg/ml where required. Erwinia mallotivora was grown in LB broth at 28 °C.

Recombinant protein cloning, expression and purification

The HrpN gene was isolated from Erwinia mallotovora. Sets of primers, termed HrpN forward (ATGAGTCTGAATACGAGTCC) and reverse (GCCGCGTCAGTTTGCTTCGT) was designed to incorporate selected restriction enzymes sites to facilitate the cloning processes. Erwinia mallotivora DNA was isolated from E. mallotivora grown in LB broth at 28°C overnight using bacterial genomic extraction kit (Sigma Aldrich). Genomic extractions were carried out according to the manufacturer’s instruction. The polymerase chain reaction (PCR) was performed using the E. mallotivora DNA as the template and specific primers that target the HrpN region. Cycle parameters for PCR included an initial incubation time of 3 min at 95°C, 30 cycles of 30 sec at 94°C, 1 min at 55°C for annealing and 1 min at 72°C for extension, and followed by final elongation for 10 min at 72°C. The PCR products were visualised by agarose gel electrophoresis, gel-purified and cloned into pGEMT (Promega) according to the manufacturer’s instruction. Transformed cells were plated out on the LB plate supplemented with 100g/ml ampicillin and 20g/ ml X-gal to allow blue and white colonies selection. The gene was then subjected to sub clone into Pet-20b expression vector and transformed into BL21 E. coli expression strain. For expression of HrpN, the PET-20b/HrpN transformed bacteria were selected on LB agar plates containing 100g/ml ampicillin. A single colony of the transformed bacteria was inoculated in 5.0ml LB medium containing appropriate antibiotic for overnight cultivation at 37°C.

Aliquots of the culture were inoculated into 50ml LB medium with 50g/ml ampicillin at 37°C until the OD600 reached 0.5. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce the expression. The expression was carried out for six hours at 37°C and the bacteria were harvested afterwards by centrifugation at 2500g for 15 minutes. The bacterial pellet was resuspended in 20mM Tris-HCl, pH 8.0, and lysed with a sonicator or treated with Bugbuster reagent (Novogen). For confirmation analysis, lysate, soluble and insoluble fractions (pellet) from each expression were analysed by SDSPAGE and Western Blotting using specific anti-His antibody. For large scale purification, the expressed HrpN cells were treated with Bugbuster reagent (Novogen) to be lysed then purified using Ni-NTA column (His tag protein purification) via the Acta Prime Chromatography System. The purified proteins were quantified using Bradford protein assay and also analyzed by SDS PAGE followed by Coomassie Brilliant Blue Staining. For Western Blot analysis, 20μg of protein from each samples were separated by 10% SDS PAGE and transferred to PVDF membrane before being probed using anti-His antibody with gentle agitation for 2h and further incubated for 2h with anti-mouse IgG alkaline phosphatase conjugate. The membrane was washed, added with the substrate solution (BCIP/NBT) and incubated until the bands appeared.

Plant Growth

Carica papaya (Eksotika I) seeds were germinated in small polystyrene cups containing potting soil. In addition to these, two months old papaya seedlings were obtained from MARDI Pontian. Johor, Malaysia. Soil rich in organic matter and nutrients with a mix of compost was used. Fertilisation and watering were conducted accordingly. The plants were continued to be grown in the greenhouse at the MARDI glasshouse house complex under glasshouse conditions.

Recombinant protein application and pathogen inoculation

A set of formulation treatments and controls were tested for their effectiveness in inducing SAR and protecting the plants against papaya dieback disease. Protein inducer treatments were carried out in 4-6 months old papaya seedling using foliar spray application solution for each seedling for three times at one week interval. Each seedling was inoculated (at the first three nodes) with 10ml of pathogen (E. mallotivora) at the concentration of 1x106 cfu one week after the third inducer treatments. Water treated plants were included as control.

Symptom evaluation

After treatments with recombinant protein and the pathogen inoculation, effects of the pathogen inoculation were evaluated through disease severity (DS) statistical analysis. After treatments with salicylic acid (SA), effects of the pathogen inoculation were evaluated for disease severity. For disease severity, index 5 (on 1 to 5 scales) on each plant was recorded according to stem blackening and the mean value was calculated. For evaluation of stem blackening, they were recorded according to the scale of 0=symptomless, 1=leaf vein blackening, 2=leaf vein blackening and slightly wilting, 3=leaf stalk wilting, 4=stem blackening and 5=plant died. Data was analysed using analysis of variance (ANOVA) followed by comparison of means using Duncan multiple range test (DMRT) [34].

Tissue collection, RNA extraction and pathogenesis-related gene analysis via RT qPCR

For molecular analysis, leaves were collected on day 20 after the first recombinant HrpN foliar application, frozen in liquid nitrogen, and stored at -80°C until further analysis. Leaf tissue was grounded to a fine powder, and RNA was extracted using GeneJet (Thermo Scientific) kit following the manufacturer’s instruction. For qPCR, 2ug of RNA was DNAse- treated to remove genomic DNA contamination and the transcripts were converted into cDNA using Biorad Reverse Transcription in accordance with the manufacturer’s protocol. The resulting cDNA was used as the template for qPCR using primers designed based on known pathogenesis-related proteins in papaya [35]. Two housekeeping genes-actin and 40SRNP-were used as the reference genes for normalization of the expression fold. SensiFast SYBR Hi-ROX kit (Bioline, USA) was used for the RT-qPCR following the manufacturer’s protocol. The experiment was carried out using Bio-RAD CFX96 real-time PCR system (Bio-Rad,USA ). The expression profiling graph was plotted using the Bio-RAD CFX96 Manager software (Bio-Rad, USA).

Results and Discussion

Cloning, expression and purification of selected recombinant protein

In this report, we attempted to look for the after-effect of foliar spraying of a T3SS protein termed HrpN from E. mallotivora, the causal agent of papaya dieback disease in Malaysia. The likelihood of activation of disease resistance mechanism via SAR against the pathogen in papaya was investigated. Formerly, Peng [36] showed that hpa1 gene of Xanthomonus oryzae pv. oryzae enhanced defence responses to diverse pathogens in tobacco. For this study, cloning of HrpN from E. mallotivora was carried out to produce recombinant HrpN. In an effort to develop a bacterial expression system for selected HrpN from E. mallotivora, gene encoding HrpN was inserted into the pET-20b(+) bacterial expression vector from Novagen. Based on the restriction enzyme digestion and sequencing analysis, we have successfully cloned HrpN gene into pET-20b(+) expression vector. Expression of the HrpN gene in pET-20b expression vector was induced in the presence of isopropyl-beta- D-thiogalactopyranoside (IPTG). The proteins of the uninduced lysates, induced lysates, the soluble fraction and the insoluble fraction were analyzed using SDS–PAGE, stained with Coomassie Blue and Western Blot analysis. The molecular weight of the expressed proteins was equivalent to the predicted molecular weight of the HrpN which was estimated to be approximately 30kDa.

Recombinant HrpN proteins were expressed as fusion proteins with an N-terminal His tag, enabling affinity purification of proteins using Nickel NTA column. To obtain larger amount of expressed HrpN, the expression was carried out in large scale (2 litre cultures) and purified 35kDa 25kDa using the Ni-NTA affinity column via Acta Prime Chromatography System. After purification, the major contaminating bands and impurities were eliminated during the affinity chromatography process. 20ug of proteins from each fraction were analyzed using SDS–PAGE, stained with Coomassie Blue and Western Blot using the anti-His antibody. Figure 1 shows the chromatogram of elution fractions of HrpN using Ni-NTA column. The fractions containing the intended HrpN recombinant proteins were either freeze-dried or stored at -80ºC for further usage. Approximately 50-100mg of pure recombinant protein was normally obtained from every 2 litre cultures of LBBroth expression culture, induced with 0.5mM IPTG, 37°C for 2 hours. The amount of pure HrpN protein was sufficient for the foliar application treatment of papaya seedlings to determine the effect of SAR inducement for increased tolerance to papaya dieback disease.

Figure 1: SDS PAGE (a) and Western Blot (b) analysis of recombinant HrpN proteins obtained through large scale expression after purification via inclusion bodies prep and Ni- NTA affinity column.(M: Protein ladder, F1-F8 : Fractions collected after Ni-NTA affinity column).

Recombinant protein treatments and pathogen inoculation/ pathogen infection assay for SAR assessment

The HrpN recombinant protein was tested to evaluate its effectiveness in inducing SAR in papaya for elevated disease resistance response and to suppress the development of papaya dieback disease. The experiment consisted of 10 replicates for each treatment and control, and was conducted at MARDI’s Biotechnology & Nanotechnology infection house using 4 monthold papaya seedlings arranged in Randomized Completely Block Design (RCBD) with two control treatments. Tap water was used to water the plants daily. Standard fertilisation and pest control programs were applied for plant maintenance. Protein inducer treatments were carried out using foliar spray application solution for each seedling for three times at one week interval. Water treated plants were included as control. The effect of HrpN protein treatment on plant vigour was assessed beforehand for two months. However, no differences in plant height, stem diameter and root mass were observed between control and HrpN- treated plants indicating there was no effect of the treatments on the plant health. To assess the protein ability to increase papaya tolerance against the papaya dieback pathogen, inoculation of ~1x 108 E. mallativora was carried out on the treated seedlings three week after the first foliar spraying for the response to disease symptoms and inducer treatments. Disease development was supervised based on quantitative assessment by assessing percentage of Disease Severity (%DS). Disease severity treatment were computed based on the formulation below using the disease symptoms scoring of 0=symptomless, 1=leaf vein blackening, 2=leaf vein blackening and slightly wilting, 3=leaf stalk wilting, 4=stem blackening and 5=plant died. The disease severity index (DSI) was computed according to the formula described by Campbell and Madden [37] and Kim [34].

Where,

DSI=Disease Severity Index

Σab=Sum of the product of assessed plants with their corresponding score scale

N=Total number of assessed plants K=Highest score scale.

Three to four days after pathogen challenge, papaya dieback disease symptom was visually rated by assessing the percentage of disease progress for the disease severity assay until 25 days post infection. In general, the HrpN formulation showed a reduced degree of symptom compared to the water control in two repeated trials. Results presented here demonstrated that the formulation increased disease tolerance to papaya dieback as previously demonstrated. The disease severity assay (DS) measure was used to indicate the effectiveness of treatments in suppressing the disease. The disease symptom was shown to develop much slower in the seedlings treated papaya plants compared to positive control treatment. Both treated and control plants started showing the stage 1 symptoms of papaya dieback disease approximately on day 4. However, the disease severity percentages were observed more on control plants when compared to treated plants. Subsequently, the severities of disease in control plants increased rapidly with 70% severity on day 14 and continue to rise until day 24 where all controls were observed to succumb to the pathogen infection. Interestingly reverse effect was observed in treated plants. Although plants in both groups exhibited the stage 1 symptom approximately on the same day post infection, the disease severities were shown to decrease in treated plants post infection with the bacterial dieback pathogen. Although initial disease symptom of brown discoloration was observed at early days post infection, all of the leaves in treated plants that showed the early symptoms started to drop between day 6 to day 10 post infection, and new shoot continued to be produced. These resulted in the decreased of severity in treated plants as shown in Figure 2.

Figure 2: Disease severity analysis for assessment of recombinant HrpN as SAR inducer.

The analysis of the disease severity demonstrated that the treated papaya plants showed significantly lower disease severity compared with positive control treatments. Based on statistical analysis, there was highly significant relationship between control and the HrpN-treated plants. The obtained result clearly revealed that the HrpN was effective in increasing resistance against papaya dieback pathogen. The use of hairpin proteins from pathogen has been shown to increase the host against the intended pathogen. Choi [38] reported enhanced disease resistance to both X. oryzae pv. oryzae and Magnaporthe grisea in rice and Arabidopsis plants that were highly expressed with hpa1 gene. An elicitor, pemG1, a hairpin gene which was isolated from M. grisea was also shown to increase disease resistance in transgenic rice containing the hairpin gene. The expression of defence related phenylalanine ammonia-lyase genes were also observed [8]. Similarly, the HrpN formulation showed promising results in inducing SAR in papaya after the papaya seedlings were applied with the protein. The development of the disease symptoms was much slower when compared to positive control treatment. The result suggests that application of SAR inducers certainly has the potential to suppress the development of papaya dieback disease.

Real Time qPCR validation analysis

Analysis of defence mechanism can provide valuable details for papaya dieback disease management strategies. This will offer valuable information for the development of durable, economical, and broad spectrum management approach for the disease. Accordingly, to determine the effect of the formulations on the expression of selected papaya defence genes expression, leaves from papayas that were applied with formulations and control plants were taken from each treatment and control replicates. Each sample has at least three biological replicates representing different individual trees. All samples were ground and stored in -80°C freezer. RNA extraction method was carried out using Plant RNA extraction kit (Thermo Scientific) following the manufacturer’s instruction. Through this method, RNAs extracted were shown to be intact and had a high concentration (Figure 3). The RNAs obtained were transcribed and used for Real Time PCR analysis. Previously, Norliza [35] showed that several pathogenesis related genes which include PR-1b, PR 1, PR1d and NPR1 have the potential to be used as SAR markers due to the increasing levels of genes expression levels few days post treatment with known SAR inducers. These genes were used to investigate the plant defence response after application with the inducers.

Figure 3: RNA obtained from treated and control plants for validation with SAR markers via Real Time PCR. C1-C3 are control untreated plants while T1-T3 are plants treated with HrpN recombinant proteins.

Figure 4: Normalized fold expression of PR1d and NPR1 in control plant (non-treated) and recombinant HrpN protein treated papaya leaf tissues.

The expression profile of two defence genes, PR1d and NPR1 that were correlated with SAR inducements in papaya in control plant (non-treated) and recombinant HrpN protein treated papaya leaves tissues was conducted with Actin and 40snp used as the reference gene for normalisation [39,40]. As shown in Figure 4, the normalised fold expression of recombinant HrpN treated papaya leaves tissues were higher than the expression in control plant tissues. By using ΔΔCT method, the increased in expression fold of ~10 to ~20 of PR1d gene in papaya plants treated with HrpN recombinant proteins in comparison of each control were observed. Seemingly, the fold increased expression of NPR1 gene was also observed in plants treated with HrpN recombinant proteins in comparison of each control with fold changes around 3-5 fold (Figure 4). Salicylic acid (SA) is a vital hormone in plant immunity and NPR1 is a gene that is triggered by SA. NPR1 is known to be involved in the SAR activation for the regulation of plant defence genes. NPR1 has been shown to induce pathogenesis related (PR) proteins after pathogen attack such as by bacteria and fungi [41]. NPR1 which contains conserved ankyrin repeat domain, a broad complex, tramtrack, and bric-à-brac/poxvirus and zinc finger (BTB/POZ) domain is the master regulator of salicylic acid mediated responses. In Arabidopsis, NPR1 was shown to control the beginning of SAR and other immune signaling pathways for basal defence and mediating crosstalk between SA along with other phytohormones. During genetic screens for mutants defective in SA responses; mutants with defects in NPR1 failed to resolve various SAR-inducing treatments, displaying little expression of pathogenesis related (PR) genes and exhibiting elevated the likelihood of infections [42,43].

Interestingly, NPR1 shares similar structural features with mammalian immune cofactor IκB, that engages in crucial roles in inflammation, immunity, cell proliferation, differentiation, and survival [44]. Norliza [40] showed that a set of PR defence related proteins were not significantly expressed in E. mallotivora infected plants through iTRAQ and quantitative Real Time PCR. These data indicated that the expression of the selected PR genes were not high enough to protect the papaya from the pathogen attack. However, upon application of recombinant HrpN, the expression fold of PR1d genes was increased to ~10 to ~20 in three different treated plants. SAR marker gene pathogenesis-related gene 1 (PR1) which was isolated from Brassica juncea and named as BjPR1 also demonstrated elevated expression in leaves of B. juncea after Alternaria brassicae infection via Quantitative real-time PCR (qRT-PCR) analysis. Furthermore, BjPR1 gene was shown to be strongly induced following SA treatments, suggesting its roles in SAR mediated plant defence [45]. From the quantitative Real Time PCR analysis, it was suggested that the application of recombinant HrpN increased the plant defence related gene expression that are related to the SAR. Furthermore, the expression pattern of the selected genes has the potential to be used in the development of molecular markers for the identification of resistant cultivars or donor varieties for molecular breeding of papaya for increased tolerance or resistance against the papaya dieback pathogen [46].

Conclusion

Erwinia mallotivora HrpN was successfully cloned and expressed in the E. coli system. Foliar application of the HrpN recombinant protein was tested to evaluate its effectiveness in inducing SAR in papaya for enhanced disease resistance to papaya dieback pathogen. Phenotypic data was taken to see if there was any effect of the recombinant protein to the papaya plants. It was concluded that recombinant protein is safe to be used as SAR chemical inducer. Control plants, which were untreated, showed faster disease infection rate when compared to treated plants as shown by the disease severity assay. It can be concluded that for positive SAR inducement, recombinant HrpN is sufficient to enhance the defence system of papaya to combat papaya dieback disease.

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Lupine Publishers | Critical Analysis of the Definition of the Meter

Lupine Publishers | Journal of Forensic Sciences

Abstract

Defining the meter is an inavoidable step towards identifying the nature and properties of physical space. It turns out that the international definition of the meter raises some questionings, partly because of the vagueness and the inconsistency of the wordings.

Introduction

The Concept of Length

Given that length is a concept, we do not measure the length of an object, but we measure what separates its ends: the result of the measurement is called length of the object .

Origin of The Meter

The definition of the length unit is the object of an impressive diachrony, as it is testified in the Manuel des Poids et Mesures published by A. Tarbé in 1840.

The uniformity of weight and measures was wished since a long time. This whish of Charlemagne, Philippe-le-Long (1321), Louis XI, François I (1540), Henri II (1557), Henri III (1575) and Louis XV (1766) had found insurmountable obstacles, owing to provincial prejudices and mainly local habits. However, there are only few abuses as much inconvenient as the diversity of measures, because it introduces uncertainty and mistrust…

The Académie des Sciences has been mandated by the constituent assembly (France, 1789-1791) for determining the weights and measures units. The Academy used the quarter of the terrestrial meridian between the equator and the north pole for basis of the metric system […] the length of the quarter of the meridian was approximately 5,132,430 toises; and the ten millionth part of this arc was almost exactly equal to 3 feet 11 lines 44/100. The impatience to decide, pushed to decree that this would be the size of the provisional meter (Law of August First 1793).

The diachrony was not caused by time; instead, it was led by tenacious searchers.

A First Definition of The Meter

The law of 18 Germinal an III (April seventh 1795) stated:

Art. 2. There will have only one standard for Weights and Measures There will have only one standard for Weights and Measurements for the enter Republic; it will be a platinum rule on which will be inscribed the meter, which has been adopted as the fundamental unit for the whole system of measurements […] it will be registred at the legislature […].

Art. 3. A model in accordance with the reference prototype will be sent in each district capital […].

Art. 5. One will name: meter, the length measure equal to the ten-millionth part of the meridian arc between the north pole and the equator (Tarbé, p. 368).

The Law of 19 frimaire an VIII (December tenth 1799) retained the final value of the meter at 3 feet 11 lines 296 thousandths (Tarbé, p. 15). We can draft a first definition of the meter from these data: The meter is a concept which corresponds to the ten millionth part of the quarter of the meridian.

Proposal for a Speed Unit

In 1983, the Conférence Générale des poids et Mesures decided:

La vitesse de la lumière dans le vide c est égale à exactement 299.792.458 mètres par seconde .

The speed of light in the vacuum c is exactly 299,792, 458 meters per second.

The speed of ligth c is a fundamental constant which is currently expressed in meter per second, m s -1. We could replace m s-1 by cel, as international unit of celerity. Then the speed of light would read : c = 299,792s,458 cel.

Definition of the meter

The same Conference of 1983 gave the meter a new definition: Le mètre est la longueur du trajet parcourue dans le vide par la lumière pendant une durée de 1/299.792.458e de seconde.

The meter is the length travelled in the vacuum by the light during a duration of 1/299,792,458th of second.

This definition is questionable for three reasons :

a) In 1983, the words meter , length , duration and second had no consistent scientific definitions.

b) Use the meter to define the speed of light, then in the wake, use the speed of light to define the meter , is a truism There will have only one standard for Weights and Measurements for the enter Republic; it will be a platinum rule on which will be inscribed the meter, which has been adopted as the fundamental unit for the whole system of measurements […]it will be registred at the legislature […].

If we refer to definitions of time and space1, it’s a nonsense: Time is a concept corresponding to what separates two states of a system. Space is a concept corresponding to what separates two systems. According to these definitions, there is no relation between time and space. In 2011, the Conférence Générale des Poids et Mesures declared: Le mètre, symbole m, est l’unité de longueur ; son amplitude est déterminée en fixant la valeur numérique de la vitesse de la lumière dans le vide à exactement 299.792.458 lorsqu’elle est exprimée en unités SI m s-1.The meter, symbol m, is the length unit; it’s amplitude is determined by setting the numerical value of the speed of the light in the vacuum at exactly 299,792,458 when it’s expressed in m s-1 SI units. This definitions does not add any substantial information to the definition of 1983; space is still depending on time.The frequency of cesium is a constant of nature, une constante de la nature CGPM [1]; hence, the meter can be defined compared to the wavelenth of cesium, which is a fundamental constant : l = c/u = (299,792, 458 cel / (9,192,631,770 Hertz). cel/Hertz has the dimension of the international length unit, which is called meter : l = 0, 032612256 m. Therefore, 1m = 30, 663319 l. The meter is equal to 30, 663319 times the wave length of More serious, the wording makes the meter dependant on the second ; with an obvious consequence : space is forced to depend on time. If we refer to our definitions of time and space, it’s a nonsense:

Time is a concept corresponding to what separates two states of a system .

Space is a concept corresponding to what separates two systems .

According to these definitions, there is no relation between time and space.

In 2011, the Conférence Générale des Poids et Mesures declared:

Le mètre, symbole m, est l’unité de longueur; son amplitude est déterminée en fixant la valeur numérique de la vitesse de la lumière dans le vide à exactement 299.792.458 lorsqu’elle est exprimée en unités SI m s-1.

The meter, symbol m, is the lengthunit ; it’s amplitude is determined by setting the numerical value of the speed of the light in the vacuum at exactly 299,792,458when it’s expressed in m s-1SI units .

This definitions does not add any substantial information to the definition of 1983 ; spaceis still depending on time.

Next Paragraph “The frequency of cesium is a constant of nature, uneconstante de la nature (cf.CGPM – 2011) ; hence, the meter can be = c/ndefined compared to the wavelenth of cesium, which is nota fundamental constant :

= (299,792,458 cel / (9,192,631,770 Hertz)

cel/Hertz has the dimension of the international length unit, which is called meter :l# 0, 032612256 m.

Therefore, 1m # 30, 663319 l

The meter is approximatelyequal to 30, 663319 times the wavelength of cesium.

The Metric System

The law of July fourth 1837 made the metric system mandatory from January first 1840, in replacement of the traditional measures Tarbé [2].

Shortly before the end of World War II, an International Economic Conference was held between the 1st and the 10th of November 1944 in Rye in New York State: among various issues, it advocated the universal adoption of the metric system Céré [3] 6th part.

It is remarkable that Anglo-Saxon countries and the countries of their former areas of influence are still not willing to comply with these provisions: the two systems are still used, with the risk of serious confusions.

In this connection, let’s remind the decree of May 8th 1790 of the French National Assembly: […] the King (Louis XVI) will be begged to write to her British Majesty, and ask her Majesty to convince the Parliament of England to contribute with the national assembly in fixing the natural unit for measures and weights […] Tarbé [1].

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Lupine Publishers | Should Reactive Oxygen Species (ROS) in Human Body be Controlled with Antioxidant Supplement?

Lupine Publishers | Journal of Food and Nutrition

Abstract

Oxidative stress in human body might cause degenerative disease which is trigger by reactive oxygen species (ROS). Antioxidant in foods or in supplements offers an ability to reduce detrimental effect of ROS and free radicals in human. However, it can only be used to maintain human health rather than to cure disease.

Abbreviations: ROS: Reactive Oxygen Species, RF: Redox Factor, PTP: Protein Tyrosine Phosphatases, ATM: Ataxia-Telangiectasia Mutated, IRP: Iron Regulatory Protein, SH: Sulfihidryl, NOXROS: NADPH Oxidase-Derived ROS

Introduction

Nutraceutical or functional food industry become fastdeveloped industry nowadays. It is likely that consumers not only looking for healthy and nutritious food, but also food with functional benefits to their health. Nutraceutical and functional food products are widely available in the market, for example, isolated nutrients, dietary supplements, herbal products, or processed foods enriched with antioxidants. One benefit that is offered from those products is an antioxidant capacity, which can help reduce the reactive oxygen species in our body.

From the biochemistry perspective, Reactive Oxygen Species (ROS) in the human body can act as double-edged sword. It plays an important role in signalling molecule reaction, but it is also responsible for the oxidative stress that can lead to degenerative disease. This essay will focus on recent studies related to the two faces role of ROS in the human body. This review also will highlight the use of antioxidant supplement in human health.

What is ROS?

Reactive Oxygen Species (ROS) are “a group of small oxygencontaining free radicals that are extremely reactive due to their unpaired valence electron” [1].There are three reactive oxygen species (ROS) form, i.e. superoxide radical, hydrogen peroxide and hydroxyl radical [2]. Reactive Oxygen Species (ROS) can cause oxidation of cells and tissues and they usually unstable, highly reactive, and energized molecule [3]. Moreover, ROS in biological systems can be formed by prooxidative enzyme systems, lipid oxidation, irradiation, inflammation, smoking, air pollutants, and glycoxidation [3].

ROS and Cell Signalling

ROS formation in the human body as a result of natural consequences of aerobic metabolism. In normal conditions, ROS can act as immune system modulation and can activate various signal transduction pathways[2]. Figure 1 showed the critical function of ROS in human metabolism. For example, PI3 kinase and Protein Tyrosine Phosphatases (PTP) play an important role in cells proliferation and survival to growth, hormone and cytokine stimulation; Redox Factor 1 (Rf1) and Nrf-2 responsible for the antioxidant and anti-inflammation regulation; Iron Regulatory Protein (IRP) can affect the iron homeostatis, and Ataxia-Telangiectasia Mutated (ATM) can regulate the DNA damage response. Additionally, ROS can influence a number of growth factors such as angiotensin in smooth muscle growth [4]. NADPH Oxidase-Derived ROS (noxROS) can kill foreign organism as part of the immune defense system in a low and intermediate concentration [1].

Figure 1: Cellular signaling pathways regulated by ROS Source: Ray [8].

ROS and Diseases

ROS in the human body can cause detrimental effects such as alteration of membrane organization and functional loss of protein, enzymes and DNA, which eventually lead to various diseases and accelerated aging [5]. For instance, 8-hydroxydeoxuguanosine can oxidize DNA, protein oxidation might cause carbonyl modifications and loss of Sulfihidryl (SH) group from protein, ratio of redox couples such as glutathione: oxidized glutathione, NADPH:NADP+, and NADH:NAD+ will tend to shift to a more pro-oxidant value. It seems that the oxidation process is depending on age. Overload oxidative stress and abundant amounts of lipid peroxidation products can produce toxic and cause detrimental effects to biomolecules and generate pathogenesis oxidative stress-related disease such as, atherosclerosis, vasospasms, cancers, trauma, stroke, asthma, hyperoxia, arthritis, heart attack, age pigments, dermatitis, cataractogenesis, retinal damage, hepatitis, liver injury, and periondontis (Figure 2).

Figure 2: Figure 2: Clinical conditions involving reactive oxygen species. Source: Lee [3].

ROS and Antioxidant

Antioxidant levels are important in promoting health [6]. Antioxidant nutraceutical with 3 to 5 servings from vegetable group and 2 to 4 servings from the fruit group (USDA/CNPP 2000) can lessen the harmful effect of ROS and free radicals; thus, it might slow the aging process [3]. However, the used of antioxidant supplement in human is much more complicated than the simple reaction of free radical scavengers. Naturally, cells are equipped with antioxidant enzymes i.e superoxide dismutase, catalase, glutathione peroxidase, glutathione disulfide reductase, glutathione-stransferase, methionine sulfoxide reductase, and peroxidase[7]. It is likely that the used dietary antioxidant supplementation might disturb the mechanism of other natural antioxidants in cell. Furthermore Lee et al. [3] reported that an antioxidant can prevent lipid oxidation if the reduction potential of a free radical scavenger is lower than a reduction of potential PUFA (600mV). For instance, ascorbic acid (vitamin C) and tocopherol (vitamin E) which have lower standard 1-electron potential (282 and 480 mV, respectively) than PUFA 600 mV, can donate a hydrogen atom to peroxyl radicals of PUFA before PUFA does it. Likewise, Hensley et al. [6] noted that the concentration of α-tocopherol by 50 to 1000mg/ day can give advantages to human; although, it has not shown on epidemiological statistics. Apparently, natural antioxidants are not sufficient to control the oxidative stress in the human body that is why antioxidant supplement can only be used to enhance the cellular antioxidant capacity. Nevertheless, at some point antioxidants supplements might be unfavorable since they could diminish the adaptive signals of ROS. Chen & Niki [5] suggested that to utilize the lipid peroxidation products so that it can have beneficial effects. They proposed that the time, amount and site of their formation should be well-controlled and programmed [5]. Thereby, the used of exogenous antioxidant should be based on the bioavailability of the products. “Bioavailability can be defined as the amount of the percentage of an ingested nutrient that is absorbed and thus available to the body for metabolic use” [3].

Conclusion

In conclusion, it seems that antioxidant supplements can be used to control reactive oxygen species in human body. This essay shows that despite the fact that reactive oxygen species can initiate cell signalling, it might be endangered human life if it available in exceeds amount. Future research into the relation between each disease with reactive oxygen mechanism and the use of antioxidant to control reactive oxygen species in human body should be performed widely. It is proposed that antioxidant supplement should be considered as an aid to maintain a health. However, the lifestyle and eating behavior is highly important factor in reducing the oxidative stress which related to reactive oxygen species. Eating fruits, vegetables, spices, herbs and beverages such as wine, tea and coffee might balance oxygen reactive species in the body. Ultimately, since it is related to human life extension, antioxidant supplement should be considered as an appropriate food supplement.

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Anti-Synepthelichorial Placenta Scfv Library Construction_Lupine Publishers

Journal of veterinary Science| Lupine Publishers

Abstract

Hens were immunized with a macerate of goat and sheep placentomes. Immunizations were repeated every 15 days. Eggs were collected over a period of 03 immunizations, IgY isolated, and its concentration measured. After 45 days of the start of immunization, chickens were euthanized, their spleens isolated and fragmented for total RNA extraction by Trizol method. Performed the synthesis of cDNA, was amplified by PCR the fragments of immunoglobulin G avian variable light chain (VL) and heavy chain (VH). Fragments of VL and VH were used to constitute the final product by recombinant PCR-overlap process. The results present the feasibility of building a library of scFv anti goats and sheep placenta opening the possibility of manufacture diagnostic kits for various physiological situations of placental trophoblast cells belonging to these species.

Introduction

The developments of the techniques of molecular biology and genetic engineering have provided the emergence of recombinant DNA technology has enabled the production of several new molecules of immunoglobulin fragments Maranhão [1]. Such biomolecules are important for the development of studies in the fields molecular biology, medicine and pharmaceuticals, and also in developing treatments for various types of diseases and diagnoses Seo et al. [2],Lynch et al. [3], such as cancer and infectious diseases Baert et al. [4]; Rocha et al. [5];Bumrela [6]; Huber et al. [7],therapies to combat rheumatoid arthritis and osteoarthritis Hughes et al. [8], against smoking Bevins et al. [9]; Peterson et al.[10];fighting drugs like methamphetamine Peterson, et al. [10] and cocaine Dickerson &Janda [11]; combating the sequelae of alcoholism Bagasra et al. [12]; combating Alzheimer’s Boado et al. [13]; Nisbetet al. [14]; detecting myocardial injury related Marti et al. [15]; diagnosis of pregnancy Payne Jr et al. [16]. Antibodies resulting from manipulation and recombination of variable fragments single chain (scFv) immunoglobulin, have been used in therapeutics and diagnostics, offering advantages over conventional antibodies Malecki et al. [17]; Wilkinson et al. [18]; Monnier et al. [19].

In ruminant animals, possessing synepthelichorial placenta (Igwebuike [20]; Pinto et al. [21]), the activity of trophoblast cells, especially the trophoblast giant cells, are related to the process of implantation of the conceptus and training of the placentome Pinto et al. [21]; Ealyet al. [22] and directly related to the recognition of the fetus by the maternal organism Shi et al. [23]; Roberts, [24]. This cellular activity induces the production and secretion of various specific molecular structures that are temporarily expressed during pregnancy Miguez et al. [25]; Machado et al. [26], but not all are known or have been isolated. Detection of these antigens or chemical markers is of great importance for breeding ruminant animals and especially for a greater understanding of the physiology and gene expression of trophoblastcells synepthelichorial placenta Roberts, [24]; Roberts et al. [27].

It is presented in this context, this work has as objective aconstruction of gene libraries of single chain variable fragments (scFv) antibody chicken (Gallus gallusdomesticus) placentome anti-ruminant animals and also to contribute to the creation of new knowledge on trophoblastcells and their effects on reproduction in animals synepthelichorial. These gene libraries will be the basis for future work, we will focus on the expression of recombinant biomolecules, which can be selected by affinity for the specific trophoblast cells in goats and sheep placentomes molecular markers.

Materials and Methods

Immunization of animals and collection of samples

Used in this study were macerated placentomas of female goats and sheep consistent with the first third of gestational development. The immunization was performed via the deep pectoral muscles at three Lhomann brown laying hens of 28 weeks old. Immunizations took place every fortnight, the first immunization was performed with the filtrate 200 mg macerate placentome goats and sheep (GSP - Goat and Sheep Placentomes) plus complete Freud’s adjuvant, 1:1. Over two immunizations were performed with the use of incomplete Freud’s adjuvant, under the same conditions for the first immunization. In the process of construction of the gene library of goat and sheep placenta antiscFv mixture of macerated placentome of female goats and sheep was used, this procedure was done this way because of the great phylogenetic closeness existing between these two species. Fifteen days after the third and final immunization, totaling a trial period of 45 days, all chickens were anesthetized using a 5:1 ratio of Ketamine (100mg/ml or 50mg/kg)and Xylazine (20mg/ml or 10mg/kg ) following the animals were euthanized by ensanguinação. The spleens of these animals were isolated, identified, and stored at -20 °C for future total RNA extraction. During the immunization eggs were collected and separated to the extraction of Y (IgY) immunoglobulins performed by using the commercial kit EGGstract ®IgY Purification System (Promega Corporation, Madison, USA), with subsequent measurement of the concentration by the method of Bradforf Bradford[28] and purification of IgY by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE).

Extraction and isolation of total RNA

Three fragments of spleen, 50mg, referring to the three chickens were used for extraction of total RNA by methodology TRIZOL reagent (Invitrogen, USA). The quantification of the concentration was carried out in a spectrophotometer (Power Wave NS2 BioTek Instruments, Inc.) at a wavelength of 260nm.

Construction of cDNA molecules and the light chain (VL) and heavy chain (VH)

The cDNA of the immunoglobulin G (IgG) was synthesized by using kit SuperScriptTM III Reverse Transcriptase (Invitrogen, USA) and dNTP Set (Fermentas Life Sciences, Maryland, USA), according to the manufacturer’s instructions. Primers used for amplification of the variable regions VL and VH were prepared by BarbasIII et al. [29].VL regions of the immunoglobulins were amplified using the sense primer CSCVK-F (5’-GTG GCC CAG GCG GCC CTG ACT CAG CCG TCC TCG GTG TC-3’); and antisense CKJo-B (5’-GGA AGA TCT AGA GGA ACC CTG TAG GAC GGT CAG G -3’). For amplification of the VH region, we used the sense primer CSCVHo-F (5’-GGT CAG AGA TCC TCT TCC TCT GTG GCC TTG ACG GAC GAG -3’) and antisense CSCG-B (5’-GCC CTG GGC CTG GCC ACT AGT GGA GAT GGA GAC GGT GAC TTC CC-3’) (Eurofins MWG Operon, Alabama, USA). The cDNA samples were pretreated with RNase A 5U/μl (Thermo Fisher SCIENTIFIC, USA), and the PCR reactions were designed to 50ng of each sample Barbas III et al. [29]. Considering the final volume of 50μl per amplification reaction, this was performed using 5U/ μl Taq DNA polymerase (Fermentas LIFE SCIENCES, Maryland, USA); 1 mM MgCl2; 2 mMdNTP Mix (Fermentas LIFE SCIENCES, Maryland, USA), 10x Taq buffer with KCl (100 mMTris-HCl pH 8.80, 500 mMKCl/Fermentas Life Sciences, Maryland, USA), 30pmol/ μl of sense and antisense primer and free water RNA and DNA in order to complete the amplification reaction 50μl. The buildings of the fragments VL and VH were made separately and each using their specific primers.

The programming of the recommended thermocycler followed by Barbas III et al. [29] beginning with an incubation at 94°C for 2 minutes. Compounds followed by 30 cycles by denaturing at 94°C for 02 minutes, annealing at 56°C for 15 seconds and extension at 72°C for 90 seconds, and the completion of the reaction cycle at 72°C for 10 minutes was used. The control reaction was performed by electrophoresis in 1% agarose gel and the result was further documented by using photodocumentator.

Construction of single-chain variable fragments (scFv) by overlap-PCR

The bands of fragments VL and VH were extracted from the agarose gel and purified separately with the aid of MinElute Gel Extraction Kit (QIAGEN). Following extraction of these products were further purified by QIAquick PCR Purification Kit (QIAGEN) and stored at -20°C. The Overlap-PCR is define the connection (or overlap) between fragments of the chain light (VL) and heavy (VH), constructed by forming a flexible peptide linker (VL-linker-VH) comprising the complementary tails of the connection between the sense primer (CSCVHo-F) heavy chain and anti- sense primer light chain (CKJo-B) used for amplification of fragments. The primers for the construction of scFv molecule of this study followed the description Barbas III et al. [29]: sense primer CSC-F (5’-GAG GAGGAGGAGGAGGAGGTG GCC CAG GCG GCC CTG ACT CAG -3’) and antisense primer CSC-B (5’-GAG GAGGAGGAGGAGGAGGAG CTG GCC GGC CTG GCC ACT AGT GGA GG-3’) (Eurofins MWG Operon, Alabama, USA). The final product was characterized Overlap-PCR with approximately 750 base pairs (bp).

The Overlap-PCR reactions were drawn for each sample 50ng of fragment VL and VH Barbas III et al. [29]. The PCR fragments overlap VL and V H has been designed to use 5U/μl Taq DNA polymerase (Fermentas LIFE SCIENCES, Maryland, USA) 1 mM MgCl2, 2 mMdNTP Mix (Fermentas LIFE SCIENCES, Maryland, USA), 10XTaq buffer with KCl (100 mMTris-HCl pH 8.80, 500 mMKCl/Fermentas LIFE SCIENCES, Maryland, USA), 30pmol/μl of sense and antisense primer and free water RNA and DNA in order to complete the reaction 50μl Overlap-PCR.

The thermocycler was programmed for Overlap PCR reaction so that the activation reaction was carried out at 94°C for 60 seconds, following 30 cycles were performed 03 times with each cycle, the first time 2 minutes 94°C, the second at 56°C for 60 seconds and a third at 72°C for 90 seconds, and the completion of reaction was conducted by one cycle at 72°C for 10 minutes. The control reaction was performed by electrophoresis on a 1% agarose gel and the results were documented through the use of photodocumentator.

Statistical Analysis

To compare the means of IgY the mixture of macerated GSPTukey’s test at 5% significance level was applied, using the statistical program ASSISTAT, version 7.6 beta/2011.

Results

Measurement of immunization

Measuring the success of immunization with GSP mixture was performed by assessing the concentration (mg/mL) of purified IgY from the yolks of immunized hens. Immunization with GSP solution showed average concentrations of 2.89±0.12, 3.90±0.15 and 4.24±0.21 mg/mL, respectively for the 1st, 2nd and 3rd immunization. Immunizations performed with the mixture of macerated GSP showed the linear regression equation y = 0.195 x + 7.74 and a coefficient of determination (R2) equal to 0.9916 (99.16%), indicating an excellent fit for the relationship between immunizations and production of IgY performed, ie more than 99% of the production of IgY was established directly by increasing the number of immunizations with a mixture GSP.

Figure 1: Purification results of immunoglobulin Y (IgY) anti-goat and sheep placentames in different stages of immunization. Polyacrylamide 6% acrilamide gel electrophoresis.MPM, molecular weight marker (kDa).

In the purification of IgY by electrophoresis on 6% polyacrylamide (SDS-PAGE) gel was identified IgY approximately 212 kDa (Figure 1). We can observe an increase in the thickness of the band related to immunoglobulins, thus confirming higher levels of concentration of IgY, which corroborates with the increased concentration of IgY.

Statistical Analysis

The data in (Table 1) corroborates the mixture homogenized with the GSP, in the course of three immunizations promoted increased immunogenicity and consequent increase in the production of IgY as a response. This analysis was provided by the use of the Tukey test at a significance level of 5%.

Table 1: Values followed by the same letter do not differ statistically among themselves. We have used the Tukey test at 5% significance level. (MSD = minimum significant difference; GM = general average; CV% = coefficient of variation in %).

Figure 2: Amplification results of the light chain (VL) and heavy chain (VH) scFv fragments of chicken Immunoglobulin G (IgG), anti-goat and sheep placentome 1% agarose gel electrophoresis.

Amplification product of the cDNA of light chain (VL) and heavy chain (VH) scFv chicken immunoglobulin G

Referring to immunization with the macerated GSP after running on 1% agarose gel, the image was identified the presence of VL bands with 350pb and VH bands with 400bp (Figure 2).

Product overlapping sequences of scFv fragments by Overlap-PCR

Also referring to mixing with the mash GSP, after Overlap PCR reaction, the formation of fragments with approximately 750pb has been identified as a result of overlapping fragments and linearization VL and VH.

Discussion

In this study, chickens were immunized with homogenized in ruminant animals placentomas more Freund’s adjuvant. As expected result was a DNA library of monoclonal antibodies (goat/sheep anti-placental) scFv fragments of immunoglobulin G from total RNA extracted from leuocyte cells from the spleen of chickens. The methodology used in this study was efficient and effective in the development and construction of goat/sheep antiplacentomescFv library. The synthesis of the molecule overlapped scFv anti-placentome placenta caprine/sheep presented as result a band of 750 bp (Figure 3), equivalent to that described by Barbas III et al.[29], Roldanet al. [30].

Figure 3: Overlap results of the light chain (VL) and heavy chain (VH) scFv fragments of chicken immunoglobulin G (IgG) anti-goat and sheep placentome (GSP). 1% agarose gel electrophoresis.

With respect to the effect of immunization and production of IgY, experimental procedures and similar results were reported by Gassmann et al. [31], in which chickens were immunized with 20 to 30 mg of mammalian protein added to Freud’s complete adjuvant. The results of 62 eggs extracted total amount of 4.0 grams of IgY and obtained as a final yield of approximately 130mg specific for a protein of mammalian cell cycle regulatory antibodies. Using the immunoblot technique identified that 20 days after immunization, IgY antibodies appeared, reaching a maximum production within 30 days after first immunization, maintaining a good level of production until 81 days, when the average was 72mg of IgY per yolk. These researchers obtained at the end of the experiment the mean concentration with standard deviation of7.44 ± 0.27 mg of IgY/mL of yolk. For Meenatchisundaram et al. [32], IgY concentrations ranged from 0.85 to 7.60 mg/mL of egg yolk, throughout the immunization period. Already to Pauly et al. [33] variations IgY were between 3.0 to 8.0 mg/ml of yolk. Chui et al. [34] reached a mean value of 5.75 mg of IgY per ml of egg yolk immunized as Barbas III et al. [29] had concentrations range from 55 to 80 mg/egg yolk and 75% purity using the same extraction kit used in this study. In this study, average concentrations of finals were obtained 3.68 ± .16 mg of IgY/mL in the yolk. One advantage of the IgY antibody production is that the required amount of antigen for high and long lasting for IgY yolk of immunized hens titration would be very low compared to immunization of other animals such as the rabbit Song et al.[35]; Hatta et al.[36]. When the result of IgY extract was applied to 6% polyacrylamide gel was characterized by bands at approximately 212kDa molecular weight. The literature describes this mass ranging from 180kDa immunoglobulin Meenatchisundaramet al. [32]; Warret al. [37]; Chalghoumiet al. [38] to 220 kDa Hamada et al. [39].

Andris-Widhopf et al. [40] have also obtained cDNA fragments of approximately 750pb as a result of Overlap PCR reaction scFv fragments. These researchers used chickens immunized with a conjugate of bovine serum albumin (BSA) plus fluorescent and hapten reported as a result of the joining VLand VH, comprising the formation of a linker sequence (VL-linker-VH). They also emphasized that the VL-linker-VHproduct, corresponds to the scFvregion of immunoglobulin, has good applicability for use as befits its use with vector transfection.

In the bursa of Fabricius, the level of the complementarity determining regions (CDRs) of scFv fragments (light and heavy chain) derived from immunoglobulin G (IgG) from chicken and poultry in general, such mechanisms occur: the gene rearrangement and gene conversion somatic hyper-mutation. These mechanisms promote increased variability in this gene fragments due to the incorporation of genes pseudo and direct, acting in the maturation cell of B lymphocytes McCormack & Thompson, [41]; Paramithiotis et al. [42]; Arakawa [43]. Thus the Overlap PCR allows the tool the final product, or the scFv fragment, a great increase in respect of gene variability, since it favors the increase of the combination of variable gene fragments, allowing the number of clones from a library of scFv generated by this method reaches to 1012units Barbas III et al. [29].

cDNA libraries are indispensable to analyze the expression and function of genes and also in the study of protein function, this expression products. Hese information can be of great importance in understanding when and how certain genes are expressed in an cell type or organism. The continuous progress in the development of constitutive techniques of scFvgene libraries and the interest in their applications suggest that these tools will continue to be an important factor for the development of new recombinant biomolecules.

Conclusion

This study was consolidated construction of gene library as a viable method for the construction of scFv recombinant biomolecules. The construction of gene libraries of recombinant scFv fragments of immunoglobulin G (IgG) chicken has great potential for use due to its high genetic variability, promoted by the mechanisms of gene rearrangement, gene conversion and somatic hyper-mutation occurring in fragments of varying chain light (VL) and heavy (VH). Thus, scFv fragments may be linked chicken techniques of molecular biology generation of biomolecules with high target-binding affinity. Caprine and ovine anti-placenta obtained by Overlap-PCR in this study biomolecules can be used to identify physiological aspects of trophoblast cells of caprine and ovine placenta, thus developing the function of these cell typespecific molecular markers.

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Lupine Publishers | Genetic Variability Studies for Economically Important Traits in Sunflower (Helianthus Annuus L.)

Lupine Publishers | Agriculture Open Access Journal

Abstract

The research was conducted in the research area of Department of Plant Breeding and Genetics (PB&G), University of Agriculture Faisalabad (UAF), Pakistan during spring 2014. The triplicated research trial was laid out following randomized complete block design (RCBD). Fifteen lines of sunflower were studied for genetic variability and association of economic traits with seed yield. The data were recorded on quantitative traits i.e. days to 50% flowering (DFF), plant height (PH), number of leaves per plant (NOL), head diameter (HD), leaf area (LA), filled achene (FA), 100 achene weight (100AW), oil contents (OC), achene yield per plant (AY/P) and qualitative traits i.e. head angle at maturity (HA), achene color(AC). The recorded data were subjected to analysis of variance, correlation and path coefficient analysis. The accessions were highly significant for all traits under study. The accessions A-2.2 showed good performance for most of studied traits. The accession B-3.1 also show better performance followed by A-4.3 and A-7.10. Genotypic correlations are higher than phenotypic correlations. 100AW, NOL, LA and HD had positive and highly significant genotypic correlations with AY/P. Genotypic correlation of AY/P was significantly negative with PH, DFF and oil contents. LA, NOL, 100AWand OC showed direct positive effect on AY/P. DFF, PH, HD and FA had negative direct effect on AY/P.

Keywords: Variability; Correlation; Path coefficient; Quantitative; Quality related traits

Abbreviations: PB&G: Plant Breeding and Genetics; RCBD: Randomized Complete Block Design; PH: Plant Height; NOL: Number of Leaves; HD: Head Diameter; LA: Leaf Area; FA: Filled Achene; AW: Achene Weight; OC: Oil Contents; AY/P: Achene Yield per Plant; HA: Head Angle; AC: Achene Color

Introduction

Oil and fats are major part of human diet, important source of energy and also play role in carrying different vitamins in body. Due to the rapid growth in population of Pakistan, requirement of edible oil has also been raised. So a gap is produced between utilization and production in Pakistan. Although the country is making huge improvement in agriculture sector but still there exist extreme shortage of edible oil. During 2015-2016 Pakistan fulfilled 17% its requirement of edible oil by domestic oil and 83% by imported oil. Both conventional and non-conventional oilseed crops are important to fill the space between utilization and production.

Sunflower (Helianthus annuus L.) belongs to non-conventional oilseed crops in Pakistan and has great ability to increase oil yield per hectare in Pakistan [1,2]. Sunflower being the 2nd most important oilseed crop of the world was introduced in Pakistan during 1960’s. Sunflower is growing twice a year in Pakistan as a major edible oilseed crop. It is grown on 0.214 million acre with 0.92 million tons seed production and 0.035 oil production. Government of Pakistan 2015-2016 [3]. Sunflower seed contains 40-50% oil contents [4]. Sunflowers commercially available varieties contain 39 to 49% oil in their seeds.

The major part of oil comprises of unsaturated fatty acids (oleic acid 20% and linoleic acid 69%), with the remainder 11% are saturated fatty acids (palmitic and stearic fatty acids) Mirza [5]. Sunflower oil is considered as premium quality oil because of its taste, high smoke point, beam colour, good nutritional quality, elevated level of unsaturated fatty acids and lack of harmful/ unstable components like erusic acid and linolenic acid. It has the sturdy oxidative stability and alpha-tocopheroles are the source of vitamin E which is anti-inflammatory agent [6]. Variations are very precious for any breeding programs [7,8]. Genetic variations existing in the genotypes can be exploited efficiently as genetic resources in breeding programs [9] Success of breeding program rely upon the extent of variation present in a germplasm for yield and yield contributing traits [10]. Correlation analysis demonstrates the association of the economically important traits with achene yield. Path coefficient analysis facilitates the division of correlation coefficients into its direct and indirect effects of economic traits on achene yield [11]. It quantifies the association of different yield components and direct or indirect effects on seed yield [12, 13].

The ultimate objective of the sunflower breeders are to increase quantity and improve quality of the oil.

Materials and Methods

The research work was performed in the research fields of Department of Plant Breeding & Genetics, University of Agriculture Faisalabad, Pakistan. Fifteen accessions of sunflower were studied as listed in Table 1. The experiment was conducted in RCBD with three replications in spring 2014. Dibbler method was used for sowing of these accessions. Row to row distance was kept 0.75m and plant to plant 0.25m. Agronomic practices were kept constant during the experiment. The data was recorded on five randomly selected guarded plants per replication of each accession for DFF, PH, NOL, HD, LA, FA, AY/P, 100AW, OC and qualitative traits i.e. HA, AC. The recorded data were analyzed for variance following the method by Steel [14]. Correlation analysis proposed by Kwon [15] and path coefficient analysis by Dewey and Lu 1957 [16] were performed for determining the association among different morphological characters and their direct and indirect effects on seed yield.

Table 1: Sunflower accessions used in the present study.

Results and Discussion

Genetic variability studies for quantitative and quality related traits

Presence of genetic variability is prerequisite for the development of new hybrids and varieties. ANOVA of different characters is presented in Table 2. Mean squares of different characters showed that accessions had noticeable differences for all the traits under study. The mean comparisons of accessions for different characters are presented in Table 3. DFF, PH,NOL, HD, LA, FA, AY/P, 100AW and OC ranges from 75-90 days, 158.17- 182.56cm, 15-32 leaves, 7.76-15.94cm, 20.04-37.15cm², 497-677 achene, 20.29-38.96g, 1.50-6.58g and 41.20-34.33% respectively. Noticeable differences were present in the accessions and selection of various economic traits may be helpful in the development of new hybrid combinations.

Table 2: Mean squares of sunflower accessions for various plant characters.

Table 3: Mean comparisons of sunflower accessions for various plant characters.

The observed results were in agreement with the results observed by different researchers [5,7,10,17-20]. Among studied accessions, A-2.2 showed good performance for NOL, HD, FA, 100AW, OC and AY/P. The accession B 3.1 showed good performance for NOL, LA, OC and AY/P. A-4.3 can be incorporate in breeding program to develop short duration, short stature high yielding hybrid. So, this breeding material can be used in breeding program for the development of short duration high yielding hybrids with short stature. Short heighted accessions may be helpful in the breeding of lodging resistant hybrids as lodging is one of the major issues of this crop. Accession A-7.10 has highest OC with good seed yield and 100AW. This accession may also be the source in production of high oil yielding hybrids.

Quality Determine Traits

Head angles of sunflower accessions are presented in the Figure 1. All the accessions showed different percentages of HA i.e. 135°, 180°, 45° and 225°. Most of the lines had three types of angles i.e. 180°, 135° and 45°. Only line A-14.13 had 180°and 135° head angle. But lines C-2.11, A-2 .5, A-4.3, B-3.1, A-7.10, A-10.1.2, A-11.1.2 and A-2.2 also showed head angle of 225°. The observed achene color for the accessions is presented in Figure 2. All the accessions possessed different percentages of achene color. All the accessions had maximum percentage of grey, black and light grey achene colour. Only line B-3.1 had black and grey achene colour. But accessions C-2.11, A-14.13, A-7.10, A-10.1.2, A-2.19 and C-2.17.1 showed a little percentage of white color achene.

Figure 1: Percentage of plants sunflower accessions for head angle.

Figure 2: Percentage of plants sunflower accessions for Achene colour.

Correlation and path coefficient analysis

Genotypic and Phenotypic correlation coefficients among quantitative traits in 15 genotypes of sunflower are showed in Tables 4 & 5 respectively. Phenotypic correlation was lesser than the genotypic correlation. This showed that expressions of traits are mainly governed by the genetics of accessions and environment had little influence on them and selection can be fruitful for the improvement of breeding program [5,10,19,21-23]. Also discussed in their results that genotypic correlation is higher than the phenotypic correlation. 100AW (0.6879), NOL (0.501), LA (0.580) and HD (0.0603) had positive and highly significant genotypic correlations with AY/P. Genotypic (-0.634) and phenotypic (-0.45) correlation of AY/P was significantly negative with oil content. Phenotypic correlation coefficients of most of the traits were nonsignificant with AY/P. Genotypic correlations of DFF (-0.363), PH (-0.53) and FA (-0.527) were negative and highly significant with achene weight [5,12,23-32] also discussed the similar results in literature. Path analyses among quantitative traits are presented in Table 6. Results of correlation and path analysis can be helpful in the direct and indirect selection of valuable traits to improve the genetic potential of breeding material. Path analysis presented direct and indirect effects of different characters on AY/P.NOL, LA(0.083), 100AW(1.8979) and OC (1.5936) showed direct positive effect on AY/P [5, 33- 35]. Also discussed that 100AW showed positive direct effect on AY/P.

Table 4: Genotypic correlation coefficients of various characters among the sunflower accessions.

Table 5: Phenotypic correlation coefficients of various characters among the sunflower accessions.

Table 6: Direct (bold diagonal) and indirect effects of various characters on achene yield per plant of sunflower accessions.

Achene yield can be increased by considering these traits. DFF (-0.1302), PH(-0.413), HD(-0.589) and FA(-0.057) had negative direct effect on AY/P. PH showed positive indirect effects through HD, NOL and DFF. HD had positive indirect effect through PH(0.157), NOL(0.1109), LA(0.034), FA(1.4132), 100AW (0.0091) and OC(2.414) [18] also reported that HD had indirect positive effect on achene yield through 100AW. NOL had positive indirect effect through LA(1.3429) and 100AW(0.953) [5,7,36,37] also discussed the indirect effect of NOL on achene yield. LA showed positive indirect effects through PH (0.2523), NOL(0.031), FA(0.0057), 100AW(1.105), DFF(0.3025) and OC(0.0348). FA showed positive indirect effect through HD (0.093) and NOL (0.074). Hundred achene weights showed positive indirect effects through PH(0.2214), NOL(0.042) and LA(2.0896) [21,38,26] also reported that 100AWs had positive indirect effect through PH, LA and NOL. OC had positive indirect effects through HD(0.055), FA(0.0028), 100AW(1.3098) and DFF (0.9848). DFF had positive indirect effects through PH(0.0841). So a criterion may be developed for the selection of economic traits [39].

Conclusion

Significant genetic variability was present in the studied breeding material which may be exploited in hybridization programs to develop high seed and oil yielding, short duration and short stature hybrids of sunflower. Accessions A2.2, B-3.1, A 4.3 and A-7.10 showed best expression of economically important traits so they might be used in the future breeding programs for the improvement of achene yield. Correlation studies explained the direction of relationship between important traits. Direct and indirect association of PH, HD, NOL, LA, FA, 100AW and OC with AY/P recommended that these traits may be used as criteria for selection of sunflower types with good yield potential [40-42]

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Lupine Publishers | Can Bio-fertilizers Produced from Agricultural Residues Be Used In Hydroponics as an Alternative to Synthetic Fertilizers?

Lupine Publishers | Agriculture Open Access Journal

Abstract

Biofertilizer is the liquid effluent obtained from aerobic or anaerobic decomposition of organic matter in the presence of water, or to be more specific, it is the remaining matter left from the decomposition of organic compounds containing both single celled or multi cellular organisms (bacteria, yeasts, filamentous fungi and algae) and the metabolites they generate. Some of the statistics relating to Brazil’s increasing dependence on the use of pesticides in agriculture are quite concerning. For example, in the last 15 years, within the state of Ceará, incidences of rare types of cancer arose to 38% above the national average. Likewise, the sale of agrochemicals in Brazil grew 190% over the last 10 years, which is more than twice the world average. Of the 50 most commonly used pesticides in the Brazil, 15 of them are actually banned in Europe. According to the last Brazilian Health Regulatory Agency (Anvisa) survey, agrochemicals have been found in 67% of the foods analyzed (25% of which were banned). In view of this alarming reality, a change of attitude is not just necessary, it is mandatory. We need to change our philosophy regarding agricultural management on a national level, one that promotes viable agricultural methodologies to produce crops and livestock, which result in a more healthful humanity and a balanced, sustainable environment.

Introduction

In the last two decades, the production of food, which requires less synthetic inputs, has attracted the attention of both research and industry. A productive and relatively inexpensive process is the use of biofertilizers prepared from the aerobic or anaerobic digestion of organic materials as a substitute for mineral-based fertilizers, in order to contribute to reduced consumption of the natural reserves of nutrients on the planet. Our goal is to reduce our nation’s dependence on agrochemicals including pesticides and synthetic, inorganic and mineral-based fertilizers. In keeping with this thinking, we have cultivated lettuces (Lactuca sativa L.) inside a greenhouse using a self-contained hydroponic system, and have successfully utilized reused agricultural matter as a substantive growth promoting solution without the need for the addition of agrochemicals (pesticides or fertilizers). The herbaceous lettuce species we chose is one of the most cultivated vegetables in the country and is of great economic importance, generating quick profitability to the producer due to its short cycle from seeding to harvest [1]. We also selected this crop as it exhibits a wide adaptability to diverse climatic conditions and is one of the most produced crops via hydroponics and thus can be grown year round.

Case Report

For the production of our specific biofertilizer, poultry litter, orange bagasse and cattle manure were combined and mixed in several concentrations. The pH, electric conductivity, salt concentration, lactic acid content, total organic carbon, nutrients (N, P, K, Ca, Mg, Cu, Fe, Mn and Zn), organic matter content, humic substances, and health quality attributes (total thermotolerant coliforms, E. coli, etc.) were assessed for each of the biofertilizer blends. Phenol, carbohydrate and amino acid concentrations were evaluated as well. Phenols are derived from plant lignins, which are cross-linked phenolic polymers and are important for plant structure and rigidity. Carbohydrates are important for the soil organic matter cycle, and are easily hydrolyzed by microorganisms - representing 10-30% of the organic carbon in the soil. Amino acids are an essential nitrogen source and are generated by the aerobic decomposition process due to the hydrolysis of various proteins. Amino acids are also very beneficial throughout the plant life cycle and are essential to numerous physiological and biochemical processes (protein synthesis, phytohormone formation, water balance regulation, ion-chelation required for plant sustenance, etc.). Urease activity was also evaluated in order to evaluate the dynamics of agricultural material transformation in biofertilizers.

After 60 days of decomposition, the biofertilizers were filtered using a tow and stored in barrels. For application in hydroponics, the biofertilizers were diluted until the electrical conductivity remained around 2500μS cm-1. The most successful results were obtained using a mixture of poultry and cattle manure in equal parts as 20% (v/v) of the total composition (80% water). It can be concluded, based upon the quantitative analysis and characterization we conducted for all of the biofertilizer formulations we produced, are all in accord with the Ministry of Agriculture, Livestock and Supply, specifications, which requires determination of the properties of all potential commercial products. The biometric results we generated for the lettuce we grew under hydroponic cultivation show that use of biofertilizers contributed positively to the productivity of the crop. When the poultry and cattle biofertilizer, which showed the best agronomic potential, was applied to the crop, lettuces were produced which exhibited the best biometric results in terms of agronomic potential [2].

After 30 days of cultivation (see illustration) lettuces grew to 28cm (12 inches) of height and fresh shoot mass reached (24.26±2.87)cm and (150.70±9.94) g, respectively (Figure 1). Recycling of organic matter has the advantage of reducing costs when compared to synthetic fertilizers, it maintains the Organic Matter (OM) cycle, and is a supplementary source of nutrients. It is noteworthy that the poultry and cattle manure mixture did not produce coliforms on the lettuces when cultivated under specified conditions.

Figure 1:

Discussion

However, at this time, these potential products would have to be further analyzed, tested and characterized to determine expiration dates, and currently there is no regulation or laws, which can be applied to the production of biofertilizers. Achieving these goals will require continued research and movement within the national legislative arena

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Lupine Publishers | Mineral Supplements of Soils

Lupine Publishers | Earth and Environment Journals

Abstract

Improving soil quality results in increased yields and benefits for farmers. Research has been carried out on the use of selected mineral waste collected in heaps to improve the quality of poor soils. The natural soil supplements obtained in this way will not only improve their quality and increase polonies but will also allow the liquidation of some of the heaps. This will contribute to the reclamation of the landscape and the environment.

Introduction

Soil and agricultural crops have been the object of multidirectional interests since ancient times. This is documented in scientific literature. Among other publications, a large part deals with those that concern soil as a source of nutrients for plants [1-5]. Another important field of research is publications on the role of soil fauna in improving soil quality [6-18]. Another research complex is geochemical research on the relationship of chemical fertilizers - improving the performance of soil with particular regard to the toxicity of additives, in particular those containing radioactive elements [19-24]. A lot of publications address the problem of the relation of fauna, and soil flora and its mineral composition [25-28] as well as the influence of mineral factors on the development of individual plant elements [4,5,29]. There are also publications on the use of organic waste for fertilization glkeb [30-33], the role of soil sun exposure in stabilizing its beneficial properties [34,35] and the use of the so-called. antagonistic plants [36,37].

A separate section is soil relations - geology [30,38-40], mineralogy - regulation of physical soil parameters including its water retention capacity [41]. Quite extensive literature concerns the use of natural minerals and rocks to improve the quality of soil and increase the size of crops from it [18,38-50]. It is in this trend that the presented publication is included, bearing in mind the impact of the proposed proceedings on possible environmental modifications [41,51-54].

Material and Methods of Research

The material for the research was taken from the profile of sediments found in the Devonian dolomite deposit exploited in the “Józefka” mine near Kielce (Figure 1). In addition, material from local heaps and granulates made from materials stored in heaps were collected for testing. In order to determine the mineralogical and petrographic characteristics of the obtained rocks, the following tests were carried out:

A. Microscopic examination in transmitted and reflected light. The Chinese production of Menij was used. The observed phenomena were documented with micrographs.

B. The analysis of the mineral composition of natural samples was made using the XRD method

C. In order to recognize the content of silty fractions and the clay minerals present in it, samples were sludged.

D. The analysis of the chemical composition of the obtained clay fraction was made using the XRD method

E. Analysis of the degree of quartz grain overlaying and SEM morphology of siliceous minerals as well as the non-standard qualitative analysis of the EDS spectrum (Figure 2.a & 2.b).

Microscopic Tests in Polarized Saint Lighting

Sand Muck

Structure: aurytowo-psamitowa, texture: compact, random Composition: quartz, min. clay, min. heavy, among which tourmaline was recognized (Figure 3). Degree of sorting of quartz grain on average sorted.

Figure 3: Microscopic photos of sandy silt. A: picture of rock at 1 polaroid, B: picture of rocks in polarized light. Visible quartz grains and brown tourmaline grain. Minerals are located in intergranular spaces.

Kaolinite Heel

Structure of silty claystone, texture, texture in places parallel, disturbed. The rock background is a very low birefringent kaolinite mass (Figure 4). In the background of the rocks there are almost exclusively clay minerals, accompanied by single quartz grains with a size of up to 20μm of quartz grain, columnar aggregates of kaolinite in places with a wormlike habit.

Figure 4: Photographs of microscopic kaolinite claystone. A: picture of the rock at 1 polaroid, B: picture of the rock in polarized light. Visible different orientation of kaolinite microaggregates manifested in the variable polarization blanking mode.

Molecular Muscovite Heel

The clay is a parallel texture made of kaolinite, smectite and muscovite (Figure 5). They are accompanied by single quartz grains.

Figure 5: Microscopic photos for kaolinite-muscovite claystone. A: picture in a non-polarized light, B: A photo in polarized light.

Figure 5.a: A: water columns

Kaolinite-Illitic Heel

Pellet structure, random texture, disturbed (flow processes) Mineral composition is a mixture of smectite and kaolinite (Figure 6). The microscopic examination shows that the overburden mainly consists of siltstones, sandy mudstones and claystone’s. The sediments are present next to quartz and clay minerals such as kaolinite and smectite also mica. Thanks to microscopic observations, several generations of quartz can be distinguished, as well as iron oxides and hydroxides responsible for the red color of the sediments.

Figure 6: Microscopic images of kaolinite-illite claystone. A: Image in natural light, B: Image in polarized light. in which the granulometric composition was determined. B: granulometric curve of the tested samples.

Analysis of the External Matrix of Natural Tests Used by the XRD Method

Due to the uncertainty of the clay components, the polarizing microscopy method was used to test the X-ray diffraction method. Analyzes of natural samples, clayey, prażed and glycolated fractions were performed (Graphs 1,1a,2, 2a,3, 3a,4,4a,5). They allowed to state that beside the detritus components in the overburden samples and heaps there are clay minerals represented by kaolinite and illite. X-ray examinations indicate that all overburden sediments and heaps occurring in the heap contain minerals beneficial for agriculture. They do not contain toxic minerals (Figure 5.a).

Graph 1: X-ray diffraction pattern of sandy silt.

Graph 1a: X-ray diffraction pattern for silt sand grain fractions. From the top: raw, roasted and glycolated preparation.

Graph 2: A diffractogram of kaolinite claystone.

Graph 2.a: X-ray diffractogram of clay fraction of kaolinite claystone. From the top: raw, roasted and glycolated preparation.

Graph 3: X-ray diffractogram of kaolinite-muscovite claystone.

Graph 3.a: X-ray diffractogram for clay fraction of clay-muscovite clay. From the top: raw, roasted and glycolated preparation.

Graph 4: X-ray diffractogram of kaolinite – illite.

Graph 4.a: X-ray diffractogram for clay fraction of clay-illite clay. From the top: raw, glycolated and roasted preparation.

Graph 5: X-ray diffraction pattern of dolomite-clay pellets containing quartz crystals.

Results Designated by Content of The Content of the Cross-Fraction on a Standing Water Post

In the case of two samples, the content of clay minerals (fraction <2 μm) in selected samples with extremely small and extremely high clay minerals were tested. They showed that the amount varies both in the overburden material and material from the heap from 3 to 64% by weight (Table 1).

Table 1.

Results of the Analysis of the Quarter of Quartz Grassing and Distribution of Minerals of Chlorine Minerals in Granulates SEM

SEM observations were made for both overburden and granulate deposits. Their distribution in pellets is of great importance as they favor the disintegration of granules and thus the dispersion of granulated material in the enriched soil. Clay minerals in overburden scales are present as aggregates of kaolinite or illite microcells (Figure 7). On the other hand, both on the heap and in the granulate, clay aggregates are degraded by micrometry (Figure 8), which means that in the process of stripping off, staying on heaps and in the granulation process, the clay aggregates disintegrate. This is a favorable phenomenon conducive to material granulation. After pouring on granules, thanks to such a construction, they undergo a faster process of foresting. thanks to which the ingredients of the granules combine with the glaze more quickly.

Figure 7: Kaolinite doline from the dolomite overburden. A: characteristic aggregates made of kaolinite tiles. B: one of the aggregates under magnification.

Figure 8: Granules, visible quartz grains (blue arrows) and dolomite (orange arrow) stuck together with clay minerals. SEM image.

SEM Images (Figures 9-11)

Figure 9: The heap of Józefek quarry. containing overburden rocks collected from the Devonian dolomites.

Figure 10: Granules with different granulation of soil supplements with different grain size, which were made from sediments accumulated in heaps.

Figure 11: Granular structure observed in the cross-section of granules. Visible dolomite grains in the clay anchor. A: magnification 10 x, B: magnification 20 x.

Summary

The performed research indicates that in the dolomite deposit of the Józefka deposit, rocks containing clay minerals from the kaolinite and illite groups as well as quartz, dolomite and calcite are present. The origin of these rocks is not fixed, but for raw material reasons, these rocks are suitable for enriching class IV-VI soils with clay minerals that can provide many ions and promote water retention in soil. Natural granules made from overburden rocks deposited on heaps and small dolomite fractions (waste) can be used as soil supplements enriching it with many elements, including magnesium. It is a beneficial stimulator of biological processes in plants. The use of the described granulates as soil supplements will increase yields, biologically strengthen growing plants and contribute to increasing harvests, and thus will increase the profits of agricultural producers. Both magnesium and other elements that will be delivered to the soil with granules will naturally become the building blocks of plant tissues. In the production and processing cycle, they will then be transferred to organisms using the bred plants. These soil supplements are completely natural and do not contain synthetic toxic substances. They are therefore environmentally friendly and human. The use of granules in the described form will not only strengthen the soil, but will also help to eliminate mineral heaps, favoring the reclamation of mining areas and restoring them to natural conditions.

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Lupine Publishers | Environmental Impacts of Hydropower and Alternative Mitigation Measures

Lupine Publishers | Agriculture Open Access Journal

Abstract

Hydropower is an important renewable energy resource worldwide. However, its development is accompanied with environmental drawbacks. The paper has reviewed the environmental drawbacks associated with hydropower projects. Change in hydrological flow regimes, deteriorating water quality, migration corridors barriers, sedimentation, greenhouse gas emission and biodiversity loss are environmental impacts of hydropower projects. Appropriate mitigation measures are required to sustainably generate hydropower. These are also discussed in the paper

Keywords: Environmental impacts; Hydropower; Mitigation measures

Introduction

There is no doubt about that the agricultural industry is the most essential one for humanity [1]. It also employs a great number of people, providing economic means to them. But it is not easy to answer whether the agricultural industry is an effective one. On the quite contrary, the industry is perhaps the least effectively managed one for the last several thousand years. As people in the world are enjoying longevity, the world consumes more and more food. Can the world’s agricultural industry feed all the people on earth? It is a vital question. If the earth capacity is limited and the crops are not produced enough, the only possible solution is to increase the productivity of the agricultural industry. In order to find ways to increase such productivity, we first have to understand why the productivity of the agricultural industry is so low. Then we can suggest how the agricultural industry changes itself to be more productive and effective. In this paper, we endeavor to answer the question from a value chain perspective.Hydropower is one of the most efficient power generation technologies, which are carbon free and use inexhaustible resources to produce the energy. The prime driver is the force of gravity and the water used to drive this power is non-destructive [1]. According to Yüksel [2] hydropower do not pollute the air we breathe in the way that the energy source does not produce any air pollutants. Unlike thermal power plants for example, there are no gaseous of fly ash emissions emitted during the production. The fact that hydropower often replace fossil-fired generation, it can therefore also be said that it is reducing the problem with acid rain and smog [1,2]. Despite all these advantages hydropower plants have, there may also be negative impacts. Lately the impact on the ecological aspects from the power plants has received attention. In the report from World Commission on Dams [3], it is stated that dams will have effects e.g. on the terrestrial ecosystem and biodiversity, the flow regime, migration of aquatic organisms, and can cause emissions of greenhouse gases. Bratrich [4] states that hydropower affects the flow regime, migration of organisms and transport of nutrients and sediments.

Abbasi [1] claim that hydropower plants causes major ecological impacts in all of the four different habitats, which are associated with the projects; the estuary into which the river flows, the downstream reaches of the dammed river, the reservoir catchment and the artificially created lake. Different research works from all corner of the world’s reported considering the negative effects of hydropower on the environments and calls for the importance of adopting of appropriate mitigation measures [5,6]. Therefore, to ensure sustainable development, various mitigation and enhancement measures have to be integrated at the early stages of project planning. Furthermore, appropriate mitigation measures not only for hydropower development that is newly planned and implemented in future, but also for the refurbishment and upgrading of hydropower plants which are currently in operation, need to be devised. The purpose of this paper is to review the impacts of hydropower on the environment and alternative mitigation measures.

Impacts of Hydropower

The hydropower projects have several impacts on the environment. Water quality decline is one of the impacts of hydropower and water quality may be affected around a hydropower plant [7]. Water discharged from a reservoir can be of a different composition to the water that is flowing into a reservoir [8]. The other impacts of hydropower plant are migrations barriers. Hydropower dams impede the flows of rivers and thereby affect the habitat of various aquatic lives [9]. Migratory animals require different environments for their different phases of their life cycle. Different stages are reproduction, production of juveniles, growth and sexual maturation. These different stages take place in different environments for different fishes [6]. Even if hydropower is a renewable energy source it is not an energy source without GHG emissions [5]. Greenhouse gas emissions can occur at three different phases hydropower plant (the construction, the operation and maintenance and the decommissioning of the plant). During the construction phase the emissions comes from the production and transport of the materials needed for the construction and from the work equipment.

In the second phase, the operation and maintenance, the emissions can come from e.g. heating or cooling systems and transportation for maintenance work [5]. Hydropower project also leads to sedimentation [10]. The natural carried sediment from the usual natural flow will be negatively affected [5,10]. Bergengren [6] states in their report that when constructing a dam or hydropower plant, changes in the hydrological regime will follow. IPCC suggest the same thing and claim in their report that changes in the hydrological regime is a significant impact from the hydropower plant. They argue that due to a hydropower plant there will be changes in water level, timing and temperature, which will affect the surrounding terrestrial and aquatic ecosystem [8]. The other biggest threats from hydropower projects are the loss of biodiversity and ecosystems that provides services that we cannot live without. The loss of valuable vegetative community types and loss of wildlife and habitats are resulted from land clearance and removal of natural vegetation [11].

Mitigation Actions

The purpose of environmental mitigation requirements at hydroelectric projects is to avoid or minimize the adverse effects of development and operation. Among the mitigation actions dam removal is becoming a more frequently used management option all around the world. Dam removal is taking into consideration for old dams in need of renovation or small dams that are no longer used or have lost most of their reservoir capacity. Another point of view is that dams and the reservoirs have many ecological effects; the disruption of the movement of different organisms is probably the most important reason for dam restoration. Thereby, dam removal makes it possible for fish migration and fish species to shift from lentic to lotic, which in turn have the ability to migrate and reproduce in free-flowing water [12]. Flow Regulations is also one the promising action for mitigating negative impacts of hydropower projects. In order to make the use of hydropower plants more compatible with the natural life of rivers, a minimum flow must be released so as to assure the preservation of the hydrological continuity of the river and the consequent conservation of natural habitat and ecological life [13]. Minimum flow release means that you allow some flow below a hydropower plant with the dual aim of maintaining current water ecological conditions and partly also for aesthetic or recreational purposes on a watercourse distance, which would mostly had remained drained otherwise [13].

The problem with sedimentation in the surroundings of the hydropower plant can be mitigated by constructing sedimentation measures such as the construction of small-scale weirs to trap the sands and the particles that later can be manually removed [14]. A direct approach to reduce the accumulation of sediment is to mechanically remove the sediments by periodic dredging [7]. Biotope Adjustments is also other migration action. Adjusting the biotope through the creation of greater environmental heterogeneity, by using such as habitat adjusting measures, the biological diversity of the benthic fauna will be strengthened [15]. Constructing migratory corridors are other mitigation actions for migrating animals. To facilitate for the migration of the species in the streams it is therefore desirable to construct corridors [11]. Fish friendly turbine technology is an emerging technology that provides a safe approach for fish passing though the turbines by minimizing the risk of injury or even death [8]. Aquatic animal Plantations is also other mitigation measures to reduce the negative effects of hydropower projects on the aquatic animals. Artificially manage the fertilization, hatching, growth and release of aquatic animal’s especially salmonid fish [16]. To introduce planted fish to protect endangered species or reintroduce species that have disappeared may be of great benefit to the environment and the biodiversity [16].

Discussion

From the above review, it is known that hydropower project have impacts on the environment. Change in hydrological flow regimes, deteriorating water quality, migration corridors barriers, sedimentation, greenhouse gas emission and biodiversity loss are environmental impacts of hydropower projects. Dam removal, flow regulations, biotope adjustment, fish plantation, sedimentation measures, constructing migration corridors and fish friendly turbine are alternative action for mitigating the negative impacts of hydropower projects on the environment [17]. Therefore, to meet the increasing demands energy and ensure sustainable development, various mitigation and enhancement measures have to be integrated at the early stages of project planning. Furthermore, appropriate mitigation measures not only for hydropower development that is newly planned and implemented in future, but also for the refurbishment and upgrading of hydropower plants which are currently in operation, need to be devised.

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