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lupine-publishers-lojpcr · 3 years ago

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Lupine Publishers | Profile of drugs used for prescription or not (selfmedication) by medical students

Lupine Publishers | LOJ Pharmacology & Clinical Research

Introduction

According to the definition of the WHO Pharmaceutical Dictionary [1] and that of the European Directive 65/65, a medicinal product is “any substance or composition having curative or preventive properties with regard to human diseases. Indeed, any substance or composition that can be administered to humans for the purpose of establishing a medical diagnosis or restoring, correcting or modifying physiological functions in humans is also considered a medicinal product [1]. However, the irrational or non-rational use of drugs (poly pharmacy, inappropriate) is a major global problem, often in inadequate dosages. These common practices are also seen with students. Thus, for Adlaf et al, the prevalence of illegal psychotropic drug use (excluding cannabis in Canada) has been estimated at 52% [2]. For Koudou, students from UFR of Bouaké, consumed at least one drug during the examination period by self-medication [3]. Although medical students are expected to have habit of good practices and use of medicines, they face the same problems of access to medicines as the general population. Our objective was to study the profile of the drugs used as prescription and/or self-medication by medical students in order to identify the reasons, risks and adverse effects of their practices.

Materials and Methods

This cross-sectional study with descriptive aim took place from May 12 to June 14, 2019, including 234 medical students from the Preparatory School of Health Sciences (EPSS) at Nangui Abrogoua University and the UFR Medical Sciences of Abidjan (UFRSM). All medical students enrolled, regardless of gender, after informed consent were submitted. Students absent during the study period were excluded. The results were analyzed with Epi info 7 software.

Results and Comments

The average age of students was 21.28 ± 4.7 years, with age variation between 15 and 33 years (Figure 1); similar to Koudou’s study on the consumption of psychostimulants (20.6 ± 2.7 years) [3] and into that of Normand et al. on the intellectual doping of pharmacy students during the examination period (21 years) [5]. Thus, students were particularly young in health sciences. Our study showed male predominance (66.2%) with sex ratio of 1.9 comparable to another study in which surroundings school and university regardless the levels of study, this sex ratio varies from 2 to 4 [6]. Most of the students in our study were in Bachelor 2 (38.5%) or Master 1 (19.2%) but they were more students in L1 (first year license) at Koudou [3]. 38.5% of these students lived with their parents, 34.2% alone and 27.4% on university campuses. While according to Koudou [3] 48.1% of students lived with parents. This difference could be explained by the larger number of students in the group 600 compared to 234 in our study. Pathological reasons for using the drugs were headache, asthenia and fever respectively 31.3%, 8.7%and 16% (Table 1). The diagnoses mentioned were first malaria (37.6%), then influenza (17.1%) and typhoid fever (11.1%). These students were self-diagnosing as a result of the knowledge acquired during the lectures, and/or internships. Our results are superimposed with those of the work of Valentin et al [7] on the prevalence and characteristics of self-medication among students aged 18 to 35 residing at the Kasapa Campus of the University of Lubumbashi.

As for drugs (prescribed or taken as self-medication), the most used were antimalarials (58%), antibiotics (13%), analgesics and antipyretics (15.8%), but also some corticosteroids and antiulcers (Table 2). These same drugs were found in Konaté’s study on self-medication in pharmacies in the city of Sikasso (Mali) [8]. Thus, 70.5% of these students practiced self-medication and only 29.5% received prescriptions (Figure 2). Self-medication is a practice to be combated because self-diagnosis and treatment can be a source of error. This tendency to self-medication has been found in Valentin et al [7] among health science students. Indeed, 45.1% of them admitted to using self-medication and 54.9% of students of other sciences. The prescription could be legible in 71% of cases. The prescribed treatment was as outpatient in 79.7% of cases. The prescriber was a doctor in 56.5% or a PhD student in 26.1% of cases.

These observations described are intended to promote habits of good prescribing. Indeed, the prescription obeys rules that must be respected and known in order to comply with the recommendations. Students who practiced self-medication 65.5% had knowledge about side effects but 34.5% knew nothing about what constituted a danger. Almost all (97.4%) of the students did not perform paraclinical examinations to confirm the diagnosis either probably due to negligence or lack of financial means. For illustration, the average cost of treatment by self-medication was 2,962 fCFA with extremes of 200 and 25,000 fCFA. While that of the prescription was 10,323 fCFA with extremes of 400 and 61,000 fCFA (Table 3). The important thing is that self-medication was less expensive than drugs prescribed by a doctor. Some African studies [8, 9] have recognized that it is due to poverty, counterfeit medicines in the market [4], the proliferation of prescribers in our centers as well as the inaccessibility to doctors in our structures. Finally, the limitations of this work could be information biases. Indeed, some students refrained from answering questions when others were limited in understanding the questionnaire.

Conclusion

Medical students use few prescription drugs, and practice more self-medication because of the costs of treatment, hence the eternal problem of accessibility to care. It is time to put into circulation the health insurance card to help students treat themselves in order to reduce self-medication.

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lupine-publishers-lojpcr · 3 years ago

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Lupine Publishers | A Brief Review on Phytochemical and Pharmacological Aspects of Andrographis Paniculata

Lupine Publishers | LOJ Pharmacology & Clinical Research

Introduction

Since the beginning of civilization, medicinal plants have been an intrinsic component of human life [1]. The conservation of ethnobotanical knowledge as part of living culture and practice between communities and the environment is essential for biodiversity conservation. The information about medicinal plants gains from various medicinal systems such as Unani, Siddha, and Ayurveda [2]. The traditional system of medicine belongs to the traditions of each country and has been passed over from generation to generation. Understanding the dynamics of traditional local knowledge of medicinal plants is important for their medicinal properties is now being developed as a source of scientific research to prove the effect of plants and generate new therapeutic resources. Medicinal plants are considered as a backbone of traditional medicine (WHO) as well as most modern medicine is also derived from medicinal plants i.e. aspirin. The medicinal plant having a rich source of components that can be used to develop and synthesize drugs. About 3.3 billion people in developing countries depend on medicinal plants on a regular basis, WHO estimated that about 80% world population rely on the medicinal plant for their primary health care. Further more, worldwide 42% of 25 top-selling drugs marketed are either directly obtained from natural sources or entities derived from plant products [3]. The quality of traditional medicine is determining its active substances produced by the plant. Andrographis paniculata is one of the important medicinal plants that is utilized throughout the world [4]. A.paniculata is an herbaceous plant of the Acanthaceae family. It is widely distributed in Southeast Asia, India, and tropical as well as in subtropical Asia. A.paniculata is also known as the “King of Bitters” since it has a highly bitter taste in all parts of the plant body [5]. Furthermore, A. paniculata is known as “Kalmegh” in India, “Chuan-Xin-Lian” in China, “Fah Tha Lai” in Thailand, “Hempedu Bumi” in Malaysia, “Senshinren” in Japan, and “green chiretta” in Scandinavian nations [6]. A.paniculata is one of the most widely used plants in Ayurvedic and Unani medicine [4]. Traditionally, A.paniculata was used in the treatment for snakebite, fever, bug bite, diabetes, malaria, and dysentery [7]. Moreover, A.paniculata is also used in the combination with other herbs and health care treatment. It is found that A.paniculata is used in more than half of the herbal formulations commercialized in India for he patic diseases [8]. Many scientific studies also have been reported regarding the medicinal properties possessed by the A.paniculata, most of which are based on traditional knowledge (Table 1). Phytochemical investigations have revealed that A. paniculata contains a wide range of chemicals. In addition, experimental evidence also reported that A.paniculata has a broad spectrum of pharmacological activity including anti-bacterial, antidiarrheal, anti-inflammatory, antiviral, antimalarial, anticancer, antimalarial, hepatoprotective, etc. In this review, we briefly discuss ethnobotanical uses, phytochemistry, and recent scientific finding pharmacological activity of the A.paniculata [6].

Table 1: Taxonomical classification of Androgrphis paniculata.

Botanical Description and Habitat

A.paniculata is native species of India, China, and Taiwan. But it is also found in Southeast Asia, tropical and subtropical Asia as well as few other nations such as Malaysia, Indonesia, Vietnam, Sri Lanka, Laos, Cambodia, Pakistan, Myanmar, and the Caribbean islands [9]. Especially, in India A.paniculata are found in Karnataka, Andhra Pradesh, Tamil nadu, Uttar Pradesh, and Madhya Pradesh. Also cultivated in Assam and West Bengal to some extent. In addition, A.paniculata are found in different habitats including forests, farms, plains, hill slopes, dry and wetlands, and wastelands [10]. A.paniculata is bitter in test, an annual herb that is abundantly branched which grows up to a height of 3.-110 cm in a humid, shady area. It has glabrous leaves that are 8.0 cm long and 2.6 cm wide, little white flowers that are rose-purple or light pink, spots on the petals, and corolla with hairs. The stem was found to be dark green in color, 0.4-1.0 m tall, 2-6 mm in diameter, quadrangular with longitudinal furrows and wings on the angles of the younger part [11] as shown in Figure 1.

Figure 1: Andrographis paniculata morphology.

Traditional Uses of Andrographis Paniculata

A.paniculata play vital importance in the Ayurvedic, Siddha, and traditional medicine systems in India [12]. For centuries, the leaves and roots of A.paniculata have been used to treat a wide range of health problems in Asia and Europe. However, the entire plant is also utilized for specific uses [13]. The plant known as “Kalmegh” in Ayurvedic literature is an essential element in the majority of Ayurvedic remedies and is officially recognized by Ayurvedic pharmacopeia. Moreover, it is used as an aperient, emollient, astringent, anti-inflammatory, diuretic, anthelmintic, carminative, and antipyretic in the Unani system of medicine [14].In India, tribal groups used this herb to cure a number of diseases such as antidote against snake bites, Banded Krait and Russell’s viper, etc. [14]. The tribal of Kheria, Khatra, Moora, and the Santal region of Bankura district, West Bengal, India utilizes an infusion of the entire plant to treat fever [15]. The extracted juice from A.paniculata leaves, alone or combined with cloves, cinnamon, and cardamom is used as a cure for flatulence, loss of appetite, griping, diarrhea in children, and irregular stool. In India during the influenza epidemic in 1919, A.paniculata was shown to be highly effective in reducing the disease progression [16]. It was also utilized by ancient Chine’s physicians to treat inflammatory diseases, colds, laryngitis, and fever, hepatitis, pneumonia, respiratory infections, tonsillitis, sores, pelvic snake bites, herpes zoster and it has been characterized as a cold property herb [13] to remove toxins and body heat. The decoction of fresh leaves of A.paniculata is used as an antihypertensive and antidiabetic in Malaysian folk medicine. Furthermore, it is advised to use it in cases of leprosy, scabies, gonorrhea, boils, chronic and seasonal fevers, and skin eruptions, due to its “blood purifying” purifying properties [4].

Phytochemistry

The aerial part (leaves and stems) of A. paniculata contains major active phytochemicals [17]. According to the survey of the literature, andrographolide is the major bioactive compound found in the A.paniculata which is a diterpene lactone that is crystalline, colorless, and has a bitter taste [9]. The leaves have the highest concentration of andrographolide about 2.39% whereas the seed has the lowest concentration about 0.58%. The quantity of the phytochemicals varies widely depending on the portion used, locality, time of harvesting, and season (Figure 2). Andrographolides are highest found immediately before the flowering season, then decline progressively [14]. Other lactones compound observed in A.paniculata is 14-deoxy-11-andrographolide, 14-deoxy-11, 12 didehydroandrographolide, andragraphan, andrographon, 14-deoxyandrographolide, neoandrographolide, deoxyandrographiside, andrographosterol, andrographiside etc. A.paniculata also contains Xanthones and quinic acid derivatives in minor concentrations. Moreover, Reddy et a. [18] reported that A.paniculata contains flavone such as 5-hydroxy-7’2’6’-trimethoxyflavone and 23-C terpenoid 14-deoxy-15-isopropylidene-11, 12- didehydroandrograholide and other flavonoid Skullcapflavone I 2’-O-glucoside, 7-Omethylwogonin, 7-Omethyldihydrowogonin and 7-O-methylwogonin 5-O-glucoside as well as diterpenoids such as isoandrographolide 14-deoxy-11, 12 didehydroandrographolide. Rao et al. [19] identified and isolated 5, 7, 20, 30-tetramethoxyflavanone and 5-hydroxy-7, 20, 30-trimethoxy flavone from the A.paniculata. A new labdane type diterpenoid which is andropanolide along with seven known diterpenoids isolated from the methanolic leaves extract of A.paniculata [20].

Figure 2: Chemical structure of major component found in A.paniculata.

Pharmacological Activity of A.Paniculata

Hepatoprotective activity

A.paniculata is widely used as a hepatoprotective and hepatostimulative agent in the Indian traditional medicine system. Traditionally the leaves aqueous extract of A.paniculata is used in the treatment of jaundice and different liver damage. Andrographolide found in the A.paniculata was protective against liver damage in rats and mice induced by carbon tetrachloride. Moreover, Andrographolide also observed significant hepatoprotective against various types of liver damage, induced by galactosamine or paracetamol [21]. The free radical scavenging activity of andrographolide has a significant hepatoprotective effect by lowering lipid peroxidation malondialdehyde product as well as by maintaining glutamic pyruvate transaminase, alkaline phosphatase, and glutathione levels in mice treated with carbon tetrachloride [22]. A.paniculata has been shown antihepatotoxic activity against plasmodium berghei K173-induced hepatic damage in mastomys natalensis [23].

Antibacterial activity

A.paniculata has been shown the antibacterial activity against a wide range of bacterial species. In vitro study found that the aqueous extract of A.paniculata shown antibacterial activity even at the low concentration (25 mg/ml) against E.coli, Shigella, Streptococci, Staphylococcus aureus, and Salmonella [24]. Another similar study leaves aqueous extract of A.paniculata reported against the methicillin- resistant S.aureus and Pseudomonas aeruginosa [25]. Furthermore, A.paniculata is also effective against HAS 1 (herpes simplex virus 1) without any cytotoxicity [26].

Antidiarrheal activity

In developing countries, Diarrhea is one of the most common diseases and it leads to the top ten causes of death among children worldwide [5]. Some drugs such as kaolin-pectin, selenium, loperamide, and bismuth have been used to treat the symptoms. However, it also causes some unfavorable side effects [5]. The study has been found that A.paniculata has significant antidiarrheal properties [27]. According to the study, an ethanolic extract of A.paniculata treated 88.3 % of acute bacillary dysentery cases and 91.3% of acute gastroenteritis cases. Furthermore, andrographolide was found to treat 91% of acute bacillary dysentery cases. The same cure rate of about 91.1% was obtained by providing a compound tablet comprising andrographolide and neoandrographolide in a 7:3 ratio. This was claimed to be more than the cure rate observed with chloramphenicol and furazolidone [28]. A.paniculata was found to be effective in curing patients with acute diarrhea and bacillary dysentery in double-blind investigation [14].

Antimalarial activity

In many tropic and subtopic countries, malaria is still a prevalent disease [14]. A.paniculata was shown to significantly suppress the growth of the Plasmodium berghei [11]. In vitro study of 50% ethanolic extract of the aerial parts (100 mg/g) shown antimalarial activity against plasmodium berghei and in vivo study in rats observed antimalarial activity after intragastric application (1g/kg body weight) [26]. It is suggested that the antimalarial effect of A. paniculata is due to the reactivation of the enzyme superoxide dismutase [5]. Another study has been reported that the crude extract of A.paniculata shown antimalarial activity against the resistant strain of Plasmodium falciparum having an IC50 value of 6mg/ml [29]. In addition, a xanthous compound isolated from the A. paniculata has been shown in vivo antimalarial activity in plasmodium infected Swiss albino mice. The results found that a significant reduction in parasitemia after treatment with a 30 mg/kg dosage [26].

Anticancer activity

Cancer is a set of disorders characterized by abnormal cell proliferation and the ability to penetrate or be spared to other regions of the body. Despite the fact that many diseases have a worse prognosis than most cancers [17]. The extract of A.paniculata having diterpenoid is significantly able to restrict cell proliferation, arrest the cell cycle and induce cell apoptosis of different cancer cells [30- 33]. Treatment of the MDA-MB-231 breast cancer cells with andrographolide extracted from A. Paniculata causes apoptosis of cancer cells and arrests the cell cycle without interfering with the normal growth of cells [34]. The study has been reported that A.paniculata exhibits potent cytotoxic activity against human epidermoid carcinoma of the skin lining of the lymphocytic leukemia cells and nasopharynx [12]. A.paniculata also shown cytotoxic effects against colon cancer cells by suppressing AKT and mTOR phosphorylation levels, resulting in ER stress-induced death [35]. Furthermore, apoptosis in colon cancer cells is induced by the andrographolide via controlling the signaling of pro-apoptotic GRP-78/IRE1/XBP-1/ CHOP [17].

Antidiabetic activity

Diabetes is a metabolic disease characterized by elevated blood sugar levels [36]. According to the WHO reports around 70 million people worldwide suffer from diabetes. Specifically in developing countries, diabetes has become a threat to human health [37]. In vivo study observed that ethanolic extract of A.paniculata exhibit the protective effect in hyperglycemic condition and also protect the tissue damage caused due to oxidative stress in a diabetic rat model produced by streptozotocin [38]. Another study conducted [39] found that oral administration of andrographolide in a dose-dependent manner reduced plasma glucose levels in diabetic rats caused by streptozotocin and wild-type rats.

Conclusion

The entire literature review indicated that Andrographis paniculata exhibits a broad range of phytochemicals and pharmacological activities. The previous study found that A.paniculata contains 50 lactane diterpenoids, 30 flavonoids, and 30 novels phytochemical isolated and identified from A.paniculata. Phytochemical study reveals that Andrographolide is a major compound found in Andrographis paniculata. It has shown a wide spectrum of pharmaceutical activity such as anti-microbial, hepatoprotective activity, anti-inflammatory activity, anti-malarial, anti-diarrheal, anti-diabetic, and cytotoxic activity. The precise information offered as a review here covers the phytochemical and pharmacological information about this plant, providing the muchneeded encouragement to use this plant in creating and sustaining a prospective means of livelihood.

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lupine-publishers-lojpcr · 3 years ago

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Lupine Publishers | Inhibition of Aortic Medial Calcification: miPEP-200b and miRNA-200b as Potential Mediators

Lupine Publishers | LOJ Pharmacology & Clinical Research

Introduction

In molecular biology, the established central dogma is the widely accepted framework of genetic information flow. This process begins with the DNA transcription from the nucleus of the cell into linear, single-stranded messenger RNA (mRNA). Ribosomes then read the mRNA strand following transportation to the cytoplasm, and translation of the code into single amino acids, which are added sequentially to the growing peptide. Once the peptide is fully synthesized, both the mRNA strand and the peptide are released from the ribosome [1]. Although this pathway has been established universally as the only way for protein synthesis, only 1.5% - 2.5% of the human genome codes for proteins [2]. The remainder of the genome produces noncoding RNAs (ncRNAs). These ncRNAs can be further classified into various subtypes; of note are miRNAs, small nucleolar RNAs (snoRNAs), long noncoding RNA (lncRNA), and circular RNA (circRNA) [3]. Although ncRNAs have always been thought to have no role in protein synthesis, many previously unknown indirect functions of ncRNA have been elucidated within the past five years. They have been shown to be involved in many settings such as macrophage activation during an immune response, abnormal expression in diabetic wounds, retinal diseases, and even endometrial physiology and disease states such as endometriosis [4-7]. Perhaps the most groundbreaking findings in the realm of ncRNAs have been their association with various human cancers [8]. Most research has been focused on exploring the epigenetics and post-transcriptional activity of ncRNA, which allows these molecules to exert regulatory effects on the expression of the genome [9]. By directly binding to mRNA strands, miRNAs are able to regulate their ability to eventually lead to protein synthesis [10]. It has been demonstrated that this process is extremely precise, allowing targeting of mRNA with high specificity. Interestingly, the miRNA family has identical 5’ regions with the 3’ region differentiating these molecules and their specificities [11]. Although protein translation and miRNAs have conventionally been thought to have such an indirect relationship, our recent discoveries suggest a much more direct engagement between the two. We have shown that the sequences in certain pri-miRNAs, more specifically pri-miR-200a and pri-miR-200b, contain ORFs that are able to be recognized by ribosomes and subsequently translated directly into protein products, miPEP-200a and miPEP-200b. Even more intriguing is the potential role of these pri-miRNA-derived peptides (miPEPs) in cancer repression [12-15].

Figure 1: Expression of miPEP-200b in prostate cells (PNT1A) and breast cells (MCF-7). MCF-7 and PNT1A cells were harvested and prepared for western blot. Total extracts were subjected to western blot analysis using miPEP-200b (antibody raised against mi-PEP-200b). 8 Kda band represents expected band for miPEP-200b.

Figure 2: Proposed mechanism of aortic medial calcification pathogenesis (dashed red line: causing decreased activity of the target, dashed green line: causing increased activity of target).

Recently we have studied the expression of miPEP-200b in mammalian cells by raising polyclonal antibodies to miPEP-200b. Our results demonstrate that miPEP-200b is expressed in both breast and prostate cells (Figure 1). These results establish the presence of pri-miRNA-encoded proteins in mammalian cells. Many studies have been conducted on the implications of the miR- 200 family in cancer development and repression; however, its association with cardiovascular pathology has not been a central area of focus. Because the miR-200 family has been shown to affect specific cellular pathways in various conditions, it would not be farfetched to speculate on its implications in cardiovascular diseases known to have abnormalities in the same cellular pathways. It was found that a single gene variant for the miR-200 family could cause increased protein kinase A (PKA) activity, with subsequent thrombocyte activation and ensuing atherosclerosis [16]. In addition, PKA has been shown to act as a promoter of human smooth muscle cell (HSMC) calcification [17]. These processes are known to be the initial steps to the eventual development of vascular calcification [18]. Previously, we have shown that pri-miRNAs of 200a and 200b code for miPEP-200a and miPEP-200b respectively and these miPEPs function like miR-200a and miR-200b suggesting that Nature preserved this duplication of functions in case of a failure in any one of their functions. As such, we hypothesize that in the same way that miR-200b plays a role in decreasing PKA activity in atherosclerotic processes, the peptide miPEP-200b (encoded by pri-miR-200b) may also act as an inhibitor of PKA-induced HSMC calcification (Figure 2). Furthermore, targeted miPEP-200b, miRNA-200b, and PKA inhibitor therapy may lead to a substantial decrease in ISH development in older patients, with an ensuing decrease in the incidence and prevalence of diastolic heart failure secondary to longstanding hypertension. In this article, we will discuss the various components that provide evidence for these potential relationships and their sequelae.

Mirna and Mipep Interaction

Although there is a significant amount of information to be discovered regarding the functions of miPEPs, certain recent important findings allow us to predict potential activities of miPEPs in relation to miRNA. Of note is the study by Lauressergues et al. which showed that in plant cells, miPEPs behave as positive feedback on miRNA production [19]. It is possible that this relationship will also be shown in human physiology under controlled experiments in the future. This relationship is quite interesting, given the scarcity of positive feedback mechanisms observed in nature. In addition, various experiments in previous years involving miRNAs that attributed their observed findings to the effects of miRNAs alone may need to be revisited. As an example, it has been demonstrated that miRNA-200a and miRNA-200b are involved in the suppression of the epithelial-to-mesenchymal transition (ETM) in certain cancer types [20]. Although miRNA itself was originally thought to be the mediator of this observation, our recent findings demonstrate that the observed ETM suppression may be a result of miRNA alone, miPEP alone or a combination of both miRNA and miPEP activity [12]. Allowing this scenario to serve as a framework (Figure 3) in many other cellular pathways, one can imagine the vast number of cellular processes that may, in fact, have a different mechanism than what was previously proposed [21,22].

Figure 3: Proposed miRNA and miPEP relationship (dashed red line: causing decreased activity of the target, dashed green line: causing increased activity of target).

PKA and Aortic Calcification

The vasculature anatomy consists of 3 main layers: tunica intima, tunica media, and tunica adventitia. In the setting of aortic wall calcification, the media layer is involved. The main component of this layer is HSMC, which aids in constriction and dilation of vessels by contracting and relaxing, respectively (Figure 4). However, during the pathological process of medial aortic calcification, these cells begin to behave as osteoblasts, evident by upregulation of several markers associated with bone synthesis, including greater alkaline phosphatase activity [23]. Although there may be several mechanisms involved in this process, one explanation is the over-activity of PKA. PKA is an enzyme found in a multitude of cell types and tissues in the body. The enzyme is activated via cyclic AMP, and following activation, it is able to phosphorylate its substrates [24]. Abnormal PKA activity, therefore, causes various downstream effects. In particular, increased PKA activity may have important pathological implications in vascular calcification. There seems to be a potential mechanism involving PKA-induced elevation of parathyroid hormone (PTH). This may then induce medial aortic calcification [25]. with one experiment involving an in vivo model of rats with elevated PTH levels causing substantial calcification of the aorta [26]. In another study, it was shown that inorganic phosphate (Pi), which acts as a stimulator of PKA, led to HMSC calcification. In this experiment, HSMC were treated with Pi, calcium levels were measured, and subsequently, PKA inhibitors were added, then calcium levels were remeasured. It was demonstrated that inhibition of PKA activity by utilizing siRNA led to a greater than 50% decrease in HSMC calcium levels [27]. Although this study utilized siRNA as the inhibitor of PKA, Magenta et al. suggest that an increased PKA activity may be observed due to a single nucleotide polymorphism (SNP) where a thymidine nucleotide was substituted by cytosine in genes coding for the miR- 200family [16]. This may suggest that the miR-200family plays a significant role in moderating PKA activity.

Figure 4: Various layers of arterial vasculature.

Aortic Calcification, Hypertension, and Heart Failure

A major sequela of vascular, specifically arterial, calcification is the development of hypertensive disease. As a result of the calcific process involving the media, the arterial system (including the aorta) becomes stiffened and loses its ability to dilate and decrease systemic vascular resistance. The resulting hemodynamic state is such that an isolated elevation in systolic blood pressure is observed, leading to both ISH and concomitant increased pulse pressure (difference between systolic and diastolic blood pressure). Although ISH has a strong association with medial calcification, its relationship with intimal atherosclerosis has not been elucidated to a significant degree [28]. With sustained, chronic hypertension, cardiac ventricular muscle cells undergo hypertrophy as a means to compensate for this increased afterload. Although this mechanism is compensatory, it is not without pathological consequence, with severe left ventricular hypertrophy (LVH) eventually leading to diastolic heart failure [29].

Discussion

Among the numerous functionalities that miRNA-200b has been shown to have [30], its suggested role in downregulating PKA activity may be an important player in preventing medial aortic calcification. This can be inferred as increased PKA activity has been associated with the deposition of bone-like material in the aortic medial layer. Although many cellular pathways may potentially be involved, one proposed mechanism is the PKA-induced increase in PTH levels. Interestingly, PTH is generally thought to be involved in bone resorption. However, it appears to have the opposite effect leading to calcification in the vasculature. Following calcification of the aorta and other arteries, the stiffening of these vessels leads to ISH. With longstanding ISH, the myocardium becomes hypertrophied as a compensatory mechanism with the eventual development of diastolic heart failure. This proposed sequence is presented in Figure 1. Furthermore, miPEP may prove to be paramount in maintaining high miRNA activity, given the positive feedback that miPEP exerts on miRNA; although this observation has only been made in plant cells, the existence of both miPEP and miRNA in humans could provide the same relationship. No such studies have been published on the potential regulatory role of miPEP on miRNA in human physiology due to the relatively recent discovery of miPEP in our previous study [12]. A very recent study showed that miRNA-8 and miPEP-8 act to produce the same end result, regardless of their exact mechanisms of action, in Drosophila [31]. These findings are quite interesting, simulating many other protective mechanisms seen in nature, such as genetic redundancy [32]. By providing two mechanisms of accomplishing the same end goal, one mechanism serves as the main actor, and the other serves as the back-up in case of an event that renders one of them nonfunctional. As such, even in the case of a knock-out polymorphism of miRNA-200b, miPEP-200b can independently regulate the function of PKA by interfering with its regulatory subunit [16] and decreasing vascular calcification.

Conclusion

If our proposed mechanism of disease progression in medial aortic calcification in the context of miRNA-200b and miPEP- 200b is shown to hold true, it will provide targets for therapeutic interventions. PKA antagonists, miRNA-200b, and miPEP-200b could be utilized with the end goal of decreasing PKA activity and decreasing vascular calcification with a subsequent decrease in the incidence and prevalence of both ISH as well as diastolic heart failure. Furthermore, there is potential for miRNA-200b and miPEP-200b levels to serve as prognostic factors for these diseases. There is reason to suggest this, as multiple studies have shown the promising potential of using miR-200 family levels as a strong prognostic marker for disease severity; low levels of the miR200 family have shown to be accurate predictors of a worse prognosis in pancreatic, lung, gastric, and bladder [33-36]. Although there is much to be discovered in this new arena involving miPEP, these findings provide an excellent starting point for future experimental designs, arming scientists with a new outlook on cellular pathways that were previously thought to behave differently.

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lupine-publishers-lojpcr · 3 years ago

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Lupine Publishers | Wish you a very Happy Thanksgiving Day

A thankful heart is the parent of all virtues. This thanksgiving may you give thanks for everything you are blesses with this happy occasion and wish you lots of happiness!

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lupine-publishers-lojpcr · 3 years ago

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Lupine Publishers | The Kinetics of SARS-Cov-2 Infections Analyzed by Means of Statistical Methods on the Basis of Official Data in Germany

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

The officially reported number of incidences of infections by SARS-CoV-2 by the German RKI were analyzed concerning periodic effects where not only the expected week-induced periodicity was found, but also for 3 ½ and 18 days. Consequences thereof were discussed.

Introduction

Infections with SARS-CoV-2 are presently one of the most important menaces for humans [1] and thus, subject of various scientific activities where impressive successes where obtained concerning vaccination and treatment and general preventive measures such as disinfections [2]. However, the formation of mutants requires a continuation of such research. Statistical methods were applied for analyzing local outbreaks and developments [3]. On the other hand, a periodicity of such processes obtained much less attention and is subject of this study.

Materials And Methods

Daily registered numbers of infections by SARS-CoV-2 were officially published in Germany by the RKI (Robert Koch Institute) [4] and were the basis of analyses. The period of March 2, 2020, until January 8, 2021, was preferred because infections were dominated by one variant of the virus so that minimal interference can be expected. The used data are reported in Figure 1. The last number of incidence was daily exponentially damped by a factor of 0.9 for filling the data up until 512 values to obtain an integer exponent of 2. This avoids artifacts by an abrupt termination. A Fourier transformation (FFT) was applied to the data to obtain the frequencies where the absolute intensities were calculated as the square root of the squares of the real and imaginary components. These somewhat abstract frequencies were transformed into the clearer periods in days (inset in Figure 1).

Results and Discussion

Periodic processes in the course of infections according to Figure 1 during the complete period were investigated by means of the Fourier transformation to obtain the frequency domain from the time domain in Figure 1. Fluctuations such as induced by local events or holidays may cause some noise but are unimportant because of no contribution to periodicity. The obtained comparably abstract frequencies were transformed to the more descriptive time of corresponding periods reported in the inset of Figure 1. One firstly finds the expected 7 days period induced by the weekly human activities; this indicates the basic viability of the concept. More interesting is the sharp peak at 3 ½ days indicating a temporarily limited process of efficient infecting. Further interesting maxima were found at 18 and at 24 days. The latter is not so pronounced as the former where there may be further maxima at even longer periods. One problem of such very long periods is the comparably low number of points because the period is the inverse of the frequency. Thus, the discussion is concentrated to the first two periods except the weekly. It seems that the infectivity is very pronounced after 3 ½ day and then decreases to lower values; special care is recommended for this short period if possible. The infectivity seems to be recovered after about 18 days; this may be problematic for mild infection with declined symptoms and might be an explanation that it was not successful to find all chains of infections even in regions of low numbers of incidences and the fact that declining proceeds very slowly in regions of high incidences.

Conclusion

Pronounced periodicities were found in the course of infections with SARS-CoV-2 and may be important for the handling in pandemic situations. There are two pronounced periodicities besides the human activity-induced weekly effect:

(i) A strong and temporarily sharp limited periodicity of 3 ½ days and

A second after 18 days. The former may be important for the handling and indicating of acute infections, whereas special care is recommended for latter to prevent a recovering of infections from persons with obviously declined infections.

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Lupine Publishers | Chemistry, Dual Use and Ethics

Lupine Publishers | LOJ Pharmacology & Clinical Research

Introduction

In the practice of chemistry, an essential ethical principle must always prevail chemistry must serve to achieve the well-being of humanity, with absolute respect for the environment. It should never, never be used against either one or the other. The exercise of chemistry inevitably involves the transformation of matter, starting from one substance and arriving at another, sometimes very easily, sometimes overcoming enormous difficulties. However, the possibility that these transformations of matter result in something unwanted, or something desired but that can be used for purposes contrary to the ideal, is great, sometimes too big [1-4] .

Duality

We might think that almost all technology can have dual uses. When the primitive human discovered that he could make fire and control it, he used it to illuminate his nights and the interior of the caves, to drive away beasts and intruders, but he also learned that he could apply it to set fire to the possessions of his enemies and thus be able to subdue them [5]. And from then to today, when our civilization use fire to cook delicacies and burn villages with napalm, has the situation changed?

Louis Fieser

Using fire. During World War II, incendiary bombs were loaded with a gel formed from crude rubber. The attack on Pearl Harbor gave the Japanese control of the most important sources of natural rubber for the manufacture of bombs. Then Colonel ME Barker, from the US Army Chemical Weapons Service, met with Dr. Louis Fieser, asking him to find a substitute for natural rubber. The requirements were various: the gel should be stable between 50ºF, for operations in tropical areas and -40ºF, for operations in cold locations; withstand storage conditions and be safe in its handling to be able to adequately fill the bombs. Fieser and his team developed a gel with excellent properties, complying with what was requested by Colonel Baker and with temperature properties and destruction capacity superior to rubber gels [6]. With military optics, a great success.

Fieser was criticized for using science for destructive purposes, against humanity. Their varied answers are synthesized in a single one, supposedly published in the first issue of Time magazine of 1968, of January 5 (Science History Institute), which says: “I have no right to judge the morality of napalm, just because I invented it”. Because he never participated in attacks using napalm, nor did he order them, he considered that his only responsibility was to have invented it [7-9]. This position of Fieser contrasts with the need for scientists, particularly chemists, to carry out our activities contemplating the possible consequences of what we produce, whether they are good or not so good. And that should also apply to the corporations and companies in charge of the mass production and distribution of these products developed by chemists. The Dow Chemical company was in charge of the manufacture of the napalm used in Vietnam (and also part of the herbicides used in that war, such as the well-known Agent Orange) and this corporation could not declare itself ignorant of the use that would be given to its product; far from it, they claimed that their product brought benefits for the acts of war that were carried out, they spoke of the merits of the war and questioned whether it was really used

indiscriminately against the civilian population (Contakes, op. cit.). What a stupid way of defending themselves!

Fritz Haber

Let’s talk about Fritz Haber, German chemist who won the Nobel Prize in Chemistry in 1918 , although it was awarded to him in 1919. The committee in charge of the selection of candidates agreed to recognize him “for the synthesis of ammonia from its elements ” [10]. The discovery by Haber allowed to use the most abundant gas in the planet’s atmosphere to produce in a fairly simple and economical way a compound that is of high social importance; since it allows, among other things, the synthesis of synthetic fertilizers with a high nitrogen content. Plants cannot use atmospheric nitrogen in their vital processes, but ammonia nitrogen is very easy to assimilate [11-13]. In this way, dependence on natural fertilizers (guano, manure, animal and human feces) is reduced and crops with much higher yields can be achieved. This discovery helped the world combat the specter of famine and Haber’s name was then linked to the concept of “bread from air” [6]. The curious thing about the matter is that Haber did not seek a welfare for humanity but the progress of his homeland. Before ammonia, most of the fertilizers used in Germany consisted of guano, which was imported from the distant lands of Chile. In this way, what drove Haber was a fervent nationalism; claimed that the meaning of Germanhood, (used by Haber) was something that would have to be adjusted “like everything great and eternal” [4,7]. The impetus for his development of the work on ammonia was based on the fact that in a state of belligerence although not of war per se (which would soon be declared politically), British ships blocked the importation of South American guano, affecting not only agriculture but also to the production of ammunition, which required large amounts of nitrates, available in guano. In September 1914, a group of experts, including Haber, undertook the task of solving this problem and first devoted themselves to the question of ammunition, which apparently was their primary motivation (Spleen CE,). At the outbreak of the Great War, Germany was prepared, in part thanks to Haber’s work, which had not been completed. He then began to think of a technology of war that would allow Germany to quickly rise to victory and decided to dedicate efforts in this direction. He focused on what he knew well, chemistry, developing the first chemical weapons. In April 1915, he himself appeared in the vicinity of Ypres to monitor the weather conditions, he spent several days until he considered that everything was in favor of the German army, and on the 22nd of that same month, he ordered an attack against the allied forces barricaded there. With the wind in his favor, blowing towards the enemy trenches, Haber ordered the release of about 150 tons of gaseous chlorine that was transported in 6,000 cylinders and that were opened almost simultaneously; the cylinders were placed along more than six kilometers of the German trench and a huge and dense cloud of greenish gas drifted towards the Allied lines [8]. Different authors mention different numbers of casualties due to this attack. For example, Harris and Paxman report 5,000 dead and 10,000 injured [11], while Spiers reports around 7,000 injured and 350 dead [2]. Haber bragged about the success of his strategy, adding that it caused a truly agonizing death [5]. And yet, he said that “all modern weapons ... owe their success to the vigor with which they temporarily weaken the psychological strength of the adversary” [4,10]. The point is, Haber was proud of the success of his attack, not caring that the Hague accords of 1899 had been violated. After the attack on Ypres, Haber was appointed captain of the German army. For him, humanity was not as important as the homeland, his homeland. To celebrate his appointment as captain, he held a great party at his home on May 1st, 1915 [3]. There something unexpected happened, closely related to Haber’s ethics.

Clara Immerwhar

Clara Immerwahr was an excellent chemist, in her own right. It is said that she was probably the first woman to obtain a doctorate in chemistry in Germany (Essex and Howes, op. Cit), but it was not easy at all. Her stay at the university was as observer, that is, she had to obtain authorization from all teachers to attend any of the classes, among many other obstacles. She finally obtained a doctorate degree in 1900. One of her teachers had been Fritz Haber but contact between them during Clara’s student days was short. His biggest approach was in a dance class. The couple met again after Clara’s graduation and married in 1901. Initially Clara participated in Haber’s projects, but little by little she was relegated to the role of wife and mother. To keep up to date, she translated articles from English into German for her husband to use and was responsible for the English edition of Fritz’s book, Thermodynamics of Technical Gas-Reactions. Clara never stopped thinking about going back to a laboratory, but when her son Hermann was born, she realized that this would be almost impossible, due to the delicate state of her son’s health. However, the differences between Haber and Clara grew deeper every day. The differences were clearly reflected in two statements that show two lines of thought, two views of scientific ethics; Clara thought that “scientific study was obliged to respect life”, while Haber argued that “In times of peace a scientist belongs to the world, but in times of war, he belongs to his country” [8]. In fact, it is mentioned that when the use of toxic substances as a method of warfare began, Clara publicly denounced that this “constituted a perversion of the ideals of science”, to which Haber responded by denouncing her as a traitor (Essex and Howes, op. cit.). So, disturbing the Haber’s reaction against his own wife, the mother of his child! Let’s go back to the big party on May 1st . Clara waited for the guests to leave, then took Haber’s service weapon, headed out into the garden, and shot herself in the chest. The one who heard the shot was her son Hermann, who found her still alive, but she died in a few moments. And to emphasize the tragedy, it seems that Haber’s orders were to go to the front immediately, so that his wife’s funeral was left to his son, who was then only thirteen years old (Vazquez, op. Cit.). This family conflict reflects two different visions of professional ethics: one decidedly nationalist, regardless of the consequences for humanity, and the other deeply human, always looking out for the well-being of all, regardless of borders and other differences. And paradoxically, this vision of ethics in favor of humanity, ended one life and deeply hurt another.

Clara and Fritz’s son Hermann was a chemist, worked in the patent area and committed suicide in 1946, after the death of his wife, who died of cancer. He apparently never recovered from having been in charge of his mother’s funeral and his father’s second marriage.

Back to Duality

Dual use processes are those that can be used to obtain substances for peaceful or harmful purposes. Dual-use chemical substances can have a legal, legitimate use and other illegal or illegitimate use. We can also find equipment and instruments that can be used in a double way; a knife can be used to cut meat and eat, or to assault people in the street. Large ultra-pure aluminum tubes, which can be used in rockets to put satellites into orbit, also make long-range missiles transporting explosives or weapons of mass destructions in their heads or use them in centrifuges to enrich uranium. In turn, the centrifugation of uranium to enrich it can lead to its use in nuclear power generation plants, or for the manufacture of atomic bombs [1]. Here we have examples of dualuse processes (centrifugation), dual-use equipment (rockets and missiles) and dual-use substances (aluminum).

Dual Use Substances

One example of a dual-use substance is isopropanol or isopropyl alcohol. It is widely used as a wound disinfectant (like ethanol or ethyl alcohol) and is widely used as an industrial solvent for the extraction of natural plant products, in inks and varnishes, for cleaning electrical components, in the manufacture of topical medicines, in cosmetics and in a wide variety of other applications. But it is also one of the raw materials for the manufacture of chemical weapons, specifically the neurotoxic agent called sarin, which has not only been used in warfare but has also been used in terrorist acts. And the case of chlorine is even more dramatic. Chlorine is the most used chemical for the purification of water, that is, to provide water suitable for human consumption from a microbiological point of view. In addition to this tremendously important use on its own, it has multiple industrial uses in plastics and drugs, to name a couple of them. But, as we have seen before, it was the first toxic agent used in a massive way in a war, that is to say, it is an indisputable chemical weapon. Is chlorine good or bad? Neither one nor the other. Its application is what can be questioned from the point of view of ethics. And it will depend on the type of ethics that is used to judge it. So, a chemist can face the ethical dilemma, when working with isopropanol or chlorine: do I make drugs to help people, or do I make weapons that are going to leave me more money? But this is not the only ethical dilemma someone may face in relation to these two products. Should the chemistry teacher respect the right to information of his students? Should he indiscriminately inform them of the relationship between beneficial isopropanol and chlorine and the evil chemical weapons? If they have never heard of this, now they know it. It is not necessary for the teacher to tell them how to do evil products, there are many Internet pages that describe step by step the chemical transformation of good into evil. Shouldn’t the teacher give that information to his students? What about their right to information? On the other hand, is it useful to inform young people and does it guarantee that chemistry misuse is avoided? Or is that achieved by hiding the information.

Uranium enrichment is the process by which the amount of fissile uranium (which decomposes by breaking its nucleus into smaller nuclei, emitting radioactive particles) is increased with respect to the amount of non-fissionable uranium found naturally in minerals. The fissile (U235) in nature is found in less than 1%, while the non-fissionable (U238 ) is found in more than 99%. If it is enriched to 10%, it is used for nuclear power plants; if it is enriched to 90% or more, it is used for armaments.

Conclusión

As we can see, the relationship of ethics and chemistry is a complicated matter. There are personal interpretations, nationalism, patriotism, particular interests and other ways to accommodate ourselves or accommodate the science to our particular interests. The author is left with the basic element mentioned by the OPCW: achievements in the field of chemistry must be used for the benefit of humanity and in defense of the environment.

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Lupine Publishers | Development and Characterization of Herbal Formulation Containing Aqueous Extract of Clitoria Ternatea Linn. (Flowers) for the Treatment of Vaginal Infection

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Among several gynecological problems, vaginal infection (vaginitis) is the most common problem that women face in their life span, caused by Candida species. Candida albicans, a fungus which is responsible for the vaginal infection and to cure it, innumerable herbs are available which were earlier frequently used by the local civilization but now their demand is increasing in global market also. Numerous studies have confirmed the use of Clitoria ternatea as an antimicrobial agent, so the present work has been undertaken to explore the anti-candida activity of the plant. Along with that, formulation of herbal cream was aimed. In this study, the herbal creams (HC1-HC8) were formulated and evaluated possessing aqueous extract of flowers of Clitoria ternatea Linn. and the results revealed the satisfactory results when compared with standard drug. The results of formulation with code HC5 were found to be very promising and excellent of all herbal cream formulated. Ensuring biological activity for any formulation is of prime interest to reveal and prove the efficacy of formulated dosage form. In view of the same, Herbal Creams (PHC) were prepared and subjected to anti-candida activities, where PHC of HC5 was evaluated for anti-Candida activity as an optimized preparation. From the results obtained, it was concluded that the PHC (HC5) have optimum anti-candida activity when compared with standard formulation (MCC) and may be used for the treatment of vaginal infection. Moreover detailed clinical approaches need to establish for the formulation of safe and effective drugs

Keywords:Clitoria ternatea; Anti-fungal; Vaginal infection

Introduction

Over the course of a lifetime, 75% of all women are likely to have at least a vaginal Candida infection and up to 45% have experiences it two or more, making it the commonest gynecologic diagnosis in primary care. As women tend to be more prone to vaginal yeast infections because of several reasons such as stress, poor nutrition, poor hygiene, lack of sleep, illness, hormonal imbalance or while pregnant or taking antibiotics. Women with immunosuppressive diseases such as diabetes and HIV infection are also at greater risk [1] and This infection also known as Vaginitis is characterized by symptoms, including discharge, odor, itching, irritation, or burning (Figure 1). Often causes by Candida species especially Candida albicans that accounts for 85% to 90% of cases [2]. From the ancient era, many plants and herbs are being used to treat various infirmities; one of them is Clitoria ternatea L, commonly known as Aparajita. In all ancient scriptures of Ayurveda, it is mentioned as one of the important herb [3]. Clitoria ternatea, also known as butterfly pea, is a perennial twinning herbaceous plant which belongs to the Fabaceae family. Major phytoconstituents of the plant responsible for several activities are flavonols namely kaempferol, 7-rhamnoside, quercetin, anthocyanins such as ternatin and delphinidin and the pentacyclic triterpenoids viz. taraxerol and taraxerone. The herb is being used for the treatment of diabetes, pain, inflammation, vaginal irritation and infections and worm infections [5].

Figure 1: Flower of Clitoria ternatea (Linn)[5].

Materials and Methods

Selection of plant material

The herb Clitoria ternatea Linn. is being used for the treatment of gynecological disorders especially vaginal infection from so long by the folks but till date no systematic investigation has been carried out to formulate the effective herbal formulation using the flower alone or in combination to treat gynecological disorders (vaginitis), therefore, this herbs were selected for the research work [6-10].

Collection and authentication of plant

The plant material selected for the present investigation was flowers of Clitoria ternatea Linn. and were collected in the months of Dec 2020 to Jan 2021 from various sites of Malwa region of Madhya Pradesh and identified & authenticated by Dr. S.N. Dwivedi, Professor and Head, Department of Botany, Janata PG College, A.P.S. University, Rewa, (M.P.) The specimen was deposited in Laboratory, with Voucher specimen No. P/CT-F/1403.

Preparation and characterization of extracts [5]

Preparation of extracts:- 250 gm of the air dried coarsely powdered flowers of Clitoria ternatea Linn. was placed in soxhlet apparatus and was extracted with distilled water until the extraction was completed (till 24 hr.). After extraction, the filtrate was evaporated, and percentage extract was determined.

Characterization of extract:- The aqueous extract of dried plant material of Clitoria ternatea Linn. (flowers) CTF were characterized for their color, odor, taste and pH and were recorded in present investigation.

Anti-candida activity of extracts

Fungal strain:- Fungal strain i.e., Candida albicans (ATTC 10231) was obtained from Medical College and was used for the present investigation.

Screening of antifungal activity by disc diffusion method:-

Preparation of disc: Disc of Whatman filter paper of one quarter inch in diameter was prepared and the same was sterilized using autoclave.

Preparation of samples entrapped disc: The accurately weighed extracts were dissolved in methanol and different stock solutions (10, 20, 30, 40, 50 μg/ml) solutions were prepared. All the dilution prepared was applied to Whatman filter paper disc using a micropipette. The discs were then dried and sterilized.

Preparation of culture plate: The Sabouraund’s agar and Mueller Hinton agar media were prepared by dissolving media in 1000ml of distilled water and sterilized by autoclaving at 121oC for 1 hour. The media were cooled and poured in sterilized Petri plates to solidify at room temperature.

Collection of fungal strains: The fungal strains (Candida albicans) were obtained from Index Medical College, Malwanchal University, Indore. The inoculums of strain were transferred to the re-cultured before starting the lab work.

Evaluation of zone of inhibition: The re-cultured fungal strains were used for antifungal evaluation. The strains were streak on the Mueller Hinton media and the drug entrapped discs were placed. For negative control, disc of distilled water and for positive control clotrimazole disc (10 μg/ml) were used. The disc of aqueous extract of dried flower of Clitoria ternatea Linn. was prepared having 10μg/ ml concentration. The petri plates were kept in incubator for 24 hrs. After 24 hrs, the petri-plates were checked for zone of inhibition. The zone of inhibition diameter was recorded with the help of zone reader scale. The zone of inhibition was calculated by subtracting diameter of sample or standard or control by diameter of disc. The more the zone of inhibition the more will be antifungal activity.

Statistical analysis: All the reading obtained were analyzed using one way analysis of variance i.e., ANOVA. Student t-test was used. The values were found to be statistically significant (*P<0.00, **P<0.01). All the values obtained were expressed as mean± standard error means (SEM).

Formulation of herbal cream[6,7]

The herbal cream was prepared using fusion method and the various steps involved in formulation of herbal cream were as described below.

Preparation of oil phase: Stearic acid, cetyl alcohol, almond oil were taken in desired quantity in porcelain dish as per the Table 1 and were melted at 700C.

Table 1: Formulation of Herbal Cream Containing Aqueous Extract of Clitoria ternatea L.

Preparation of aqueous phase: Aqueous extract of dried plant material of Clitoria ternatea Linn. glycerol, methyl paraben, triethanolamine and water as per Table 1 were taken in another porcelain dish and were heated at 700C.

Addition of aqueous phase to oil phase: The aqueous phase was added to the oil phase with continuous stirring at room temperature. Perfume was added at last and the formulation was transferred in a suitable container.

Physical evaluation: The prepared creams were evaluated for physical characteristics by evaluating clarity and transparency which was determined through visual inspection by observing the sample in light at white background (Tables 2 & 3).

Table 2: Antifungal Activity of Clitoria ternatea Linn.

Table 3: Results of Evaluation Parameters of Formulated Herbal Creams.

Determination of pH

pH of the prepared formulations was determined using digital pH meter. The pH meter was first calibrated, and zero reading was recorded. The samples were diluted with water to make suspension and were taken in the beaker and the readings were taken from calibrated electrode. The procedure was repeated, and reading were taken in triplicate and recorded.

Determination of viscosity

The viscosity of the herbal cream was determined by Brookfield Viscometer using spindle no. 1 working at 20 rpm at temperature 4oC and 37oC. About 15ml of the samples from each batch was taken in beaker and spindle was immersed in the formulation (Figure 2). The reading was recorded at initial and after rotation at different temperature. The reading was recorded thrice.

Figure 2: Drug Release of Formulations and Standard.

Determination of homogeneity

Homogeneity of various formulations was tested by visual observation and reported. The formulations were also evaluated for presence of any aggregates or gritty particles (Tables 4 & 5).

Table 4: Drug Content of Herbal Cream containing Aqueous Extract of Clitoria ternatea L. (Flowers).

Table 5: In-Vitro Drug Release of Herbal Cream Containing Aqueous Extract of Clitoria ternatea L.

Determination of spreadibility

The formulations were placed on the glass slide and the empty glass slide was place on the top of cream containing slide. The herbal cream placed between slides was pressed to form thin uniform layer. The two slides were fixed and on the upper glass slide, the weight of 20 ± 0.5 g of the weight was tied. Due to weight, both the slides were separated, and the time required to separate the two slides, i.e., the time in which the upper glass slide moved over the lower plate was taken as measure of spread ability. The three readings were recorded, and mean time was taken. The spread ability was calculated using the formula:

S= m x l/t

Where,

m = weight tide to upper slide

l = length moved on the glass slide

t = time taken.

Determination of wetness

The prepared herbal creams were determined for wetness by applying on skin surface. The amount of cream taken from each batches must be the quantity sufficient to apply at 10 mm2 area of skin surface.

Determination of type of smear

The prepared herbal cream was applied on the skin surface and after the application the type of film or smear formed on the skin was recorded. The amount of cream taken from each batches were quantity sufficient to apply at 10 mm2 area of skin surface.

Determination of emolliency

The prepared formulation was checked for emolliency, slipperiness and amount of residue left after the application of cream. To determine emollinecy, the sufficient quantity of cream was taken from each batches and applied at 10 mm2 area of skin surface and the results were noted.

Determination of Type of Emulsion

Dilution test: The prepared herbal cream was diluted with oil or water depending upon the type of emulsion whether o/w or w/o the results obtained were noted down.

Dye solubility test: The prepared herbal cream was mixed with a water-soluble dye i.e., amaranth and was observed under the microscope. The results obtained were interpreted.

Determination of drug content: The drug content of the herbal creams was estimated using UV-Visible spectrophotometer. Nearly about 1g of the formulation was taken in 50 ml of volumetric flask and the solution was made up to mark with methanol. The solution was shaken and filtered through what man filter paper and about 0.1ml of the filtrate was further diluted to 10ml with solvent and estimated at suitable wavelength.

In vitro drug release/dissolution test: The semi permeable dialysis membrane bag (7cm long) was prepared, and the herbal cream was placed in the membrane. The dialysis bag was then suspended in 50ml of ethanol: water (1:1) at temperature 37°C ± 0.5°C in water bath. About 1ml of sample was withdrawn from the membrane at predetermine regular intervals (2, 4, 6 and 8 hr.) and the fresh equal volume was replaced simultaneously. The samples were withdrawn, diluted and analyzed by UV Visible spectrophotometer at suitable λmax for herbal cream containing aqueous extract of Clitoria ternatea Linn. The experiment was repeated thrice, and the cumulative drug release was calculated from the reading.

Anti-Fungal activity of optimized formulation: The antifungal activity (anti-candida activity) of optimized herbal cream and standard cream formulations was determined by disc diffusion method and results obtained for Zone of inhibition were compared. Marketed Clotrimazole cream (MCC) 1% w/w IP was used as a standard.

Results and Discussion

The herbal cream containing aqueous extract of Clitoria ternatea flowers was prepared and evaluated on various parameters. All the preparations gave satisfactory results on all the defined parameters and the results are shown in Table 2. From the results of drug content of formulated herbal cream containing aqueous extracts of dried plant material of Clitoria ternatea Linn. (Flowers) CTF and different proportion of excipients, it was found that HC5 has maximum drug content in combination therefore, HC5 were evaluated for anti-Candida activity. From the results (Table 3) obtained it was concluded that the PHC (HC5) have optimum and significant anti-candida activity when compared with standard formulation (MCC) and hence, it may be used for the treatment of gynecological disorders (vaginitis). Moreover detailed clinical approaches need to establish for the formulation of safe and effective drugs.

Conclusion

Candida albicans, a fungus which is responsible for the vaginal infection. Several literatures have suggested the effectiveness of Clitoria ternatea as antifungal agent for the treatment of vaginal infections. To prove it scientifically, anti-candida activity of aqueous extract of selected plant were evaluated. The zone of inhibition of extract on Candida albicans were investigated and results indicated that the selected extract have significant anti-candida activity when compared with standard drug Clotrimazole. From the results obtained, it was concluded that the extracts of selected herb have optimum anti-candida activity and may be used for the treatment of gynecological disorders (vaginitis) (Table 6). The results of formulation with code HC5 were found to be most promising and excellent among all herbal creams formulated when compared with standard formulation (MCC) and may be used for the treatment of vaginal infection. However detailed research study needs to be done to develop this as an effective formulation.

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Lupine Publishers | Happy American Independence Day Wishes

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Lupine Publishers|The Effect of Body Mass Index on Components Size in Patients With Total Knee Replacement

Abstract

Introduction: Nowadays, one of the most common practices in orthopedic surgery is the Total Knee Arthroplasty. Logistic equipment, including prostheses, is the cornerstone of this surgical procedure. Clearly having a general overview of the size of consumable prostheses, the surgeons will act more confidently.

Materials and Method: This descriptive-analytic study employed a census method based on the inclusion criteria of the medical records of all patients who underwent knee replacement surgery in Besat Hospital of Hamedan in 1395 (2016-17) in terms of body mass index, age, gender, and size of tibial and femoral components. Data were analyzed by SPSS software version 16.

Results: A total of 214 patients were included: 21.5% male and 78.5% female. The mean age of patients was 67.95 years, and the mean body mass index was 27.98 kg/m2. As for the assessment of the size of components, most tibial components were of medium size (49.5%) and femoral components were of small size (65.9%). It was seen that there was an inverse correlation between tibial component and body mass index (BMI), and a direct and significant correlation between tibial component and height and weight in terms age and gender (p<0.05). Nevertheless, the correlation between femoral component and weight was statistically significant only at ages under 65 years and it was indirect (p=0.02).

Conclusion: Determining factors of the size of components used in joint replacement surgery include body mass index (BMI), weight and height of the patients for tibial surgeries, and weight of the patients for femoral ones.

Keywords: Body mass index (BMI); tibial component; femoral component

Abbreviations: OA: Osteoarthritis; BMI: body mass index; TKA: Total knee arthroplasty

Introduction

gradually causes various deformities in knee joint and makes it painful while walking. It also disrupts skeletal alignment and increases the risk of Osteo arthritisfalling and fractures. The disease usually affects both limbs and this mutuality can exacerbate functional disorders [8]. Osteoarthritis causes major health and social problems in the elderly and brings a heavy economic burden on the health and social welfare system [9]. Progression of the disease in this weight-bearing joint leads to significant disability and reduced functional capacity in a wide range of daily activities. The main causes of this functional decline in the patients are weakness of the quadriceps muscle, deep-seated perturbation and poor stability [10,11,12]. Common therapies for reducing pain and improving patients’ function include lifestyle changes and daily habits, rehab Rehabilitation and medicine therapy [13]. Patients with symptoms of pain, joint instability, reduced range of motion and deformity, and lack of response to conservative treatment need to have a surgery, which may include knee joint replacement. In spite of reducing the patients’ pain and improving the quality of their life, this surgery is a costly process with relatively high complications. Therefore, accurate preoperative planning for reducing costs and probable complications is so important for these patients. The planning procedure includes the preparation of medicine, equipment and operating room. Logistic equipment, including prostheses, is the cornerstone of this surgical procedure. Clearly having a general overview of the size of consumable prostheses, the surgeons will act more confidently. Since the results of various studies regarding body mass index, component size and surgical complications are controversial and sufficient studies have not been done in this field, this study aimed to examine the effect of BMI on the size of components used in patients undergoing knee joint replacement surgery.

Method

This descriptive-analytic study examined all patients suffering from severe knee joint osteoarthritis who had came to the orthopedic clinic and after clinical examination, had undergone arthroplasty surgery. The required data for patients’ medical records and surgical description were extracted and recorded in the checklist. Preoperative data, including preoperative examinations, lateral and patellar radiographies, and lower limb alignment were documented. Preoperative counseling was done and its information was recorded. In the alignment graph, the angle between the mechanical axis of femur and tibia was measured. The BMI of the patients after recording height and weight was calculated using SPSS software. The patients transferred to the operating room and received anesthetic injections. Their consciuosness was assessed and the size of tibial and femoral component used for them was measured by the the classic anterior midline approach after incising tibia and femur. Finally, the joint replacement was completed using a cement prosthesis (ZIMMER, NEXGEN LPS, WARSAW, US), and the day after surgery, patients were prepared for recovery and discharged with appropriate equipment. The common size of the femoral component used for patients was 8. To facilitate the analysis, the sizes 1 to 3 were classified in the small group, the sizes 4 and 5 in the medium group and the sizes 6 to 8 in the large group. Moreover, body mass index (BMI) was classified in very fat (35≤), fat (34.9-30), normal (24.9-20), and thin (20>), based on the WHO standard categorization. Then, the relationship between BMI and size of measurements was measured using statistical analysis. The inclusion criteria of the study were all patients with severe knee joint osteoarthritis who were candidates for joint replacement surgery, along with their satisfaction for participation in the study, and exclusion criteria were the incompleteness of the data recorded in the case, the patients’ discontent and their unwillingness to co operatation.

Statistical analysis

The collected data were entered into computer and analyzed using SPSS version 16.0 statistical software. Quantitative data were described using mean and standard deviation; qualitative data were described in the form of tables, charts and frequency ratios. In the analytical section, Pearson correlation coefficient (PCC) was used to determine the correlation between quantitative variables and the size of tibial and femoral components; Spearman’s correlation coefficient was used to determine the correlation between ranked variables and the size of tibial and femoral components. The significance level in this study was considered as 0.05.

Results

Descriptive data of the 214 patients examined in the study are as follows

In terms of gender, the highest frequency belongs to the female group of 168 (78.5%) and the frequency of male group is 46 (21.5%). In terms of age, mean age of the participants of this study was 67.95 years. The minimum age was 32 years and maximum age was 94 years. The most frequent ages among patients undergoing knee joint replacement surgery was 60 to 69 years (46.26%). As for BMI of the patients undergoing knee joint replacement surgery, the mean body mass index was 27.89 kg/m2 (range 17.58-44 kg/ m2; standard deviation 4.49 kg/m2). The highest frequency was in the overweight group (25-29.9) (Table 1).

In terms of component size frequency, most tibial components were of medium size (49.5%) and most femoral components were of small size (65.9%). For used tibial components (small-mediumlarge) classified by various categories of BMI, no statistically significant rank correlation was observed between the size of used tibial component and BMI (P=0.097). According to the results of one way ANOVA test, a statistically significant difference was observed between the mean BMI and the size of used tibial component (P=0.005). The results of Tukey follow-up test showed that the mean BMI in small component (P=0.006) and in medium component (P=0.013) were significantly more than the mean BMI in large component (Table2).

Table 2: Comparison of mean BMI in terms of the type of used tibial component.

For used femoral components (small-medium-large) classified by various categories of BMI, no statistically significant rank correlation was observed between the size of used femoral component and BMI (P=0.570) (Table3).

According to the results of one way ANOVA test, no statistically significant difference was observed between the mean BMI and the size of femoral component (P=0.156). As for the relation between the size of tibial and femoral component and weight in terms of gender and age, a direct and significant was observed between tibial component and weight in terms age and gender (p<0.05). Nevertheless, the correlation between femoral component and weight was statistically significant only at ages under 65 years (p=0.02) (Table 4).

Moreover, for the relation between the size of tibial and femoral component and height in terms of gender and age, a direct and significant was observed between tibial component and heightin terms age and gender (p<0.05). Nevertheless, the correlation between femoral component and height was not statistically significant (p<0.05) (Table 5).

Discussion

Knee joint is one of the main joints of human body which is usually affected by various progressive degenerative and inflammatory diseases, which ultimately lead to the destruction of articular cartilage (arthrodial cartilage) and its dysfunction [1]. Nowadays, knee joint replacement arthroplasty is a helpful method for the patients with severe knee joint In this study, there was no statistically significant difference between mean BMI and the size of femoral component. Also, the correlation between femoral component with height was not statistically significant. There was an inverse correlation between the size of tibial component and BMI and a significant correlation between the size of tibial component and height and weight in terms of gender and age. There was no statistically significant correlation between the size of femoral component and BMI and height, but a significant and direct correlation was observed between the size of the femoral component and weight at the age under 65 years. According toa study by Sershon et al. about the effect of body mass index on the accuracy of measuring the size of components (templating), there was no significant relation between between body mass index and measuring the size of femur acetabular components [14]. Similarly, this study didn’t find any statistically significant relation between body mass index and the size of femoral component. However, there was a statistically significant and direct correlation between the size of the femoral component and weight at the age under 65 years. The results of one-year research of Stickles et al. on patients with knee joint surgery showed that in terms of postoperative satisfaction, there was no difference between the obese and non-obese patients [15]. Another study by Collins et al. in 2012 regarding the effect of obesity on the need for re-surgery and patient’s satisfaction after knee replacement, revealed that 9 months after the surgery, obese (BMI>30) and non-obese patients’ level of satisfaction was good. There was no need to re-surgery among two groups, however obese patients showed a lower functional performance scores than non-obese patients [16]. Jackson et al. conducted a study in 2009 which followed535 patients for 9 years. The results showed that obese patients had lower functional performance scores than non-obese patients [17]. Two different studies conducted by Nunes et al. examined the effective factors on the final outcome of Total knee arthroplasty (TKA) during 3 years and 7 years follow-up respectively [18,19]. McElroy et al. developed a structured review in 2013 and their patients were followed up for at least 5 years. The results showed that BMI>30 had lower functional performance scores and lower complications and only few of them used partial prosthesis [20]. A study by Isa et al. in 2013 found that obesity had no significant effect on TKA outcomes. In this study, 210 of 174 patients were obese. According to the gained results, obese patients showed more complications than non-obese patients (10.5% vs. 3.8%). In terms of other outcomes, there was no significant difference between the two groups [21].This study, in contrary to the mentioned studies, didn’t examine the realtion between patients’ body mass index and their postoperative satisfaction. Nevertheless, there was a statistically significant correlation between body mass index and the size of tibial component. The fact that the mean body mass index has an inverse correlation with the size of tibial component but a significant and direct correlation with the height, is probably due to its inverse correlation with the square of the height, that is, the higher the height, the smaller will be the body mass index. However, as mentioned by previous studies, the body mass index not only determines the size of tibial component, it is also a variable predictor of the long-term result of tibia and femur joint replacement surgery. Those with a higher mass body index are more likely to give pressure to the prosthesis and feel more pain and discomfort. In result, they are more likely to have need to re-surgery.

Conclusion

In patients undergoing knee joint replacement surgery, BMI and height don’t affect the size of femoral component, but at ages under 65 year, size of this component increases with weigh gain. Size of tibial component has an inverse correlation with the mean body mass index (BMI) and has a direct correlation with height and weight. Moreover, the results of this study showed that the increase in BMI is predominantly due to an increase in body fat mass.

Declarations

Ethics approval and consent to participate The study was conducted under the auspices of the ethical board of Hamadan University of Medical Sciences and registered with the number IR. 1396.286 in the Research Ethics Committee of the University of Medical Sciences.

Acknowledgements

There was no conflict of interest by any of the authors including financial and personal relationships with other people or organizations that inappropriately influenced this study.There has been no financial support with the project.

Limitations of the study

This study examines the results of just one health center. It is likely that the patients who have not visited this center would be different from those who participated in the study. It is recommended to conduct a similar study with a larger sample.

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Lupine Publishers | A Hmsh2 c.2332 T>A Mutation and Membrane-hMSH2/ TCRγδ/NKG2D-Mediated Cytotoxicity of Human Vγ9vδ2 T Cells in Ovarian Serous Cystadenocarcinoma

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Membrane human MutS homologue 2 (mhMSH2) is a well-characterized endogenous ligand for human Vγ9Vδ2 T cells, but its germline gene expression in mhMSH2-overexpressing ovarian serous cystadenocarcinoma (OSC) cells and tissues is unclear. Herein, we discovered a silent hmsh2 c.2332 T>A mutation in the OSC HO8910 cell line (GenBank accession no.MG674653) and identified serval novel soluble forms of hMSH2 protein from its whole cell protein extracts and culture supernatant. The mhMSH2/ TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells were validated in mhMSH2/6-overexpressing SKOV3 cells. Positive expression of cytoplasmic and/or membrane hMSH2/6, cytoplasmic and/or nuclear hMSH3 and complete loss of nuclear expression of hMSH2 was observed in all examined ovarian cancer tissues. The clinical significance of the novel hmsh2 c.2332 T>A mutation, the soluble full-length and truncated forms of hMSH2 proteins, and the complete loss of nuclear hMSH2 protein expression in OSC development and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further elucidated.

Keywords: Hmsh2; Mutation; Membrane hMSH2; Vγ9Vδ2 T cells; Ovarian serous cystadenocarcinoma.

Abbreviations: hMSH2: Human MutS Homologue 2; MMR: Mismatch Repair; M: membrane; EBV: Epstein-Barr virus; OSC: Ovarian Serous Cystadenocarcinoma; ATCC: American Type Culture Collection; IHC: Immunochemistry; mAb: monoclonal Antibody; FCM: Flow Cytometry; PFA: Paraformaldehyde; FITC: Fluorescein Isothiocyanate; DAPI: 4’,6-diamidino-2-phenylindole; LS: Lynch Syndrome.

Introduction

Human MutS homologue 2 (hMSH2), a critical element of the DNA mismatch repair (MMR) system, is normally localized in the nucleus of host cells and dimerizes with hMSH3 or hMSH6 (hMSH3/6) to form complexes that participate in DNA damage and cell apoptotic signaling. Inherited or acquired defects in the hmsh2 gene or protein lead to dysfunctional correction of errors in DNA synthesis and duplication, the cell cycle and Ig isotype switching of B cells and thus have a close association with the genesis of many types of tumors [1,2]. In our published study, we reported a broad overexpression of membrane hMSH2 (mhMSH2) in a series of epithelial tumor cell lines and Epstein-Barr virus (EBV)- transformed B lymphoid cells [3]. mhMSH2 overexpressed on malignant cells was subsequent characterized as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells, a subset of innate immune cells that play a crucial role in antitumor/antiviral immunity and autoimmunity [3-5]. The induction of ectopic mhMSH2 expression on renal carcinoma cells by oxidative stress via the p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways with interleukin-18 promotion was subsequently revealed, but the systemic expression of hmsh2 gene in mhMSH2- overexpressing carcinomas is still unknown [6]. There are 238,700 estimated new cases of ovarian cancer and 151,900 deaths per year worldwide, and thus, this disease is the fifth most common cause of female cancer-related death in the United States in 2018 [7,8]. Among the 4 subtypes of ovarian cancer (high-grade serous, endometrioid, clear cell ovarian carcinomas and non-epithelial subtypes), Ovarian Serous Cystadenocarcinoma (OSC) is the most common and lethal type in women [9]. Previously, we reported the unusual ectopic mhMSH2 expression on the human OSC cell lines HO8910 and SKOV3 and the specific binding of mutated mhMSH2 on SKOV3 cells to synthesized OT3 peptides of human Vδ2 TCR [3,10]. In this work, we further elucidated the hmsh2 gene mutation and ectopic subcellular protein expression in HO8910 cells and the mhMSH2-mediated recognition and cytolytic effects of Vγ9Vδ2 T cells towards SKOV3 cells. The abnormal subcellular distribution of hMSH2/3/6 in different subtypes of ovarian cancer tissues was demonstrated by immunochemistry (IHC).

Materials and Methods

Cell lines, culture medium and tumor tissues

HO8910 cells (OSC) were maintained in RPMI-1640 complete medium (Invitrogen, Shanghai, China). SKOV3 (OSC) and HK-2 (proximal tubular cell line derived from normal kidney) cells were cultured in 10% FBS DMEM/F12 medium (Niuyin, Beijing, China). All cell lines were purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences and confirmed to have no mycoplasma contamination before use. Expansion and subset separation of effector human Vγ9Vδ2 T cells were carried out as we previously described [3]. Ovarian cancer tissues belonging to different histotype (OV-1, serous adenocarcinoma; OV-2, embryo sinus carcinoma; OV-3, cyst-myxomatous adenoma) were freshly obtained from Peking Union Hospital with informed consent provided by the patients. The use of human tissues for research was also approved by the ethics committee of the Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou Institute of Pediatrics (2015020917).

Gene sequencing of hmsh2 in HO8910 cells

Appropriate numbers of HO8910 cells in the logarithmic growth phase were collected for total mRNA extraction and cDNA reverse transcription. The target hmsh2 gene was amplified using fragment PCR as we described in Subcellular hMSH2 expression is aberrant in membrane-hMSH2-overexpressing cervical, lung and gastric cancer cell lines and tissues (article in progression). Three PCR products with predicted lengths (990, 1250 and 1549bp) were purified and introduced into the pGEM-T easy vector (Promega, USA). Recombinant plasmids were positively selected and characterized by Not I digestion before gene sequencing. Human msh2 variants in HO8910 cells were screened by alignment with the full-length hmsh2 sequence (NM_000251) using Laser gene 7 software.

Soluble full-length and truncated forms of hMSH2 in the whole cell extracts and culture supernatant of HO8910 cells

Soluble forms of hMSH2 protein in the complete protein extracts and the cell culture supernatant of HO8910 cells were simultaneously separated by SDS-PAGE and blotted with antihMSH2 McAb (65021-1 Ig, 1:500, Proteintech Group, USA) and with goat anti-mouse IgG/HRP secondary antibody (1:5000, Zhongshan Jinqiao Company, China). 𝛽-actin was blotted as endogenous reference control (MW 43 kDa).

Growth curves of HO8910 cells

For growth curves of both OSC cell lines, appropriate numbers of trypsin-digested HO8910, SKOV3 and HK-2 cells were planted in 24-well plates in triplicate and cultured for 8 consecutive days in a 5% CO2 atmosphere at 37°C. Growth curves of the examined cell lines were obtained by using the cell counting method. The expansion rates of target OSC cells were compared with that of HK-2 control cells.

Quantitative real-time PCR for hmsh2 mRNA expression in HO8910 cells

The human msh2 gene transcription levels in HO8910, SKOV3 and HK-2 cells were comparatively analyzed by qRT-PCR. The results were processed with Sequence Detector Version 1.2 (Applied Biosystems, USA) and Sigma Plot 11.0 as we previously described [3].

Ectopic membrane, cytoplasmic and nuclear expression of hMSH2 in HO8910 cells

The membrane, cytoplasmic and nuclear hMSH2 expression in HO8910, SKOV3 and HK-2 cells were further investigated by laser confocal microscopy with different fixation reagents. The target cells (2-3×105) were cultured overnight on pre-autoclaved glass cover slips in a 24-well format, properly fixed with 4% paraformaldehyde (PFA) or ice-cold methanol for 10-15 min and blocked with 0.5% BSA for 30 min at 4°C before labeling with specific anti-hMSH2 (N- 20, Santa Cruz Biotechnology, USA) polyclonal antibody or rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Zhongshan Jinqiao Company, Beijing, China). The nuclei of the examined tumor cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma, USA) before being mounted on a Leica DMIRE2 inverted microscope (objective, 40×; numerical aperture, 1.25).

Participation of mhMSH2/TCRγδ/NKG2D in Vγ9Vδ2 T cell-mediated anti-OSC immunity

For further analysis of the role of mhMSH2 in human Vγ9Vδ2 T cell-mediated anti-OSC immunity, effector Vγ9Vδ2 T cells were incubated with SKOV3 cells at different ratios (effector: target [E: T] 20:1, 10:1, 5:1 and 2.5:1). Cytotoxicity was measured with the LDH method, as we previously described [3]. For cytotoxicity blockade assays, target OSC cells were pretreated with anti-hMSH2 (N20, Santa Cruz Biotechnology), anti-NKG2D (149810, R&D Systems) and anti-TCRγδ (B1.1, Immunotech, France) antibodies before incubation with effector Vγ9Vδ2 T cells at different E:T ratios (20:1, 10:1, 5:1 and 2.5:1). The blockade cytotoxicity was measured and compared to that of the rabbit IgG blockade control group. Target SKOV3 cells were stained with anti-hMSH2, anti-hMSH3 and anti-hMSH6 antibodies to confirm the cell surface expression of hMSH2/3/6 antigens before cytotoxicity and antibody-blocking cytotoxicity assays.

Subcellular hMSH2/3/6 Distribution In OSC Tissues

For IHC analyses, freshly collected ovarian cancer tissues were classically made into paraffin sections (3-4 μm in thickness) after proper neutral formalin fixation. The biopsies were heated at 60°C overnight and gradient dehydrated with methanol before antigen retrieval in boiled citrate buffer, followed by overnight incubation with purified mouse anti-hMSH2 monoclonal antibody (mAb) (clone G219-1129,1:200), mouse anti-hMSH3 mAb (clone 52, 1:20), mouse anti-hMSH6 mAb (clone 44, 1:30) (BD Pharmingen, USA) or isotype-matched mIgG1 at 4°C in a moist environment after 3% H2O2 treatment. The coloration was developed with PV9000 reagents (Zhongshan Jinqiao Company, Beijing, China) and captured with a Leica DM3000 imaging system.

Statistical Analysis

GraphPad Prism 7.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis and data plotting. Data are presented as the mean ± SD. Differences between/among groups were compared with Student’s t-test (two-tailed) or one-way ANOVA, P<0.05 was considered statistically significant.

Results

Hmsh2 gene sequencing and soluble full-length and truncated hMSH2 protein blotting

The PCR products of hmsh2 in HO8910 cells were 990, 1250 and 1549 bp in length (Figure 1A). By aligning the spliced full gene sequence with hmsh2 (NM_000251), we observed a point mutation (hmsh2 c.2332 T>A) in the HO8910 cell line (Figure 1B). It was a silent mutation that did not alter the primary structure of hMSH2 protein. The gene sequencing data of mutated hmsh2 in HO8910 cells were submitted to GenBank (Accession number: MG674653) for release. No additional gene mutation of hmsh2 was found in other mhMSH2-overexpressing epithelial tumor cell lines (data not shown here). By SDS-PAGE separation and western blots, several soluble forms of hMSH2 proteins were identified from the whole HO8910 cell protein extract [including the full-length form (MW ~105 kDa) and four truncated variants (MW~70, ~62, ~55 and ~40 kDa)] and the cell culture supernatant (MW~88, ~68 kDa) (Figure 1C).

Hmsh2 gene transcription and ectopic cytoplasm/ membrane expression

The growth and proliferation of both OSC cell lines were depicted with growth curves determined by cell counting assays. Both HO8910 cells and SKOV3 cells achieved rapid growth on day 5 and expanded much faster than the normal control HK-2 cells (Figure 2A). The mRNA expression of hmsh2 was slightly decreased in SKOV3 cells but substantially elevated in HO8910 cells compared with HK-2 cells (Figure 2B). Further detection of the ectopic membrane overexpression of hMSH2 on target OSC cells with laser confocal microscopy showed that both HO8910 and SKOV3 displayed different degrees of green fluorescence on the cell surface after labelling with a specific anti-hMSH2 antibody. The cell surface green fluorescence was much stronger on SKOV3 cells than on HO8910 cells. No green fluorescence was observed on normal control HK-2 cells (Figure 2C, the upper panels of pictures). The subcellular distribution of hMSH2 in HO8910 and SKOV3 cells was further determined by confocal microscopy with ice-cold methanol fixation. The membranes of both cells displayed different degrees of green fluorescence. The density of green fluorescence in the cytoplasm and the nuclei of SKOV3 cells was much stronger than that of HO8910 cells. There was no green fluorescence observed in HK-2 cells (Figure 2C, the lower panels of pictures).

Previously, we identified the membrane-overexpressed hMSH2 on several epithelial tumor cell lines as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells [3,6]. Herein, mhMSH2-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells towards target OSC cells were confirmed by cytotoxicity and independent specific antibody blockade cytotoxicity assays. mhMSH2 overexpression on OSC targets was validated with flow cytometry (FCM) before cytotoxicity and cytotoxicity blockade assays (Figure 3A). At E:T ratios of 20:1, 10:1, 5:1 and 2.5:1, the cytotoxic efficiency of effector human Vγ9Vδ2 T cells against target SKOV3 cells was 53%, 26%, 23% and 12%, respectively (Figure 3B). The ectopic mhMSH2-mediated recognition and cytotoxicity could be strongly blocked by specific anti-hMSH2, anti-NKG2D or anti-TCRγδ antibodies at different E:T ratios, indicating the participation of mhMSH2 in Vγ9Vδ2 T cell-mediated anti-OSC immunity by NKG2D/γδ TCR recognition (Figure 3C).

Subcellular distribution of hMSH2/3/6 in OSC tissues

As shown in Table 1, all ovarian cancer cells displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, while loss of nuclear expression of hMSH2 was observed in all 3 categories of ovarian cancer tissues. Strong and clear cytoplasmic and/or membrane hMSH2/3/6 expression was strikingly observed in ovary cyst-myxomatous adenoma nests (Figure 4A). Total loss of nuclear expression of hMSH6 was found in 66.67% (2/3) of the examined ovarian cancer tissues (Figure 4A). hMSH2/3/6 expression showed substantial heterogeneity among individual ovarian cancer patients (Figure 4B).

Discussion

Human msh2 is one of the most crucial genes involved in the DNA MMR pathway and was strongly associated with increased tumor mutational burden in a multivariate analysis [11]. Recent studies have shown that high hMSH2 expression is significantly associated with smoking, while low hMSH2 expression is an indicator of MMR deficiency in lung adenocarcinoma [11]. The high expression of hMSH2 accompanied by increased PD-L1 expression and CD8+ T cell infiltration therefore leads to the development of a prominent immunotherapy-responsive microenvironment for lung adenocarcinoma and acts as a potential surrogate biomarker of tumor mutational burden to identify immune checkpoint blockade responders in this disease [11]. Moreover, recent studies revealed that MSH2-MSH6 played a crucial role in activation-induced deaminase-initiated antibody diversity by recognizing uracil(s) in the Ig gene loci to generate DNA breaks [12]. Many studies have identified mutations in hmsh2 as diagnostic and/or prognostic factors in carcinomas not only in the US and Canada but also in the Middle East and China [13-15]. Human msh gene mutations also have strong potential as novel candidate triple-negative breast cancer predisposition genes and are closely associated with acute adverse events and survival in rectal cancer patients receiving postoperative chemoradiotherapy [16-19]. Pathogenic or likely pathogenic germline mutations in pms2, msh2 or msh6 were detected in 0.5% (6/1,179) of lung cancer patients [18]. In this study, we screened a novel mutation in the gene sequence of hmsh2 at c.2332 T>A in the OSC cell line HO8910. This is a silent mutation that theoretically does not result in a change to the amino acid sequence of hMSH2 protein or to the phenotype of HO8910 cells. However, a rapid expansion and a strong increase in hmsh2 mRNA expression were observed in this cell line compared with the control cell line. Moreover, several soluble full-length or truncated variants of hMSH2 proteins were observed in the whole cell protein extract and the culture supernatant of HO8910. We deduce that these altered biological characteristics of HO8910 cells and the genesis of OSC might be linked to the hmsh2 c.2332 T>A variation, as evidence showed that germline mutations of hmsh2 and its family members were closely associated with the pathogenesis of Lynch syndrome (LS). For example, hmsh2 c.2152 C>T alteration has been recently reported as a founder mutation in Portugal; its high proportion implies combined screening for this mutation and some other previously reported founder mutations will be helpful in the genetic testing of Portuguese families with suspected LS [19]. The occurrence and clinical significance of hmsh2 c.2332 T>A and soluble full-length and truncated forms of hMSH2 proteins in clinical OSC patients remain to be clarified in the future.

Compared with HO8910 cells, SKOV3 cells displayed stronger ectopic membrane and nuclear expression of hMSH2 in laser confocal microscopy and FCM analyses, suggesting better membrane anchoring and a more efficient nuclear importing system in SKOV3 cells. The ability of the notably overexpressed mhMSH2 on SKOV3 cells to promote human Vγ9Vδ2 T cell-mediated recognition and cytotoxicity via TCRγδ/NKG2D receptors towards target OSC cells was later validated by independent cytotoxicity and specific antibody blockade cytotoxicity assays, providing further evidence for mhMSH2 functioning as an OSC-associated self-antigen (ligand) for human Vγ9Vδ2 T cells in anti-ovarian cancer immunity [10]. In the absence of information/data demonstrating that the observed mutation impacts function, one cannot determine if the mutation is physiologically relevant. In further investigations on the subcellular distribution of hMSH2 and its companion proteins in ovarian cancer tissues, we found that all examined ovarian cancer specimens (serous adenocarcinoma, embryo sinus carcinoma, cystmyxomatous adenoma) displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, and total loss of nuclear expression of hMSH2/6 commonly occurred in almost all of the ovarian cancer tissues. Considering the high hmsh2 gene transcription and the loss of nuclear distribution of hMSH2 protein in HO8910 cells, we hypothesized that the nuclear importing system of hMSH2 and/ or hMSH6 protein was dysfunctional. By contrast, recently, it has been reported that a high overall rate (16.2%) of MMR deficiency was surprisingly observed in ovarian endometrioid carcinoma and was significantly associated with increased IFOG (International Federation of Obstetrics and Gynecology) grade and CD8+ intraepithelial lymphocyte infiltration but not with cancer-specific death [19]. These findings suggest a promising future in which loss of nuclear expression of hMSH2 and/or hMSH6 protein may function as effective screening/diagnostic markers or therapeutic targets for different subtypes of ovarian cancers and as endogenous immune ligands not only for Vγ9Vδ2 T cells [20]. We expected that a human cell-based assay system for functional testing of hMSH2 and its family members will facilitate the identification of highrisk ovarian carcinoma patients and the generation of individual autoantigen-targeted immunotherapies for OSC.

Conclusion

In this study, we identified a silent hmsh2 c.2332 T>A mutation (GenBank accession no. MG674653) along with serval novel soluble truncated forms of hMSH2 variants in HO8910 cells and validated the mhMSH2/TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells against mhMSH2/6-overexpressing SKOV3 cells. We also demonstrated the abnormal subcellular distribution of hMSH2/3/6 in the examined ovarian cancer tissues. The clinical significance of the critical findings in OSC genesis and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further investigated.

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Lupine Publishers | Comparative in Silico Analysis of Antigenic Proteins “Nucleocapsid Protein “of SARS-Cov-2 from Different Geographical Locations

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

SARS-CoV-2 Nucleocapsid protein considered as a vaccine target. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 in different geographical locations considered as an important factor in order to produce a global vaccine for the pandemic. The present investigation relies on the analysis of the similarities and differences of the SARS-CoV-2 Nucleocapsid protein from various geographical locations, considering certain protein parameters, as well as the study of the alignment of the protein sequence of amino acids.

Keywords: Nucleocapsid protein; SARS-CoV-2; geographical locations; bioinformatics

Introduction

SARS-CoV-2 is a single-stranded, positive-sense RNA virus with a 30 kb genome, one of the largest among RNA viruses [1]. The viral envelop of SARS-CoV-2, contains many proteins such as spike protein, and glycoprotein [2]. Another protein incorporated with the viral genome is nucleocapsid protein which is responsible for protection the virus from the host cell environment [3]. The SARSCoV- 2 spike protein is being used as the principal antigen target in vaccine progress. However, the multifaceted molecular details of viral entrance may lead to obstacles with the vaccine reaction [4]. The nucleocapsid (N) protein is a significant antigen for coronavirus, which contribute to RNA package and virus particle discharge [5]. After infection, the N protein enters the host cell along with the viral RNA to facilitate its replication and to process the assembly and release of the virus. SARS-CoV N comprises two distinct RNA-binding domains (N-terminal domain and C-terminal domain) connected by a poorly structured linkage region. Due to positive amino acids, SARS-CoV N-terminal domain and C-terminal domain have been documented to bind to the viral RNA genome [6]. Serological diagnosis has shown that the unique antibodies to the N protein in the serum of SARS patients have a higher sensitivity and longer persistence than those of other structural SARS-CoV proteins. In addition, anti-N antibodies have been observed at an early stage of infection with a high specificity. Thus, any information obtained from the study of this protein, whether in vivo or in vitro, will improve our understanding of COVID-19 and enable us to develop better biological agents for the treatment or diagnosis of diseases [7]. It is becoming clearer how important this protein is, to the multiple stages of the viral life cycle. This studie provide valuable and timely insights specific to the SARS-CoV-2 N protein, a vaccine target that has some distinct advantages over other possible SARSCoV- 2 antigens according to geographical location. Because of the conservation of the N-protein sequence, the increased awareness of its genetics and biochemistry regarding different geographical location, the N-protein SARS-CoV-2 should be considered as a global vaccine candidate for SARS-CoV-2. The present investigation relies on study the similarities and difference of Nucleocapsid protein “of SARS-CoV-2 from different geographical locations considering some protein parameters as well as study the alignment of amino acid sequence of protein.

Materials and Methods

Sequences, alignment, and construction of phylogenetic tree

Amino acids sequences for the Nucleocapsid protein of SARSCoV- 2 from different geographical locations were obtained from the National Center for Biotechnology Information database. The accession numbers of the corresponding database entries and isolation source are listed in Table 1. Multiple sequence alignment of Nucleocapsid protein of SARS-CoV-2 was performed in order to find the evolutionary relationships between sequences from different geographical location to identify shared patterns. Sequences were multiply aligned Jalview software version 2.8 [8]. Phylogenetic trees were constructed with neighbour-joining using MEGA [9]. A distance matrix was generated using the model building Jones-Taylor-Thornton [10]. Graphical way of representing and visualizing consensus data developed of amino acid multiple sequence alignment developed were displayed according to method described by Tom Schneider and Mike Stephens [11].

Computation of amino acid composition and molecular weight of protein sequences

Estimation of the amino acid composition and molecular weight was determined using Isoelectric Point Calculator (IPC), a web service and a standalone program for the accurate estimation of protein and peptide characteristic [12].

Solvent content of protein crystals

From the currently available sequence, of the solvent content of Nucleocapsid protein. The fraction of the crystal volume occupied by solvent was calculated according to Matthews [13].

Results

Table 2 is displayed the chemical composition of the Nucleocapsid protein “of SARS-CoV-2 from different geographic location. The preset data revealed that amino acid composition of the tested 13 sequences showed high similarity except for the protein sequence from Spain. Figure 1 represented the overall height of the stack indicates the amino acid sequence conservation at each position of protein, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. In general, sequence logo of Nucleocapsid protein provides that almost the sequence from different location did not differ from each other and revealed high similarity. Regarding Phylogeny estimation for Nucleocapsid protein of SARS-CoV-2 from different geographical locations. Neighbour-joining tree constructed using Mega 10.1.8. The multiple sequence alignments are depicted in Figure 2. Constructed phylogenetic tree is depicted in Figure 3. The current results indicated that; Neighbour-joining tree of Nucleocapsid protein of SARS-CoV-2 displays the comparison of amino acid sequences of Nucleocapsid protein from different location. The results shows that the phylogenetic analysis rooted by two clusters (Figure 3). Cluster 1 represents France, Australia, China, India, South Africa, Morocco, United Kingdom and Spain. In the Cluster 1 Spain considered as the main root. Cluster 2 represents USA, Canada, Argentina, Saudi Arabia and Nigeria. The main root of the cluster 2 is Canada. Figure 4 shows Matthews coefficient , the current results revealed that the parameter value equal to 0.68 for all locations except for Spain its value reaches 0.85. Figure 5 shows the results for Solvent content of protein crystals were near 80 % for all geographical locations but has been observed that the value calculate for Spain was the lowest equal to 44%. As showed in Table 3 molecular weight of Nucleocapsid protein “of SARS-CoV-2 from different geographic location ranges from 36759.93 to 45696.7 Dalton, exhibited low variability in molecular weight.

The current study demonstrated that the Nucleocapsid protein analysis from different location shared a common primary amino acid composition which resulted in a uniform sequence alignment. Previously reports declared that alignments are a powerful way to compare related protein sequences. They can be used to capture various facts about the sequences aligned, such as communal evolutionary descent or shared structural function [14]. The obtain composition information of SARS-CoV-2 Nucleocapsid protein revealed high similarity in different geographic location with mild variability in amino acid concentration. The study compared aminoacid distributions between different locations. When looking at overall amino acid frequencies it could be recognized that byand- large, amino acid frequencies in all investigated sequences mirrored to each other except for the sequence obtained from Spain. The biggest differences arose in all amino acids except for Histidine, Isoleucine, Tryptophan and valine. According to Jackson et al. [15] there are several patterns of sequence variation that are consistently seen in natural proteins. The first steps in a macromolecular structure determination of protein are to detect the count of molecules in the crystallographic asymmetric unit. The crystal volume per unit of protein molecular weight, known as Matthews coefficient. A substantial percentage of the protein crystals volume is employed by solvent ranged from 27% to 78%, with the most common value being about 43% [16]. The Matthews Coefficient and solvent content are calculated for Nucleocapsid protein “of SARS-CoV-2 from different geographic location which revealed almost the same results or all locations except for Spain. Water plays an important role in the structure of biomolecules and often influences protein function [17]. Water molecules not only affect protein folding, but also mediate biological processes such as enzymatic reactions and molecular recognition. Information about the fraction of water (solvent) plays a significant role in the X-ray structure determination process [18]. The present results revealed that solvent content for Nucleocapsid protein of SARS-CoV-2 from different geographical locations were almost similar except for Spain.

Conclusion

In the current study, the variation of Nucleocapsid protein of SARS-CoV-2 composition and its alignment were investigated for different location, it revealed high similarity for each other and showed a uniform sequence alignment with mild variability in amino acid concentration. The crystal volume per unit of protein molecular weight of Nucleocapsid protein “of SARS-CoV-2 revealed almost the same results or all locations except for Spain

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Lupine Publishers | Alignment of SARS-Cov-2 Spike Protein Compared with Ebola, and Herpes Simplex Viruses in Human Murine and Bats

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Our analytical approach consisted in sequence alignment analysis of spike protein in different viruses, followed by construction of phylogenetic tree. Additionally, we investigated some commotional parameters on the protein sequence determining chemical composition as well as estimated PI .Our observation revealed significant difference in S protein between the 3 tested viruses in different species These differences may have significant implications on pathogenesis and entry to host cell.

Introduction

Viruses are inert outside the host cell which are unable to generate energy. As obligate intracellular parasites, during replication, they are fully reliant on the complex biochemical machinery of eukaryotic or prokaryotic cells. The central purpose of a virus is to transport its genome into the host cell to allow its expression by the host cell [1]. Binding of a single surface glycoprotein of virus to its host receptor promotes pH-dependent conformational variations once within the endosome acidic environment, thereby transporting the viral bilayer in closeness with the host cell membrane to promote fusion [2]. The structural physiognomies of virus coats (capsids) are extremely appropriate to virus propagation. The coat must enclose and protect the nucleic acid, flexible against interference be talented of broaching the outer wall of a target cell and provide a confident pathway for attending nucleic acid into the target cell. Hollow spikes on the capsid fulfill the latter two roles, from which it has been deduced that they must have unusually high strength and stiffness in axial compression [3]. The spike protein (S protein) is a type I transmembrane protein, a sequence of amino acids ranging from 1,160 for avian infectious bronchitis virus (IBV) and up to 1,400 amino acids for feline coronavirus [4]. Spike proteins assemble to create the distinctive “corona” or crown-like look in trimers on the surface of the virion. The ectodomain of all CoV spike proteins portions the same organization in two domains: a receptor-binding N-terminal domain called S1 and a fusionresponsible C-terminal S2 domain The variable spike proteins (S proteins) reflect CoV diversity, which have developed into forms that vary in their receptor interactions and their reaction to different environmental triggers of virus-cell membrane fusion [5]. A notable peculiarity between the spike proteins of diverse coronaviruses is whether it is cleaved or not during assembly and exocytosis of virions [6]. The entry Herpesvirus and membrane fusion to the host cell equire three virion glycoproteins, gB and a gH/gL heterodimer, that function as the “core fusion machinery” [7]. The viral envelope of Ebola virus contains spikes consisting of the glycoprotein (GP) trimer. These viral spike glycoprotein docks viral particles to the cell surface, endosomal entry, and membrane fusion [8]. Acute respiratory syndrome coronavirus (SARS-CoV) is a zoonotic pathogens that traversed the species barriers to infect humans. These coronaviruses hold a surface-located spike (S) protein that recruits infection by mediating receptor-recognition and membrane fusion [9]. Bioinformatics plays an important role in all aspects of protein analysis, including sequence analysis, and evolution analysis. In sequence analysis, several bioinformatics techniques can be used to provide the sequence comparisons. In evolution analysis, we use the technique like phylogenetic trees to find homologous proteins and identified the most related taxa. With bioinformatics techniques and databases, function, structure, and evolutionary history of proteins can be easily identified [10]. Several bioinformatics methods can be used in sequence analysis to provide sequence comparisons.

Materials and Methods

Sequences, alignment, and construction of phylogenetic tree

Amino acids sequences for the spike protein of Covid-19, Ebola, and herpes simplex viruses from bats, murine as well as Homo sapiens were taken from the National Center for Biotechnology Information database. The accession numbers of the corresponding database entries and species names are listed in Table 1 and Figure 1. Sequences were aligned with by CLUSTALW [11]. Selection of conserved blocks was performed using GBlocks (version 0.91 to eliminate divergent regions and poorly aligned positions using standard settings according to Castresana [12]. The Akaike information criterion (AIC) were performed, using a maximum-likelihood (ML) starting tree to estimates the quality of each model, relative to each of the other models (Table 2). The most suitable model was WAG+G+F [13]. Then phylogenetic tree was constructed with neighbor-joining and maximum likelihood algorithms using MEGA version 5 [14]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 100 repetitions [15].

Computation of the theoretical pI (isoelectric point) of protein sequences

Estimation of the isoelectric point (pI) based on the amino acid sequence was determined using isoelectric Point Calculator (IPC), a web service and a standalone program for the accurate estimation of protein and peptide pI using different sets of dissociation constant (pKa) values [16]. Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. For each model, AICc value (Akaike Information Criterion, corrected), Maximum Likelihood value (lnL), and the number of parameters (including branch lengths) are also presented [1]. Non-uniformity of evolutionary rates among sites may be modeled by using a discrete Gamma distribution (+G) with 5 rate categories and by assuming that a certain fraction of sites is evolutionarily invariable (+I). Abbreviations: TR: General Time Reversible; JTT: Jones-Taylor-Thornton; rtREV: General Reverse Transcriptase; cpREV: General Reversible Chloroplast; mtREV24: General Reversible Mitochondrial.

Results

In order to unravel the phylogenetic relationship of Spike protein between the different taxa, a phylogenetic consensus tree was constructed using the Bayesian Inference (BI) and Maximum Likelihood (ML) methods (Figure 1). The present results revealed that the identity between different taxa was nonsignificant, However alignment of human Coronavirus (Covid-19) and Bat Coronavirus revealed identity equal to 57.98% followed by Murine Coronavirus which displayed 27.61% when compared by human Coronavirus. The chemical composition of the tested protein is illustrated in Table 3. Figures 2 & 3 show the correlation plots between the theoretical isoelectric points for spike protein of different corona virus in different species. The current results displayed that estimated pI of spike protein sequence in three investigated viruses in different species ranges from 5.59 to 8.08. With highest value for spike protein in herpesvirus of bat. The Estimated charge over pH range of the investigated were listed in Table 4. The current results revealed that the behavior of s protein in different species exhibited different estimated charge at different pH ranges. Some S protein reveled low negative charge as pH increases such as Ebola Virus of bat while S protein of Corona virus in murine showed high negative charge as pH elevated.

Discussion

One aspect that may provide some insight into the interactions of the S protein is the electrostatic potential it generates. the affinity constant for the receptor-binding domain (RBD) of viron protein potentially contributing to its transmission efficiency [17]. The S protein remains predominantly in the closed conformation to mask its receptor-binding domains (RBDs), thereby impeding their binding. To bind with ACE2, the S protein transforms into its open conformation, revealing its binding interface [18]. The present study relies on a comparative investigation, regarding the identity of spike protein of 3 viruses (Covid-19, Ebola, and herpes simplex) to identify the most related taxa. Our investigation revealed high similarities of Spike protein of coronaviruses in human and bats [19]. The computed amino acid composition of spike protein. Several residues showed a significant difference between the compositions in spike proteins in the three investigated virus in different species. This result reveals the importance of specific residues in these classes of proteins. The polar uncharged residues, especially Serine, Asparagine, and Glycine, have higher occurrence in spike protein of murine, which are important for the folding, stability, and function of such class of proteins [20]. Our analysis revealed that the Coronavirus spike protein of murine may be more efficient in discovering suitable vaccine for Coronavirus. The S protein amino acids variations among different coronaviruses such as (SARS, herpes and ebola). The SARS-CoV-2 virus shares 57.98% of its genome with the other bat coronaviruses. The sequence identity in the S protein bat coronavirus appears to be the closest relative of SARS-CoV-2. Our results in accordance with Rothan [21]. The isoelectric point (pI) is the pH value at which a molecule’s net charge is zero [22]. Information about the isoelectric point is important because the solubility and electrical repulsion are lowest at the pI. Hence, the tendency to aggregation and precipitation is highest. In the case of viruses, the value thus provides information about the viral surface charge in a specific environment [23]. In polar media, such as water, viruses possess a pH-dependent surface charge [23]. This electrostatic charge governs the movement of the soft particle in an electrical field and thus manages its colloidal behavior, which plays a major role in the processes of virus entry. The pH value at which the net surface charge changes its sign is called the isoelectric point and is a characteristic parameter of the virion in equilibrium with its atmosphere in water chemistry [24]. For some viruses the attachment influences that encourage binding to accommodating cells are extremely definite, but the arrangement of actions that activates viral entry is only now establishment to be understood. The charge of attachment protein may play an important role in attachment and entry of virus [25]. The current results revealed that the pH affect the net charge of S protein of different taxa with different behavior. All the investigated taxa exhibit increases in negative charge as the pH increased except for the Ebola virus form Bats which showed unstable behavior regarding the S protein charge.

Conclusion

In our study, we have investigated the variation of pHdependent changes in charges of a protein. The current results revealed that the pH affect the net charge of S protein of different taxa with different behavior.

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Lupine Publishers | A De-Novo SCN2A Mutation Identified in a Chinese Patient With Epilepsy: A Case Report

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Objective: To investigate the genetic causes of epilepsy in a 5-year-old Chinese female patient.

Methods: Clinical diagnosis and next-generation sequencing.

Results: The patient carries a de-novo heterozygous missense mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene. This mutation was evaluated as a pathogenic mutation based on the standards and guidelines of ACMG (American College of Medical Genetics and Genomics) and clinical research publications.

Conclusion: The de-novo heterozygous mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene is the genetic cause of the epilepsy for the patient. So far, this mutation of SCN2A gene is the first reported in the worldwide overall populations.

Keywords: Chinese; Epilepsy; SCN2A gene

Abbreviations: Whole Exome Sequencing (WES); Sanger sequencing; American College of Medical Genetics and Genomics (ACMG)

Introduction

The SCN2A gene encodes the voltage-gated sodium channel Na(v)1.2, which plays an important role in the initiation and conduction of action potentials. SCN2A is expressed in axon initiation segments and at nodes of Ranvier in myelinated nerve fibers during early development, and is later expressed in unmyelinated axons [1]. Mutations in the SCN2A gene, cause a variety of neuropsychiatric syndromes with different severity ranging from self-limiting epilepsies with early onset to developmental and epileptic encephalopathy with early or late onset and intellectual disability (ID), as well as ID or autism without seizures [2-4]. So far, more than 700 mutations in SCN2A were reported to be associated with epilepy [5]. The aim of the present study was to detect and report genetic causes of a 5-year-old Chinese female patient with epilepsy. The patient was found to have a de-nove mutation (c.3686 A>T p.Asp1229Val) in the SCN2A gene that has not reported in the previous studies.

Materials and Methods

Clinical diagnosis

A 5-year-old female Chinese patient who was born with no family history of seizures or other types of neurological diseases, has experienced epileptic spasms since she was 1.5 years old. The patient had fallen down while she was walking, and her head contacted the ground, but she did not loss consciousness at the time. On the same day, the patient began to nod her head continuously, with each occurance lasting approximately several seconds in length. Since then, her head nods occurred in clusters daily, and up to 30 to 40 times a day at the worst cases. The patient was carried to the hospital and was diagnosed with infantile spasms. Her symptoms of head nodding was relieved after treatment with intravenous immunoglobulin (pH4), Sodium valproate oral solution and Topiramate. The patient took Sodium valproate oral solution and Topiramate after her discharge from the hospital. However, she still had the head nods which occurred at random intervals. After she turned 2-years-old, the head nods did not occur again, but she began to have epilepsy symptoms such as loss of consciousness, twitching of the limbs, spitting out white foam et al. The epilepsy happened approximately once a month and lasted approximately 1 to 2 minutes in length at each occurence. The patient visited the hospital again and the Clonazepam was added to her medications [6]. Her symptoms of epilepsy were being controlled well and did not happen within a 8 month period. However, in April of 2019, when the patient was 5 years old, her head nods occurred again without any obvious causes. This time, the head nods happened in the early morning, and 4～5 times everyday. The patient was admitted to our hospital (the Second Hospital of Shanxi Medical University). The following tests were performed for the patient: physical examinations, brain MRI, and electroencephalogram.

Molecular test

In order to study the cause of the disease, whole exome sequencing was performed for the patient. Furthermore, Sanger sequencing was used to verify the variant for the patient and her parents. Sequencing data was analyzed by using numerous bioinformatics’ softwares, the pathogenicity of the mutation was evaluated based on the standards and guidelines of ACMG (American College of Medical Genetics and Genomics), Clinvar database, OMIM (Online Mendelian Inheritance in Man), HGMD (Human Gene Mutation Database), and clinical research papers published in scientific journals.

Results and Analysis

Clinical data analysis

The patient was admitted to our hospital in April of 2019. The patient appeared normal under physical examinations. She has a clear mind, but only can pronunce simple words. She had poor orientation, cognition, memory, calculation, and attention span. Brain MRI showed that the symmetrical brain hemispheres on both sides, and the structures of gray and white matter were normal. No other abnormalities were found in the brain parenchyma (Figure 1). The electroencephalogram of the patient showed that the slowwave activity were in the awake period (Figure 2A). Total epileptic discharge during sleep period (Figures 2B & 2C). The patient was given intravenous human immunoglobulin and Dexamethasone for immunotherapy. The medications she has had were also adjusted to Levetiracetam tablets, Clonazepam, Topiramate, and Sodium valproate oral liquid. No recurrence of the head nods after 3 month of the treatment. Figure 2D showed that no obvious abnormal discharge was found in the patient’s return visit on Oct. 2020.

Molecular biological data analysis

In order to identify the causes of the patient’s seizures, we conducted Whole Exome Sequencing (WES) based on Nextgeneration sequencing for the patient. a heterozygous missense mutation in SCN2A gene (c.3686 A>T p.Asp1229Val) reference transcript, NM_001040143) was detected in the patient. This variant was further confirmed by Sanger Sequencing, and not detected in either of the healthy parents, which indicated that this variant was de novo (Figure 3A). No other pathogenic variants were detected in more than approximately 800 genes that were defined by the OMIM database as related with epilepsy syndromes for this patient (Figures 3B & 3C). The mutation c.3686 A>T (p.Asp1229Val) either not being recorded in any clincial disease-related database (Clinvar and HGMD), nor in Human genome databases (1000 Genome and Genome mutation frequency database). The function prediction databases (SIFT and polyphen, etc.) predicted this mutation to be damaging. This variant was evaluated as a pathogenic mutation based on the standards and guidelines of ACMG.

Discussion

Pathogenic variants in SCN2A are reported in a spectrum of neurodevelopmental disorders including developmental and epileptic encephalopathies, benign familial neonatal-infantile seizures, episodic ataxia, and autism spectrum disorder and intellectual disability with and without seizures [1]. More than 1000 mutaions of SCN1A gene were reported in the Clinvar database, including deletion, duplication, indel, insertion and single nucleotide types. Around 70% of those variations was evaluated as responsible for the occurences of Benign familial infantile seizures or early infantile epileptic encephalopathy 11. The single nucleotide mutations were the most frequently reported mutations and were 84% in the total mutation types. However, only about 8.5% of the single nucleotide mutations were due to de-nove mutations in the SCN2A gene. Comparing with the 65% of the de-novo rate in the SCN1A gene, the rate of de-novo in the SCN2A gene is significantly lower [6]. We evaluated WES data from 332 Chinese patients with epilepsy and identified the one de-nove missense mutation in the SCN2A in this patient and the mutation was evaluated as the cause of the disease. Generally, the de-novo mutations of SCN2A gene often lead to severe phenotype with developmental delay [3]. The patient in our case reported here is only 5 years old, but she had epilepsy syndromes for more than three years and had more severe symptoms such as poor orientation, cognition, memory, calculation, and attention et al. The mutation of the SCN2A gene we detected (c.3686 A>T p.Asp1229Val) has not been recorded in the Clinvar database as of today [6]. Our report provided further evidence for the cause of epilepsy from a genetic level. We predict the result will be immediately useful for the clinical interpretation of SCN2A variants, and also provides deeper insights for SCN2A mutations associated with the broad clinical spectrum of seizures.

Declarations

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Second Hospital of Shanxi Medical University. Written informed consent was obtained from individual or guardian participants. This study was supported in the part by grants from the Epilepsy Research Fund (UCB No. 2014007) from China antiepileptic Association.

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Lupine Publishers | Risk of Antimicrobial Resistance Development from Pet Animals to Humans: Case of Enterobacteriaceae Family

Lupine Publishers | LOJ Pharmacology & Clinical Research

Antimicrobials are vital instruments for the treatment of contagious bacteriological infections in pet animals, as well as in humans. The demise of the effectiveness of antimicrobial substances can honestly deal with pet animal health and human health. A necessity for the enhancement of innovative antimicrobials for the treatment of multiresistant infections, specifically those caused by Gram-negative bacteria, has been recognized in human medicine, and an imminent subsequent demand in veterinary medicine is required. A distinctive feature associated with antimicrobial resistance and the risk of resistance development in pet animals is their close interaction in conjunction with humans. This generates chances for interspecies transmission of resistant bacteria. This review aims to recapitulate the current information on the use and indications for the Enterobacteriaceae family in pet animals and the spread of antimicrobial resistance among pet animals and their owners. The critical antimicrobial resistance microbiological threats from pet animals that directly or indirectly may cause adverse health effects in humans are carbapenemase-producing Enterobacteriaceae bacteria such as Escherichia coli, Klebsiella spp., Enterobacter spp. and Salmonella spp.

Keywords: Antimicrobial resistance; Antibiotics; Public health; Microbiology

Introduction

Throughout the last half of the century, the quantity of pet animals in contemporary civilization has considerably augmented, and a modification in their public part has arisen [1]. Awareness of their welfare has grown because of the close interaction between pets and their owners [2]. Humans could develop antimicrobialresistant bacteria or the consequent resistance genes from food animals [3,4] and interaction with their pet animals [5,6]. Enterobacteriaceae and multidrug-resistant Gram-negative bacteria have become apparent in healthy and sick pets, suggesting a possible threat of the spread of these bacteria to humans from contaminated or inhabited pet animals [7]. Additionally, there is also the possibility to transfer resistance genes vice versa. To evaluate the hazards contained by the framework of treatments for new antimicrobials for pet animals, could arise a necessity for further data requirements concerning antimicrobial resistance [8]. Antimicrobials are used commonly in the routine procedure for medicinal and preventative reasons in pet animals [9]. Nevertheless, antimicrobial intake data for dogs and cats are repeatedly deficient and typically addressed to drug company trades [10]. Even if trade data provide a challenging assessment of antimicrobial consumption’s enormity, data on the utilization of antimicrobials in various species are deficient [11]. Pet animal sales of antimicrobials are a small percentage of the global sales example of animals’ antimicrobial agents.

Various antimicrobial products approved for human use are also used in pet animals in the treatment of the “cascade” [12]. Prevalent use of broad-spectrum antimicrobials has been described in pet animal practice in Europe (Figure 1). The most used antimicrobials for dogs and cats are β-lactams, for example, amoxicillin and amoxicillin combined with clavulanic acid [13]. Shortage of verified diagnosis might take the lead to the abuse of antimicrobials [14]. Antimicrobial management has occurred to treat disorders in which effectiveness has not been demonstrated, for instance diarrhea in dogs for which antimicrobial treatment is generally not proposed [15,16]. Multidrug resistance bacteria have been described in pet animals, every so often cruelly conceding the therapy result. Since restricted reconnaissance and understanding of the zoonotic transmission of antimicrobial resistance between humans and pet animals, the level of spread and significance for public health is inadequately appreciated [17]. Within the next, the critical drug-resistant bacteria are assessed and the indication for their transmission among humans and their pet animals.

Enterobacteriaceae as a Significant Public Health Concern in Human Medicine

Go to

Representatives of the Enterobacteriaceae family comprise numerous species, for instance, Escherichia coli, Enterobacter spp., Salmonella spp., and Klebsiella spp. [18]. Countless bacteria belonging to these species are symbiotic organisms of the digestive tract [19]. Increasing antimicrobial resistance among Enterobacteriaceae is evolving as an essential public health apprehension in human medicine. Enterobacteriaceae, which produce ESBLs, extended-spectrum cephalosporinases, and plasmid-mediated AmpC β-lactamases (ESBLs) are particularly important. Around are quite a few articles on ESBL-producing bacteria in pet animals [6]. Escherichia coli, Salmonella spp., Enterobacter spp., and Klebsiella spp. as Potential Hazards Over the years, E. coli was reported continuously from the first recorded case in Japan in pet animals, followed by the emergence in humans in following years, to the incidence in ESBL-producing uropathogenic E. coli from pet animals in Spain [20]. Since that time, the number of statements relating to E. coli ESBLs in pet animals has grown precipitously [21]. CTX-M enzymes have developed a swiftly expanding family of ESBLs in bacteria from human infections. In pet animals, equally clinical and commensal isolates of E. coli frequently generate CTX-M type β-lactamases [22]. E. coli has lately become known as a global pandemic replicate in humans [23]. Descriptions of clinical infections in animals caused by E. coli are merger, which may be since its detection requires genotypic techniques. Various clinical E. coli isolates from pet animals are like human clinical E. coli isolates founded on their virulence genotype, and resistance characteristics, etc. Many E. coli strains such as ST156, ST405, ST410, and ST648 could be found both in humans and their pets [24]. The detection of duplicates in humans and dogs and cats may suggest their transmission through direct contact. Such transmission could likewise be a related component to the prompt and effective spreading of E. coli, even though, between humans, the incredibly critical transmission path is almost certainly "hand to hand" [25]. Salmonella spp. have been correlated with epidemics of nosocomial intestinal infections in pet animals in veterinary clinics and an animal sanctuary-some of the outburst as well engaged veterinary organization and other people in connection with pets [26].

In cases like this, pet animal sanctuaries could work as foci of transmission for Salmonella spp. among humans and animals if acceptable control measures are not provided [27]. Information on antibiotic resistance phenotypes and genotypes of Salmonella spp. in animals and humans in different countries and geographic regions is necessary to combat the spread of resistance [28]. This will improve the understanding of antibiotic resistance epidemiology, tracing new emerging pathogens, assisting in disease treatment, and enhancing the prudent use of antibiotics. However, the extent of antibiotic resistance in foodborne pathogens and humans in many developing countries remains unknown [29]. Among 25526 recorded isolates of salmonellae, 5086 isolated from humans, and 20440 from animals in 1994 and 1997 in France, the antibiotic resistance phenotype was determined for all human and 5336 animal isolates. In Salmonella enterica serovar typhimurium, one of the two most frequently isolated serovars from humans as well as animals, resistance to ampicillin was observed in 61% of both human and animal isolates in 1994 and in 73% of human and 53% of animal isolates in 1997.

During these periods, resistance to co-amoxiclav was between 45% and 66% for both types of the isolate. Resistance to ampicillin was associated with resistance to streptomycin, spectinomycin, sulphonamide, tetracycline and chloramphenicol in over 70% of isolates [30]. Resistance to ampicillin as well as co-amoxiclav never exceeded 7% in Salmonella enteritidis. While Salmonella hadar was practically absent among the human isolates in 1994, this serovar was the third most frequent in 1997, and at that time, 92% were resistant to nalidixic acid. Among the animal S. hadar isolates, the prevalence of resistance to nalidixic acid increased from 3% in 1994 to 72% in 1997. None of these isolates manifested high-level resistance to ofloxacin. The levels of resistance to aminoglycosides (<3%) and trimethoprim-suphamethoxazole (<14%) remained practically unchanged in all three serovars. The resistance markers of 463 ampicillin-resistant S. typhimurium isolated in 1997 were determined. Among the 24 phenotypes observed, six multiresistant phenotypes, representing 82% of these isolates (as compared with 80% in 1994), were associated with the PSE-1 gene typically found in the lysotype DT104 of this serovar [30]. Being as by E. coli, extended-spectrum cephalosporinases and plasmid-mediated AmpC β-lactamases producing strains of Salmonella spp. are of disquiet [24]. Antibiotic of the cephalosporin type licensed for use in veterinary medicine resistance was detected in more than 10% of cats and 21% of dog Salmonella spp. isolates with detected β-lactamases, respectively [31]. The main issue and problem are that there is limited knowledge of ESBLs in other Enterobacteriaceae of pet animals. Klebsiella spp. from the human epidemic clone was isolated from dogs and cats in Spain [32]. It was found to be highly resistant to aminoglycosides due to the ArmA methyltransferase. The emergence and clonal spread of Klebsiella spp. in dogs were first reported in Germany [33]. While in Singapore, an analysis of 186 diagnostic reports collected from a veterinary clinic between 2014 to 2016 showed that sick companion animals could carry bacteria of significance to human health [34]. Among the 186 specimens submitted, 82 showed polymicrobial growth (45%, 82/186), and in total, 359 bacteria were isolated. Of the 359 bacteria reported, 45% (162/359) were multi-drug resistant, and 18% (66/359) were extended-spectrum-β-lactamase species. Resistance to broad-spectrum antibiotics was also observed among individual species. Namely, methicillin-resistance among Staphylococcus pseudintermedius (63%, 32/51) and Staphylococcus aureus (50%, 4/8); fluoroquinolone-resistance among Escherichia coli (40%,17/42) and carbapenem-resistance among Klebsiella pneumoniae (7%, 2/30) were noted [34].

This analysis suggests that sick pets may contribute to the pool of clinically relevant antibiotic-resistant bacteria and play a role in the spread of antibiotic resistance. Antibiotic-resistant bacteria such as Klebsiella pneumoniae are common in the digestive tract and upper respiratory tract of animals and humans [35]. Several studies have shown that this bacterium is found in humans and in animals, one of which is pigs that are known to be a reservoir for the spread of this bacteria [36]. Not only in pigs, but this antibiotic-resistant bacterium is also known to be found in other food-producing animals, as well as in pet animals. Many cases of Klebsiella pneumoniae in humans have been reported, but Klebsiella pneumoniae in humans related to animals or strains related to animals and humans were also reported [37]. Control and prevention are needed to prevent the spread of antibiotic-resistant bacteria from animal to animal, animal to human and vice versa, and the surrounding environment.

Conclusion

In humans, the control of resistance is based on hygienic measures: prevention of cross-contamination and decreased antibiotic usage. In animals kept together, sanitary measures, such as prevention of oral-fecal contact, are hardly achievable. Consequently, lessening the need for antibiotics is the only possible way of managing resistance in pet animals. This can be achieved by improving pet animal welfare systems and eradicating or vaccinating against infectious diseases. Furthermore, eliminating antibiotics as preventive measures in pet animals would decrease antibiotic use and minimize transmission from animal to human resistance. This would not only diminish the public health risk of dissemination of resistant bacteria or resistant genes from pets to humans but would also be of significant importance in maintaining the efficacy of antibiotics in human medicine and veterinary medicine as well. A more extensive study to better understand the extent of distribution and the factors affecting antibiotic-resistant bacteria’s transmission to and from pets is more than necessary.

Acknowledgment

This research was supported by COST Action “European Network for Optimization of Veterinary Antimicrobial Treatment”, grant number CA18217.

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Lupine Publishers | Comparison of the Efficacy of Plaque Removal of Listerine Smart Rinse Kids and Vi– One Junior Fluoridated Mouthwash in Children Aged 6 To 10 Years

Lupine Publishers | Interventions in pediatric Dentistry: Open Access Journal

Abstract

Objectives: In this study, a comparative study was done on the effects of Vi-One and Listerine fluoridated mouthwashes on the reduction of dental plaque in pediatric patients between 7-11 years of age in dental clinics of Sepideh and apple in Shiraz, Iran. Listerine Smart Rinse Kids is a product of the United States and Vi-One Junior mouth wash is the domestic production of the country at Rozhin Corporation. This research was conducted by Mohammad Karimi and Hassan Dehghan in 2018-2019.

Material and Methods: In this study, 100 individuals were selected and divided into two groups of 50. During the study, no other method of controlling the plaque was used. In this method, the first group first used Vi-One mouthwash for 10 days and after two weeks of rest and minimizing dental plaque, they used Listerine for 10 days. While the second group used Listerine first, then they applied Vi- One in the same way. The results of this review were then evaluated.

Results: The mean of plaque index in total mouth and in the posterior teeth area with the use of Listerine Smart Rinse Kids was lower than that of Vi-One Junior mouth rinse. In another words, Listerine had a better effect on plaque removal than the Vi-One mouthwash in the posterior mandibular region.

Conclusion: The results show that although Listerine mouthwash had a better effect on dental plaque removal, none of the two mouthwashes had a significant difference in effects on maxillary and mandibular jaws.

Keywords: Dental Plaque; Vi- One, Listerine, Fluoridated Mouthwash; Periodontal Diseases; Plaque Index

Introduction

Currently, dental caries and gingivitis are common oral and dental diseases in this country. One element that can prevent tooth decay is Fluoride. In the oral health program of the country, fluoride mouth wash 2% was used to prevent dental caries in elementary school students all over the country [1]. In the other hand, dental plaque is an important factor in the formation of dental caries and periodontal diseases. Leo and his colleagues have identified dental plaque as the main cause of gingivitis [2]. With the use of mouthwashes, one can control the dental plaque, chemically [3,4]. In fact, mechanical plaque removal is one of the most common and effective methods for preventing caries and inflammation of the gum [5]. Fluoride mouthwash usage is contraindicated in children younger than six years of age due to the risk of swallowing and causing systemic toxicity and fluorosis [6-8]. Symptoms of acute oral fluoride toxicity in children include severe nausea, vomiting, hyper salivation, abdominal pain, and diarrhea [9]. In severe or fatal cases, these symptoms can be followed by convulsions, cardiac arrhythmias, and coma [10- 12]. Laboratory and animal data have shown that prevention the accumulation of plaque and consequently, reduction in dental plaque can be achieved when fluorides is applied topically which inhibits the bacterial multiplication [13]. The fluoride from mouth rinse is retained in dental plaque and saliva to help prevent dental caries [14]. In one review, the average caries reduction in nonfluoridated communities attributable to fluoride mouth rinse was 31% [15]. Another study in Sweden reported that the use of fluoride mouthwash along with brushing has a significant effect in decreasing of dental caries [16]. Listerine Smart Rinse Kids has been used for the purpose of this study. This product is an alcoholfree mouthwash. The ingredients include Sodium fluoride 0.02% (0.01% w/v fluoride ion), Water, Sorbitol, flavor, phosphoric acid, Sucralose, Cetylpyridinium chloride, disodium phosphate, sodium saccharin, menthol, blue 1 and green 3 [17]. One study reported that use of this mouthwash can strengthen teeth 99% better than brushing alone [18]. Another source indicated that it gives 12- hour cavity protection [19]. Vi-one Junior Mouthwash is specially designed for children. This mouth rinse contains Sodium fluoride 0.05%, Cetylpyridinium chloride 0.05% and Disodium phosphate agents. The respective flavors contain sugar-free and harmless sweetener. This brand also is an alcohol-free product [20]. The purpose of this study was to compare the efficacy of two types of mouthwashes, one the domestic mouthwash (Vi-One Junior) and the other, the brand name Listerine Smart Rinse kids fluoridated mouthwash in the removal of the dental plaque.

Material and Methods

This study was a cross-over clinical trial. The eligibilities for entering in our study were as follow:

a) Children having at least 20 teeth with no large restorative area.

b) No history of periodontal Diseases.

c) Not having any Prosthodontic or Orthodontic appliances.

The condition for withdrawal from the study, if there was any sign of reactions to any of these mouth rinses. There was no obligation to have any food regimen.

The study population consisted of 100 patients who were in a 50-member group. Before taking oral mouthwash, plaque index was minimized, and all subjects underwent tooth scaling at the beginning and, if necessary, teeth polishing were done before taking mouthwash. Oral hygiene was assessed via a plaque index. First, in both groups, the Silness-Löe plaque index was recorded. It is an Index for evaluating the thickness of the plaque in the gingival region, which measures the thickness of plaque on all surfaces (M, B, D, and L) [21].

Coding for the plaque index was carried out according to the criteria [22]:

a) Code 0: No plaque

b) Code 1: A film of plaque is adhering to the free gingival margin and adjacent area of the tooth. The plaque may be seen in situ only after application of disclosing solution or by using the probe on the tooth surface.

c) Code 2: Moderate accumulation of soft deposits can be seen with the naked eye within the gingival pocket, the tooth, or gingival margin.

d) Code 3: Abundance of soft matter can be seen within the gingival pocket and/or on the tooth, and gingival margin.

In this index, each tooth is divided into four surface area but in our purposes in the present study, we modified the surfaces area from 4 to 6; thus, we have three surfaces in the buccal area (Mesiobuccal, Midbuccal, and Distobuccal) and three surfaces in the lingual area (Mesiolingual, Midlingual and Distolingual). The first group used Vi-One & Listerine mouthwash (kids mouthwash), for 10 days in the following way. Needless to say, this process was supervised by parents at home. The kids have to gargle 5 cc ’s of Vi-One mouthwash 2 times per day for 30 seconds, and during this period of time, no other plaque control methods and tooth brushing should be used. At the end of the period of 10 days, the plaque index was recorded again. Then, the subjects were given a week to rest and stop using the mouthwash while they had permission to start brushing like before. Again, the plaque index was minimized for patients with polishing the teeth, and they used Listerine mouthwash for 10 days. In the same way, 5 cc ‘s of the mouthwash twice daily was used for 30 seconds, and at the end of the one-week period, the plaque was recorded. For the second group, in the first 10 days, mouthwash. Listerine was prescribed and in the second 10 days, the Vi-One mouthwash was applied. All procedures were performed according to the above pattern.

Results

Paired T-test was used for statistical analysis of the findings. The findings showed when Listerine Smart Rinse Kids was used; the mean of plaque index in all area of the mouth (especially in the mandibular jaw and the posterior region) was significantly less than the time Vi-One was applied. However, there was no significant efficacy difference between the use of both types of mouthwash in the upper jaw and the anterior region.

There was no significant difference between the mean plaque index in Vi-One mouthwash between upper and lower jaw, and there was no significant difference between the maxillary and lower jaw in the case of Listerine Smart Rinse Kids either. The presence of this indicator in both types of mouthwash in the anterior region was significantly less than the posterior region. The mean and standard deviation of the plaque index in both groups, as well as in different regions of the mouth, are listed in Table 1.

Discussion

In general, the anti-plaque properties of mouthwashes are completed through bactericidal and bacteriostatic effects, separation of microorganisms from dental surfaces, loosening of joints to these surfaces or lowering of the surface tension of the tooth [2,21,23]. Some mouthwashes can be useful for preventing tooth decay or periodontitis [6,24]. Furthermore, mouthwashes are recommended for children and adolescents with orthodontic appliances or adults who need deep cleansing (such as curettage) [6]. These types of mouthwashes were generally used before and after surgery (especially Chlorhexidine) and have a very positive effect on the treatment of gum and ulcerative inflammation [6]. Use of this type of mouthwash should not last longer than 2 to 3 weeks due to some side effects such as staining the teeth and soft tissue staining, increased calculus deposition, unpleasant taste, burning sensation, and mucosal irritation [6]. It’s time to use this mouthwash after brushing and before bedtime, and it’s best not to eat anything after half an hour after use. Fluoride-containing mouthwashes are another type of mouth rinse that has a fairly large use. These mouthwashes have a significant effect on teeth strengthening. Fluoride in the mouthwash cause bonding with enamel and dentin, and with bonding with calcium and phosphorus, they form Fluorapatite, which is more resistant to caries than Hydroxyapatite. Fluorides also accelerate the mineralization, repair the decayed teeth surfaces, and help to increase the reverse processing of damaged tooth surface area [25,26]. Fluoride also reduces the effect of oral bacteria on teeth. It is done by interfering with the function and formation of the microorganisms. The best fluoride mouthwash protects the teeth against the acids which are produced by dental plaques. “Neglecting the oral hygiene of children leads to the accumulation of plaque and as a consequent the formation of dental calculus which will have a devastating effect on the both child’s gums and teeth” [27].

In one study, the statics showed an alcohol-free mouthwash containing a combination of 0.075% CPC and 0.05% Na F produces statistically significant reductions in dental plaque and gingivitis after three and six months compared to baseline [28]. In another research, Jessica E. Koopman, et al argued that the oral microbial community displayed remarkable resilience towards the disturbances it was presented with. The effects of the fluoride mouthwash on the microbial composition were trivial [29]. On the other side, in another study, the research showed that all four fluoride mouth rinses were effective in decreasing the plaque levels of S. Mutans [30]. In this study, we investigated the effect of two mouthwashes of Listerine Smart Rinse Kids and Vi-One in which Vi-One mouthwash in the posterior region was less efficient than the Listerine mouthwash, and the interesting point that most kids mentioned the taste of Listerine was more acceptable. Given that the contents of sodium fluoride were equal in both mouthwashes, due to the fact that Listerine mouthwash was more acceptable than the mouthwash, it could be related to the other materials present in this product which can be a part of the manufacturer’s secrets. This difference in taste can be a factor in the effect of improving Listerine’s efficacy in the posterior regions.

Conclusion

Listerine Smart Rinse Kids had a better effect on plaque removal than the Vi-One mouthwash in the posterior mandibular region. Both types of mouthwash had a better effect on the anterior region than the posterior region, but none of the two mouth rinses had a different effect on the maxillary and lower jaw. Although many popular types of mouthwash may help to control dental plaque and gingivitis, they should only be used as an adjunct to other oral hygiene measures such as brushing and flossing. Fluoride mouthwashes should be encouraged in children above the age of 6 with a high risk of caries

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