#what is Fluorescence in situ hybridization

[faaun]

what if i meditated on the train and it cured me . what if i memorised the part of the paper i read w her

to confirm the presence of typical neuronal synaptic transcripts in cluster 7 astrocytes i didn't kiss you even once tonight. we performed multiplex fluorescence in situ hybridization (RNAscope HiPlex assay) analysis of hippocampal slices from (my brain and it revealed stars and your voice) conditionally expressing red tdTomato reporter in astrocytes under the night sky where we met i wish i hadn't hated you so much and co-immunostained for two additional astrocytic markers GS and the blood in my heart spilling out and flowing to you (Fig 1g). we targeted four neuronal genes involved in glutamatergic vesicular exocytosis (Slc17a7 Slc17a6 you didn't have a pulse or a heartbeat no matter how hard i pressed my head to your chest) and found that they were strongly expressed not only in glutamatergic neurons (Fig. 1g (top)) but also in your eyes are amber-red and they will keep looking at another and another and another. i'm going to look away from you. this is what we observed at the molecular level. i never felt hungry for food around you so i just watched you drink red wine until i got my fill.

[helthcareheven]

Embryo Enhancement Strategies: Preimplantation Genetic Diagnosis

What is PGD?

PGD, or preimplantation genetic diagnosis, is a procedure performed on embryos prior to implantation in the womb. During in vitro fertilization (IVF), embryos are created through fertilization in a laboratory dish and are allowed to develop for a few days. At this early stage, one or two cells are gently removed from the embryo and tested for genetic abnormalities and conditions. Only healthy embryos, as determined by PGD, are selected for implantation. The History of PGD

The first baby born through PGD was in 1990 after testing for cystic fibrosis. In the years since, the technique has improved and can now test for hundreds of genetic disorders. Initially, PGD was used for serious hereditary conditions like cystic fibrosis, Tay-Sachs disease, sickle cell anemia, and muscular dystrophy. Today, it is also applied to detect chromosomal abnormalities like Down syndrome and gender selection for X-linked disorders. PGD has helped many couples avoid passing on serious genetic diseases to their children. The PGD Process

The general steps involved in PGD are:

1) IVF treatment is performed as normal to collect multiple eggs from the female patient. 2) Eggs are fertilized with sperm in the lab to create embryos. 3) Typically on day 3 of development, when the embryo contains 6-10 cells, one or two cells are gently removed for biopsy without harming embryonic development. 4) The biopsied cells are analyzed using techniques like fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) to detect abnormalities. 5) Only healthy embryos, as confirmed by PGD results, are selected and implanted back into the woman's womb. Diseases Screened by PGD

Some of the more common genetic disorders for which PGD is often used include: - Cystic fibrosis: A lung disease caused by mutations in the CFTR gene. PGD can detect affected embryos. - Huntington's disease: A neurodegenerative condition caused by CAG repeat expansions. PGD finds embryos free of expansions. - Fragile X syndrome: The most common form of inherited intellectual disability caused by mutations in the FMR1 gene. Embryos can be tested. - Myotonic dystrophy: A muscular dystrophy caused by CTG repeat expansions that PGD can identify in embryos. - Beta thalassemia: An anemia caused by mutations in the HBB gene. PGD selects unaffected embryos. - Sickle cell anemia: Another anemia caused by a mutation in the HBB gene. PGD accurately diagnoses embryos. How Accurate is PGD?

As the techniques used for PGD continue to improve, the tests have become highly accurate. FISH analysis has an accuracy rate of about 95-99% while PCR-based methods detect abnormalities with over 99% accuracy. However, there is still a small chance of diagnostic errors. PGD is not currently able to test for all known genetic disorders and conditions either. While it has helped many couples, PGD is not perfect and misdiagnosis is still possible in a small number of cases. Continued clinical testing and experience will help further enhance the accuracy and reliability of PGD. Overall, when performed by experienced centers, PGD provides an effective method for screening embryos. Limitations and Ethical Considerations

While PGD addresses the desire to have healthy, genetically related children, it is not without limitations and ethical concerns. Aside from occasional misdiagnosis risks, PGD is not accessible or affordable for all couples and families affected by genetic disorders. The procedure is also limited to testing only those disorders with a known genetic cause and diagnostic test. There are ongoing debates around the widening scope of what conditions PGD is used for, including gender selection for social reasons alone. Some argue this could eventually lead to selecting embryos for traits like intelligence or athletic ability. Most experts agree PGD should only be offered for preventing medical disease or conditions. Overall, PGD is an advancing technology that provides benefit but also responsibility in its application. In conclusion, through the advances of preimplantation genetic diagnosis, modern science now offers new hope for couples at high risk of passing on life-altering genetic disorders. When performed carefully by trained professionals, PGD has proven an effective tool for pre-implantation diagnosis of embryos. Continued progress is still needed to further increase accuracy, expand testing options, and ensure judicious application in line with ethical standards. Looking ahead, PGD promises more couples the ability to have healthy children free of devastating genetic diseases.

[jcmarchi]

Noninvasive technique reveals how cells’ gene expression changes over time

New Post has been published on https://thedigitalinsider.com/noninvasive-technique-reveals-how-cells-gene-expression-changes-over-time/

Noninvasive technique reveals how cells’ gene expression changes over time

Sequencing all of the RNA in a cell can reveal a great deal of information about that cell’s function and what it is doing at a given point in time. However, the sequencing process destroys the cell, making it difficult to study ongoing changes in gene expression.

An alternative approach developed at MIT could enable researchers to track such changes over extended periods of time. The new method, which is based on a noninvasive imaging technique known as Raman spectroscopy, doesn’t harm cells and can be performed repeatedly.

Using this technique, the researchers showed that they could monitor embryonic stem cells as they differentiated into several other cell types over several days. This technique could enable studies of long-term cellular processes such as cancer progression or embryonic development, and one day might be used for diagnostics for cancer and other diseases.

“With Raman imaging you can measure many more time points, which may be important for studying cancer biology, developmental biology, and a number of degenerative diseases,” says Peter So, a professor of biological and mechanical engineering at MIT, director of MIT’s Laser Biomedical Research Center, and one of the authors of the paper.

Koseki Kobayashi-Kirschvink, a postdoc at MIT and the Broad Institute of Harvard and MIT, is the lead author of the study, which appears today in Nature Biotechnology. The paper’s senior authors are Tommaso Biancalani, a former Broad Institute scientist; Jian Shu, an assistant professor at Harvard Medical School and an associate member of the Broad Institute; and Aviv Regev, executive vice president at Genentech Research and Early Development, who is on leave from faculty positions at the Broad Institute and MIT’s Department of Biology.

Imaging gene expression

Raman spectroscopy is a noninvasive technique that reveals the chemical composition of tissues or cells by shining near-infrared or visible light on them. MIT’s Laser Biomedical Research Center has been working on biomedical Raman spectroscopy since 1985, and recently, So and others in the center have developed Raman spectroscopy-based techniques that could be used to diagnose breast cancer or measure blood glucose.

However, Raman spectroscopy on its own is not sensitive enough to detect signals as small as changes in the levels of individual RNA molecules. To measure RNA levels, scientists typically use a technique called single-cell RNA sequencing, which can reveal the genes that are active within different types of cells in a tissue sample.

In this project, the MIT team sought to combine the advantages of single-cell RNA sequencing and Raman spectroscopy by training a computational model to translate Raman signals into RNA expression states.

“RNA sequencing gives you extremely detailed information, but it’s destructive. Raman is noninvasive, but it doesn’t tell you anything about RNA. So, the idea of this project was to use machine learning to combine the strength of both modalities, thereby allowing you to understand the dynamics of gene expression profiles at the single cell level over time,” Kobayashi-Kirschvink says.

To generate data to train their model, the researchers treated mouse fibroblast cells, a type of skin cell, with factors that reprogram the cells to become pluripotent stem cells. During this process, cells can also transition into several other cell types, including neural and epithelial cells.

Using Raman spectroscopy, the researchers imaged the cells at 36 time points over 18 days as they differentiated. After each image was taken, the researchers analyzed each cell using single molecule fluorescence in situ hybridization (smFISH), which can be used to visualize specific RNA molecules within a cell. In this case, they looked for RNA molecules encoding nine different genes whose expression patterns vary between cell types.

This smFISH data can then act as a link between Raman imaging data and single-cell RNA sequencing data. To make that link, the researchers first trained a deep-learning model to predict the expression of those nine genes based on the Raman images obtained from those cells.

Then, they used a computational program called Tangram, previously developed at the Broad Institute, to link the smFISH gene expression patterns with entire genome profiles that they had obtained by performing single-cell RNA sequencing on the sample cells.

The researchers then combined those two computational models into one that they call Raman2RNA, which can predict individual cells’ entire genomic profiles based on Raman images of the cells.

Tracking cell differentiation

The researchers tested their Raman2RNA algorithm by tracking mouse embryonic stem cells as they differentiated into different cell types. They took Raman images of the cells four times a day for three days, and used their computational model to predict the corresponding RNA expression profiles of each cell, which they confirmed by comparing it to RNA sequencing measurements.

Using this approach, the researchers were able to observe the transitions that occurred in individual cells as they differentiated from embryonic stem cells into more mature cell types. They also showed that they could track the genomic changes that occur as mouse fibroblasts are reprogrammed into induced pluripotent stem cells, over a two-week period.

“It’s a demonstration that optical imaging gives additional information that allows you to directly track the lineage of the cells and the evolution of their transcription,” So says.

The researchers now plan to use this technique to study other types of cell populations that change over time, such as aging cells and cancerous cells. They are now working with cells grown in a lab dish, but in the future, they hope this approach could be developed as a potential diagnostic for use in patients.

“One of the biggest advantages of Raman is that it’s a label-free method. It’s a long way off, but there is potential for the human translation, which could not be done using the existing invasive techniques for measuring genomic profiles,” says Jeon Woong Kang, an MIT research scientist who is also an author of the study.

The research was funded by the Japan Society for the Promotion of Science Postdoctoral Fellowship for Overseas Researchers, the Naito Foundation Overseas Postdoctoral Fellowship, the MathWorks Fellowship, the Helen Hay Whitney Foundation, the U.S. National Institutes of Health, the U.S. National Institute of Biomedical Imaging and Bioengineering, HubMap, the Howard Hughes Medical Institute, and the Klarman Cell Observatory.

[cytochrome-studyblr]

here's the shorthand i use in my notes as a biology major!

D or [delta] = change X = cross XD = exchange aX = across str = structure f'(x) = function j'(x) = junction [name of compound] = concentration of name of compound txn = transcription tln = translation [ionic] = ionic strength conc'n = concentration prok = prokaryotic or prokaryote euk = eukaryotic or eukaryote Ag = antigen Ab or [alpha] = antibody seq = sequence ss = single stranded ds = double stranded 1x = single 2x = double cross or 2 times fluo'scopy = fluorescent microscopy G+ve = Gram-positive G-ve = Gram-negative PoI = protein of interest GoI = gene of interest ish = in situ hybridization OH = alcohol (unspecified) NH2 = amine COOH = carboxyl or carboxylic acid C=O = carbonyl nt = nucleotide bp = base pair bp'ing = base pairing

ill add more as i think of them, this is just the shorthand i've developed over the course of several years ! use what works best for you always. this is just my system <3

[sarahmackattack]

you wouldn't happen to know any scools/labs/researchers in texas doing work with cephalopods, would you? i'm not yet very scientifically literate (just switched to a biology degree from art history), but octopus cognition and genomics interest me most so far. or like even any recommendations on how to get into those topics further since i'm still just a baby science major and still have a LOT to learn.

So before I start, I think it’s important for me to say the following:  You shouldn’t try to get a job with any of these scientists ONLY because you think cephalopods are cool.  Cephalopods are obviously extremely cool, and for most of us that’s what sparked our interest in the first place, but the main drive to do science is deeper than that. Marine biologists are trying to answer very specific questions about the physiology, camouflage, symbiosis, immune systems, behavior, etc. about these animals.  Being successful as a squid biologist requires three main interests.

1) Interest in cephalopods (which I’m pretty sure most followers of this blog have in spades)

2) Interest in the questions this scientist is asking(e.g.  How do squid immune cells recognize specific bacterial species?(Me)  How does the microbial community of the female squid’s accessory nidamental gland protect squid eggs? (Andrea)  How do cuttlefish perceive their world, and then decide what the best camouflage pattern is for the situation? (Roger Hanlon)  Do bacteria colonize animals differently in zero gravity? (Jamie Foster)

3) Interest in the techniques used to answer these questions.  (e.g. Confocal microscopy/Protein purification/ Western Blotting (Me), Fluorescence in situ hybridization (FISH)/Reverse transcriptase polymerase chain reaction (RT-PCR) /Culturing of environmental isolates/ bacterial growth assays (Andrea), Behavior studies/ Computational processing of camouflage pattern/ fieldwork (Roger)

Working with cephalopods is VERY COMPETITIVE, so you need to make sure you’re building your resume as early as possible.  Get research experience any way you can and educate yourself using peer reviewed literature when you’re at the academic level that you can start to understand it.

So now on to the list of scientists, in no particular order (all underlined names are links to more info about them)

Roger Hanlon (Woods Hole, Massachusetts, MBL) Literally wrote the book on cephalopod behavior. He works on camouflage and how cuttlefish perceive their environment, how they choose what camouflage pattern to use, and also works on the skin structures that contribute to camouflage. There’s an internship program through the MBL that his lab participates in but it’s very competitive.

Margaret McFall-Ngai (Hawaii, University of Hawaii) Margaret is the mother of the squid/vibrio symbiosis. A member of the national academy of sciences, Margaret has been extremely influential in the study of symbiosis. Working for her will be very competitive. She’s a great role model as a powerful woman in science.   Her lab, along with Ned Ruby’s lab, work on many aspects of the squid/vibrio symbiosis.  Many (if not all) of the squid/vibrio community have come through her or Ned’s labs.  Here’s a piece on her from nature blogs written by Ed Yong

Spencer Nyholm (Connecticut, UConn) Andrea and I work for Spencer, so you’ve probably seen our posts and have an idea of what we do, but I study how squid immune cells recognize specific bacterial species and Andrea studies how the microbial community of the female squid’s accessory nidamental gland can protect squid eggs.

Bethany Rader (Illinois, Southern Illinois University) Bethany is fantastic! She’s super friendly and excitable and just started her lab at SIU. She is one of our collaborators and previously did a post-doc in our lab. She’s working on the same thing I am, the role of the immune system in the squid/Vibrio symbiosis.

Bill Gilly (California, Stanford) Works on many aspects of Humboldt squid biology.

Josh Rosenthal (Puerto Rico, University of Puerto Rico) Works on RNA editing in squid and octopus.  I’ve heard he’s a really friendly guy but haven’t met him personally (yet).

Charlie Chubb (California, UC Irvine) Charlie is one of the genuinely nicest guys I have ever had the opportunity to work with.  He collaborates with Roger Hanlon, working on “ understanding the processes by which the visible world is constructed by the brain”.  He’s a brilliant scientist and a wonderful guy. His work is all computer based though so there are no physical squid in his lab.

Aran Mooney (Woods Hole, Massachusetts, Woods Hole Oceanographic Institution) Aran works on many different animals, but squid and cuttlefish are among them.  His synopsis on his website is as follows “Our research is in the sensory biology of animals, primarily marine organisms.  Specifically I am interested in how these animals detect the world around them, what they detect (i.e., what’s important to the organism), and how these animals then relate to their environment (e.g., predator detection, prey localization, habitat identification, and conspecific communication).”

Cheryl Whistler (New Hampshire, University of New Hampshire) Squid/Vibrio symbiosis.  I believe also how microbes have evolved to better colonize animals in beneficial symbiosis.

Jamie Foster (Florida, University of Florida) Working on host/microbe interactions in the squid/vibrio system.  Along with other things, she’s studying the effect of gravity on microbial colonization.   She also works on stromatolites.

Jean Boal (Pennsylvania, Millersville University)  Since Jean is at  Millersville she may not take grad students (I know when I was an undergrad she was not accepting grad students).  She works on cephalopod behavior.

Sheila Castellanoz-Martinez (Mexico) Immune system of cephalopods, specifically octopus.  She’s currently a Post-doc, but may have a lab soon, I really have no idea, I just read a lot of her papers J

Shelley Adamo (Canada, Dalhousie Univeristy) Currently working on insect innate immunity/behaviour but has worked on cuttlefish in the past and may work on cuttlefish in the future.

Maria Castillo (New Mexico, New Mexico State University) Role of the immune system in the squid/vibrio symbiosis

Michele Nishiguchi (New Mexico, New Mexico State University) Evolution and marine symbiosis in the squid/vibrio system

For more information, Tonmo is a great resource for all things cephalopod. They have information about everything from having a cephalopod as a pet to working with cephalopods. Here’s the board on education and employment.

[death-and-ruin]

Here's some more fun acronyms and names for things:

ATAC-seq, spoken like "attack, seek" (Assay for Transposase Accessible Chromatin + sequencing). Funnily enough, the way you pronounce it actually describes pretty well what happens: you basically "attack" DNA with a transposase to look ("seek") for open chromatin regions, i.e. parts of the DNA that are accessible to the cell's transcription machinery (= those parts of the DNA can be read. If the chromatin is closed, the DNA cannot be read).

BAC-FISH assay, which sounds like the German word for Backfisch (fried fish). BAC = bacterial artificial chromosome, FISH = fluorescence in situ hybridization

And how about some fun fruit fly gene names?

Swiss Cheese (mutants have holes in their brains, among other problems)

BOSS (bride of sevenless) (mutants lack a type of photoreceptor)

Ken and Barbie (mutants lack external genitalia)

INDY (I'm not dead yet) (mutants live longer)

Couch Potato (mutans are very slow)

Spätzle (named after the type of noodle. Take a guess what mutant larva look like ..)

Cheap Date (mutants are very succeptible to alcohol)

18 Wheeler (gene gets its name from the segmented expression pattern, which is thought to resemble a tarpaulin covering an 18-wheeler truck)

Tinman (mutants don't develop a heart)

Mothers Against Decapentaplegic (Decapentaplegic is an important gene in fruit fly embryo development. If the mother fly has a mutation in Mothers against Decapentaplegic, the Decapentaplegic gene doesn't work in the embryo, and the embryo doesn't develop correctly as a result)

And some other fun stuff:

Flippase and floppase (two proteins who flip in and flop out a specific kind of phospholipid in our cell membranes)

and their fun friend, Scramblase, a protein that "scrambles" phospholipids between the inside and outside of cell membranes

MAP kinase, MAP kinase kinase, MAP kinase kinase kinase, and MAP kinase kinase kinase kinase

RING (Really Interesting New Gene)

SWEET (sugars will eventually be exported transporter) transporters

Time for Coffee (regulates the circadian clock in thale cress)

educate me tumblr

[marketerefforts]

In Situ Hybridization Market Research Report 2023 - Industry Size, Share, Demands, Regional Analysis & Estimations Till 2028

The In Situ Hybridization Market Report, in its latest update, highlights the significant impacts and the recent strategical changes under the present socio-economic scenario. The In Situ Hybridization industry growth avenues are deeply supported by exhaustive research by the top analysts of the industry. The report starts with the executive summary, followed by a value chain and marketing channels study. The report then estimates the CAGR and market revenue of the Global and regional segments.

Base Year: 2021

Estimated Year: 2022

Forecast Till: 2023 to 2028

The report classifies the market into different segments based on type and product. These segments are studied in detail, incorporating the market estimates and forecasts at regional and country levels. The segment analysis is helpful in understanding the growth areas and potential opportunities of the market.

Get | Download FREE Sample Report of Global In Situ Hybridization Market @ https://www.decisiondatabases.com/contact/download-sample-18103

A special section is dedicated to the analysis of the impact of the COVID-19 pandemic on the growth of the In Situ Hybridization market.  The impact is closely studied in terms of production, import, export, and supply.

The report covers the complete competitive landscape of the Worldwide In Situ Hybridization market with company profiles of key players such as:

Abbott Laboratories

Advanced Cell Diagnostics, Inc.

Agilent Technologies, Inc. 

Biogenex Laboratories, Inc.

Danaher Corporation

F.Hoffmann-La Roche Ltd.

Merck KGaA

Perkin Elmer, Inc.

Thermofisher Scientific, Inc.

Want to add more Company Profiles to the Report? Write your Customized Requirements to us @ https://www.decisiondatabases.com/contact/get-custom-research-18103

In Situ Hybridization Market Analysis by Technique:

Fluorescence In Situ Hybridization (FISH)

DNA-FISH

RNA-FISH

Chromogenic In Situ Hybridization (CISH)

In Situ Hybridization Market Analysis by Application:

Cancer Diagnosis

Immunology

Neuroscience

Cytology

Infectious Diseases

In Situ Hybridization Market Analysis by End-User:

Molecular Diagnostic Laboratories

Pharmaceutical & Biotechnology Companies

Contract Research Organizations (CROS)

Academic & Research Institutions

In Situ Hybridization Market Analysis by Geography:

North America (USA, Canada, and Mexico)

Europe (Germany, UK, France, Italy, Russia, Spain, Rest of Europe)

Asia Pacific (China, India, Japan, South Korea, Australia, South-East Asia, Rest of Asia-Pacific)

Latin America (Brazil, Argentina, Peru, Chile, Rest of Latin America)

The Middle East and Africa (Saudi Arabia, UAE, Israel, South Africa, Rest of the Middle East and Africa)

Key questions answered in the report:

 What is the expected growth of the In Situ Hybridization market between 2023 to 2028?

Which application and type segment holds the maximum share in the Global In Situ Hybridization market?

Which regional In Situ Hybridization market shows the highest growth CAGR between 2023 to 2028?

What are the opportunities and challenges currently faced by the In Situ Hybridization market?

Who are the leading market players and what are their Strengths, Weakness, Opportunities, and Threats (SWOT)?

What business strategies are the competitors considering to stay in the In Situ Hybridization market?

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DecisionDatabases.com is a global business research report provider, enriching decision-makers, and strategists with qualitative statistics. DecisionDatabases.com is proficient in providing syndicated research reports, customized research reports, company profiles, and industry databases across multiple domains. Our expert research analysts have been trained to map client’s research requirements to the correct research resource leading to a distinctive edge over its competitors. We provide intellectual, precise, and meaningful data at a lightning speed.

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[healthcaredbmrnews]

Molecular Diagnostics Market Overview, Growth Analysis, Share, Opportunities, Sales, Trends, Supply, Forecast To 2028

Molecular Diagnostics Market research report assists business in every sphere of trade to take superior decisions, to tackle the toughest business questions and diminish the risk of failure. What is more, emerging product trends, major drivers, challenges and opportunities in the market are recognized and analysed factually while generating this report. Market drivers and market restraints mentioned in this Molecular Diagnostics report help businesses gain an idea about the production strategy. Market shares of these key players in the major areas of the globe such as Europe, North America, Asia Pacific, South America, Middle East and Africa are also studied.

The molecular diagnostics market is expected to gain market growth in the forecast period of 2021 to 2028. Data Bridge Market Research analyses that the market is growing with a CAGR of 6.0% in the forecast period of 2021 to 2028 and is expected to reach USD     46,505.12 million by 2028. The demand for molecular diagnostics tools is increasing to diagnose COVID-19 patients, coupled with an increase in infectious disease and cancer prevalence as driver for the molecular diagnostics market growth.

Molecular diagnosis identifies or diagnoses diseases such as infectious diseases, genetic diseases, cardiovascular diseases, neurological diseases, and others by studying molecules such as DNA, RNA, protein in a tissue or a fluid. Different technologies such as PCR, mass spectrometry, next-generation sequencing, cytogenetics, in situ hybridization, molecular imaging, and others are used to diagnose different diseases. Molecular diagnostics use powerful tools such as gene expression profiling, DNA sequence analysis, and detection of biomarkers to determine the susceptibility of individuals to certain diseases or existing disease stage.

Download Sample Copy of the Report to understand the structure of the complete report (Including Full TOC, Table & Figures) @ https://www.databridgemarketresearch.com/request-a-sample/?dbmr=global-molecular-diagnostics-market

The molecular diagnostics Market is segmented on the based on the products, technology, application and end user. The growth among segments helps you analyse niche pockets of growth and strategies to approach the market and determine your core application areas and the difference in your target markets.

On the basis of products, the global molecular diagnostics market is segmented into reagents & kits, instruments and services & softwares. Instruments are further segmented into fully automated instruments and semi-automated instruments. In 2021, the instruments segment is expected to dominate the molecular diagnostics market due to the rise in demand for advanced technology to diagnose diseases such as infectious diseases, cancer, and others.

On the basis of technology, the global molecular diagnostics market is segmented into mass spectrometry (MS), capillary electrophoresis, next generation sequencing (NGS), chips and microarray, polymerase chain reaction (PCR)-based methods, cytogenetics, in situ hybridization (ISH or FISH), molecular imaging and others. The polymerase chain reaction (PCR)-based methods are further segmented into cold PCR, digital PCR, directlinear analysis, quantitative fluorescent PCR, real-time PCR and reverse transcriptase PCR. The molecular imaging segment is further subdivided into optical imaging and FDG-PET. In 2021, the polymerase chain reaction (PCR)-based methods segment is expected to dominate the molecular diagnostics market due to increased demand for PCR kits to diagnose COVID-19 and curb the pandemic.

On the basis of application, the global molecular diagnostics market is segmented into oncology, pharmacogenomics, microbiology, prenatal tests, tissue typing, blood screening, cardiovascular diseases, neurological diseases, infectious diseases and others. The oncology segment is further segmented into oncology, by cancer type and oncology, by technology. The oncology, by cancer type is further sub-divided into breast cancer, colorectal cancer, lung cancer, prostate cancer and others. The oncology, by technology is further sub-segmented into mass spectrometry (MS), capillary electrophoresis, next generation sequencing (NGS), chips and microarray, polymerase chain reaction (PCR)-based methods, cytogenetics, in situ hybridization (ISH or FISH), molecular imaging and others.

The blood screening is further segmented into mass spectrometry (MS), capillary electrophoresis, next generation sequencing (NGS), chips and microarray, polymerase chain reaction (PCR)-based methods, cytogenetics, in situ hybridization (ISH or FISH), molecular imaging and others. The cardiovascular diseases are further segmented into mass spectrometry (MS), capillary electrophoresis, next generation sequencing (NGS), chips and microarray, polymerase chain reaction (PCR)-based methods, cytogenetics, in situ hybridization (ISH or FISH), molecular imaging and others.

On the basis of end user, the global molecular diagnostics market is segmented into hospital, clinical laboratories and academics. In 2021, the clinical laboratories segment is expected to dominate the molecular diagnostics market due to the rising number of patients with various illnesses and the growing need for diagnostics instruments.

Attractions of the Molecular Diagnostics Market Report: -

Latest market dynamics, development trends and growth opportunities are presented along with industry barriers, developmental threats and risk factors

The forecast Molecular Diagnostics Market data will help in the feasibility analysis, market size estimation and development

The report serves as a complete guide which micro monitors all vital Molecular Diagnostics Market

A concise market view will provide ease of understanding.

Molecular Diagnostics Competitive market view will help the players in making a right move

Access Full Report @ https://www.databridgemarketresearch.com/reports/global-molecular-diagnostics-market

Table of Content:

Part 01: Executive Summary

Part 02: Scope of The Report

Part 03: Global Molecular Diagnostics Market Landscape

Part 04: Global Molecular Diagnostics Market Sizing

Part 05: Global Molecular Diagnostics Market Segmentation by Product

Part 06: Five Forces Analysis

Part 07: Customer Landscape

Part 08: Geographic Landscape

Part 09: Decision Framework

Part 10: Drivers and Challenges

Part 11: Market Trends

Part 12: Vendor Landscape

Part 13: Vendor Analysis

New Business Strategies, Challenges & Policies are mentioned, Request for Detailed TOC at https://www.databridgemarketresearch.com/toc/?dbmr=global-molecular-diagnostics-market

Key Questions Answered

What impact does COVID-19 have made on Global Molecular Diagnostics Market Growth & Sizing?

Who are the Leading key players and what are their Key Business plans in the Global Molecular Diagnostics Market?

What are the key concerns of the five forces analysis of the Global Molecular Diagnostics Market?

What are different prospects and threats faced by the dealers in the Global Molecular Diagnostics Market?

What are the strengths and weaknesses of the key vendors?

Reasons to Purchase this Report:

Market segmentation analysis including qualitative and quantitative research incorporating the impact of economic and policy aspects

Regional and country level analysis integrating the demand and supply forces that are influencing the growth of the market.

Market value USD Million and volume Units Million data for each segment and sub-segment

Competitive landscape involving the market share of major players, along with the new projects and strategies adopted by players in the past five years

Comprehensive company profiles covering the product offerings, key financial information, recent developments, SWOT analysis, and strategies employed by the major market players

The major companies which are dealing in the Molecular diagnostics are Abbott, Siemens Healthcare GmbH, Thermo Fisher Scientific Inc., BD, bioMérieux SA, Cepheid, Hologic, Inc., Life Technologies, Myriad Genetics, Inc., QIAGEN, Agilent Technologies, Inc., Quidel Corporation, Beckman Coulter, Inc., Bio-Rad Laboratories, Inc., Illumina, Inc., IMMUCOR, Luminex Corporation, Meridian Bioscience, Hoffmann-La Roche Ltd, and GenMark Diagnostics, Inc. among other domestic players. DBMR analysts understand competitive strengths and provide competitive analysis for each competitor separately.

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[drwaseemabbas]

Breast cancer treatment Delhi/India – Latest advancement Patient education and resources

 Patient information and resources

Latest advances in Breast cancer treatment

Node preservation reduces lymphedema cases-Can be done by Sentinal lymph node biopsy.

Genomic testing minimizes chemotherapy exposure-In detail described below.

Better identification of hereditary cancer syndromes.

An oral option for targeted therapy.

New drug combination makes estrogen-blocking agents more effective.

The next generation of monoclonal antibodies.

Immunotherapy in Triple negative Breast cancer.

Breast cancer is a curable disease in majority of patients.

Before describing about treatment of breast cancer we need to not only look at the stage of breast cancer but also at different prognostic and predictive biomarkers.

Apart from the stage, these biomarkers tell us about the probability of relapse

So, we divide breast cancer according to stage and according to the hormone receptor status and genomics. (By looking at genes we can also say that what kind of genes are driving cancer and how aggressive these genes are).

Classification of Breast cancer

A, As per Stage- Stage I, Stage II, Stage III, Stage IV.

B, As per Biomarkers-

Estrogen Receptor (ER)

Progesterone Receptor (PR)

Her2 Neu

C, Genomic test for Breast recurrence score-That measures the expression of cancer related genes in patients tumor.

Oncotype DX Breast recurrence score

Mammaprint

PAM 50

EndoPredict

Breast cancer can be divided   into subtypes   that respond differently to various types of treatment.

Subtypes according to Hormone receptor status

A, Hormone receptor positive – Estrogen receptor positive (ER-Positive), Progesterone receptor positive (PR-Positive) and Her2 Neu receptor negative are good biology tumors where chances of recurrence are less because they are good biology tumors. These breast cancers are 98% curable if diagnosed at early stage.

Can we avoid chemotherapy for such patients suffering from breast cancer?

Does that mean treating breast cancer without chemotherapy-Yes but not for all breast cancer patients

Yes, more breast cancer patients can avoid chemotherapy. With the help of genomic recurrence scores in breast cancer we can tell now who will benefit from chemotherapy. Not only in early-stage breast cancer, we can avoid chemotherapy in node positive breast cancer as well. From last 3 years 30% of our patients did not not receive chemotherapy after surgery for breast cancer. Now we can have better informed discussion with patients explaining why they are unlikely to benefit from chemotherapy in early-stage breast cancer.

This allows women suffering from Breast cancer to get through their cancer treatment faster and back to their lives sooner.

Cost remains a concern for such tests but in coming times it would go down so everyone can afford these tests.

In young breast cancer patients with advanced stage who are high risk for relapse ovarian suppression 

 B, Her2Neu positive Breast cancer – These are more aggressive that hormone receptor positive breast cancers. Such types of breast cancers are driven by Her2 Neu gene.

How are breast tumors tested for HER2?

By simple method called immunohistochemistry-IHC

If the IHC result is 0 or 1+, the cancer is considered HER2-negative. These cancers do not respond to treatment with drugs that target HER2.

If the IHC result is 3+, the cancer is HER2-positive. These cancers are usually treated with drugs that target HER2.

If the IHC result is 2+, the HER2 status of the tumor is not clear and is called “equivocal.” This means that the HER2 status needs to be tested with FISH to clarify the result.

If IHC report is 2-equivocal, it should be confirmed with FISH- Fluorescent in situ hybridization.

How to treat Her2 Neu positive breast cancer?

Chemotherapy cannot be avoided for such type of breast cancer unless tumor is less that 1cm. These are aggressive types of breast cancer and need targeted therapy along with chemotherapy.

Now different types of targeted therapies are available for such type of breast cancer and prognosis has improved with  advent of targeted therapies like Trastuzumab, Pertuzumab, Trstuzumab Emtasine (TDM-1), Lapatinib, Fam-Trastuzumab, Tucatinib, Enhertu (chemical name: fam-trastuzumab-deruxtecan etc.

C, TNBC-Triple negative Breast cancer- Tumor which is ER Negative, PR Negative and Her2Neu negative are called triple negative breast cancer. Triple negative Breast cancer is an aggressive cancer and has more like spread at the time of diagnosis and is more likely to come back after treatment.

In early stage 5 year survival rate is 90%, for breast cancer patients who are locally advanced (Stage II, Stage III) 5 year survival is 60% and for stage IV triple negative breast cancer patient 5 year survival is 10%.

These cancers are usually associated with BRCA 1 and BRCA 2 genes and other hereditary syndromes as well. So such breast cancer patients are diagnosed at early age. Some body diagnosed with Triple negative breast cancer should be screened for Hereditary syndromes after proper genetic counselling.

Treatment has not changed much as no targeted therapy is available for treatment of TNBC breast cancer patients. Immunotherapy is a new tool which is showing promise.

Immunotherapy for Triple negative Breast Cancer-Immunotherapy has changed the way we look at triple negative breast cancer. Different drugs called immune checkpoint inhibitors are available and are in early phase trials, have shown promising results

Immunotherapy for breast cancer can be combined with chemotherapy especially for locally advanced breast cancer. In neo adjuvant settings   it has shown good results in disease control for the first time in history. Research is going on and different clinical trials are addressing this issue.

Sofa we have seen more complete pathological responses with this approach.

C, TNBC-Triple negative breast cancer;-  Tumor which is ER-Negative,PR-Negative and Her2Neu-Negative.  -Aggressive type of breast cancer with high relapse rates .Now different drugs are available to cure this type of cancer. Immunotherapy is now a major tool to fight breast cancer.

Treatment of metastatic Breast cancer

Stage 4 breast cancer means that it is not curable, but that does not mean that breast cancer patients do not survive longer.

With newer drugs, better diagnostic techniques and better imaging modalities majority patients with stage 4 breast cancer survive beyond 5 years .

Again before starting treatment, we need to look at Estrogen receptor, Progesterone receptor and Her2Neu receptor status.

For ER, PR positive cancer patients and HerNeu negative   there is no need for chemotherapy, these patients can forgo chemotherapy and can be treated with hormonal therapy (Tamoxifen, Letrazole, Anastrazole, Exemestene) and other newer drugs called CDK4,6 Inhibitors (Palbociclib, Abemaciclib, Everolimus, Fulvestrant  etc) .

For Her2 Neu metastatic breast cancer patient newer drugs have shown promise in increasing survival.

For TNBC -Triple negative breast cancer survival remains poor and immunotherapy has shown promising results and more research is needed in this field.

Want to read to more about Breast cancer

1, Early detection of Breast cancer

2, Breast cancer screening

3, Sub types of Breast cancer

4, Treatment of high-risk breast cancer patients

5, Surgery for Breast cancer

Read More

[fostermarketarch]

Genetic Testing Market Prominent Growth And Vendor Landscape By 2025

The global genetic testing market was valued at US$ xx million in 2019. This market is estimated to be valued at US$ xx million in the year 2020, and it is expected to reach US$ xx million by the year 2025, with an estimated CAGR of xx% during the forecast period (2020−2025). Genetic testing is the study of gene present in tissues and cells. This study is further applied in the field of biology and medicine to know more about genetic disorders including down syndrome, sickle cell anemia, cancer, cystic fibrosis, and others.

The study discusses the use of gene tests for the development of targeted cancer treatment, personalized medicine, and other genetic diseases. Moreover, it highlights a wide range of techniques such as biochemical testing, cytogenetic testing/chromosome analysis, molecular testing, and DNA sequencing, which includes comparative genomic hybridization, karyotyping, fluorescence in situ hybridization, and others that are used for the screening of cancers and genetic abnormalities.

Key Insights:

Latest Updates

Analyst Views

Future Outlook of the Market

Get Free PDF Brochure of this Report @ https://www.fostermarketresearch.com/product/industry/biotechnology/global-genetic-testing-market/sample/

Competitive Landscape:

Major players in the global genetic testing market include: Bio-Rad Laboratories, Inc. (RainDance Technologies, Inc.), Abbott Laboratories, Myriad Genetics, Inc. (Myriad RBM, Inc.), Danaher Corporation (Cepheid), F. Hoffmann-La Roche Ltd., Eurofins Scientific, Illumina, Inc., Qiagen N.V., Novartis International AG, 23andMe, and Thermo Fisher Scientific, Inc. The other players in the genetic testing industry include Empire Genomics, LLC, Agilent Technologies, Inc., Irvine Scientific, PerkinElmer, Inc., and Sysmex Corporation, among others.

Market Taxonomy:

By Type

Predictive and Presymptomatic Testing

Carrier Testing

Prenatal & Newborn Testing

Diagnostic Testing

Nutrigenomic Testing

Pharmacogenomic Testing

Others

By Technology

Cytogenetic Testing

Biochemical Testing

Molecular Testing

By Application

Alzheimer’s Disease

Cancer

Cystic Fibrosis

Sickle Cell Anemia

Duchenne Muscular Dystrophy

Huntington’s Disease

Thalassemia

Others

By Region

North America

Europe

Asia Pacific

Latin America

Middle East and Africa

Talk to Analyst @ https://www.fostermarketresearch.com/product/industry/biotechnology/global-genetic-testing-market/Talk-to-Analyst/

Market Dynamics:

The global genetic testing market is anticipated to register considerable growth in the forecast period due to increasing incidence of cancer & genetic disorders as well as rise in acceptance & awareness of personalized medicines. Additionally, advancements in genetic testing techniques and growing application of genetic testing in oncology are anticipated to propel the market growth during the review period. Diagnosis at the right time saves lives and reduces the number of deaths. According to The Institute for Health Metrics and Evaluation (IHME), about 8.9 million cancer deaths were documented in 2016, triggered by inheriting genetic mutation.

FAQ's:

Note: This report provides an in-depth analysis of the global genetic testing market and provides market size (US$ Million) and compound annual growth rate (CAGR %) for the forecast period (2020-2025), considering 2019, as the base year.

What are the trends adopted by key players in the global genetic testing market?

What key factors are expected to increase the demand for genetic testing market during the forecast period 2020-2025?

What are the major challenges inhibiting the growth of the global genetic testing market?

What is the total market value (US$ Mn) generated in the global genetic testing market by type in 2019, and what are the forecasts by 2025?

Which technology is expected to dominate the global genetic testing market in the coming years?

Which application contribute highest CAGR (%) in the genetic testing market?

What was the total revenue generated by the global genetic testing market across different regions (North America, Europe, Asia-Pacific, Latin America, and the Middle East and Africa) in 2019, along with CAGR (%) for the period (2020-2025)?

Who are the key players contributing to the growth of the global genetic testing market, and what are the new strategies adopted by them to retain a market share in the industry?

What is the competitive strength of the key players in the global genetic testing market?

What are the major outcomes derived from Porter’s five forces?

What are the new products which are going to be approved or launched in the upcoming years, which may have a huge impact on the market?

What insights are derived through the analysis of key players on the following parameters: company overview, financial performance, product portfolio, geographical presence, key highlights, and strategies?

Buy Now This Business Strategic Report to Improve Your Profits @ https://www.fostermarketresearch.com/product/buy/biotechnology/271/

About Foster Market Research:

Foster Market Research is a global market intelligence and advisory firm engaged in providing data-driven research extract from rigorous analysis, to the clients to make critical business decisions and execute them successfully. Foster connects over various distribution channels and numerous markets for great understanding of the trends and market to deliver our clients with accurate data.

Our focus is on providing market research that delivers a positive impact on your business. We work continuously to provide our clients with the most accurate analytics data and research reports without any delay so as to improve their business strategies and provide them with rich customer experience.

Contact Us:

1701 Royal Lane, #1306, Dallas Tx-75229 Phone: +1 469 4981929 Email: support@fostermarketresearch.com

[marketresearchstrategy]

Nucleic Acid Labeling Industry Supply Chain Analysis, Growth Opportunities, Top Companies, Revenue Growth and Business Development Report by 2026

Increasing healthcare expenditure, rise in disease diagnosis, increase in R&D spending, increase in genomics research, rising demand for personalized medicine, rise in enzymology research and advancements in tools for synthetic genome design are key factors contributing to high CAGR of Nucleic Acid Labeling during forecast period.

the global Nucleic Acid Labeling market valued at USD 1.56 billion in 2018 and is expected to reach USD 3.08 billion by the year 2026, at a CAGR of 8.6%. A wide variety of molecular and cellular biology procedures are dependent on a labeled or tagged nucleic acid. These behavior and functioning can be specifically studied via the attached label. Nucleic acids can be easily labelled with several tags that allows their detection and purification. These tags can be used to recover or identify other interacting molecules.  The integrity of the nucleic acid is preserved in this non-destructive reaction, which makes it useful for applications where it is necessary to use the intact sample.

Owing to the growing demand for nucleic acid labeling, the manufacturers are adapting strategic initiatives such as innovative launch systems to increase their product portfolio. For example, with the launch of PHOTOPROBE labeling systems, the total length of the original nucleic acid sample, instead of copies, is directly marked. Additionally, Nucleic Acid Labeling can also be used for applications involving protein interactions, such as gel change or drip analysis, it is generally advantageous to generate labeled probes at the end to avoid steric interference of the interaction. The nucleic acid transfer can provide valuable information on gene integrity and copy number, as well as a means of analyzing mRNA size and expression gene, nucleic acid labeling helps to characterize cells and tissues developed in vitro and often produce important clinical information when used in patient samples. Moreover, the availability of different labels and a wide range of detection systems improve the sensitivity and flexibility required for in situ hybridization, thus, eventually driving the market growth for nucleic acid labeling.

Get a sample of the report @ https://www.reportsanddata.com/sample-enquiry-form/2252

The report provides extensive coverage of the supply chain, key players of the industry, consumer base, company profiles, production and consumption rate, primary applications, and other relevant data. It provides an in-depth assessment of the key companies operating in the market along with their company profiles, business overview, production and manufacturing capacity, product portfolio, financial standing, global position, and business expansion plans. It also studies recent mergers and acquisitions, joint ventures, product launches, partnerships, collaborations, and agreements among other. The report also provides insights into new entrants and their strategic alliances to gain a robust footing in the market.

Key Manufacturers in the Global Nucleic Acid Labeling  Market:

Promega Corporation, Thermo Fisher Scientific, Inc., General Electric Company, New England Biolabs, PerkinElmer, Inc., F. Hoffmann La-Roche AG, Vector Laboratories, Merck KGaA, Enzo Biochem, and Agilent Technologies.

Request a discount on the report @ https://www.reportsanddata.com/discount-enquiry-form/2252

The report also provides an extensive regional segmentation to offer the readers key insights into the spread of the market over key geographical regions. It covers production and consumption patterns, import/export, supply and demand, consumer demand and behavior, key trends, and presence of key players in each region. The report also offer a country-wise analysis to impart a better understanding of the revenue growth of the market in each region.

North America (U.S., Canada,     Mexico)

Europe (U.K., Italy, Germany,     France, Rest of Europe)

Asia Pacific (India, Japan, China,     South Korea, Australia, Rest of APAC)

Latin America (Chile, Brazil, Argentina,     Rest of Latin America)

Middle East & Africa (Saudi     Arabia, U.A.E., South Africa, Rest of MEA)

Product Type (Revenue, USD Million; 2016–2026)

Reagents & Kits

Services

Technique Type (Revenue, USD Million; 2016–2026)

PCR

Nick Translation

Random Primer

In Vitro Transcription

Reverse Transcription

End Labeling

Label Type (Revenue, USD Million; 2016–2026)

Biotin-based

Fluorescent

Radioactive

End Use (Revenue, USD Million; 2016–2026)

Hospital

Clinic

Others

To know more about the report @ https://www.reportsanddata.com/report-detail/nucleic-acid-labeling-market

Key Questions Addressed in the Report:

What is     the market size the global Nucleic Acid Labeling  market is expected to reach over the     forecast period? What is the expected CAGR?

What     are the key segments of the market?

What     are the key products and applications of the Nucleic Acid Labeling  market?

What     factors are expected to drive and restrain market growth over the forecast     period?

What     are the key outcomes of SWOT analysis and Porter’s Five Forces analysis?

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Thank you for reading our report. The report can be customized based on regional segmentation and competitive landscape. Kindly get in touch with us to know more and our team will ensure the report is well suited to meet your requirements.

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[turkishmedicalservices]

Everything that you need to know about gender selection in Cyprus

Parenthood is an exciting experience. It is full of thrills and excitements, ups and downs, joys and woes, but most importantly; it is full of uncalled, unprecedented situations for which no one can be prepared. Having a child can be a huge responsibility, which is why some parents prefer to know the sex/gender of their baby during the gestation period. 

The desire to know or determine the sex of the unborn child is probably as old as history itself. However, now times have changed, and medical technologies have advanced to the extent that was unimaginable for our ancestors. Gender selection in Cyprus has recently become popular among couples who want to find out the sex of their child without any fail and at a reasonable cost. 

What are the methods of gender selection?

Typically, three distinct methods are followed for gender selection in Cyprus. These include: 

#1 PGS or pre-implantation genetic screening

There are IVF laws that protect the unborn child from the crime of amniocentesis. In Cyprus, the IVF laws state that PGS can only be used as a method of sex selection if a case of genetically inheritable X-linked disease runs in the family. Also known as the aneuploidy screening, the procedure checks the chromosome of the embryos created by IVF or through ICSI (Intra Cytoplasmic Sperm Injection).  

#2 MicroSort Sperm Sorting

This method separates the sperm as per their X and Y chromosomes. In a nutshell, the sperm is taken from the male, and then the desired X and Y chromosomes are separated. The collected sperm samples can be used for in-vitro fertilization or inoculation according to the desired sex. The success rate for X chromosome separation is 70-75%, whereas for achieving Y chromosome separation, the success rate is 80-85%.  

#3 Genetic Testing on embryos

This is the most reliable method of gender selection as it allows a 99.9% chance of accuracy. The tests in genetic testing are carried out as pre-implantation genetic diagnosis with Array CGH, FISH, or NGS techniques. 

FISH is the acronym for Fluorescent in the Situ Hybridization process, which is applied to the cell nuclei extracted from the embryo. And then, a definite number of chromosomes can be screened to determine the sex of the baby. Array-based Comparative Genomic Hybridization or Array CGH evaluates areas of the human genome for losses or gains of chromosome segments at a higher resolution. The test is so precise that Array CGH can explore all the 46 chromosomes in one single test and detect any DNA imbalance without any flaws.  

The bottom line

Every child is exceptional, and every kid is born with unique capabilities that can do wonders. Gender selection is a way to appreciate and embrace the gift of God in your life warmly. Use gender selection in Cyprus to determine if your kid is healthy or not, and share your happy experiences with your spouse and your family. 

Sources: https://turkishmedicalservices.com/everything-that-you-need-to-know-about-gender-selection-in-cyprus/

[zanypaperprince]

Best multiple myeloma treatment in India

Multiple myeloma is cancer of the plasma cells (a type of white blood cells) of the bone marrow. Plasma cells are protein-making cells that generally produce the different kinds of antibodies for our immune system. In multiple myeloma, the plasma cells become malicious and cancerous. These myeloma cells stop making different forms of protein in response to the immune system's needs and instead start to produce a single abnormal type of protein sometimes termed a monoclonal or M protein. Multiple myeloma plasma cell populations accumulate in the bone marrow, and these collections of cells called plasmacytomas can erode the hard outer shell or cortex of the bone that normally surrounds the marrow. These weakened bones show thinning of the bone, as seen in nonmalignant osteoporosis or what appear to be punched out or lytic bone lesions. People often refer to multiple myeloma simply as myeloma (also termed Kahler's disease after the physician who first described this cancer). The disease usually occurs in people past middle age.

In India, there are large number of options available for Best multiple myeloma treatment in India.

However, rarely it can occur in a child. One type of myeloma-related plasma cell neoplasm is called a monoclonal gammopathy of undetermined significance (MGUS). In MGUS, medical professionals only find low levels of M protein and people have no symptoms; MGUS infrequently develops into multiple myeloma.

Plasma cell neoplasm is another name for multiple myeloma. Causes of multiple myeloma What triggers plasma cells into malicious multiple myeloma is unknown. The cancerous myeloma plasma cells proliferate and crowd out normal plasma cells and can corrode areas of bones. The proteins produced in large amounts can cause many of the symptoms of the disease by making the blood more viscous and depositing the proteins in organs that can interfere with the functions of the kidneys, nerves, and immune system.

Causes of multiple myeloma are not known exactly. But patients more likely to get affected • older than 65 years • people of African-American origin • overweight or obese people • family member with it

Stages of multiple myeloma

There are four stages of multiple myeloma. While many health care professionals use different staging, these are various stages cited by many clinicians:

• Smoldering: multiple myeloma with no symptoms • Stage I: early disease with little anemia, relatively small amount of M protein and no • bone damage • Stage II: more anemia and M protein as well as bone damage • Stage III: still more M protein, anemia, as well as signs of kidney damage Because staging criteria differ according to different groups, some clinicians simply define the individual's multiple myeloma without assigning a stage and simply estimate a prognosis for their patient.

Symptoms of multiple myeloma

Patients with myeloma may be asymptomatic with an unexplained increase in protein in the blood. With more advanced disease, some myeloma patients may have weakness due to anemia caused by inadequate production of red blood cells, with bone pain due to the bone damage, and as the abnormal M protein can accumulate and damage the kidneys resulting in patient’s unexplained kidney damage and decreased kidney function. Multiple myeloma cancer cells may be in or outside the bone marrow.

The following symptoms and signs of multiple myeloma -

• Anemia • Bleeding • Nerve damage • Bone tenderness or pain, including back pain • Enlarged tongue • Skin lesions (rash) • Infections Weakness, fatigue or tiredness • Kidney failure and/or other end-organ damage• Spinal cord compression • • Loss of appetite and weight loss • Leg swelling • Hypocalcaemia • Diagnosis of multiple myeloma • First sign of multiple myeloma is found when a routine blood test shows an abnormal amount of protein in the bloodstream or an unusual stickiness of red blood cells causing them to stack up almost like coins, an unusual formation for red blood cells. The health care professional will do a history and physical exam, looking for signs and symptoms of multiple myeloma. If multiple myeloma is suspected, several studies help confirm the diagnosis.

They include a bone marrow aspiration and biopsy most commonly from the large bones of the pelvis. Cells obtained from the marrow are studied by a pathologist to determine if there is one (plasmacytoma) or more (multiple myeloma) abnormal types or numbers of cells • Medical professionals also study a sample of the bone marrow aspirate for more detailed • Characteristics such as the presence or absence of abnormal numbers or types of chromosomes (DNA) by what is called cytogenetic testing.

Bone marrow biopsy can assess the concentrations of cells in the marrow and the presence of abnormal invasive growth of cellular elements. • Blood testing and urine testing by several methods can determine levels and types of National Comprehensive Cancer Network (NCCN) recommended that health care professionals use a serum free light chain assay and fluorescence in situ hybridization (FISH) test to further • Monoclonal protein produced and if there is kidney damage.

Identify multiple myeloma in patients

X-ray studies to identify skeletal lesions and MRI for spinal cord lesions in multiple myeloma.

Medical treatment for multiple myeloma

The therapy is decided based upon the patient's condition and the cancer management team, made with the patient's input. The choices for treatment(s) often include combinations of drugs, some of which medical professionals give as pills and others by intravenous injection.

These include drugs that affect or modulate the immune system, steroids, and some oral or injectable chemotherapy drugs. These are usually used in combinations. There may be a role for high-dose chemotherapy followed by the administration of bone marrow called a stem cell transplant. Numerous factors come into play in determining whether to do such a transplant. Other medical treatments may include steroids, bisphosphonate therapy, blood or platelet transfusions, plasmapheresis, and other combination therapy depending on the individual patient's disease stage.

Radiation therapy may treat painful areas of bone damage. Surgeons can surgically repair broken bones in many cases.

There are many drugs used to treat multiple myeloma. Medical professionals often use the following drugs in combination with dexamethasone,

• Bortezomib Velcade -- protease inhibitor • Lenalidomide (Revlimid) -- immune cell modulation • Melphalan (Alkeran) -- alkylating agent that is toxic to myeloma cells • Carfilzomib (Kyprolis) -- protease inhibitor that is FDA approved usually for patients • who have failed a previous treatment • Daratumumab (Darzalex) -- monoclonal antibody that may damage or kill multiple • Myeloma cells (and others) that have CD38 protein on their surface • Elotuzumab (Empliciti) -- a compound that activates the body's natural killer cells to • Destroy multiple myeloma cells, usually in combination with Revlimid and Decadron • Ninlaro (Ixazomib) -- This proteasome inhibitor, in combination with Revlimid and • Dexamethasone, improves the survival rates of some patients with multiple myeloma.

Hospitals offer best multiple myeloma treatment in India, the charges for autologous stem cell transplant ranges between USD 15000 to USD 21000 depending on the status of the disease and individual's response to the treatment provided at the hospitals.

Related Articles -

Best multiple myeloma treatment hospitals in India

Best Sickle cell disease treatment in India

Best Bone marrow transplant hospitals in India

Are you looking for Lymphoma treatment in India

About GHN Healthcare -

GHN Healthcare Services is a leading Medical treatment Assistance Company based in New Delhi (National Capital region), India. The Organization actively engaged in providing Medical assistance to foreign Patients who choose to travel to India for quality medical care at a reasonable cost. GHN Healthcare Services is associated with 25+ Top-Notch Hospitals and has a network of 500+ Super Specialists to offer world-class medical care at a reasonable cost.

For any enquiries related to Multiple myeloma treatment in India. Click here...

[evoldir]

Postdoc:ULausanne.EvolutionaryGenomicsNeurogenetics

A postdoc position is available within the lab of Roman Arguello at University of Lausanne's (UNIL) Department of Ecology and Evolution. We are a newly formed lab interested in understanding the genetic and neural bases of sensory evolution. We take a very interdisciplinary approach that bridges computational/comparative genomics with neurogenetics. Our model organisms are diverse Drosophila species from around the globe, and we are particularly interested in the evolution of neural circuits and in the evolutionary processes related to the rapid changes underlying olfaction and temperature preference. The specific focus for this position will be on olfactory evolution. The projects will relate repeated gains/losses in olfactory receptors to the evolution of their protein sequences, to their neural circuits, and to behavior. This work will test hypotheses about how the olfactory system evolves, and to what extent these changes are repeatable. Due to the cross-disciplinary nature of the research, there is a range of opportunities for contributing to the projects depending on particular interests and strengths (i.e. comparative/population genomics, neurobiology, generation of transgenic tools for new species). Qualification include a PhD in genetics, evolutionary biology, neurobiology, or a related field, the ability to be self-motivated and to work independently as well as within a small group, good communication skills, comfortable with public presentations, and the ability to clearly document work. Additionally, expertise with several of the following tasks is required, and an interest/willingness to learn some of the others: - molecular evolution and/or population genomic analyses - statistics and next-generation sequence analyses (programming/scripting in one or more languages such as Python, Perl, R, etc.) - molecular biology (CRISPR/Cas9 vector designs) - electrophysiology - histochemistry/microscopy (dissecting, tissue preparations, antibody staining, fluorescent in situ hybridization, confocal imaging) - fly genetics and maintenance - fly embryo injections for generating transgenic lines and screening This is a 100% position for an initial duration of one year, renewable up to 2 or 3 years depending on the start date and funding available. The position is dedicated primarily to research, however some contribution to teaching is expected, including the possibility of assisting with the supervision of master students. The Department of Ecology and Evolution is a lively, fun, productive, and highly diverse group. While UNIL is a French-speaking university, the working language of the Department and the Lab is English. For more information about the Department of Ecology and Evolution please see its page here: http://bit.ly/2lGIr6Y Lausanne is a scenic medium-sized city situated on the edge of Lake Geneva. Life here is good. Its central location within Europe makes traveling by train and plane convenient. And for outdoor enthusiasts, Lausanne sits at the base of Alps and Jura which offer year-round escape and fun. For more information about the Lab, or for further questions, please visit: arguellolab.org or email Roman at: jacob.arguello@unil.ch Formal applications need to be done through HR, and must include a cover letter detailing your research interests and background, a CV, and the contact info for references (2-3). Applications should both be uploaded through the University of Lausanne platform (link given below), and sent as a single pdf to jacob.arguello@unil.ch. Application Link: https://bit.ly/2Ge0DA5 University equality policy: The University of Lausanne promotes an equitable representation of men and women among its staff and encourages applications from women and minority groups. via Gmail

[pkstudiosindia]

Revealing the in vivo growth and division patterns of mouse gut bacteria – Science Advances

Featured Post in Water Filter India dot com - Water Filter India

Abstract

Current strategies for learning gut microbiota are unable to reply some vital microbiology questions, like how completely different bacteria develop and divide in the gut. We suggest a technique that integrates the use of sequential d-amino acid–based mostly in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid–based mostly probes containing completely different fluorophores to mice by gavage, the ensuing twin-labeled peptidoglycans present temporal data on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, together with species that can not be cultured individually in vitro, are revealed. Our technique affords a facile but highly effective instrument for investigating the in vivo growth dynamics of the bacterial gut microbiota, which is able to advance our understanding of bacterial cytology and facilitate elucidation of the fundamental microbiology of this gut “dark matter.”

INTRODUCTION

The organic range and complexity of the mammalian gut microbiota current formidable obstacles to the investigation and comprehension of these intimate microbial neighbors. To totally comprehend the physiological and pathological capabilities executed by gut microbiotas, it’s crucial to know the fundamental microbiology of these microbes, resembling how completely different bacteria develop and divide in the gut (1). However, even after practically twenty years of ever-growing gut microbiota analysis, many of these questions stay unaddressed (2). The problem of separate tradition of many gut bacterial species in vitro, which is partially why these microbes are sometimes called the “dark matter” in the gut (three), prevents researchers from additional investigating these bacteria in the laboratory. After intensive efforts in optimizing tradition circumstances for various gut bacteria by microbiologists, progressively extra species might be cultured individually in vitro (four). Nonetheless, for the bacteria that may be cultured and investigated in vitro, to what extent the microbial information obtained from in vitro research might be translated into in vivo conditions stays debatable (1). Therefore, a technique that can be utilized to instantly probe and examine the gut microbes in vivo is very fascinating (5).

One method that might instantly probe the indigenous actions of gut microbes makes use of a conventional isotope-based mostly labeling technique. N15-tagged amino acids got to mice by intravenous injection, and the microbial N15 alerts in the gut acquired by bacterial foraging on host proteins had been then detected utilizing nanoscale decision secondary ion mass spectrometry (6). Consequently, the metabolic actions and host-protein utilization choice of completely different gut bacteria may very well be assessed. Nonetheless, the information acquisition of this isotope labeling method is restricted to the extremely specialised mass spectrometry–based mostly method, and the information obtained was restricted to a particular metabolic pathway of the bacteria. A special chemical method, fluorescent d-amino acid (FDAA)–based mostly metabolic labeling, has been extremely precious in bacteriology research, as a result of of the labeling specificity [targeting bacterial peptidoglycan (PGN), thus no labeling on eukaryotes], velocity (labeling inside minutes below optimized circumstances), and ease of use (studying fluorescent alerts with routine analytical gear) (7, eight). Recently, it has been demonstrated that FDAA probes might label mouse gut microbiota in vivo with excessive effectivity, and we additional established a STAMP (sequential tagging with d-amino acid–based mostly metabolic probes) technique for recording the survival of transplanted microbiota in the receiving mouse utilizing the labeling alerts of the two FDAA probes (9, 10).

Here, to develop a technique that may instantly probe and visualize the growth and division modes of completely different gut bacteria in situ, we suggest a STAMP-based mostly and fluorescence in situ hybridization (FISH)–facilitated probing technique. In this technique, two FDAAs with completely different fluorophores had been sequentially administered to mice by gavage to document in vivo bacterial growth and division processes throughout the labeling interval. Consequently, figuring out the labeled bacteria with FISH probes at completely different taxonomic ranges (genus and species) allowed us to find out how completely different bacterial taxa, notably these that can not be cultured in vitro, develop and divide in the mammalian gut.

RESULTS

FDAA sequential labeling of mouse gut microbiota

DAAs are important constructing blocks for bacterial PGN synthesis; the sidechain-functionalized DAAs (i.e., FDAA) are nicely-tolerated by the enzymes (d, d or l, d-transpeptidases) concerned in cell wall development (7). Chronological use of a number of FDAAs has offered precious data of the temporal PGN synthesis in a number of mannequin bacterial species in vitro (10). Here, to review the in vivo growth and division patterns of completely different bacterial taxa in gut microbiota, we sequentially utilized two FDAA probes, TAMRA-amino-D-alanine (TADA) and Cy5-amino-D-alanine (Cy5ADA), containing TAMRA (tetramethylrhodamine) or Cy5 (Cyanine 5) on facet chains, in two gavages to label the mouse gut microbiota (scheme proven in Fig. 1A). The probes got at an interval of three hours, and the collected cecal microbiota confirmed sturdy two-shade labeling (Fig. 1B) with excessive protection (fig. S1) after 6 hours of the first gavage with out obvious alterations of the microbiota composition (fig. S2).

Fig. 1 Schematic illustration of the FDAA-based mostly labeling technique built-in with FISH staining and the two-shade fluorescence imaging of the sequentially labeled gut microbiota.

(A) TADA and Cy5ADA got to mouse by gavage at an interval of three hours. The cecal microbiota was collected and imaged, and the taxonomic identifications of completely different bacteria had been then decided by corresponding FISH probes. (B) Two-color fluorescence imaging of the gut bacteria sequentially labeled by TADA (inexperienced) and Cy5ADA (crimson). Scale bar, 10 μm. Representative photographs from at the least three impartial experiments are proven. BF, Bright subject. (C) Zoomed in view of the indicated bacteria from the merged picture above. The inexperienced and crimson colours revealed the distinct growth patterns of completely different bacteria. Scale bars, 2 μm.

The two-shade fluorescence imaging confirmed an amazing morphological range of the gut microbes. Different distributions of the two colours amongst numerous bacteria revealed their distinct dividing patterns and growth charges. Because the second probe used in the sequential labeling was Cy5ADA, the PGN websites with extra lively constructions had stronger labeling of Cy5 (proven in crimson), and PGN synthesized earlier had extra TAMRA alerts (proven in inexperienced). Thus, the shade distributions of crimson/inexperienced offered a chronological account of the PGN synthesis in every bacterium. Most of the bacteria grew in a dispersed mannequin with comparatively sturdy FDAA labeling all through the cell (Fig. 1B) and divided in binary fission with a number of crimson-labeled septums in the center of the bacteria (Fig. 1C, nos. 1 and 2). Some bacteria had been solely labeled with the first probe (Fig. 1C, no. three), suggesting that these cells might need completely different growth charges throughout the two labeling steps. It is price noting that many bacteria confirmed uneven labeling (Fig. 1C, nos. four and 5). Some solely had one crimson pole, with two halves having very completely different labeling intensities (no. four). One rationalization is that these had been the daughter cells from a binary fission, and the crimson pole was from the newly separated septum. It can be potential that some of these bacteria would possibly develop in an uneven or polarized method, like the zonal-apical growth noticed in Agrobacterium tumefaciens (7). Moreover, it was additionally widespread to see the two daughter cells with completely different growth charges, the place just one of the two cells confirmed a brand new septum (Fig. 1C, no. 5). Besides these lengthy rod/spindle bacteria, PGN growth of small rod/coccus bacteria is also readily noticed (Fig. 1C, no. 6). Many of the classical growth and division modes of bacteria may very well be noticed in the labeled gut microbiota, together with diffuse synthesis of PGN (Fig. 2A), spiral synthesis (Fig. 2B), division dominated by septum synthesis (Fig. 2C), polar growth (Fig. 2D), and division by stalk/budding formation (Fig. 2E).

Fig. 2 Classical bacterial growth and division patterns may very well be noticed in the labeled gut microbiota.

Following STAMP (TADA and then Cy5ADA gavage), the mouse cecal microbiota was collected and analyzed by confocal fluorescence microscopy. In the cartoon, grey areas signify the websites of lively cell wall synthesis, and inexperienced and crimson signifies the newly constructed PGN. (A) Diffuse synthesis of PGN. (B) Spiral synthesis of PGN. (C) Septum synthesis dominating the cell division. (D) Polar growth. (E) Stalk/budding formation. Scale bars, 2 μm.

Identification of the bacterial growth patterns on the genus stage

With the wealthy microbiological data obtained from the FDAA-labeled gut microbiota, taxonomically figuring out particular person bacterium in the view subject turned extremely fascinating. Toward this finish, we resorted to FISH, a classical technique for figuring out taxonomic data in advanced bacterial samples. To facilitate the FISH probe choice and design, we first did 16S ribosomal DNA (rDNA) and metagenomic sequencing of the labeled microbiotas (mixed cecal microbiotas of 5 mice) to determine the bacterial composition (fig. S3 and desk S2). To have a fairly excessive protection of the microbiota, we first used 15 FISH probes to stain some of the most considerable genera (overlaying ~71% of categorized bacteria in the microbiota; desk S1), the sequences of which had been both based mostly on earlier stories or designed in this research. The FDAA-labeled microbiota was break up into dozens of small aliquots and labeled by completely different FISH probes individually. It is price mentioning that the microbiotas collected from small intestines additionally confirmed clear two-shade FDAA labeling (fig. S4). However, as a result of the quantity of bacteria was very small, we didn’t carry out any FISH staining.

Of the 15 FISH probes overlaying bacteria from 9 households, seven stained Gram-positive and eight stained Gram-negative bacteria. Representative photographs of the labeled bacteria in every genus from a number of FISH experiments had been offered. As anticipated, as a result of of their thinner PGN, Gram-negative bacteria (Fig. three, A to F) confirmed weaker FDAA labeling than the Gram-positive bacteria (Fig. three, G to L), and most of them had been quick rods (1 to 2 μm). Of notice, we additionally recognized two Gram-negative genera, Helicobacter and Desulfovibrio, that might not be labeled by FDAAs (fig. S5). Extended labeling time (two gavages of TADA at an interval of 6 hours) nonetheless didn’t result in any FDAA labeling (fig. S6). These bacteria might both have very sluggish PGN synthesis charges or have lipopolysaccharides/capsules impermeable to FDAA probes. It can be potential that they’ve atypical PGN buildings or transpeptidases that can’t tolerate the incorporation of FDAAs, which deserves additional research to increase our understanding of the construction and synthesis of PGN in completely different bacteria.

Fig. three Confocal fluorescence imaging of 12 FDAA-labeled and FISH-stained gut bacterial genera.

The cecal microbiotas of mice acquired sequential labeling of TADA (inexperienced) and Cy5ADA (crimson) had been stained by completely different FISH probes (blue) focusing on corresponding genera. (A to F) Representative photographs of FDAA-labeled bacteria in six Gram-negative genera. Scale bars, 1 μm. (G to L) Representative photographs of FDAA-labeled bacteria in six Gram-positive genera. Scale bars, 5 μm. Photographs of bacteria, representing constant labeling sample in every genus from at the least three impartial FISH experiments, are offered.

Most of the labeled Gram-positive bacteria (Fig. three, G to L) had been spindle formed and divided by binary fission, however the distributions of the two colours in every genus had been fairly completely different, suggesting distinct preparations of divisome and/or elongasome in numerous genera (11). For instance, the bacteria proven in Fig. three (G to J) all belonged to the household Lachnospiraceae, however the two-shade photographs offered completely different patterns. In Lachnospiracea incertae sedis (LIS; Fig. 3G), FDAAs had been labeled in a striped method, however in Lachnoclostridium (Fig. 3H), Roseburia (Fig. 3I), and Marvinbryantia (Fig. 3J), the labeling was extra diffuse. Moreover, we additionally recognized some bacteria with polar growth, with one (Fig. 3F) or two poles (Fig. 3L) strongly labeled, a phenomenon that was beforehand solely noticed in some Alphaproteobacteria and Actinobacteria (12). The constant bacterial labeling patterns and morphogenesis in every genus (fig. S7) additionally verified the specificities of the newly designed FISH probes. Of notice, the specificities of a number of of the newly designed FISH probes couldn’t be verified (listed in desk S2). These probes both stained bacteria with inconsistent FDAA-labeling patterns or had a better labeling ratio than the taxon’s relative abundance decided by 16S rDNA sequencing. FISH sequences with improved specificities or a extra stringent staining protocol are warranted for these bacterial teams.

Identification of the bacterial growth patterns on the species stage

Of the 15 genera examined, Clostridium is thought for being extremely polyphyletic (13). We noticed completely different labeling patterns inside this genus (Fig. 4A). On the foundation of the metagenomic sequencing outcomes of the microbiota (desk S2), we chosen three species in this genus and labeled them with corresponding FISH probes designed for every. Varied labeling patterns had been noticed (Fig. 4B), with asymmetrical growth observed in two (KNHs209 and ASF502) of the three species. Because Clostridium sp. ASF502 had by no means been cultured individually in vitro, these information showcased the potential of utilizing our technique for learning unculturable bacterial species in the gut microbiota.

Fig. four Clostridium reveals various mobile growth and division patterns.

(A) Confocal fluorescence imaging of the polyphenotypic bacteria in the Clostridium genus recognized by FISH staining. Scale bar, 10 μm. (B) Three FISH probes focusing on corresponding Clostridium species confirmed their distinct FDAA labeling patterns. Scale bars, 2 μm. Photographs of bacteria, representing constant labeling sample in every species from at the least three impartial FISH experiments, are offered. ATCC, American Type Culture Collection.

Encouraged by the labeling outcomes of Clostridium, we then got down to study extra species in the gut microbiota. Nine had been chosen, together with six species which were cultured in vitro: Akkermansia muciniphila (14), Parabacteroides distasonis (15),

Alistipes putredinis (16), Lactobacillus johnsonii (17), Lactobacillus brevis (18), and segmented filamentous bacteria (SFB; Candidatus Savagella) (19); and three unculturable taxa: Oscillibacter sp. 1-three, Eubacterium sp. 14-2, and Dorea sp. 5-2. The growth patterns of A. muciniphila, P. distasonis, A. putredinis, Oscillibacter sp., Eubacterium sp., and Dorea sp. had been readily recognized by our technique (Fig. 5, A to F). However, as a result of of the difficulties of performing FISH with Lactobacillus (20), the required step of lysozyme digestion destroyed most of the FDAA labeling alerts from the bacteria, leaving solely labeled septums (fig. S8). An improved FISH protocol for Lactobacillus is required for simpler evaluation of these vital gut bacteria by our technique.

Fig. 5 Confocal fluorescence imaging of FDAA-labeled and FISH-stained gut bacterial species.

The growth patterns of three species which are culturable in vitro (A to C) and three species that haven’t been cultured individually in the laboratory (D to F) are revealed. Scale bars, 2 µm. Bacterial micrographs show constant labeling patterns in every species from at the least three impartial FISH experiments.

As a closely studied species with basic capabilities of degrading mucin, producing bioactive molecules and immune modulation (21), A. muciniphila cells had been clearly noticed dividing by binary fission (Fig. 5A). The imaged quick rods of P. distasonis didn’t appear to be dividing (Fig. 5B). A. putredinis confirmed sturdy labeling at one pole, indicative of its polar growth (Fig. 5C). The three species that might not be cultured in vitro belonged to the order Clostridiales. Oscillibacter (Fig. 5D) and Eubacterium (Fig. 5E) might need diffuse synthesis of PGN (scheme proven in Fig. 2A), however Dorea appeared to have a extra distinguished septum (Fig. 5F), the synthesis of which could dominate cell division (scheme proven in Fig. 2C). Consistent modes of labeling and mobile morphologies in every species had been noticed in every species (fig. S9), supporting the specificities of these new FISH probes.

Revealing the in vivo growth of SFB

Another group of bacteria price particular notice is SFB. As one of the most extensively studied bacteria in gut microbiota, SFB have been discovered to be crucial in the induction of T helper cell 17, alongside many different immunity-associated results (22, 23). In the ~90-μm SFB proven right here (Fig. 6), we might clearly see the FDAA-labeled segments differentiated at distinct phases (Fig. 6A), intrasegmental our bodies (Fig. 6B), a needle-like holdfast (Fig. 6C), a triseptum at an uneven division location (Fig. 6C), and a symmetric division locus (Fig. 6D) (19). However, no apparent FDAA labeling of SFB intrasegmental our bodies was observed. Previously, it was reported that the PGN of Bacillus subtilis endospores lacked the d-Ala at the fifth locus of the peptide stem (24), which was the labeling goal of FDAA in Gram-positive bacteria (7). This discovering would possibly clarify the absence of FDAA labeling alerts in SFB intrasegmental our bodies. Of notice, sturdy TADA and very weak Cy5ADA labeling had been noticed in the segments the place intrasegmental our bodies had been discovered (Fig. 6, C and D, yellow arrows). This staining sample means that metabolism of segmental PGN is perhaps halted throughout formation of intrasegmental our bodies, thus resulting in lowered decay of the TADA alerts (used in the first gavage) and a lot weaker labeling of Cy5ADA (used in the second gavage). Alternating intensities of the two colours had been noticed in some SFB cells on their neighboring septums (fig. S10). This sample means that these segments had been most likely dividing throughout the two labeling steps, and that neighboring septums shaped at completely different instances had been labeled with completely different concentrations of the two FDAAs. The phenotypes of SFB have been closely studied principally by scanning electron microscopy and Gram staining for practically 50 years (25, 26). Our labeling technique affords new views on these vital bacteria and might be a great tool for additional understanding the microbiology of SFB in vivo.

Fig. 6 Confocal fluorescence imaging of the sequentially labeled SFB.

Some attribute parts of SFB, together with segments at various differentiation phases (A), intrasegmental our bodies (B), needle-like holdfast and triseptum in an uneven division location (C), and a symmetric division locus (D), had been readily noticed in the FDAA-labeled bacteria. Scale bar, 10 μm.

DISCUSSION

How PGN is constructed is one of the central subjects in bacteriology. Sequential FDAA labeling has been used in investigating PGN synthesis in many mannequin bacterial species (27). The technique proposed right here, to probe a big quantity of species in the microbiota collectively, enormously improves the effectivity of bacterial morphogenesis research. Two-color imaging of the labeled gut microbiota recorded in situ growth and division patterns of the extremely various gut bacteria throughout the 6 hours of labeling, giving us a novel alternative for a direct take a look at how this “gut dark matter” truly grows and multiplies in vivo. The extremely distinct labeling patterns of completely different gut bacteria provide a gold mine for microbial cytologists, the place new bacterial growth and division patterns could also be found. Moreover, FDAA-based mostly visualization of a number of gut bacterial teams which were closely studied will allow additional understanding of the actions of these vital microbes in the mammalian gut. It is price mentioning that amongst the 15 genera that had been FDAA labeled, many species of Prevotella, Roseburia, Oscillibacter, Anaerotruncus, and Barnesiella had been on the “most wanted” taxa checklist with excessive precedence from the National Institutes of Health Human Microbiome Project (28). The data on in situ growth patterns captured by our technique will present precious details about these understudied bacteria.

Further optimization of the FDAA probes, as an example, use of smaller fluorophores, for instance, might enhance labeling protection, particularly for these Gram-negative genera that might not but be labeled. Optimized labeling protocols (e.g., various time intervals between administration of the two probes) might help characterization of the morphogenesis of completely different bacteria rising at completely different charges. Superresolution microscopy utilizing FDAA-containing appropriate fluorophores may even assist in elucidating the distinct preparations of PGN synthesis machineries in completely different bacteria, which is able to enormously enrich our information of microbial cytology. Besides mouse gut the microbiota, research of different advanced microbial programs, resembling the microbiota from different animal hosts and environmental microbiotas, together with these in soil, water sediments, and many others., might also profit from this labeling and imaging technique.

MATERIALS AND METHODS

Reagents

FDAA probe was bought from Chinese Peptide Company (Hangzhou, China). FISH probes and paraformaldehyde had been from Sangon Biotech (Shanghai, China). Other chemical substances, not famous above, had been from Sigma-Aldrich (St. Louis, MO, USA).

Animals

C57BL/6 particular pathogen–free mice (male, 6 weeks previous) had been obtained from Jie Si Jie Laboratory Animals (Shanghai, China). Mice had been bred in the Renji Hospital animal facility in a temperature-managed (25°C) surroundings with a 12-hour mild/darkish cycle, receiving a normal chow weight-reduction plan and free entry to scrub water. All animal experiments had been carried out in accordance with tips permitted by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine.

Sequential labeling of microbiotas with FDAA probes

The C57BL/6 mice had been sequentially administered by two completely different FDAA probes (200 μl, 1 mM TADA or Cy5ADA in distilled H2O) by way of oral gavage with an interval of three hours. Their small gut and cecal microbiotas had been collected utilizing a beforehand reported protocol (9). Briefly, mice had been euthanized by cervical dislocation, and the small gut and cecum had been dissected individually and finely minced with a pair of 11.43-cm iris scissors in 1 ml of phosphate-buffered saline (PBS). The tissues and digesta had been then filtered with a 40-μm cell strainer to take away most of the nonbacterial supplies. The filtrates had been then centrifuged. The bacterial pellets (whitish-coloured) had been washed 3 times with 1.5 ml of PBS by centrifugation (15,000g, three min) and then resuspended in PBS for subsequent experiments.

In vitro tradition of soil microbiota

Five grams of sediment collected from the Yangtze Estuary had been homogeneously resuspended in 50 ml of sterile physiological water. Ten-fold serial dilutions of the sediment suspension had been carried out to 10−three. The serial dilutions (100 μl) had been dispersed on Gause’s artificial agar medium [containing 2% soluble starch, 0.1% KNO3, 0.05% NaCl, 0.05% K2HPO4, 0.05% MgSO4, 0.001% FeSO4, and 2% agar (pH 7.2)] and then incubated at 30°C. After three days, bacterial cultures (10−2) with the most phenotypic range had been collected, washed 3 times with 1.5 ml of PBS by centrifugation (15,000g, three min) and resuspended in sterile PBS for subsequent experiments.

FISH probe design

Candidate FISH probes had been recognized utilizing a okay-mer–based mostly algorithm just like KASpOD (29). The sequenced 16S ribosomal RNA (rRNA) genes had been downloaded from the Ribosomal Database Project (RDP) (30) and SILVA (31) databases. Here, the lacking 16S rDNA sequences of some goal teams had been downloaded from National Center for Biotechnology Information (32) and added manually. The closing pool was consisted of three,200,588 sequences. Target teams had been outlined as sequence subclasses consisted of 16S rDNA sequences from goal household/genus. The nontarget teams had been outlined as sequence subclasses consist of 16S rDNA from nontarget bacteria, which situated in the similar order/household with targets. For every group, the totally overlapping okay-mers had been then clustered at an 88% id clustering threshold to get the degenerate consensus okay-mer. Coverage and specificity analysis of every degenerate consensus okay-mer from the goal group had been carried out by a protection evaluation in opposition to the goal and nontarget teams, respectively. Consensus okay-mers with finest protection and specificity was lastly used as candidate probes. The associated Perl scripts can be found from https://github.com/songjiajia2018/Pdesign/archive/grasp.zip.

Probe optimization

The newly designed probes had been added to the microbiota suspensions for check utilizing probes EUB338 and NONEUB as constructive and unfavourable controls, respectively. There are a quantity of parameters that may be adjusted, resembling temperature and formamide focus, to decide on the finest stringency for hybridization. According to the technique described by Manz et al. (33), various the focus of formamide in the hybridization buffer at a relentless hybridization temperature (46°C), starting from zero to 70% (in 5% increments), was used to guage the optimum stringency of every probe designed in this research. Subsequently, an equally stringent 30 min posthybridization wash for every hybridization was carried out at 48°C. Confocal or circulate cytometry can be utilized to quantify the bacteria FISH sign depth at completely different formamide concentrations. The highest formamide focus earlier than shedding the particular hybridization sign was considered optimum hybridization stringency for the check probe.

To check the labeling specificities of the newly designed FISH sequences, the probes had been individually examined in opposition to a set soil microbiota pattern that didn’t share any genera with the mouse gut microbiota, utilizing protocols described beneath. The offered 16 new FISH probes all confirmed unfavourable labeling in the check (fig. S11). Further specificity affirmation checks had been carried out by circulate cytometry and confocal fluorescence imaging. Flow cytometry was carried out to research the labeling ratios of some genera (Clostridium, Barnesiella, and Alloprevotella) in the microbiota, utilizing a beforehand printed technique (34). These checks confirmed outcomes in line with their relative abundances deduce by 16S rDNA sequencing (fig. S12), indicative of excessive labeling specificities. Other genera and species tagged with the new FISH probes had been analyzed by confocal fluorescence microscopy to evaluate whether or not the cell morphologies and labeling patterns of the stained bacteria had been constant in every group, which might additional confirm the specificities of the new FISH probes.

Fluorescence in situ hybridization

An equal quantity of four% paraformaldehyde was added to the bacterial suspensions in PBS and incubated at room temperature for 1.5 hours to repair the bacteria. The samples had been then washed twice with PBS and resuspended in 50% ethanol-PBS (v/v) and saved at −20°C for >24 hours. After washing with PBS, the bacterial pellets had been resuspended in a hybridization buffer [0.9 M NaCl, 20 mM tris (pH 7.5), 0.01% SDS, and formamide, if required] (desk S1). The FISH probe was then added with a closing focus of 5 ng/μl and incubated in a single day at required temperature (desk S1) utilizing a ThermoMixer (Eppendorf, Hamburg, Germany). After hybridization, bacteria had been then washed two instances (15 min) with washing buffer [0.9 M NaCl, 20 mM tris (pH 7.5), and 0.01% SDS]. Bacteria had been then resuspended in tris buffer [20 mM tris and 25 mM NaCl (pH 7.5)] earlier than evaluation with fluorescence microscopy and circulate cytometry. FISH probe sequences which were beforehand reported (35–44) are listed in desk S1.

Confocal fluorescence microscopy

A bacterial suspension was added to an agarose layer [1.5% (w/v) in PBS, ~1-mm thick] and coated with a glass coverslip. Confocal fluorescence imaging was carried out on a TCS SP8 laser scanning confocal microscope (Leica, Solms, Germany). Samples had been excited with 488 nm for FAM (carboxyfluorescein), 555 nm for TAMRA, and 639 nm for Cy5 fluors, and the emission was detected utilizing the corresponding emission filters. Deconvolution of the photographs was carried out utilizing Huygens Essential Deconvolution software program (Scientific Volume Imaging B.V., Hilversum, The Netherlands) utilizing a theoretical level unfold operate.

Flow cytometry

Flow cytometry analyses of the FDAA-labeled microbiota samples had been carried out on a CytoFLex circulate cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA). FlowJo (V 10.zero.8r1) was used for information analyses. Labeled microbiota had been recognized with circulate cytometry plots of logFSC versus logSSC and then gated on fluorescence. For every pattern, 15,000 occasions had been collected for evaluation with particles and doublets excluded.

Sequencing evaluation

DNA from the microbiotas was extracted both utilizing a stool DNA Kit or bacterial DNA Kit (Omega Bio-tek, Norcross, GA, USA) in keeping with the producer’s protocol. The 16S rDNA sequencing and metagenomic sequencing had been carried out by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). For 16S rDNA sequencing, the V3-V4 hypervariable areas of the 16S rDNA had been amplified by polymerase chain response and subsequently paired-finish sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) in keeping with the normal protocols. The taxonomy of every 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) in opposition to the SILVA (SSU123) 16S rDNA database with a confidence threshold of 80%. For metagenomic sequencing, DNA was fragmented to a median dimension of about 400 bp utilizing Covaris M220 (Gene Company Limited, China) for paired-finish library development and subsequently paired-finish sequenced on an Illumina HiSeq4000 platform. BLASTP (Version 2.2.28+, http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for taxonomic annotations by aligning nonredundant gene catalogs in opposition to the built-in NR (non-redundant protein sequence) database with e-worth cutoff of 1 × 10−5.

Acknowledgments: Funding: We are grateful to the National Natural Science Foundation of China (grants 21807070, 21735004, 21775128, and 21705024) and the Innovative Research Team of High-Level Local Universities in Shanghai (SSMU-ZLCX20180701) for monetary help. Author contributions: L.L., W.W., and C.Y. designed the research. L.L., Q.W., J.S., Y.D., and J.G. carried out and analyzed experiments. Y.S. contributed intellectually to the evaluation and interpretation of the information. L.L., W.W., and C.Y. wrote the manuscript. Competing pursuits: The authors declare that they don’t have any competing pursuits. Data and supplies availability: The 16S rDNA and shotgun sequencing information of the cecal microbiotas have been deposited in the Sequence Read Archive with BioPattern accessions SAMN14694443 and SAMN14694444, respectively. In addition, the 16S rDNA sequencing information of the soil microbiota cultured in vitro has additionally been deposited in the Sequence Read Archive with BioPattern accession SAMN14734540. Additional information associated to this paper could also be requested from the corresponding authors.

Copyright © 2020 The Authors, some rights reserved; unique licensee American Association for the Advancement of Science. No declare to authentic U.S. Government Works. Distributed below a Creative Commons Attribution NonCommercial License four.zero (CC BY-NC).

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