Understanding PathoGenetix Delivers Bacterial Identification System to FDA

PathoGenetix has delivered an early commercial version of the RESOLUTION™ Microbial Genotyping System to the U.S. Food and Drug Administration (FDA). The FDA is leasing the RESOLUTION System as part of a three-year collaborative agreement to evaluate the rapid bacterial identification technology for use in FDA foodborne illness outbreak investigation and response.

The RESOLUTION System, based on PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as enriched food and clinical samples. The bacterial strain information provided by the RESOLUTION System is comparable to pulsed field gel electrophoresis (PFGE), the current gold standard for pathogen typing in foodborne illness outbreak investigation and response.

Identifying the pathogen strain that is causing a foodborne illness outbreak is a critical step in defining the extent of the outbreak, determining the food involved, finding the original source of the contamination and defining the scope of a product recall. The ability of the RESOLUTION System to derive useful pathogen strain and serotype information directly from a complex mixture, and to shorten the time for pathogen typing to just five hours, could allow for quicker decisions affecting public health.

Current microbial identification techniques such as PFGE and whole genome sequencing (WGS) require a cultured isolate as input, and advanced, time-consuming laboratory processes for preparation and processing of food samples. Analysis of the patterns created by PFGE, or the extensive data generated by WGS, can be complex and add significantly to the time required to identify the pathogen strain and serotype. Because the PathoGenetix system is culture independent, and fully automated from sample preparation to final report, it has the potential to greatly reduce the time, complexity and skill-level required to identify foodborne pathogens in hospital and public health labs monitoring foodborne outbreaks.

We need to know Why Is Lactate Dehydrogenase (LDH) Release A Good Measure For Cytotoxicity?

When treated with a cytotoxic compound, living cells may face one of two fates. They could either stop growing and dividing, or die through either of two distinct processes - necrosis or apoptosis. Basically, cells undergoing necrosis (accidental cell death) swell and lose membrane integrity before shutting down and releasing their intracellular contents into the surrounding environment. This type of cell death is usually triggered by external factors such as toxic chemical or traumatic physical events.

On the other hand, cells undergoing apoptosis (normal or programmed cell death) go through a series of well-defined events such as the shrinking of the cytoplasm, cleavage of DNA into smaller fragments, etc. before being engulfed by white blood cells.

When the cell membranes are compromised or damaged in any way, lactate dehydrogenase (LDH), a soluble yet stable enzyme found inside every living cell, is released into the surrounding extracellular space. Since this only happens when cell membrane integrity is compromised, the presence of this enzyme in the culture medium can be used as a cell death marker. The relative amounts of live and dead cells within the medium can then be quantitated by measuring the amount of released LDH using a colorimetric or fluorometric LDH cytotoxicity assay.

Other enzymes such as adenylate kinase and glucose-6-phosphate may also be used to measure cytotoxicity but these enzymes are not stable and lose their activity during cell death assays.

Understanding How to extract biologically active proteins from the cells

Extraction of proteins from cells and tissue of organisms is the first step towards isolation of proteins. The extracellular matrix needs to be removed or digested in case of tissue, the cell wall needs to be digested for organisms like bacteria, yeast and plants, and the cell membrane needs to be disrupted to release the proteins in solution. Traditionally, physical methods for disruption of cells and tissues are employed to release cellular proteins including sonication, french press, homogenization, manual grinding or using blenders.  Although one is able to get the active proteins, these physical methods have several limitations:

• Expensive
• Cumbersome due to use of heavy equipment
• Reduced yield as the sample is processed through several steps
• Batch to batch variation in protein yield due variability and handling

Mechanical methods can sometimes denature proteins as these methods are harsh and some of them generate heat and can denature protein if appropriate cooling of sample is not done.

Chemical methods for cell disruption using lysis buffers with ionic detergents can be used to release proteins; however they can denature the protein.  Non-ionic detergents offer advantage over lysis buffer and ionic detergents as they are mild and the proteins are not denatured. Therefore, nowadays popular methods for extraction of biologically active proteins from cells or tissues use non-ionic detergents along with other additives, such as enzymes for cell wall disruption and addition of protease inhibitors to inhibit proteases. This method may involve mild mechanical methods, including brief homogenizing or vortexing depending upon the cell or tissue type.

You know PathoGenetix Delivers Bacterial Identification System to MRIGlobal for Evaluation

PathoGenetix™, Inc., developer of an automated system for rapid bacterial strain typing, announced today that it has delivered and installed an early commercial version of the RESOLUTION™ Microbial Genotyping System to MRIGlobal, an independent contract research organization. MRIGlobal has purchased the RESOLUTION System as part of a U.S. government-funded project, and will be evaluating use of the System for identification and strain typing of specific organisms using MRI-developed assays.

The RESOLUTION System is based on PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, which enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as environmental, clinical and enriched food samples. Initially developed to detect bio-threat pathogens in environmental samples under a five-year, $50-million contract through the Department of Homeland Security, the breakthrough GSS technology isolates and analyzes DNA direct from complex mixtures—without the need for a pure culture isolate. The strain type information provided by GSS is comparable in resolution to pulsed field gel electrophoresis (PFGE), one of the current gold standards for pathogen identification.

MRIGlobal and PathoGenetix have collaborated for several years in the ongoing evaluation of the GSS technology for biodefense applications. MRIGlobal has purchased a RESOLUTION System as part of a U.S. government-funded project, and will be evaluating the system for use in the rapid identification of specific organisms, using MRIGlobal-developed assays. The RESOLUTION instrument will be used to build a database for identification of select microorganisms. As one of the nation’s leading research institutes, MRIGlobal conducts programs in the areas of national security and defense, life sciences, energy and the environment, agriculture and food safety, and engineering and infrastructure.

What are Protease Inhibitors and Cocktails?

Protease inhibitors and protease inhibitor cocktails can best be described as biological and/or chemical compounds that bind reversibly or irreversibly withany proteasespresent in the solution. Since proteases are naturally released following cell or tissue lysis, these compounds are added to the lysate to protect them from being degraded and preserve the nature of the proteins of interest for subsequent experimentations.

Types of Protease Inhibitors

Protease inhibitors are available individually or as concentrated cocktails. Individual protease inhibitors are best used in applications where the protein being purified is a protease and when performing proteolytic assays of already purified proteins. Some of the most common protease inhibitors include AEBSF, aprotinin, chymostatin and PMSF (for serine proteases), E-64 (for cysteine proteases), EDTA/EGTA and phosphoramidon (for metalloproteases), bestatin (for some aminopeptidases), and pepstatin (for aspartic proteases). Leupeptin can be used to inhibit the action of both serine and cysteine proteases while ALLN can be used to inhibit calpain I and II and other neutral cysteine proteases.

Protease inhibitor cocktails, on the other hand, contain multiple protease inhibitors in appropriate relative amounts so they can ideally be used for most protein work. By using cocktails, you don't have to determine which inhibitors to use and the amount needed to get the job done. You also reduce the risk of human and pipetting errors which may seriously compromise the results of your research.

A number of protease inhibitor cocktails can inhibit a wide range of proteases from cell and tissue samples of animal (both mammals and insects), plant, yeast and bacterial origin. Some broad spectrum protease inhibitor cocktails are also compatible with 2D gel electrophoresis and mass spectrometry. You can likewise get protease inhibitor sets which contain 12 ready-to-use inhibitors design your own protease inhibitor cocktails, supplement your existing cocktails and/or screen for specific protease classes.

How Are They Made Recombinant Proteins?

After choosing an appropriate vector and host that will satisfy the requirements of your experiment, you need to go through the following steps to create your recombinant protein.

Recombinant Proteins - Some Common Applications

As mentioned earlier, this technology has far reaching applications. Here are some of them.

• Medicine. Recombinant proteins are used to produce insulin, human growth hormone (HCH, somatotropin), blood clotting factor VIII (usually administered to patients with hemophilia) and hepatitis B vaccine. In addition, this technology is also responsible for developing the methods for diagnosing HIV infections.

• Biological research. Recombinant DNA can be used in mapping genes and as reagents in laboratory experiments.
   
• Agriculture. The technology was used to develop Golden rice, a variety of rice that aims to reduce vitamin A deficiency. It was also used to develop insect-resistant and herbicide-resistant crops.
   
• Food processing. Several food additives such as chymosin, an enzyme used in the manufacture of cheese, are now being produced using this technology.

Understanding What is the role of detergents in the study of membrane proteins?

Detergents are amphipathic molecules that can be used to extract, solubilize, and manipulate (disrupt or form) membrane proteins from biological membranes for subsequent biochemical and physical characterization. In addition, detergents can be used to control protein crystallization, prevent nonspecific binding in affinity purification and immunoassay procedures, and act as additives in electrophoresis.

Due to their unique structure, detergents can act as excellent solubilizing agents. They have a polar, hydrophilic (water-loving) head group which extends from a long hydrophobic (water-fearing) tail. In aqueous solutions, the hydrophilic heads interact with the hydrogen bonds of the water molecules while the hydrophobic tails aggregate to form highly organized spherical structures called micelles upon reaching a certain concentration (known as the Critical Micelle Concentration or CMC). In non-aqueous solutions, detergents form reverse micelles instead.   

The average size and shape of micelles is affected by the type, size, and stereochemistry of the surfactant and the solvent environment. And although the concentration of micelles increases as you add more detergent to the solution, the concentration of detergent monomers stays constant above the CMC.  

So, how do they solubilize protein membranes? As you may already know, biological membranes have the same amphipathic properties as detergents. They have a charged polar head connected to two hydrophobic tails and form a bilayer (the hydrophobic tails are sandwiched between two faces of polar head groups). Found between these layers are your proteins and lipids.  

You cannot release these proteins in aqueous solutions since they are tightly held in the lipid bilayer by the hydrophobic interactions between the lipid tails and hydrophobic protein domains. However, you can extract your target proteins by using the most appropriate detergent solution.  

What is RNA-Seq no longer required

Epigenetics refers to external modifications to DNA that do not change the DNA sequence but can control gene expression, the “on” and “off” status of genes. Epigenetic alterations can be influenced by several elements such as age and environment/lifestyle, and many aberrant modifications can lead to several diseases like cancer and neurodevelopmental disorders.

DNA methylation is a key mechanism of epigenetic regulation, where a methyl group is added to the cytosine (C) or adenine (A) nucleotides in the DNA molecule; in humans, the most common DNA methylation is in CpG dinucleotides. Histone modification is another key mechanism and corresponds to alterations (methylation or acetylation) in histones, the core element of nucleosomes where DNA sequences are wrapped around.

Many bioinformatics tools have been developed to assess the role of epigenetic regulation in gene expression, namely high throughput methods for methylation arrays, CHIP-Sequencing, gene expression microarray and RNA-Sequencing. Quantitative models based on epigenetic information are however needed to accurately predict the up or down regulation in gene expression.

In this study, a new machine learning-based model to predict gene expression as a consequence of epigenetic modification was developed in a lung cancer context. This model analyzed a large set of data on histone modification, CpG methylation, and genomic information, allowing the accurate prediction of differential RNA expression in lung cancers. The team used publicly available data from The Cancer Genome Atlas (TCGA) Project (Illumina Infinium HumanMethylation450K Beadchip CpG methylation array data from paired lung cancer and adjacent normal tissues) and the ENCODE project (histone modification marker CHIP-Seq data). A comprehensive list of 1,424 characteristics was analyzed, including nucleotide composition and conservation, histone H3 methylation modification and CpG methylation.

How does SMALL RNA SEQUENCING works?

Non-coding small RNAs (ncRNA, smallRNA) and microRNAs (miRNA) are key regulators of gene expression, heterochromatin formation and nucleus organisation. Their action is dependent on tissue and environment and, through various mechanisms, they have a major influence on cancer and numerous diseases.

Several platforms within the France Génomique network will accept requests for small RNA studies or microRNA studies (Pasteur, Strasbourg, Montpellier, Nice, Paris centre, CEA/IG-CNG).
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Starting with total RNA, the small RNAs are purified on acrylamide gel and used to prepare sequencing libraries. In this step, a sequence specific adapter is added to both ends of the RNA molecule, RNA is converted into double-stranded cDNA, and amplification is performed. The libraries are then quality controlled on a Bioanalyser (Agilent) and quantified on a QuBit (Invitrogen). The resulting sequences will be of native RNA (directional RNA-Seq).

Do you konw Control DNA for Epigenetic

Techniques used for methylation detection and analysis require high quality control DNA for comparison to sample DNA. In vitro methylated DNA using a CpG methyltransferase gives us the opportunity to specifically generate positive control DNA for methylation studies. Alternatively, mammalian DNA can also be used for methylation studies like bisulfite conversion, methyl sensitive PCR (MSP), methyl-DNA immunoprecipitation (Me-DIP) or by methyl-CpG binding domain (MBD)-based capture techniques.

What Is Epigenetics?

If all cells are created from the same genetic material, why are there so many different cell types? Listen to Sriharsa Pradhan, Research Division Head, RNA Biology at NEB, as he describes how DNA is methylated and how this affects the path of reading the DNA code the same way an obstruction would derail a train off its tracks.

How Mitochondrial Sequencing works

Mitochondrial DNA sequencing is a useful tool for researchers studying human diseases such as diabetes, certain cancers, and mechanisms of aging. Mitochondrial DNA sequencing is also used in population genetics and biodiversity assessments. Targeted mitochondrial DNA sequencing can be used to detect mutations present in some copies of the mitochondrial genome (heteroplasmic mutations). Finally, mitochondrial DNA sequencing is important for human identification and forensics applications.

Rapid resequencing of mitochondrial genomes for disease research and biodiversity assessment is accomplished using Ion Torren next-generation sequencing.

Mitochodrial sequencing using Ion Torrent™ next-generation sequencing enables:

• Capacity of 16 samples per run with barcoding
• Accurate variant calling, especially in hypervariable regions of mitochondria

Highly targeted mitochondrial DNA sequencing is performed by gold-standard Sanger sequencing, using Applied Biosystems genetic analyzers. The simple workflow can be performed in less than 5 hours using the Applied Biosystems 3500 Genetic Analyzer.

You now SNP Genotyping by Fragment Analysis?

SNP genotyping identifies single nucleotide polymorphisms (SNPs) that are common DNA variants present across the human genome. SNPs have been shown to be responsible for differences in genetic traits, susceptibility to disease, and response to drug therapies.

Genotyping of SNPs has become extremely important to researchers working to understand and treat disease. SNPs occur approximately once every 100 to 300 bases and can be detected by various different techniques such as RFLP, SSCP, sequencing, and more.

Go to the Publications & Literature tab below to learn some of the many ways our customers use the SNaPshot®  Multiplex System for their research.

Amazing SNP Genotyping & Variant Detection by Sequencing

Variant Detection by Sanger Sequencing
Sanger sequencing is the gold-standard sequencing technology, making it ideal for confirmation of novel variants. Designing sequencing experiments for variant detection is easier than ever with our new Primer Designer™ tool for Sanger sequencing (currently available in US & Canada), containing >300,000 predesigned primer pairs for PCR and sequencing.

Variant Detection by Ion Torrent™ Next-Generation Sequencing
With industry-leading performance and gold-standard accuracy, Sanger sequencing with Applied Biosystems® genetic analyzers serve as your trusted partner for confirming NGS results.

What is Ion semiconductor sequencing

Now, more laboratories can adopt powerful next-generation sequencing technology to get answers faster —with increased throughput, higher accuracy, and the simplest workflow from sample preparation through to data analysis. With the Ion Torrent™ Personal Genome Machine® (PGM) System, human disease researchers can detect variants by targeted gene sequencing in cancer and genetic disorders, and microbiologists can easily type a bacteria or virus, or characterize novel microbes.

The combination of Ion AmpliSeq™ target technology, an Ion PGM™ System, and fully automated template preparation from the upcoming Ion Chef™ System**, offers the simplest and fastest* workflow for gene panel sequencing. Get meaningful results in just half the time of the alternative, with highly multiplexed Ion AmpliSeq™ DNA panels, the walk away solution for preparing sequence-ready chips from libraries provided by the Ion Chef™ System, and faster sequencing on the Ion PGM™ System. From sample to results, there are no centrifugation steps, and half the pipetting steps and hands on time of the alternative NGS

Amazing RNAi

RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms. At Thermo Fisher Scientific, we have developed two types of small RNA molecules that function in RNAi: short interfering RNA (siRNA) molecules and microRNAs (miRNA). Thermo Fisher Scientific offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications. Your choice of tool depends on your model system, the length of time you require knockdown, and other experimental parameters.

Effectively knock down gene expression using our proprietary Silencer® Select, Stealth RNAi™, and Silencer siRNAs, Analyze miRNA function with mirVana™ miRNA mimics and inhibitors
Find a complete solution for successful in vivo RNAi experiments including the highest nuclease stability Ambion

Five Simple Tips to Future Proof Your Loan

In the event that you are in the marketplace of mortgage, we’ve got some handy tips for you to make sure your preparation is as beneficial and stable as possible.

Tip#1. Assess the mortgage loan and the current
Tip#2. Refinance mortgage loan to make sure it suits your requirements
Tip#3. Try some management methods and techniques
Tip#4. Check and control against the unexpected financial shortfalls
Tip#5. Prepare for known and unknown factors

Tips To Not Hiring Bad Bankruptcy Attorneys

If you are facing the situation of hiring bankruptcy attorney and you don’t know how to have the right attorney for your particular case, then here are 5 tips to avoid hiring bad attorney.

– Tip #1: Do not wait – just take action!
– Tip #2: Ask around – people around you!
– Tip #3: Go to the court!
– Tip #4: Schedule the consultation!
– Tip #5: Don’t confuse quality with price!

Tips To Hire Small Business Attorneys

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It’s important for a business owner to have attorney for representing the interests when the requirements arise. When you are facing the potential law agencies, you will need some tips to support you to find a proper attorney for your small business case.

TIP#1 – SPECIALIZATION.
TIP#2 – FEE STRUCTURE.
TIP#3 – ADVISORY TEAM.
TIP#4 – ACCESSIBILITY.
TIP#5 – REFERRALS.

Do more searches on the internet or ask your local legal agencies for further details about this if you are interested in the term business attorney.

How many type of Business Liability Insurance

You may be pondering what kind of business liability insurance to purchase. The way of your business and also the sort of administrations you give will help focus your needs. Take in more about the most well-known sorts underneath:

General business liability insurance: Covers guarantees because of wounds, mischances and carelessness. A general liability arrangement can shield you against expenses that outcome from real harm, property harm, therapeutic costs, legitimate expenses, judgments, and individual damage cases, for example, criticism and defamation. On the off chance that you lease or lease your space, business general liability, or CGL insurance, might likewise give scope to harm to that working environment. Contingent on your circumstance, you may need premises liability insurance.

Professinoalliability insurance: Also known as lapses and exclusions insurance, "E and O" or negligence, this scope is accessible for business proprietors who need insurance against a scope of cases including mistakes and carelessness. Certain callings, for example, doctors and chiropractors, are obliged to buy negligence insurance with a specific end goal to practice in their state. In the event that your state does not oblige this scope, it may command that a few experts, including legal counselors, unveil their absence of expert liability insurance.


Product liability insurance: This scope is fundamental for companies that make, circulate, or offer an item since they're regularly obligated for its wellbeing. This kind of insurance aides keep a monetary misfortune if the item is deficient and causes damage or mischief to the purchaser. The measure of item liability scope you require differs by the seriousness of your organization's dangers.

There are numerous different sorts of liability insurance too, and a few callings need particular sorts of liability assurance. For instance, corporate officers frequently buy "executives and officers" liability insurance to ensure the organization if a ranking staff part makes a move or creates an impression that outcomes in a liability claim.