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By Jennifer Sokolowski, MD, PhD.
Human induced pluripotent stem cells (iPSCs) can be used to create models of human organ systems and are useful for a) ascertaining the mechanisms underlying pathological conditions and b) developing and testing therapeutics. For example, studies have used iPSCs from human patients with diseases like Alzheimer's and Parkinson's to test the influence of pathological isoforms of proteins as well as the efficacy of genetic rescue.1,2
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By Jamshed Arslan, Pharm. D., PhD.
Colorectal cancer stem cells are a rare subpopulation of colorectal cancer cells that can self-renew and initiate and sustain tumor growth when transplanted into an animal host.1,2 Colorectal cancer stem cells are identified by CD133, CD166, CD44, nuclear-beta-catenin, ALDH1, EphB2 and...
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By Victoria Osinski
Inflammation results from stimuli signaling damage or infection. The immune system inflammatory response can be beneficial or harmful depending on the type and duration of stimuli. The source, structure, and abundance of these stimuli vary quite a bit. One major category of inflammatory stimulation, or "signal 0s" is the family of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).1,2 These patterns are found on bacterial cell walls, DNA, lipoproteins, carbohydrates, or other structures. While many DAMPs and PAMPs have been identified, they stimulate...
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By Christina Towers, PhD
Is p62 a good indicator of autophagic flux? The short answer: Yes … but … SQSTM1 encodes the cargo adaptor protein, p62, which interacts with autophagic substrates and delivers them to autophagosomes for degradation. In the process, p62 is itself degraded and when autophagy is induced, a corresponding decrease in p62 levels is observed. Congruently, when autophagy is potently inhibited, i.e. genetic knock out of ATG core autophagy proteins like ATG5 or ATG7, p62 accumulates in the cell. These changes can be observed by immunofluorescent staining, western blotting, and immunohistochemistry...
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By Jamshed Arslan, Pharm. D., PhD.
Cancers in the brain often come from tumors elsewhere in the body. Several adaptive mechanisms influence brain metastasis, such as blood brain barrier leakage that can be induced by stroma or disseminated tumor cells. The brain tumor microenvironment is complex, but the metastasizing cancer cells share common molecular features. To define such molecular characteristics of brain metastasis in vivo, a team led by Dr. Don Nguyen of Yale University School of Medicine developed an advanced RNA sequence-based approach....
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By Michalina Hanzel, PhD
In this final instalment of our three blog-posts series on major cellular mechanisms responsible for neurodegenerative disorders, we will explore the processes of neuroinflammation and microglial activation. Previously, the role of autophagy in the clearance of aggregation-prone proteins in the context of neurodegenerative diseases was discussed in...
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By Christina Towers, PhD
Autophagic flux is defined as the amount of cellular material degraded and recycled through the process of autophagy, whereby cells break down and discard waste. Briefly, the autophagy process involves the formation of an autophagosome, which engulfs cytoplasmic materials. Fusion of the autophagosome with acidic lysosomes mediates the degradation of cellular contents. The process involves over 30 core autophagy proteins (...
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By Beth Melson, MS
Antibiotic resistance is a global threat to public health. Widespread, inappropriate use of antibiotics, such as to treat viral infections or promote growth in livestock, has led to increased incidence of antibiotic resistance1. Resistance is costly to both human health as well as the economy1, and thus the identification of new, effective antibiotics is a public health priority. One solution to this problem is to repurpose commercially available, FDA-approved drugs to target host cell processes that would...
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By Michalina Hanzel, PhD
In this second installment of our three blog-posts series on major cellular mechanisms responsible for neurodegenerative disorders, we will explore the processes of mitochondrial dysfunction and oxidative stress. Previously, the role of autophagy in the clearance of aggregation-prone proteins in the context of neurodegenerative diseases was discussed in Mechanisms of Neurodegeneration:...
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By Jamshed Arslan, Pharm. D., PhD.
Squamous cell carcinoma is the most common cancer in the oral cavity.1 The tumor surface biofilms in oral cancers contain high levels of aerobic and anaerobic microorganisms.1,2 Periodontal pathogens can activate epithelial Toll-like receptors (TLRs) on tumor cells to promote oral squamous cell carcinoma (OSCC) progression. In this regard, TLR2 is known to recognize micro-organisms in the oral cavity by forming a heterodimer with TLR1 or TLR6...
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Kristy R. Howell, PhD
The cellular recycling process known as autophagy may be induced by a variety of conditions including reduced nutrient availability, serum starvation and pharmacological agents (e.g., Rapamycin (mTOR)). Upon autophagy induction, a process of increased sequestration of dysfunctional cellular components into autophagosomes ensues. The autophagosome fuses...
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By Michalina Hanzel, PhD
In a series of three blog posts I will briefly explore the major cellular mechanisms responsible for many neurodegenerative disorders. The first, and perhaps the most apparent, is the accumulation of misfolded, aggregate-prone proteins, as a result of a failure of the autophagy system, which leads to the loss of neuronal structure and function and eventual neuronal loss.
Autophagy is a conserved cellular process used to eliminate obsolete proteins and cell components by degradation and...
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By Jamshed Arslan, Pharm. D., PhD.
Colon cancer is responsible for over 600,000 deaths per year worldwide. Colon cancer can be classified into two categories: mismatch repair (MMR)-deficient and MMR-proficient cancers.1 MMR-deficient colon cancer shows high CD8+ cytotoxic T-lymphocyte (CTL) and low Th17 cells, while MMR-proficient colon cancer shows an opposite trend. MMR-proficient colon cancer is associated with poor prognosis and is resistant to anti-cancerous immune checkpoint inhibitors. In other words, outcome of immune therapy is...
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By Christina Towers, PhD
Autophagy is a recycling process that relies on the formation of a unique organelle termed an autophagosome. An elegant way to monitor autophagy is through various microscopy techniques to visualize and quantify the formation and turnover of these double membrane structures. Some techniques are more quantitative than others, each accompanied by advantages as well as caveats.
This technique is the only tool that can reveal all of the dynamic stages of a...
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By Victoria Osinski
Identifying cellular subsets by flow cytometry requires careful and thorough planning in order to ensure the correct subset of cells are identified and isolated. While generally thought of as antibody-secreting cells, an expanding body of research proves that B cells are a heterogeneous population. Flow cytometry can be used to identify many of these B cell subsets, so long as important nuances and technical approaches are considered.
Before subsetting B cells, steps must be...
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By Jamshed Arslan, Pharm. D., PhD.
Atherosclerosis is a chronic inflammatory condition in which plaques of fats and other substances slowly buildup on the inner walls of arteries to restrict blood flow. In atherosclerosis-associated ailments like obesity, hypertension and type II diabetes, intercellular tight junctions are impaired, leading to increased gut permeability. This means that products of intestinal microbiota like lipopolysaccharide from Gram-negative bacteria can seep into the blood stream.
A pro-atherogenic role of lipopolysaccharide has been suggested. For example,...
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By Jamshed Arslan, Pharm. D., PhD.
Toll-like receptors (TLRs) are microbe-sensing proteins that act as first responders to danger signals. TLRs help the intestinal epithelial cells (IECs) recognize commensal bacteria from the harmful/foreign ones. Polymorphisms and variants of TLRs have been linked with immune system’s attack on commensal bacteria, leading to inflammatory bowel disease and colorectal cancers. The endogenous pattern of TLR expression in IECs and the functional outcome of TLR signaling in vivo are not well defined due to various technical difficulties. Thanks to the researchers in institutes in California and...
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By Jamshed Arslan, Pharm. D., PhD.
Gut microbiome modulates various disorders even outside the gut. For example, the microbiome is linked to amyloid-β deposition and the consequent inflammation in Alzheimer’s dementia. Altering gut microbiome through probiotics and/or exercise has been hypothesized to improve memory in Alzheimer’s disease (AD). A multi-continental collaboration of scientists tested this hypothesis on AD mice and found that probiotics and exercise can retard AD progression, at least partly by changing the microbiome.
The researchers used Framelim, a product...
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By Jennifer Sokolowski, MD, PhD.
Microglia play a critical role in pruning neurons and synapses during homeostatic maintenance in the adult brain.1 A recent study by Ayata et al. (2018) identified regional differences in clearance and found a role for epigenetic control via polycomb repressive complex 2 (PRC2) in adult rodents.2
Ayata et al. defined a cell clearance phenotype by labeling microglia with iba1 and CD68 and evaluating morphology, cell volume, and CD68+ lysosome content. They also...
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By Christina Towers, PhD
Autophagy is a catabolic process used to breakdown and recycle damaged proteins and organelles. It is a multistep process that, in its simplest form, consists of 4 steps: initiation, phagophore nucleation, elongation, and fusion – resulting in a double membrane structure called an autophagosome that, upon fusion with the acidic lysosome, facilitates the degradation of its contents. LC3B (and its family members) are incorporated into the elongating autophagophore and are necessary for efficient...
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By Christina Towers, PhD
Autophagy is a stress-induced cellular recycling process that plays an important physiological role in many diseases. It is induced by a variety of stimuli, both intracellular and extracellular, and is a tightly regulated process with over 30 core autophagy proteins necessary for efficient autophagosome formation and fusion with lysosomes to facilitate degradation1. One of the most common ways to...
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By Jamshed Arslan, Pharm. D., PhD.
Western blotting involves three steps: 1) separating proteins by size through gel electrophoresis 2) transferring separated proteins to a solid support (typically, a PVDF membrane) and 3) using antibody to mark your protein(s) of interest in the mixture of proteins. The second step of moving negatively charged proteins from the gel to a blotting membrane (PVDF) can be performed in dry or wet conditions.
Dry conditions are famous for easiness and speed. Reagents, power packs and wiring involved in dry transfers are less messy and therefore, potentially less hazardous. They allow blotting of several...
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By Christina Towers, PhD
Autophagy is the catabolic process that degrades cytoplasmic material via the lysosome. The process of macroautophagy was originally characterized in yeast, where the core autophagy-related proteins (ATGs) were identified. In this system, the multistep process of phagophore initiation and elongation as well as the conjugation machinery that mediate efficient autophagosome completion were determined and cloned to assess function. The mechanisms of...
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By Jamshed Arslan Pharm.D., PhD.
Kinome describes kinases, and kinomics refers to the kinase signaling. Studying the effects of reagent (exogenously applied growth factor or inhibitor) on kinase activity is a common experiment in cell, developmental and integrative biology laboratories. This blog offers a cost-effective and robust method of obtaining adequate amount of sample for kinomic analysis from mammalian culture cells like HEK cells after treatment with reagent. Using this method, you can easily get over 50 μg protein sample in 50 μl volume from a confluent well of a standard 6-well plate with 0.5 x 106...
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By Christina Towers, PhD
Autophagy facilitates the degradation and recycling of damaged cytoplasmic material. The multistep process includes a double membrane structure called an autophagosome that engulfs proteins and organelles and then fuses with lysosomes to facilitate their breakdown. Autophagy is tightly regulated by nutrient availability and is negatively regulated by the robust nutrient sensor mammalian target of rapamycin (mTOR). Starvation, either by glucose deprivation or amino acid depletion, causes a marked increase in autophagy, best quantified by an increase in...
