Notes

Protean function of epigenetic systems as well as their affect within controlling the Tregs in TME.
26, 0.95-1.68, p-het = 0.039) and CIMP-high (OR = 2.01, 1.40-2.88) compared to CIMP-low/negative CRC (OR = 1.50, 1.22-1.85, p-het=0.101). Current smoking seemed more strongly associated with sessile serrated pathway (CIMP-high + BRAF-mut; OR = 2.39, 1.27-4.52) than with traditional pathway CRC (MSS + CIMP-low/negative + BRAF-wt; OR = 1.50, 1.16-1.94) and no association was observed with alternate pathway CRC (MSS + CIMP-low/negative + KRAS-wt; OR = 1.08, 0.77-1.43). No heterogeneity was observed in alcohol consumption association by molecular subtypes. CONCLUSIONS In this large case-control study, smoking was more strongly associated with MSI-high and KRAS-wt CRC and with cases showing features of the sessile serrated pathway. Association patterns were less clear for alcohol consumption.PURPOSE RNA-seq is a promising approach to improve diagnoses by detecting pathogenic aberrations in RNA splicing that are missed by DNA sequencing. RNA-seq is typically performed on clinically accessible tissues (CATs) from blood and skin. RNA tissue specificity makes it difficult to identify aberrations in relevant but nonaccessible tissues (non-CATs). We determined how RNA-seq from CATs represent splicing in and across genes and non-CATs. METHODS We quantified RNA splicing in 801 RNA-seq samples from 56 different adult and fetal tissues from Genotype-Tissue Expression Project (GTEx) and ArrayExpress. We identified genes and splicing events in each non-CAT and determined when RNA-seq in each CAT would inadequately represent them. We developed an online resource, MAJIQ-CAT, for exploring our analysis for specific genes and tissues. RESULTS In non-CATs, 40.2% of genes have splicing that is inadequately represented by at least one CAT; 6.3% of genes have splicing inadequately represented by all CATs. A majority (52.1%) of inadequately represented genes are lowly expressed in CATs (transcripts per million (TPM) 10). CONCLUSION Many splicing events in non-CATs are inadequately evaluated using RNA-seq from CATs. MAJIQ-CAT allows users to explore which accessible tissues, if any, best represent splicing in genes and tissues of interest.Covalent loading or directional binding of biomolecules on gold nanoparticles (AuNPs) could lead to better results than simple direct adsorption for an enhanced ELISA application. The use of Mini-Parasep solvent-free (SF) without ether or ethyl acetate for the clean and efficient concentration of protozoa cysts, it is a single-use device for in vitro diagnostic use only. In this work, we used Mini-Parasep SF for the detection of giardia cysts in comparison to direct smear and Merthiolate-Iodine Formaldehyde Concentration (MIFC) technique in addition to its use in antigen detection by AuNPs biomolecule loading using rabbit polyclonal antibodies (pAb) against purified Giardia antigen (PGA). As a result, Mini-Parasep SF was the most effective method for Giardia cyst detection and regarding optimization of Mini-Parasep antigen detection, our data showed increased sensitivity and specificity of nano-sandwich ELISA to 92% and 94% respectively and increased positive predictive value (PPV) and negative predictive value (NPV) to 88.64% and 95.91% respectively. In conclusion, this research provides that Mini-Parasep SF concentrator enhanced Giardia cyst detection and improved antigen preparation for AuNPs sandwich ELISA in giardiasis diagnosis. The advantages of this method are the short assay time and the raised accuracy of antigen detection providing concentrated samples without the risk of solvent use and being a disposable Mini-Parasep it helps in giardia antigen purification as well as raising the sensitivity and specificity of ELISA through binding AuNPs.Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Selleckchem U0126 Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.Crohn's disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn's disease. A detailed protocol for the induction of Crohn's disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.
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