RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of ...gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.
Highly Multiplexed Subcellular RNA Sequencing in Situ Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan ...
Science (American Association for the Advancement of Science),
03/2014, Letnik:
343, Številka:
6177
Journal Article
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Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe ...fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.
Prior data have suggested that suboptimal antibiotic prescribing in the emergency department (ED) is common for uncomplicated lower respiratory tract infections (LRTI), urinary tract infections ...(UTI), and acute bacterial skin and skin structure infections (ABSSSI). The objective of this study was to measure the effect of indication-based antibiotic order sentences (AOS) on optimal antibiotic prescribing in the ED.
This was an IRB-approved quasi-experiment of adults prescribed antibiotics in EDs for uncomplicated LRTI, UTI, or ABSSSI from January to June 2019 (pre-implementation) and September to December 2021 (post-implementation). AOS implementation occurred in July 2021. AOS are lean process, electronic discharge prescriptions retrievable by name or indication within the discharge order field. The primary outcome was optimal prescribing, defined as correct antibiotic selection, dose, and duration per local and national guidelines. Descriptive and bivariate statistics were performed; multivariable logistic regression was used to determine variables associated with optimal prescribing.
A total of 294 patients were included: 147 pre-group and 147 post-group. Overall optimal prescribing improved from 12 (8%) to 34 (23%) (P < 0.001). Individual components of optimal prescribing were optimal selection at 90 (61%) vs 117 (80%) (P < 0.001), optimal dose at 99 (67%) vs 115 (78%) (P = 0.036), and optimal duration at 38 (26%) vs 50 (34%) (P = 0.13) for pre- and post-group, respectively. AOS was independently associated with optimal prescribing after multivariable logistic regression analysis (adjOR, 3.6; 95%CI,1.7–7.2). A post-hoc analysis showed low uptake of AOS by ED prescribers.
AOS are an efficient and promising strategy to enhance antimicrobial stewardship in the ED.
Here, we present a generalized method of guide RNA “tuning” that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ ourmethodology to ...generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.
Methotrexate (MTX) is an immunomodulator used in pediatric inflammatory bowel disease (IBD) maintenance regimens. However, MTX use is associated with liver toxicity. We aimed to systematically review ...and meta-analyze the incidence of hepatotoxicity with MTX use among children with IBD.
We searched MEDLINE, EMBASE, Web of Science, and Cochrane Central Register of Controlled Trials databases from 1946 to April 2013 for cohort studies and collected information about the study design, IBD treatment results, and hepatotoxicity. Pooled proportions of toxicity with 95% confidence interval (CI) were estimated using a random-effects model.
Twelve high-quality studies were included in this review. Fifty-seven of 457 patients treated with MTX developed varied degrees of abnormal liver biochemistry. The pooled proportion of patients with abnormal liver biochemistry was 10.2% (95% CI 5.4%-18.5%) across all studies included in the meta-analysis. Due to hepatotoxicity, dose reductions were required in 6.4% (95% CI 4.3%-9.5%), whereas 4.5% (95% CI 2.8%-7.2%) of patients required discontinuation.
Hepatotoxicity after the use of MTX among IBD patients was a relatively common event. Monitoring for hepatotoxicity is strongly recommended, as discontinuation of MTX may be necessary in a significant proportion of children.
Background
Magnetic resonance enterography (MRE) is increasingly relied upon for noninvasive assessment of intestinal inflammation in Crohn disease. However very few studies have examined the ...diagnostic accuracy of individual MRE signs in children.
Objective
We have created an MR-based multi-item measure of intestinal inflammation in children with Crohn disease — the Pediatric Inflammatory Crohn’s MRE Index (PICMI). To inform item selection for this instrument, we explored the inter-rater agreement and diagnostic accuracy of individual MRE signs of inflammation in pediatric Crohn disease and compared our findings with the reference standards of the weighted Pediatric Crohn’s Disease Activity Index (wPCDAI) and C-reactive protein (CRP).
Materials and methods
In this cross-sectional single-center study, MRE studies in 48 children with diagnosed Crohn disease (66% male, median age 15.5 years) were reviewed by two independent radiologists for the presence of 15 MRE signs of inflammation. Using kappa statistics we explored inter-rater agreement for each MRE sign across 10 anatomical segments of the gastrointestinal tract. We correlated MRE signs with the reference standards using correlation coefficients. Radiologists measured the length of inflamed bowel in each segment of the gastrointestinal tract. In each segment, MRE signs were scored as either binary (0-absent, 1-present), or ordinal (0-absent, 1-mild, 2-marked). These segmental scores were weighted by the length of involved bowel and were summed to produce a weighted score per patient for each MRE sign. Using a combination of wPCDAI≥12.5 and CRP≥5 to define active inflammation, we calculated area under the receiver operating characteristic curve (AUC) for each weighted MRE sign.
Results
Bowel wall enhancement, wall T2 hyperintensity, wall thickening and wall diffusion-weighted imaging (DWI) hyperintensity were most commonly identified. Inter-rater agreement was best for decreased motility and wall DWI hyperintensity (kappa≥0.64). Correlation between MRE signs and wPCDAI was higher than with CRP. AUC was highest (≥0.75) for ulcers, wall enhancement, wall thickening, wall T2 hyperintensity and wall DWI hyperintensity.
Conclusion
Some MRE signs had good inter-rater agreement and AUC for detection of inflammation in children with Crohn disease.
Spatial Sequencing: A Perspective Turczyk, Brian M; Busby, Michele; Martin, Allison L ...
Journal of biomolecular techniques,
07/2020, Letnik:
31, Številka:
2
Journal Article
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Fluorescent spatial sequencing brings next-generation sequencing into a new realm capable of identifying nucleic acids in the cell's natural environment. For the first time, scientists are able to ...multiplex the assignment of specific locations to hundreds of transcriptional targets and lay the foundation for understanding how genetic changes control the fate of each cell within the tissue microenvironment. In this perspective, we discuss the capabilities of fluorescent spatial sequencing in the context of other spatial imaging technologies and describe how these new technologies offer a data-rich, multiomic solution to many research applications. Fluorescent spatial sequencing has opened options for exploring many fundamental questions in biology, helping us gain a better understanding of cell and tissue development and disease progression.
Background In the war against the Taliban, Canada was the lead North Atlantic Treaty Organization (NATO) nation to provide medical and surgical care to NATO soldiers, Afghanistan National Army ...soldiers, Afghanistan Nation Police, civilians working in and outside Kandahar Airfield and Afghanistan civilians at the Role 3 Multinational Medical Unit (R3MMU) from February 2006 to October 2009. Methods We obtained data from the Joint Theatre Trauma Registry between May 1 and Oct. 15, 2009; 188 patients were admitted to the R3MMU intensive care unit (ICU). We analyzed the ICU data according to types and causes of trauma, mechanical ventilation prevalence, ICU medical and surgical complications, blood products utilization, length of stay in the ICU and mortality. Results The admitting services were general surgery (35%), neurosurgery (29%), orthopedic surgery (18%) and internal medicine (3%). Improvised explosive devices (46%) and gunshot wounds (26%) were the main causes of ICU admissions. The mean injury severity score for all patients admitted to the ICU was 37, and 81% of ICU patients required mechanical ventilation for a mean duration of 3 days. The main ICU complications were coagulopathy (6.4%), aspiration pneumonia (4.3%), pneumothorax (3.7%) and wound infection (2.7%). The following blood products were most used: packed red blood cells (55%), fresh frozen plasma (54%), platelets (29%) and cryoprecipitate (23%). The average length of stay in the ICU was 4.3 days, and the survival rate was 93%. Conclusion The high survival rate suggests that ICU care is a necessary and vital resource for a trauma hospital in a war zone.
There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage ...fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.
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