SPLASH: A statistical, reference-free genomic algorithm unifies biological discovery.

Today's genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), which directly analyzes raw sequencing data, using a statistical test to detect a signature of regulation: sample-specific sequence variation. SPLASH detects many types of variation and can be efficiently run at scale. We show that SPLASH identifies complex mutation patterns in SARS-CoV-2, discovers regulated RNA isoforms at the single-cell level, detects the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a unifying approach to genomic analysis that enables expansive discovery without metadata or references.

DIVE: a reference-free statistical approach to diversity-generating and mobile genetic element discovery.

Diversity-generating and mobile genetic elements are key to microbial and viral evolution and can result in evolutionary leaps. State-of-the-art algorithms to detect these elements have limitations. Here, we introduce DIVE, a new reference-free approach to overcome these limitations using information contained in sequencing reads alone. We show that DIVE has improved detection power compared to existing reference-based methods using simulations and real data. We use DIVE to rediscover and characterize the activity of known and novel elements and generate new biological hypotheses about the mobilome. Building on DIVE, we develop a reference-free framework capable of de novo discovery of mobile genetic elements.

Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

[WITHDRAWN] SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery.

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2022/497555. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2022.06.24.497555.

Cause-specific mortality among patients with cirrhosis in a population-based cohort study in Ontario (2000-2017).

BACKGROUND: Although patients with cirrhosis are at increased risk of death, the exact causes of death have not been reported in the contemporary era. This study aimed to describe cause-specific mortality in patients with cirrhosis in the general population. METHODS: Retrospective cohort study using administrative health care data from Ontario, Canada. Adult patients with cirrhosis from 2000-2017 were identified. Cirrhosis etiologies were defined as HCV, HBV, alcohol-associated liver disease (ALD), NAFLD, or autoimmune liver disease/other with validated algorithms. Patients were followed until death, liver transplant, or end of study. Primary outcome was the cause of death as liver-related, cardiovascular disease, non-hepatic malignancy, and external causes (accident/self-harm/suicide/homicide). Nonparametric analyses were used to describe the cumulative incidence of cause-specific death by cirrhosis etiology, sex, and compensation status. RESULTS: Overall, 202,022 patients with cirrhosis were identified (60% male, median age 56 y (IQR 46-67), 52% NAFLD, 26% alcohol-associated liver disease, 11% HCV). After a median follow-up of 5 years (IQR 2-12), 81,428 patients died, and 3024 (2%) received liver transplant . Patients with compensated cirrhosis mostly died from non-hepatic malignancies and cardiovascular disease (30% and 27%, respectively, in NAFLD). The 10-year cumulative incidence of liver-related deaths was the highest among those with viral hepatitis (11%-18%) and alcohol-associated liver disease (25%), those with decompensation (37%) and/or HCC (50%-53%). Liver transplant occurred at low rates (< 5%), and in men more than women. CONCLUSIONS: Cardiovascular disease and cancer-related mortality exceed liver-related mortality in patients with compensated cirrhosis.

A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain.

Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain.

SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery.

Today's genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a new unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), an approach that directly analyzes raw sequencing data to detect a signature of regulation: sample-specific sequence variation. The approach, which includes a new statistical test, is computationally efficient and can be run at scale. SPLASH unifies detection of myriad forms of sequence variation. We demonstrate that SPLASH identifies complex mutation patterns in SARS-CoV-2 strains, discovers regulated RNA isoforms at the single cell level, documents the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a new unifying approach to genomic analysis that enables an expansive scope of discovery without metadata or references.

Macrophage depletion blocks congenital SARM1-dependent neuropathy.

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Features of fibrosis regression abound in "non-cirrhotic" patients with resected hepatocellular carcinoma.

Cirrhosis is a major risk factor for developing hepatocellular carcinoma (HCC). However, many surgically resected HCCs are presumably non-cirrhotic. The dynamic nature of chronic liver disease leads to periods of hepatic repair and fibrosis regression. We hypothesize that most resected HCCs, including those from non-cirrhotic patients, exhibit features of fibrosis regression in their background liver, suggesting previously more advanced liver disease. We reviewed the histology of 37 HCC resections performed between 2005-2020, including 30 from non-cirrhotic patients. The non-neoplastic liver was evaluated for features of liver disease and of the hepatic repair complex (HRC). CD34 immunohistochemistry was performed as a marker of sinusoidal capillarization. CD34 staining was evaluated manually and also by a digital image classifier algorithm. Overall, 28 cases (76%) had a high number of fibrosis regression and hepatic repair features (≥4 out of 8 features). Amongst the 30 non-cirrhotic patients, 21 (70%) showed a high number of repair features. Relative CD34 expression was increased in cases with a high number (≥4) of HRC features versus a low number (≤3) of features (p = 0.019). High HRC cases were more likely to exhibit nodular circumferential CD34 staining (p = 0.019). Our findings suggest that most resected HCC from non-cirrhotic patients display features of fibrosis regression in their background liver. Thus many, if not most, HCC patients who are "non-cirrhotic" may in fact have regressed cirrhosis. This finding reinforces that patients with regressed cirrhosis continue to be at high risk for HCC.

Disentangling glial diversity in peripheral nerves at single-nuclei resolution.

The peripheral nerve contains diverse cell types that support its proper function and maintenance. In this study, we analyzed multiple peripheral nerves using single-nuclei RNA sequencing, which allowed us to circumvent difficulties encountered in analyzing cells with complex morphologies via conventional single-cell methods. The resultant mouse peripheral nerve cell atlas highlights a diversity of cell types, including multiple subtypes of Schwann cells (SCs), immune cells and stromal cells. We identified a distinct myelinating SC subtype that expresses Cldn14, Adamtsl1 and Pmp2 and preferentially ensheathes motor axons. The number of these motor-associated Pmp2+ SCs is reduced in both an amyotrophic lateral sclerosis (ALS) SOD1G93A mouse model and human ALS nerve samples. Our findings reveal the diversity of SCs and other cell types in peripheral nerve and serve as a reference for future studies of nerve biology and disease.

Recovery From Dialysis-Treated Acute Kidney Injury in Patients With Cirrhosis: A Population-Based Study.

RATIONALE & OBJECTIVE: The decision to initiate kidney replacement therapy (KRT) for acute kidney injury (AKI) in cirrhosis remains controversial because it is unclear which patients will benefit. We sought to characterize factors associated with recovery from KRT-treated AKI in patients with cirrhosis to inform shared clinical decision-making. STUDY DESIGN: Population-based retrospective cohort study. SETTING & PARTICIPANTS: Adult patients from Ontario, Canada, identified using administrative data to have cirrhosis at the time of hospital admission with AKI (based on serum creatinine level) who were treated with KRT (January 1, 2009, to December 31, 2016) and followed up until the end of 2017. EXPOSURES: Demographic characteristics and comorbidities before admission. OUTCOMES: Kidney recovery defined as the absence of KRT for at least 30 days. ANALYTICAL APPROACH: The cumulative incidences of kidney recovery, death, and liver transplant were calculated at 1, 3, 6, and 12 months, and independent predictors of kidney recovery were evaluated using Fine and Gray competing risk regression models that generated subdistribution hazards ratios (sHRs). RESULTS: Overall, 722 patients were included (median age, 61 [interquartile range, 54-68] years; Model for End-Stage Liver Disease (MELD)-Na score, 26 [interquartile range, 22-34]; 66% were male; 52% had viral hepatitis, 25% nonalcoholic fatty liver disease, 18% alcohol-associated liver disease). The cumulative incidences of kidney recovery at 1, 3, 6, and 12 months were 3%, 22%, 25%, and 26%, respectively. Higher MELD-Na score (sHR per 5 units greater, 0.72 [95% CI, 0.65-0.80]), acute-on-chronic liver failure (sHR, 0.61 [95% CI, 0.43-0.86]), and sepsis (sHR, 0.57 [95% CI, 0.41-0.81]) were associated with a lower hazard of kidney recovery, whereas those on a liver transplant waitlist (sHR, 3.10 [95% CI, 1.96-4.88]) and who were admitted to a teaching hospital (sHR, 1.48 [95% CI, 1.05-2.08]) were more likely to experience kidney recovery. LIMITATIONS: Observational design, AKI etiology not identified. CONCLUSIONS: Kidney recovery from KRT occurred in only one quarter of patients and was very unlikely after 3 months. These findings provide information regarding prognosis that may guide decisions regarding KRT initiation and continuation.

Objective Structured Assessment of technical skill (OSATS) in the Surgical Skills and Technology Elective Program (SSTEP): Comparison of peer and expert raters.

BACKGROUND: A major challenge with Competency Based Medical Education (CBME) is that of increased assessment burden on faculty. To reduce this burden, the accuracy and reliability of peer-assessment for surgical skills requires further exploration. METHODS: Forty-two second year medical students were video recorded while performing a simple interrupted suture and an instrument tie. Four novice raters underwent a short training session on the use of the Objective Structured Assessment of Technical Skills (OSATS) checklists. Videos of the suturing task were then independently assessed by the four novice raters and two expert raters on two occasions. Agreement between novice and expert rater scores was calculated using the intraclass correlation coefficient (ICC). RESULTS: For both simple interrupted suturing (ICC = 0.78, CI = 0.66-0.86, p < 0.001) and instrument ties (ICC = 0.87, CI = 0.80-0.92, p < 0.001), there was good agreement between novice and expert raters. CONCLUSIONS: Novice raters can be taught to use the OSATS checklists to assess peers on simple suturing and instrument tying tasks.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Recent developments in filtration media and respirator technology in response to COVID-19.

Abstract: The COVID-19 pandemic triggered a surge in demand for N95 or equivalent respirators that the global supply chain was unable to satisfy. This shortage in critical equipment has inspired research that addresses the immediate problems and has accelerated the development of the next-generation filtration media and respirators. This article provides a brief review of the most recent work with regard to face respirators and filtration media. We discuss filtration efficiency of the widely utilized cloth masks. Next, the sterilization of and reuse of existing N95 respirators to extend the existing stockpile is discussed. To expand near-term supplies, optimization of current manufacturing methods, such as melt-blown processes and electrospinning, has been explored. Future manufacturing methods have been investigated to address long-term supply shortages. Novel materials with antiviral and sterilizable properties with the ability for multiple reuses have been developed and will contribute to the development of the next generation of longer lasting multi-use N95 respirators. Finally, additively manufactured respirators are reviewed, which enable a rapidly deployable source of reusable respirators that can use any filtration fabric.

RNA splicing programs define tissue compartments and cell types at single-cell resolution.

The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10X Chromium data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes MYL6 and RPS24. These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in SAT1. Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.

Spectrally accurate quantitative analysis of isotope-labeled compounds.

Calculated profile mode mass spectrometry (MS) data are fitted to lineshape-calibrated liquid chromatography LC/MS data using a Multiple Linear Regression (MLR) model to quantitate the relative concentrations of stable or radiolabeled compound mixtures. This alternative approach significantly improves the precision and accuracy over existing MS methods while providing the much-needed statistical diagnostics on the goodness-of-fit model and thus reliability of the quantitative results obtained. Test compound data containing S/Cl atoms have been measured with either stable deuterium labeling or radioisotope uniform 14 C labeling onto an aromatic ring. Since the entire relative distribution of variously labeled compounds is automatically obtained through this approach, it is feasible to directly calculate the Specific Activity (SA) from such mass spectral analysis without radioactivity detection and the usual standard curve quantitation. The applicability of this approach to systematically and accurately accommodate and account for incomplete labeling chemistry or other impurities is also discussed, with wide-ranging implications including metabolic flux, HDX (Hydrogen/Deuterium Exchange), and quantitative proteomics.

Sensory Nerves Regulate Transcriptional Dynamics of Lymph Node Cells.

The nervous system plays important roles in homeostasis and inflammatory responses in tissues. However, the regulation of lymph nodes (LN) by nerves remains largely unknown. Huang et al. demonstrate that LNs are innervated by unique peptidergic nociceptors that signal to various endothelial, stromal, and immune cell types in LNs.

Las compresiones torácicas mecánicas frente a las manuales en la parada cardiaca / Mechanical versus manual chest compressions for cardiac arrest

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