Polymorphisms in the yeast galactose sensor underlie a natural continuum of nutrient-decision phenotypes.

In nature, microbes often need to "decide" which of several available nutrients to utilize, a choice that depends on a cell's inherent preference and external nutrient levels. While natural environments can have mixtures of different nutrients, phenotypic variation in microbes' decisions of which nutrient to utilize is poorly studied. Here, we quantified differences in the concentration of glucose and galactose required to induce galactose-responsive (GAL) genes across 36 wild S. cerevisiae strains. Using bulk segregant analysis, we found that a locus containing the galactose sensor GAL3 was associated with differences in GAL signaling in eight different crosses. Using allele replacements, we confirmed that GAL3 is the major driver of GAL induction variation, and that GAL3 allelic variation alone can explain as much as 90% of the variation in GAL induction in a cross. The GAL3 variants we found modulate the diauxic lag, a selectable trait. These results suggest that ecological constraints on the galactose pathway may have led to variation in a single protein, allowing cells to quantitatively tune their response to nutrient changes in the environment.

Accurate somatic variant detection using weakly supervised deep learning.

Identification of somatic mutations in tumor samples is commonly based on statistical methods in combination with heuristic filters. Here we develop VarNet, an end-to-end deep learning approach for identification of somatic variants from aligned tumor and matched normal DNA reads. VarNet is trained using image representations of 4.6 million high-confidence somatic variants annotated in 356 tumor whole genomes. We benchmark VarNet across a range of publicly available datasets, demonstrating performance often exceeding current state-of-the-art methods. Overall, our results demonstrate how a scalable deep learning approach could augment and potentially supplant human engineered features and heuristic filters in somatic variant calling.

Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals.

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild, or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the spike antigen using a novel TCR-binding algorithm. The predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools in the unexposed donors. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. However, our findings suggest that SARS-CoV-2 reactive T-cells are likely to be present in many individuals because of prior exposure to flu and CMV viruses.

Reliability of mecA in Predicting Phenotypic Susceptibilities of Coagulase-Negative Staphylococci and Staphylococcus aureus.

The mecA gene is commonly used to identify resistance in Staphylococcus aureus, but historically is not used for coagulase-negative staphylococci (CoNS). Analysis of 412 staphylococcal blood cultures (2014-2018) revealed that the absence of mecA had high concordance (100%) with oxacillin susceptibility for S. aureus and CoNS alike.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

Hemorrhage Attenuates Neutrophil Recruitment in Response to Secondary Respiratory Infection by Pseudomonas Aeruginosa.

Neutrophil recruitment into the lung airspaces plays an important role in the containment and clearance of bacteria. Hemorrhagic shock, a complication of traumatic injury, induces immune dysfunction that compromises host defense and frequently leads to secondary infection. The objective of the current study was to determine whether prior hemorrhage impacts neutrophil recruitment in response to secondary Pseudomonas aeruginosa. Experiments were performed using a mouse model (C57BL/6) of respiratory infection by P. aeruginosa (strain PA103, 3 × 10 colony-forming units [CFUs]) that is delivered by intratracheal inhalation 24 h after hypovolemic hemorrhagic shock (fixed mean arterial blood pressure at 35 mmHg for 90 min, Ringer's lactate infused as fluid resuscitation). By postmortem flow cytometry analyses of bronchoalveolar lavage fluid, we observe that prior hemorrhage attenuates the entry of neutrophils into the lung airspaces in response to P. aeruginosa. The reduction in neutrophil recruitment occurs in an amplified inflammatory environment, with elevated lung tissue levels of interleukin 6 and C-X-C motif ligand 1 in mice receiving hemorrhage prior to infection. As compared to either insult alone, outcome to sequential hemorrhage and respiratory infection includes enhanced mortality. The effect of prior hemorrhage on clearance of P. aeruginosa, as determined by quantifying bacterial CFUs in lung tissue, was not statistically significant at 24 h postinfection, but our data suggest that further inquiry may be needed to fully understand the potential impact of hemorrhagic shock on this process. These results suggest that changes in neutrophil recruitment may contribute to the immune dysfunction following hemorrhagic shock that renders the host susceptible to severe respiratory infection.

Surviving Deadly Lung Infections: Innate Host Tolerance Mechanisms in the Pulmonary System.

Much research on infectious diseases focuses on clearing the pathogen through the use of antimicrobial drugs, the immune response, or a combination of both. Rapid clearance of pathogens allows for a quick return to a healthy state and increased survival. Pathogen-targeted approaches to combating infection have inherent limitations, including their pathogen-specific nature, the potential for antimicrobial resistance, and poor vaccine efficacy, among others. Another way to survive an infection is to tolerate the alterations to homeostasis that occur during a disease state through a process called host tolerance or resilience, which is independent from pathogen burden. Alterations in homeostasis during infection are numerous and include tissue damage, increased inflammation, metabolic changes, temperature changes, and changes in respiration. Given its importance and sensitivity, the lung is a good system for understanding host tolerance to infectious disease. Pneumonia is the leading cause of death for children under five worldwide. One reason for this is because when the pulmonary system is altered dramatically it greatly impacts the overall health and survival of a patient. Targeting host pathways involved in maintenance of pulmonary host tolerance during infection could provide an alternative therapeutic avenue that may be broadly applicable across a variety of pathologies. In this review, we will summarize recent findings on tolerance to host lung infection. We will focus on the involvement of innate immune responses in tolerance and how an initial viral lung infection may alter tolerance mechanisms in leukocytic, epithelial, and endothelial compartments to a subsequent bacterial infection. By understanding tolerance mechanisms in the lung we can better address treatment options for deadly pulmonary infections.

Identification of Patients At-Risk of QT Interval Prolongation during Medication Reviews: A Missed Opportunity?

The prolongation of the QT interval is a relatively rare but serious adverse drug reaction. It can lead to torsade de pointes, which is potentially life-threatening. The study's objectives were: determine the use of QT interval-prolonging drugs in an elderly community-dwelling population at risk of medication misadventure and identify recommendations regarding the risk of QT interval prolongation made by pharmacists when performing medication reviews. In a retrospective evaluation, 500 medication review reports from Australian pharmacists were analysed. In patients taking at least one QT interval-prolonging drug, the individual risk of drug-induced QT interval prolongation was assessed. Recommendations of pharmacists to avoid the occurrence of this drug-related problem were examined. There was a high prevalence of use of potentially QT interval-prolonging drugs (71% patients), with 11% of patients taking at least one drug with a known risk. Pharmacists provided specific recommendations in only eight out of 35 patients (23%) with a high-risk score and taking drugs with known risk of QT interval prolongation. Pharmacists' recommendations, when present, were focused on drugs with known risk of QT interval prolongation, rather than patients' additional risk factors. There is a need to improve knowledge and awareness of this topic among pharmacists performing medication reviews.

Coinfection With Influenza A Virus and Klebsiella oxytoca: An Underrecognized Impact on Host Resistance and Tolerance to Pulmonary Infections.

Pneumonia is a world health problem and a leading cause of death, particularly affecting children and the elderly (1, 2). Bacterial pneumonia following infection with influenza A virus (IAV) is associated with increased morbidity and mortality but the mechanisms behind this phenomenon are not yet well-defined (3). Host resistance and tolerance are two processes essential for host survival during infection. Resistance is the host's ability to clear a pathogen while tolerance is the host's ability to overcome the impact of the pathogen as well as the host response to infection (4-8). Some studies have shown that IAV infection suppresses the immune response, leading to overwhelming bacterial loads (9-13). Other studies have shown that some IAV/bacterial coinfections cause alterations in tolerance mechanisms such as tissue resilience (14-16). In a recent analysis of nasopharyngeal swabs from patients hospitalized during the 2013-2014 influenza season, we have found that a significant proportion of IAV-infected patients were also colonized with Klebsiella oxytoca, a gram-negative bacteria known to be an opportunistic pathogen in a variety of diseases (17). Mice that were infected with K. oxytoca following IAV infection demonstrated decreased survival and significant weight loss when compared to mice infected with either single pathogen. Using this model, we found that IAV/K. oxytoca coinfection of the lung is characterized by an exaggerated inflammatory immune response. We observed early inflammatory cytokine and chemokine production, which in turn resulted in massive infiltration of neutrophils and inflammatory monocytes. Despite this swift response, the pulmonary pathogen burden in coinfected mice was similar to singly-infected animals, albeit with a slight delay in bacterial clearance. In addition, during coinfection we observed a shift in pulmonary macrophages toward an inflammatory and away from a tissue reparative phenotype. Interestingly, there was only a small increase in tissue damage in coinfected lungs as compared to either single infection. Our results indicate that during pulmonary coinfection a combination of seemingly modest defects in both host resistance and tolerance may act synergistically to cause worsened outcomes for the host. Given the prevalence of K. oxytoca detected in human IAV patients, these dysfunctional tolerance and resistance mechanisms may play an important role in the response of patients to IAV.

Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.

Integrated nanoscale deterministic lateral displacement arrays for separation of extracellular vesicles from clinically-relevant volumes of biological samples.

Extracellular vesicles (EVs) offer many opportunities in early-stage disease diagnosis, treatment monitoring, and precision therapy owing to their high abundance in bodily fluids, accessibility from liquid biopsy, and presence of nucleic acid and protein cargo from their cell of origin. Despite their growing promise, isolation of EVs for analysis remains a labor-intensive and time-consuming challenge given their nanoscale dimensions (30-200 nm) and low buoyant density. Here, we report a simple, size-based EV separation technology that integrates 1024 nanoscale deterministic lateral displacement (nanoDLD) arrays on a single chip capable of parallel processing sample fluids at rates of up to 900 µL h-1. Benchmarking the nanoDLD chip against commonly used EV isolation technologies, including ultracentrifugation (UC), UC plus density gradient, qEV size-exclusion chromatography (Izon Science), and the exoEasy Maxi Kit (QIAGEN), we demonstrate a superior yield of ∼50% for both serum and urine samples, representing the ability to use smaller input volumes to achieve the same number of isolated EVs, and a concentration factor enhancement of up to ∼3× for both sample types, adjustable to ∼60× for urine through judicious design. Further, RNA sequencing was carried out on nanoDLD- and UC-isolated EVs from prostate cancer (PCa) patient serum samples, resulting in a higher gene expression correlation between replicates for nanoDLD-isolated EVs with enriched miRNA, decreased rRNA, and the ability to detect previously reported RNA indicators of aggressive PCa. Taken together, these results suggest nanoDLD as a promising alternative technology for fast, reproducible, and automatable EV-isolation.

Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

PURPOSE: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments. PROCEDURES: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing. RESULTS: The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking. CONCLUSIONS: MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents.

PURPOSE: Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo. PROCEDURES: By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r 2) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain. RESULTS: At 7T, r 2 relaxivity of bare MEs was higher (250 s-1 mM-1) than that of conventional SPIO (178 s-1 mM-1). Optimized in vitro loading of MEs into 231BR cells yielded 1-4 pg iron/cell (compared to 5-10 pg iron/cell for conventional SPIO). r 2 relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence. CONCLUSIONS: Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r 2 relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

Move it or Lose it. Is it Reasonable for Older Adults with Osteoarthritis to Continue to Use Paracetamol in Order to Maintain Physical Activity?

Osteoarthritis is a common progressive disease in older adults, and those affected often have impaired physical function, co-existing disease states, and reduced quality of life. In patients with osteoarthritis, pain is reported as a primary cause of mobility limitation, and guidelines recommend a mix of pharmacologic and non-pharmacologic strategies for pain management. The benefits of exercise in the management of osteoarthritis are well established; however, pain appears to be the biggest barrier to patients engaging in, and adhering to, physical activity programs. Attitudes towards the use of pain medications differ widely, and lack of efficacy or fear of side effects may lead to sub-therapeutic dosing. Furthermore, a recent review suggesting that short-term paracetamol use is ineffective for osteoarthritis has added to the confusion. This narrative review investigates limitations of current medications, summarizes patient attitudes toward the use of analgesics for osteoarthritis pain (with a focus on paracetamol), and explores the uptake of physical activity for osteoarthritis management. Evidence suggests that, despite clear guidelines, symptoms of osteoarthritis generally remain poorly managed. More research is required to investigate clinical outcomes in patients with osteoarthritis through optimized medication plans to better understand whether longer-term analgesic use in conjunction with physical activity can assist patients to overcome mobility limitations.

Novel MRI Contrast Agent from Magnetotactic Bacteria Enables In Vivo Tracking of iPSC-derived Cardiomyocytes.

Therapeutic delivery of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) represents a novel clinical approach to regenerate the injured myocardium. However, methods for robust and accurate in vivo monitoring of the iCMs are still lacking. Although superparamagnetic iron oxide nanoparticles (SPIOs) are recognized as a promising tool for in vivo tracking of stem cells using magnetic resonance imaging (MRI), their signal persists in the heart even weeks after the disappearance of the injected cells. This limitation highlights the inability of SPIOs to distinguish stem cell viability. In order to overcome this shortcoming, we demonstrate the use of a living contrast agent, magneto-endosymbionts (MEs) derived from magnetotactic bacteria for the labeling of iCMs. The ME-labeled iCMs were injected into the infarcted area of murine heart and probed by MRI and bioluminescence imaging (BLI). Our findings demonstrate that the MEs are robust and effective biological contrast agents to track iCMs in an in vivo murine model. We show that the MEs clear within one week of cell death whereas the SPIOs remain over 2 weeks after cell death. These findings will accelerate the clinical translation of in vivo MRI monitoring of transplanted stem cell at high spatial resolution and sensitivity.