invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis.

INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

The unusual gene architecture of polyubiquitin is created by dual-specific splice sites.

BACKGROUND: The removal of introns occurs through the splicing of a 5' splice site (5'ss) with a 3' splice site (3'ss). These two elements are recognized by distinct components of the spliceosome. However, introns in higher eukaryotes contain many matches to the 5' and 3' splice-site motifs that are presumed not to be used. RESULTS: Here, we find that many of these sites can be used. We also find occurrences of the AGGT motif that can function as either a 5'ss or a 3'ss-previously referred to as dual-specific splice sites (DSSs)-within introns. Analysis of the Sequence Read Archive reveals a 3.1-fold enrichment of DSSs relative to expectation, implying synergy between the ability to function as a 5'ss and 3'ss. Despite this suggested mechanistic advantage, DSSs are 2.7- and 4.7-fold underrepresented in annotated 5' and 3' splice sites. A curious exception is the polyubiquitin gene UBC, which contains a tandem array of DSSs that precisely delimit the boundary of each ubiquitin monomer. The resulting isoforms splice stochastically to include a variable number of ubiquitin monomers. We found no evidence of tissue-specific or feedback regulation but note the 8.4-fold enrichment of DSS-spliced introns in tandem repeat genes suggests a driving role in the evolution of genes like UBC. CONCLUSIONS: We find an excess of unannotated splice sites and the utilization of DSSs in tandem repeats supports the role of splicing in gene evolution. These findings enhance our understanding of the diverse and complex nature of the splicing process.

Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Application of single cell multiomics points to changes in chromatin accessibility near calcitonin receptor like receptor and a possible role for adrenomedullin in the post-shock lung.

Introduction: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the "priming" effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS. Methods: Hypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8-12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest. Results: Sham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment. Conclusion: High pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of "priming" and "cellular pre-activation/pre-disposition" processes within the lung microenvironment.

Left Ventricular Septal Avulsion in the Setting of Blunt Cardiac Injury.

A 23-year-old man sustained blunt cardiac injury after a motor vehicle collision resulting in left ventricular septal avulsion, ruptured chordae tendineae, and moderate to severe tricuspid regurgitation that necessitated operative intervention. The patient underwent successful resection of a prolapsed avulsed septal wall segment and concomitant tricuspid valve repair.

Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

Deep RNA sequencing of intensive care unit patients with COVID-19.

COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.

Investigating the impacts of H2O2 treatment on gills of healthy Atlantic salmon reveals potential changes to mucus production with implications on immune activity.

Current treatment strategies for relevant infectious diseases in Atlantic salmon (Salmo salar L.) include the use of low salinity or freshwater bathing. However, often availability is restricted, and hydrogen peroxide (H2O2) is used as an alternative. The potential impacts of H2O2 on fish mucosal tissues, especially the gills therefore need to be considered. In this study the mucosal and immunological effects of H2O2 treatment on the gills of healthy Atlantic salmon were examined by gene expression (qPCR) and immunohistochemistry (IHC) investigating T-cell, B-cell, and mucin activity. Healthy fish were treated with H2O2 and sampled at different times: 4 h, 24 h and 14 days post-H2O2 treatment (dpt) (total n = 18) to investigate the effect of holding time and H2O2 treatment. Treatment with H2O2 resulted in up-regulation of markers for T-cell activity and anti-inflammatory response and down-regulation of mucin expression in the gills at 14 dpt compared to fish sampled prior to treatment (0h; n = 5 fish). These findings were supported by IHC analysis, which despite being highly variable between samples, showed an increase in the number of CD3+ T cells at 14 dpt in 50% of treated fish compared to pre-treatment fish. The results from this study suggest that H2O2 treatment does not immune compromise healthy Atlantic salmon after 14 dpt (i.e., post-recovery) but modulates gill immune activity and disrupts the mucus covering of the gills. However, further studies are required to determine whether the effects observed are related to H2O2 treatment in isolation or other variables such as holding time or environmental factors.

A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus.

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus.

Efficacy testing of an immersion vaccine against Aeromonas salmonicida and immunocompetence in ballan wrasse (Labrus bergylta, Ascanius).

The development of effective vaccines is a critical step towards the domestication of emerging fish species for aquaculture. However, traditional vaccine delivery through intraperitoneal (i.p.) injection requires fish to reach a minimum size and age and therefore cannot provide protection at early developmental stages when infection may occur. This study investigated the effectiveness of immersion vaccination with respect to immunocompetence in a cleaner fish species (ballan wrasse, Labrus bergylta, Ascanius) used in Atlantic salmon farming as an alternative means to control sea lice. The species is susceptible to atypical strains of Aeromonas salmonicida (aAs) at early life stages (<15 g), when i.p. vaccination is not applicable. While immersion vaccination is currently used in commercial hatcheries, the optimal fish size for vaccination, and efficacy of the vaccine delivered by this route has not yet been established. Importantly, efficacy depends on the capability of the species immune system to recognise antigens and process antigens to trigger and produce an adaptive immune response, (process known as immunocompetence). In this study, the efficacy of a polyvalent autogenous vaccine administered by immersion in juvenile ballan wrasse and the subsequent immune response induced was investigated after prime and booster vaccination regimes. In addition, temporal expression (0-150 days post hatch) of adaptive immune genes including major histocompatibility complex (MHC II CD74 molecule) and immunoglobulin M (IgM) was assessed using quantitative PCR (qPCR). Prime and/or boost vaccination by immersion of juvenile ballan wrasse (0.5 g and 1.5 g corresponding to 80 and 170 days post hatch (dph), respectively) did not provide significant protection against aAs vapA V after bath challenge under experimental conditions. Despite no evident protection >80 dph, MHC II and IgM transcripts were first reported at 35 and 75 dph, respectively, suggesting a window of immunocompetence. The results provide important new information on the onset of adaptive immunity in ballan wrasse and highlight that immersion vaccination in the species for protection against aAs should be performed at later developmental stages (>1.5 g) in the hatchery.

In silico analysis of AhyI protein and AI-1 inhibition using N-cis-octadec-9z-enoyl-l-homoserine lactone inhibitor in Aeromonas hydrophila.

AhyI is homologous to the protein LuxI and is conserved throughout bacterial species including Aeromonas hydrophila. A. hydrophila causes opportunistic infections in fish and other aquatic organisms. Furthermore, this pathogennot only poses a great risk for the aquaculture industry, but also for human public health. AhyI (expressing acylhomoserine lactone) is responsible for the biosynthesis of autoinducer-1 (AI-1), commonly referred to as a quorum sensing (QS) signaling molecule, which plays an essential role in bacterial communication. Studying protein structure is essential for understanding molecular mechanisms of pathogenicity in microbes. Here, we have deduced a predicted structure of AhyI protein and characterized its function using in silico methods to aid the development of new treatments for controlling A.hydrophila infections. In addition to modeling AhyI, an appropriate inhibitor molecule was identified via high throughput virtual screening (HTVS) using mcule drug-like databases.The AhyI-inhibitor N-cis-octadec-9Z-enoyl-l-Homoserine lactone was selected withthe best drug score. In order to understand the pocket sites (ligand binding sites) and their interaction with the selected inhibitor, docking (predicted protein binding complex) servers were used and the selected ligand was docked with the predicted AhyI protein model. Remarkably, N-cis-octadec-9Z-enoyl-l-Homoserine lactone established interfaces with the protein via16 residues (V24, R27, F28, R31, W34, V36, D45, M77, F82, T101, R102, L103, 104, V143, S145, and V168), which are involved with regulating mechanisms of inhibition. These proposed predictions suggest that this inhibitor molecule may be used as a novel drug candidate for the inhibition of auto-inducer-1 (AI-1) activity.The N-cis-octadec-9Z-enoyl-l-Homoserine lactone inhibitor molecule was studied on cultured bacteria to validate its potency against AI-1 production. At a concentration of 40 µM, optimal inhibition efficiency of AI-1 was observedin bacterial culture media.These results suggest that the inhibitor molecule N-cis-octadec-9Z-enoyl-l-Homoserine lactone is a competitive inhibitor of AI-1 biosynthesis.

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

The immune landscape of SARS-CoV-2-associated Multisystem Inflammatory Syndrome in Children (MIS-C) from acute disease to recovery.

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening disease occurring several weeks after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Deep immune profiling showed acute MIS-C patients had highly activated neutrophils, classical monocytes and memory CD8+ T-cells, with increased frequencies of B-cell plasmablasts and double-negative B-cells. Post treatment samples from the same patients, taken during symptom resolution, identified recovery-associated immune features including increased monocyte CD163 levels, emergence of a new population of immature neutrophils and, in some patients, transiently increased plasma arginase. Plasma profiling identified multiple features shared by MIS-C, Kawasaki Disease and COVID-19 and that therapeutic inhibition of IL-6 may be preferable to IL-1 or TNF-α. We identified several potential mechanisms of action for IVIG, the most commonly used drug to treat MIS-C. Finally, we showed systemic complement activation with high plasma C5b-9 levels is common in MIS-C suggesting complement inhibitors could be used to treat the disease.

Efficient Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Exhaled Breath.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease, coronavirus disease-2019 (COVID-19), in some patients. Samples for SARS-CoV-2 testing are typically collected by nasopharyngeal swab, with the virus detected by PCR; however, patients can test positive for 3 months after infection. Without the capacity to assay SARS-CoV-2 in breath, it is not possible to understand the risk for transmission from infected individuals. To detect virus in breath, the Bubbler-a breathalyzer that reverse-transcribes RNA from SARS-CoV-2 particles into a sample-specific barcoded cDNA-was developed. In a study of 70 hospitalized patients, the Bubbler was both more predictive of lower respiratory tract involvement (abnormal chest X-ray) and less invasive than alternatives. Samples tested using the Bubbler were threefold more enriched for SARS-CoV-2 RNA than were samples from tongue swabs, implying that virus particles were being directly sampled. The barcode-enabled Bubbler was used for simultaneous diagnosis in large batches of pooled samples at a lower limit of detection of 334 genomic copies per sample. Diagnosis by sequencing can provide additional information, such as viral load and strain identity. The Bubbler was configured to sample nucleic acids in water droplets circulating in air, demonstrating its potential in environmental monitoring and the protective effect of adequate ventilation.

Serological responses to koi herpesvirus (KHV) in a non-cyprinid reservoir host.

Koi herpesvirus (KHV) is a highly contagious virus that causes KHV disease (KHVD) inducing high mortality in carp and koi (Cyprinus carpio L.). In the late stage, latency occurs with very low, often non-detectable virus concentrations, which represents a challenge for virus detection. After validation according to OIE recommendations, an antibody ELISA was established to recognize antibodies of C. carpio against KHV infection. In this study, the ELISA was modified to detect anti-KHV antibodies from a non-cyprinid fish. Experimentally infected rainbow trout (Oncorhynchus mykiss) were able to transmit KHV to naïve carp at two different temperatures, demonstrating their potential as a reservoir host. At 20°C, KHVD was induced in carp but not at 15°C. Unexpectedly, rainbow trout developed humoral response against KHV at both temperatures. In contrast to carp, at 15°C trout produced neutralizing antibodies but not at 20°C. While antibodies obtained from infected carp sera reacted in a similar way against all KHV, antibodies from rainbow trout sera reacted differently to the same isolates by ELISA. The data show that even when non-cyprinid fish species are infected with KHV, they can produce antibodies that differ from those observed in carp.

Emerging therapeutic targets for sepsis.

Introduction: Sepsis is characterized by a dysregulated host response to infection. Sepsis-associated morbidity/mortality demands concerted research efforts toward therapeutic interventions which are reliable, broadly effective, and etiologically based. More intensive and extensive investigations on alterations in cellular signaling pathways, gene targeting as a means of modifying the characteristic hyper and/or hypo-immune responses, prevention through optimization of the microbiome, and the molecular pathways underlying the septic immune response could improve outcomes.] Areas covered: The authors discuss key experimental mammalian models and clinical trials. They provide an evaluation of evolving therapeutics in sepsis and how they have built upon past and current treatments. Relevant literature was derived from a PubMed search spanning 1987-2020.Expert opinion: Given the complex nature of sepsis and the elicited immune response, it is not surprising that a single cure-all therapeutic intervention, which is capable of effectively and reliably improving patient outcomes has failed to emerge. Innovative approaches seek to address not only the disease process but modify underlying patient factors. A true improvement in sepsis-associated morbidity/mortality will require a combination of unique therapeutic modalities.

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius).

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time-consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V-specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost-effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

Deep RNA Sequencing of Intensive Care Unit Patients with COVID-19.

PURPOSE: COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. METHODS: Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (>100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. RESULTS: We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. CONCLUSIONS: Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies. TAKE HOME MESSAGE: Deep RNA sequencing provides a novel diagnostic tool for critically ill patients. Among ICU patients with COVID-19, RNA sequencings can identify gene expression, pathogens (including SARS-CoV-2), and can predict mortality. TWEET: Deep RNA sequencing is a novel technology that can assist in the care of critically ill COVID-19 patients & can be applied to other disease.

Unjustified Administration in Liberal Use of Tranexamic Acid in Trauma Resuscitation.

BACKGROUND: Early administration of tranexamic acid (TXA) has been widely implemented for the treatment of presumed hyperfibrinolysis in hemorrhagic shock. We aimed to characterize the liberal use of TXA and whether unjustified administration was associated with increased venous thrombotic events (VTEs). METHODS: We identified injured patients who received TXA between January 2016 and January 2018 by querying our Level 1 trauma center's registry. We retrospectively reviewed medical records and radiologic images to classify whether patients had a hemorrhagic injury that would have benefited from TXA (justified) or not (unjustified). RESULTS: Ninety-five patients received TXA for traumatic injuries, 42.1% were given by emergency medical services. TXA was considered unjustified in 35.8% of the patients retrospectively and in 52% of the patients when given by emergency medical services. Compared with unjustified administration, patients in the justified group were younger (47.6 versus 58.4; P = 0.02), more hypotensive in the field (systolic blood pressure: 107 ± 31 versus 137 ± 32 mm Hg; P < 0.001) and in the emergency department (systolic blood pressure: 97 ± 27 versus 128 ± 27; P < 0.001), and more tachycardic in emergency department (heart rate: 99 ± 29 versus 88 ± 19; P = 0.04). The justified group also had higher injury severity score (median 24 versus 11; P < 0.001), was transfused more often (81.7% versus 20.6%; P < 0.001), and had higher in-hospital mortality (39.3% versus 2.9%; P < 0.001), but there was no difference in the rate of VTE (8.2% versus 5.9%). CONCLUSIONS: Our results highlight a high rate of unjustified administration, especially in the prehospital setting. Hypotension and tachycardia were indications of correct use. Although we did not observe a difference in VTE rates between the groups, though, our study was underpowered to detect a difference. Cautious implementation of TXA in resuscitation protocols is encouraged in the meantime. Nonetheless, adverse events associated with unjustified TXA administration should be further evaluated.