Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

Suppression of the host antiviral response by non-infectious varicella zoster virus extracellular vesicles.

Varicella zoster virus (VZV) reactivates from ganglionic sensory neurons to produce herpes zoster (shingles) in a unilateral dermatomal distribution, typically in the thoracic region. Reactivation not only heightens the risk of stroke and other neurological complications but also increases susceptibility to co-infections with various viral and bacterial pathogens at sites distant from the original infection. The mechanism by which VZV results in complications remote from the initial foci remains unclear. Small extracellular vesicles (sEVs) are membranous signaling structures that can deliver proteins and nucleic acids to modify the function of distal cells and tissues during normal physiological conditions. Although viruses have been documented to exploit the sEV machinery to propagate infection, the role of non-infectious sEVs released from VZV-infected neurons in viral spread and disease has not been studied. Using multi-omic approaches, we characterized the content of sEVs released from VZV-infected human sensory neurons (VZV sEVs). One viral protein was detected (immediate-early 62), as well as numerous immunosuppressive and vascular disease-associated host proteins and miRNAs that were absent in sEVs from uninfected neurons. Notably, VZV sEVs are non-infectious yet transcriptionally altered primary human cells, suppressing the antiviral type 1 interferon response and promoting neuroinvasion of a secondary pathogen in vivo. These results challenge our understanding of VZV infection, proposing that the virus may contribute to distant pathologies through non-infectious sEVs beyond the primary infection site. Furthermore, this study provides a previously undescribed immune-evasion mechanism induced by VZV that highlights the significance of non-infectious sEVs in early VZV pathogenesis. IMPORTANCE: Varicella zoster virus (VZV) is a ubiquitous human virus that predominantly spreads by direct cell-cell contact and requires efficient and immediate host immune evasion strategies to spread. The mechanisms of immune evasion prior to virion entry have not been fully elucidated and represent a critical gap in our complete understanding of VZV pathogenesis. This study describes a previously unreported antiviral evasion strategy employed by VZV through the exploitation of the infected host cell's small extracellular vesicle (sEV) machinery. These findings suggest that non-infectious VZV sEVs could travel throughout the body, affecting cells remote from the site of infection and challenging the current understanding of VZV clinical disease, which has focused on local effects and direct infection. The significance of these sEVs in early VZV pathogenesis highlights the importance of further investigating their role in viral spread and secondary disease development to reduce systemic complications following VZV infections.

Fine-Tuning of CD8(+) T Cell Mitochondrial Metabolism by the Respiratory Chain Repressor MCJ Dictates Protection to Influenza Virus.

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.

A unified mechanism for intron and exon definition and back-splicing.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes.

Specialized interferon action in COVID-19.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

Multiomics Signatures of Coagulopathy in a Polytrauma Swine Model Contrasted with Severe Multisystem Injured Patients.

Trauma-induced coagulopathy (TIC) is a leading contributor to preventable mortality in severely injured patients. Understanding the molecular drivers of TIC is an essential step in identifying novel therapeutics to reduce morbidity and mortality. This study investigated multiomics and viscoelastic responses to polytrauma using our novel swine model and compared these findings with severely injured patients. Molecular signatures of TIC were significantly associated with perturbed coagulation and inflammation systems as well as extensive hemolysis. These results were consistent with patterns observed in trauma patients who had multisystem injuries. Here, intervention using resuscitative endovascular balloon occlusion of the aorta following polytrauma in our swine model revealed distinct multiomics alterations as a function of placement location. Aortic balloon placement in zone-1 worsened ischemic damage and mitochondrial dysfunction, patterns that continued throughout the monitored time course. While placement in zone-III showed a beneficial effect on TIC, it showed an improvement in effective coagulation. Taken together, this study highlights the translational relevance of our polytrauma swine model for investigating therapeutic interventions to correct TIC in patients.

Agnostic identification of plasma biomarkers for postpartum hemorrhage risk.

BACKGROUND: Postpartum hemorrhage is difficult to predict, is associated with significant maternal morbidity, and is the leading cause of maternal mortality worldwide. The identification of maternal biomarkers that can predict increased postpartum hemorrhage risk would enhance clinical care and may uncover mechanisms that lead to postpartum hemorrhage. OBJECTIVE: This retrospective case-control study employed agnostic proteomic profiling of maternal plasma samples to identify differentially abundant proteins in controls and postpartum hemorrhage cases. STUDY DESIGN: Maternal plasma samples were procured from a cohort of >60,000 participants in a single institution's perinatal repository. Postpartum hemorrhage was defined as a decrease in hematocrit of ≥10% or receipt of transfusion within 24 hours after delivery. Postpartum hemorrhage cases (n=30) were matched by maternal age and delivery mode (vaginal or cesarean) with controls (n=56). Mass spectrometry was used to identify differentially abundant proteins using integrated peptide peak areas. Statistically significant differences between groups were defined as P<.05 after controlling for multiple comparisons. RESULTS: By study design, cases and controls did not differ in race, ethnicity, gestational age at delivery, blood type, or predelivery platelet count. Cases had slightly but significantly lower predelivery and postdelivery hematocrit and hemoglobin. Mass spectrometry detected 1140 proteins, including 77 proteins for which relative abundance differed significantly between cases and controls (fold change >1.15, P<.05). Of these differentially abundant plasma proteins, most had likely liver or placental origins. Gene ontology term analysis mapped to protein clusters involved in responses to wound healing, stress response, and host immune defense. Significantly differentially abundant proteins with the highest fold change (prostaglandin D2 synthase, periostin, and several serine protease inhibitors) did not correlate with predelivery hematocrit or hemoglobin but identified postpartum hemorrhage cases with logistic regression modeling revealing good-to-excellent area under the operator receiver characteristic curves (0.802-0.874). Incorporating predelivery hemoglobin with these candidate proteins further improved the identification of postpartum hemorrhage cases. CONCLUSION: Agnostic analysis of maternal plasma samples identified differentially abundant proteins in controls and postpartum hemorrhage cases. Several of these proteins are known to participate in biologically plausible pathways for postpartum hemorrhage risk and have potential value for predicting postpartum hemorrhage. These findings identify candidate protein biomarkers for future validation and mechanistic studies.

Admission Viscoelastic Hemostatic Assay Parameters Predict Poor Long-Term Intracerebral Hemorrhage Outcomes.

BACKGROUND: Viscoelastic hemostatic assays (VHAs) provide more comprehensive assessments of coagulation compared with conventional coagulation assays. Although VHAs have enabled guided hemorrhage control therapies, improving clinical outcomes in life-threatening hemorrhage, the role of VHAs in intracerebral hemorrhage (ICH) is unclear. If VHAs can identify coagulation abnormalities relevant for ICH outcomes, this would support the need to investigate the role of VHAs in ICH treatment paradigms. Thus, we investigated whether VHA assessments of coagulation relate to long-term ICH outcomes. METHODS: Patients with spontaneous ICH enrolled into a single-center cohort study receiving admission Rotational Thromboelastometry (ROTEM) VHA testing between 2013 and 2020 were assessed. Patients with previous anticoagulant use or coagulopathy on conventional coagulation assays were excluded. Primary ROTEM exposure variables were coagulation kinetics and clot strength assessments. Poor long-term outcome was defined as modified Rankin Scale ≥ 4 at 6 months. Logistic regression analyses assessed associations of ROTEM parameters with clinical outcomes after adjusting for ICH severity and hemoglobin concentration. RESULTS: Of 44 patients analyzed, the mean age was 64 years, 57% were female, and the median ICH volume was 23 mL. Poor 6-month outcome was seen in 64% of patients. In our multivariable regression models, slower, prolonged coagulation kinetics (adjusted odds ratio for every second increase in clot formation time 1.04, 95% confidence interval 1.00-1.09, p = 0.04) and weaker clot strength (adjusted odds ratio for every millimeter increase of maximum clot firmness 0.84, 95% confidence interval 0.71-0.99, p = 0.03) were separately associated with poor long-term outcomes. CONCLUSIONS: Slower, prolonged coagulation kinetics and weaker clot strength on admission VHA ROTEM testing, not attributable to anticoagulant use, were associated with poor long-term outcomes after ICH. Further work is needed to clarify the generalizability and the underlying mechanisms of these VHA findings to assess whether VHA-guided treatments should be incorporated into ICH care.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Zoster-Associated Prothrombotic Plasma Exosomes and Increased Stroke Risk.

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk.

Mass Spectrometry-Based Atlas of Extracellular Matrix Proteins across 25 Mouse Organs.

The extracellular matrix (ECM) is a critical non-cellular component of multicellular organisms containing a variety of proteins, glycoproteins, and proteoglycans which have been implicated in a wide variety of essential biological processes, including development, wound healing, and aging. Due to low solubility, many ECM proteins have been underrepresented in previous proteomic datasets. Using an optimized three-step decellularization and ECM extraction method involving chaotrope extraction and digestion via hydroxylamine hydrochloride, we have generated coverage of the matrisome across 25 organs. We observe that the top 100 most abundant proteins from the ECM fractions of all tissues are generally present in all tissues, indicating that tissue matrices are principally composed of a shared set of ECM proteins. However, these proteins vary up to 4000-fold between tissues, resulting in highly unique matrix profiles even with the same primary set of proteins. A data reduction approach was used to reveal related networks of expressed ECM proteins across varying tissues, including basement membrane and collagen subtypes.

Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability.

OBJECTIVE: We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca2+) influx as a mechanism of trauma-induced endothelial permeability. SUMMARY BACKGROUND DATA: Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca2+ signaling may play a role. METHODS: Ex vivo plasma from injured patients with "Low Injury/Low Shock" (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and "High Injury/High Shock" (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca2+ channel. Live cell fluorescence microscopy and ECIS were used to assess cytosolic Ca2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain (MLC) phosphorylation and actomyosin contraction. RESULTS: Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca2+ increase. Cytosolic Ca2+ increase, MLC phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca2+ present. High Injury/High Shock plasma did not induce endothelial permeability without extracellular Ca2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca2+ influx and permeability. CONCLUSIONS: This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca2+ influx via the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca2+-targeted therapies and interventions in the care of severely injured patients.

Omics Signatures of Tissue Injury and Hemorrhagic Shock in Swine.

OBJECTIVE: Advanced mass spectrometry methods were leveraged to analyze both proteomics and metabolomics signatures in plasma upon controlled tissue injury (TI) and hemorrhagic shock (HS)-isolated or combined-in a swine model, followed by correlation to viscoelastic measurements of coagulopathy via thrombelastography. BACKGROUND: TI and HS cause distinct molecular changes in plasma in both animal models and trauma patients. However, the contribution to coagulopathy of trauma, the leading cause of preventable mortality in this patient population remains unclear. The recent development of a swine model for isolated or combined TI+HS facilitated the current study. METHODS: Male swine (n=17) were randomized to either isolated or combined TI and HS. Coagulation status was analyzed by thrombelastography during the monitored time course. The plasma fractions of the blood draws (at baseline; end of shock; and at 30 minutes, 1, 2, and 4 hours after shock) were analyzed by mass spectrometry-based proteomics and metabolomics workflows. RESULTS: HS-isolated or combined with TI-caused the most severe omic alterations during the monitored time course. While isolated TI delayed the activation of coagulation cascades. Correlation to thrombelastography parameters of clot strength (maximum amplitude) and breakdown (LY30) revealed signatures of coagulopathy which were supported by analysis of gene ontology-enriched biological pathways. CONCLUSION: The current study provides a comprehensive characterization of proteomic and metabolomic alterations to combined or isolated TI and HS in a swine model and identifies early and late omics correlates to viscoelastic measurements in this system.

Functional and molecular determinants of right ventricular response to severe pulmonary hypertension in a large animal model.

Right ventricular (RV) failure is the major determinant of outcome in pulmonary hypertension (PH). Calves exposed to 2-wk hypoxia develop severe PH and unlike rodents, hypoxia-induced PH in this species can lead to right heart failure. We, therefore, sought to examine the molecular and structural changes in the RV in calves with hypoxia-induced PH, hypothesizing that we could identify mechanisms underlying compensated physiological function in the face of developing severe PH. Calves were exposed to 14 days of environmental hypoxia (equivalent to 4,570 m/15,000 ft elevation, n = 29) or ambient normoxia (1,525 m/5,000 ft, n = 25). Cardiopulmonary function was evaluated by right heart catheterization and pressure volume loops. Molecular and cellular determinants of RV remodeling were analyzed by cDNA microarrays, RealTime PCR, proteomics, and immunochemistry. Hypoxic exposure induced robust PH, with increased RV contractile performance and preserved cardiac output, yet evidence of dysregulated RV-pulmonary artery mechanical coupling as seen in advanced disease. Analysis of gene expression revealed cellular processes associated with structural remodeling, cell signaling, and survival. We further identified specific clusters of gene expression associated with 1) hypertrophic gene expression and prosurvival mechanotransduction through YAP-TAZ signaling, 2) extracellular matrix (ECM) remodeling, 3) inflammatory cell activation, and 4) angiogenesis. A potential transcriptomic signature of cardiac fibroblasts in RV remodeling was detected, enriched in functions related to cell movement, tissue differentiation, and angiogenesis. Proteomic and immunohistochemical analysis confirmed RV myocyte hypertrophy, together with localization of ECM remodeling, inflammatory cell activation, and endothelial cell proliferation within the RV interstitium. In conclusion, hypoxia and hemodynamic load initiate coordinated processes of protective and compensatory RV remodeling to withstand the progression of PH.NEW & NOTEWORTHY Using a large animal model and employing a comprehensive approach integrating hemodynamic, transcriptomic, proteomic, and immunohistochemical analyses, we examined the early (2 wk) effects of severe PH on the RV. We observed that RV remodeling during PH progression represents a continuum of transcriptionally driven processes whereby cardiac myocytes, fibroblasts, endothelial cells, and proremodeling macrophages act to coordinately maintain physiological homeostasis and protect myocyte survival during chronic, severe, and progressive pressure overload.

Hemoglobin Concentration Impacts Viscoelastic Hemostatic Assays in ICU Admitted Patients.

OBJECTIVES: Low hemoglobin concentration impairs clinical hemostasis across several diseases. It is unclear whether hemoglobin impacts laboratory functional coagulation assessments. We evaluated the relationship of hemoglobin concentration on viscoelastic hemostatic assays in intracerebral hemorrhage (ICH) and perioperative patients admitted to an ICU. DESIGN: Observational cohort study and separate in vitro laboratory study. SETTING: Multicenter tertiary referral ICUs. PATIENTS: Two acute ICH cohorts receiving distinct testing modalities: rotational thromboelastometry (ROTEM) and thromboelastography (TEG), and a third surgical ICU cohort receiving ROTEM were evaluated to assess the generalizability of findings across disease processes and testing platforms. A separate in vitro ROTEM laboratory study was performed utilizing ICH patient blood samples. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Relationships between baseline hemoglobin and ROTEM/TEG results were separately assessed across patient cohorts using Spearman correlations and linear regression models. A separate in vitro study assessed ROTEM tracing changes after serial hemoglobin modifications from ICH patient blood samples. In both our ROTEM (n = 34) and TEG (n = 239) ICH cohorts, hemoglobin concentrations directly correlated with coagulation kinetics (ROTEM r: 0.46; p = 0.01; TEG r: 0.49; p < 0.0001) and inversely correlated with clot strength (ROTEM r: -0.52, p = 0.002; TEG r: -0.40, p < 0.0001). Similar relationships were identified in perioperative ICU admitted patients (n = 121). We continued to identify these relationships in linear regression models. When manipulating ICH patient blood samples to achieve lower hemoglobin concentrations in vitro, we similarly identified that lower hemoglobin concentrations resulted in progressively faster coagulation kinetics and greater clot strength on ROTEM tracings. CONCLUSIONS: Lower hemoglobin concentrations have a consistent, measurable impact on ROTEM/TEG testing in ICU admitted patients, which appear to be artifactual. It is possible that patients with low hemoglobin may appear to have normal viscoelastic parameters when, in fact, they have a mild hypocoagulable state. Further work is required to determine if these tests should be corrected for a patient's hemoglobin concentration.