Vascular Proteome Responses Precede Organ Dysfunction in a Murine Model of Staphylococcus aureus Bacteremia.

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected, clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular cross talk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of Staphylococcus aureus sepsis by combining in vivo labeling of the endothelial cell surface proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. These responses include abundant vascular shedding, dysregulation of the intrinsic pathway of coagulation, compartmentalization of the acute phase response, and abundant upregulation of glycocalyx components. Vascular cell surface proteome changes were also found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggest a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis. IMPORTANCE Sepsis is a life-threatening response to infection that results in immune dysregulation, vascular dysfunction, and organ failure. New methods are needed for the identification of diagnostic and therapeutic targets. Here, we took a systems-wide approach using data-independent acquisition (DIA) mass spectrometry to track the progression of bacterial sepsis in the vasculature leading to organ failure. Using a murine model of S. aureus sepsis, we were able to quantify thousands of proteins across the plasma and parenchymal and vascular compartments of multiple organs in a time-resolved fashion. We showcase the profound proteome remodeling triggered by sepsis over time and across these compartments. Importantly, many vascular proteome alterations precede changes in traditional correlates of organ dysfunction, opening a molecular window for the discovery of early markers of sepsis progression.

Endothelial Heparan Sulfate Mediates Hepatic Neutrophil Trafficking and Injury during Staphylococcus aureus Sepsis.

Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

Proteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsis.

Sepsis is a life-threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction, organ failure and death. Here we provide a semi-quantitative atlas of the murine vascular cell-surface proteome at the organ level, and how it changes during sepsis. Using in vivo chemical labeling and high-resolution mass spectrometry, we demonstrate the presence of a vascular proteome that is perfusable and shared across multiple organs. This proteome is enriched in membrane-anchored proteins, including multiple regulators of endothelial barrier functions and innate immunity. Further, we automated our workflows and applied them to a murine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis to unravel changes during systemic inflammatory responses. We provide an organ-specific atlas of both systemic and local changes of the vascular proteome triggered by sepsis. Collectively, the data indicates that MRSA-sepsis triggers extensive proteome remodeling of the vascular cell surfaces, in a tissue-specific manner.

ApoC-III ASO promotes tissue LPL activity in the absence of apoE-mediated TRL clearance.

Hypertriglyceridemia results from accumulation of triglyceride (TG)-rich lipoproteins (TRLs) in the circulation and is associated with increased CVD risk. ApoC-III is an apolipoprotein on TRLs and a prominent negative regulator of TG catabolism. We recently established that in vivo apoC-III predominantly inhibits LDL receptor-mediated and LDL receptor-related protein 1-mediated hepatic TRL clearance and that apoC-III-enriched TRLs are preferentially cleared by syndecan-1 (SDC1). In this study, we determined the impact of apoE, a common ligand for all three receptors, on apoC-III metabolism using apoC-III antisense oligonucleotide (ASO) treatment in mice lacking apoE and functional SDC1 (Apoe-/-Ndst1f/fAlb-Cre+). ApoC-III ASO treatment significantly reduced plasma TG levels in Apoe-/-Ndst1f/fAlb-Cre+ mice without reducing hepatic VLDL production or improving hepatic TRL clearance. Further analysis revealed that apoC-III ASO treatment lowered plasma TGs in Apoe-/-Ndst1f/fAlb-Cre+ mice, which was associated with increased LPL activity in white adipose tissue in the fed state. Finally, clinical data confirmed that ASO-mediated lowering of APOC-III via volanesorsen can reduce plasma TG levels independent of the APOE isoform genotype. Our data indicate that apoE determines the metabolic impact of apoC-III as we establish that apoE is essential to mediate inhibition of TRL clearance by apoC-III and that, in the absence of functional apoE, apoC-III inhibits tissue LPL activity.

Epidermis and Enamel: Insights Into Gnawing Criticisms of Human Bitemark Evidence.

Critics describe forensic dentists' management of bitemark evidence as junk science with poor sensitivity and specificity and state that linkages to a biter are unfounded. Those vocal critics, supported by certain media, characterize odontologists' previous errors as egregious and petition government agencies to render bitemark evidence inadmissible. Odontologists acknowledge that some practitioners have made past mistakes. However, it does not logically follow that the errors of a few identify a systemic failure of bitemark analysis. Scrutiny of the contentious cases shows that most occurred 20 to 40 years ago. Since then, research has been ongoing and more conservative guidelines, standards, and terminology have been adopted so that past errors are no longer reflective of current safeguards. The authors recommend a comprehensive root analysis of problem cases to be used to determine all the factors that contributed to those previous problems. The legal community also shares responsibility for some of the past erroneous convictions. Currently, most proffered bitemark cases referred to odontologists do not reach courts because those forensic dentists dismiss them as unacceptable or insufficient for analysis. Most bitemark evidence cases have been properly managed by odontologists. Bitemark evidence and testimony remain relevant and have made significant contributions in the justice system.

Bite-Mark and Pattern Injury Analysis: A Brief Status Overview.

Bite marks are one component of forensic investigation requiring subjective interpretation for determining unknown source evidence to a putative suspect. Recent scrutiny has lead to questions about the scientific validity of patterned evidence, bite-mark analysis in particular, and its role in judicial proceedings. This article discusses some issues that persist in forensic circles and the difficulties surrounding the field of bite-mark analysis that inherently must employ human subjectivity in its execution of duty.

Vitamin D treatment modulates organic dust-induced cellular and airway inflammatory consequences.

Exposure to organic dusts elicits airway inflammatory diseases. Vitamin D recently has been associated with various airway inflammatory diseases, but its role in agricultural organic dust exposures is unknown. This study investigated whether vitamin D reduces organic dust-induced inflammatory outcomes in cell culture and animal models. Organic dust extracts obtained from swine confinement facilities induced neutrophil chemokine production (human IL-8, murine CXCL1/CXCL2). Neutrophil chemokine induction was reduced in human blood monocytes, human bronchial epithelial cells, and murine lung slices pretreated with 1,25-(OH)(2) D(3) . Intranasal inhalation of organic dust extract induced neutrophil influx, and CXCL1/CXCL2 release was also decreased in mice fed a relatively high vitamin D diet as compared to mice fed a low vitamin D diet. These findings were associated with reduced tracheal epithelial cell PKCα and PKCε activity and whole lung TLR2 and TLR4 gene expression. Collectively, vitamin D plays a role in modulating organic dust-induced airway inflammatory outcomes.

Toll-like receptor 2 regulates organic dust-induced airway inflammation.

Organic dust exposure in agricultural environments results in significant airway inflammatory diseases. Gram-positive cell wall components are present in high concentrations in animal farming dusts, but their role in mediating dust-induced airway inflammation is not clear. This study investigated the role of Toll-like receptor (TLR) 2, a pattern recognition receptor for gram-positive cell wall products, in regulating swine facility organic dust extract (DE)-induced airway inflammation in mice. Isolated lung macrophages from TLR2 knockout mice demonstrated reduced TNF-α, IL-6, keratinocyte chemoattractant/CXCL1, but not macrophage inflammatory protein-2/CXCL2 expression, after DE stimulation ex vivo. Next, using an established mouse model of intranasal inhalation challenge, we analyzed bronchoalveolar lavage fluid and lung tissue in TLR2-deficient and wild-type (WT) mice after single and repetitive DE challenge. Neutrophil influx and select cytokines/chemokines were significantly lower in TLR2-deficient mice at 5 and 24 hours after single DE challenge. After daily exposure to DE for 2 weeks, there were significant reductions in total cellularity, neutrophil influx, and TNF-α, IL-6, CXCL1, but not CXCL2 expression, in TLR2-deficient mice as compared with WT animals. Lung pathology revealed that bronchiolar inflammation, but not alveolar inflammation, was reduced in TLR2-deficient mice after repetitive exposure. Airway hyperresponsiveness to methacholine after dust exposure was similar in both groups. Finally, airway inflammatory responses in WT mice after challenge with a TLR2 agonist, peptidoglycan, resembled DE-induced responses. Collectively, these results demonstrate that the TLR2 pathway is important in regulating swine facility organic dust-induced airway inflammation, which suggests the importance of TLR2 agonists in mediating large animal farming-induced airway inflammatory responses.

The use of full spectrum digital photography for evidence collection and preservation in cases involving forensic odontology.

Photography often represents the best method to collect and preserve evidence in forensic cases. This is especially true in forensic odontology with cases involving dental identification, human abuse and, perhaps most significantly, bitemark cases. Basic visible light photography is adequate in most dental identification cases; however, full spectrum digital photography is best utilized to collect all available evidence in cases of human abuse and bitemarks. This paper will discuss the types of photographic evidence that should be collected with various forensic odontological cases and the specific techniques utilized in full spectrum forensic digital photography. The use of full spectrum photography captures the forensic injuries using special techniques recording the injuries in each of the four resultant events that occur when light strikes skin.

Analysis of a quantitative trait locus for seizure susceptibility in mice using bacterial artificial chromosome-mediated gene transfer.

PURPOSE: Previous quantitative trait loci (QTL) mapping studies from our laboratory identified a 6.6 Mb segment of distal chromosome 1 that contains a gene (or genes) having a strong influence on the difference in seizure susceptibility between C57BL/6 (B6) and DBA/2 (D2) mice. A gene transfer strategy involving a bacterial artificial chromosome (BAC) DNA construct that contains several candidate genes from the critical interval was used to test the hypothesis that a strain-specific variation in one (or more) of the genes is responsible for the QTL effect. METHODS: Fertilized oocytes from a seizure-sensitive congenic strain (B6.D2-Mtv7a/Ty-27d) were injected with BAC DNA and three independent founder lines of BAC-transgenic mice were generated. Seizure susceptibility was quantified by measuring maximal electroshock seizure threshold (MEST) in transgenic mice and nontransgenic littermates. RESULTS: Seizure testing documented significant MEST elevation in all three transgenic lines compared to littermate controls. Allele-specific RT-PCR analysis confirmed gene transcription from genome-integrated BAC DNA and copy-number-dependent phenotypic effects were observed. CONCLUSIONS: Results of this study suggest that the gene(s) responsible for the major chromosome 1 seizure QTL is found on BAC RPCI23-157J4 and demonstrate the utility of in vivo gene transfer for studying quantitative trait genes in mice. Further characterization of this transgenic model will provide new insight into mechanisms of seizure susceptibility.

Acupuncture normalizes the release of accumbal dopamine during the withdrawal period and after the ethanol challenge in chronic ethanol-treated rats.

Many studies have shown that acupuncture can contribute to the biochemical balance in the central nervous system and maintenance or recovery of homeostasis. It is well known that chronic administration of ethanol may produce depletion or sensitization of extracellular dopamine levels in the nucleus accumbens. The present study was designed to investigate the effects of acupuncture on chronic ethanol-induced changes in extracellular dopamine levels in the nucleus accumbens shell (using in vivo microdialysis in unanesthetized rats). Male Sprague-Dawley rats were treated with 3 g/kg/day of ethanol (20%, w/v) or saline by intraperitoneal injection for 21 days. Following 72 h of ethanol withdrawal, acupuncture was applied at bilateral Shenmen (HT7) points for 1 min. Different group of rats using the same paradigm of ethanol treatment were acupunctured at the same points after the systemic ethanol challenge (3 g/kg, i.p.). Acupuncture at the specific acupoint HT7, but not at control points (PC6 or tail) significantly prevented both a decrease of extracellular dopamine levels in the nucleus accumbens during ethanol withdrawal and an increase in accumbal dopamine levels induced by the ethanol challenge. These results provided strong evidence that stimulation of the specific acupoint HT7 helps to normalize the release of dopamine in the mesolimbic system following chronic ethanol treatment.

Effect of acupuncture on behavioral hyperactivity and dopamine release in the nucleus accumbens in rats sensitized to morphine.

Acupuncture as a therapeutic intervention has been used for the treatment of many functional disorders including substance abuse. However, there are still many unanswered question about the basic mechanism underlying acupuncture's effectiveness in the treatment of drug addiction. Repeated injection of psycostimulants or morphine can produce behavioral and neurochemical sensitization and have been used as a model for studying drug addiction. The present study was designed to investigate the effect of acupuncture on repeated morphine-induced changes in extracellular dopamine levels using in vivo microdialysis and repeated morphine-induced behavioral changes. Male Sprague-Dawley rats were treated with saline or increasing doses of morphine (10, 20 and 40 mg/kg, s.c., twice daily for 3 days). Following 15 days of withdrawal, acupuncture was applied at bilateral Shenmen (HT7) points for 1 min after the systemic challenge with morphine HCl (5 mg/kg, s.c.). Results showed that acupuncture at the specific acupoint HT7, but not at control points (TE8 and tail) significantly decreased both dopamine release in the nucleus accumbens and behavioral hyperactivity induced by a systemic morphine challenge. These results suggest that the therapeutic effect of acupuncture on morphine addiction occurs through inhibition of neurochemical and behavioral sensitization to morphine.

Confirmation of a major QTL influencing oral morphine intake in C57 and DBA mice using reciprocal congenic strains.

C57BL/6 (B6) and DBA/2 (D2) mice exhibit disparate behavior when tested for voluntary morphine intake in a two-bottle choice drinking paradigm with B6 mice consuming 10 times more drug than D2 mice. Previous genetic mapping studies identified a locus, Mop2, on the proximal part of chromosome 10 that explained over half of the genetic variance in this mouse model of opioid self-administration. We constructed a set of reciprocal congenic strains between B6 and D2 mice in which the proximal portion of chromosome 10 has been introgressed from one strain onto the background of the other. We tested mice from this pair of reciprocal strains together with progenitor B6 and D2 mice in a two-bottle choice drinking paradigm with morphine and quinine. The results showed that introgression of chromosome 10 alleles from the B6 strain onto a D2 genetic background increased voluntary morphine intake four-fold compared to progenitor D2 mice. Preference for morphine was also increased significantly in D2.B6-Mop2 mice compared to progenitor D2 mice. Conversely, introgression of chromosome 10 alleles from the D2 strain onto a B6 genetic background decreased morphine intake by half compared to progenitor B6 mice in B6.D2 -Mop2 mice; however, high morphine preference was maintained in this congenic strain most likely due to strong quinine aversion. When quinine was eliminated from the control bottle, morphine preference in B6.D2-Mop2 mice was decreased significantly relative to B6 and D2.B6-Mop2 mice. Overall, these data confirm the existence of a gene(s) on chromosome 10 proximal to D10Mit124 that has a strong influence on the difference in morphine drinking behavior between B6 and D2 mice.

Lessons learned from the WTC disaster: a first-person account.

Sept. 11, 2001, was a day of learning for the world. We learned oceans do not isolate the United States from acts of terrorism. Prior to hijackers flying commercial airplanes into the Pentagon and World Trade Center towers, forensic investigators had never dealt with a mass fatality incident of this magnitude on the mainland soil of the U.S. during non-wartime conditions. During the process of gearing up for the task of locating and identifying the victims, forensic dentists also learned a number of things. Established techniques for disaster management were tested to their extreme limits. Before and while this multi-functional effort was taking place, we familiarized ourselves with what techniques worked best under the immense pressure and tedious nature this job presented. Some of these accepted and now "tried and true" protocols are presented in this article.