Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Differences in Acute Graft-Versus-Host Disease (GVHD) Severity and Its Outcomes Between Black and White Patients.

Acute graft-versus-host disease (GVHD) is a significant complication following hematopoietic stem cell transplantation (HCT). Although recent advancements in GVHD prophylaxis have resulted in successful HCT across HLA barriers and expanded access to HCT for racial minorities, less is known about how race affects the severity and outcomes of acute GVHD. This study examines differences in the clinical course of acute GVHD and the prognostic value of GVHD biomarkers for Black and White recipients. We conducted a retrospective analysis of patients in the Mount Sinai Acute GVHD International Consortium (MAGIC) database who underwent HCT between 2014 and 2021 to describe the difference in clinical course of acute GVHD and significance of GVHD biomarkers between Black and White recipients. We used propensity score matching to generate a 1:3 matched cohort of 234 Black patients and 702 White patients with similar baseline characteristics. In the first year after HCT Black patients experienced a higher cumulative incidence of grade III-IV acute GVHD (17% versus 12%, P = 0.050), higher nonrelapse mortality (NRM; 18% versus 12%, P = .009), and lower overall survival that trended toward statistical significance (73% versus 79%, P = .071) compared to White patients. The difference in NRM in the first year was even greater among Black patients who developed GVHD than White patients (24% versus 14%, P = .041). The distribution of low, intermediate, and high MAGIC biomarker scores at the time of treatment was similar across racial groups (P = .847), however, Black patients with high biomarker scores experienced significantly worse NRM than White patients (71% versus 32%, P = .010). Our data indicate that Black patients are at a higher risk of NRM following HCT, primarily from a higher incidence of severe GVHD. Serum biomarkers at treatment initiation can stratify patients for risk of NRM across races, however Black patients with high biomarker scores had a significantly greater NRM risk. These results suggest a need for strategies that mitigate the higher risk for poor GVHD outcomes among Black patients.

Inpatient Outcomes of Cerebral Venous Thrombosis in Patients With Malignancy Throughout the United States.

Background and Purpose: Cerebral venous thrombosis (CVT) is associated with a high degree of morbidity and mortality. Our objective is to elucidate characteristics, treatments, and outcomes of patients with cancer and CVT (CA-CVT). Methods: The 2016-2019 National Inpatient Sample (NIS) database was queried for patients with a primary diagnosis of CVT. Patients with a currently active diagnosis of malignancy (CA-CVT) were then identified. Demographics and comorbidities were compared between CA-CVT and CVT patients. Subgroup analyses explored patients with hematopoietic cancer and non-hematopoietic cancers. Stroke severity and treatment were explored. Inpatient outcomes studied were discharge disposition, length of stay, and mortality. Results: Between 2016 and 2019, 6,140 patients had a primary diagnosis code of CVT, and 370 (6.0%) patients had a coexisting malignancy. The most common malignancy was hematopoietic (n=195, 52.7%), followed by central nervous system (n=40, 10.8%), respiratory (n=40, 10.8%), and breast (n=40, 10.8%). These patients tended to be older than non-CA-CVT and were more likely to have coexisting comorbidities. CA-CVT patients had higher severity scores on the International Study of Cerebral Vein and Dural Sinus Thrombosis Risk Score (ISCVT-RS) and increased complications. In a propensity-score matched cohort, there were no differences in inpatient outcomes. Conclusion: Malignancy occurs in 6% of patients presenting with CVT and should be considered a potential comorbidity in instances where clear causes of hypercoagulabilty have not been identified. Malignancy was linked to higher mortality rates. Nonetheless, after adjusting for the severity of CVT, the outcomes for inpatients with cancer-associated CVT were comparable to those without cancer, indicating that the increased mortality associated with malignancy is probably due to more severe CVT conditions.

TASER 7 and TASER 10 Probe Penetration Into Human Skull Simulants.

ABSTRACT: The older generation TASER probes X26 and X2 have been shown to be capable of piercing the skull with their tips. With the introduction of the new TASER 7 and the far more powerful TASER 10, concerns have arisen as to whether these weapons might penetrate the skull more deeply and thus prove to be potentially lethal. For this reason, we tested the penetration capacity of these weapons on polyurethane-gelatine-buckskin head simulants at different firing distances. The striking speeds and striking angles were documented with a high-speed camera, and the piercing depths were recorded by computed tomography. None of the probes penetrated the skull, but their tips did; TASER 7 probe tips pierced up to 5.6 mm, whereas TASER 10 probe tips pierced up to 10.4 mm. The TASER 7 probes fared better with regard to penetration depth at shorter firing distances; on the other hand, the TASER 10 probes pierced more deeply at distances of 3 to 4 m, with their flight stability improving after the first 2 m. Our results imply that TASER 7 and TASER 10 probes are not to be expected to cause great harm or even death when striking the head.

The resisted torsional stress test in diagnosis of femoral stem loosening in uncemented total hip arthroplasty - first description and retrospective study.

INTRODUCTION: Aseptic femoral stem loosening is among the most common causes for revision in total hip arthroplasty (THA). We describe a simple clinical test that triggers pain in the proximal femur in patients with a loose stem. A previously described passive rotation test was associated with a poor sensitivity. The resisted torsional stress test (RTST) was used for several years in our hospital, and this is the first description of its reliability. METHODS: We retrospectively reviewed our database of uncemented stem revisions. Preoperative clinical reports were searched for data on the RTST. A positive RTST was defined as sharp pain felt at the stem level with active internal rotation against a passive external rotation impulse in 90° hip flexion. The definition of stem fixation (fixed vs. loose) was made by readout of the surgery reports. RESULTS: The RTST was reported in 83 cases and was positive in 32 of the 43 stems, which were found loose intraoperatively and in 9 of the 40 stems, which were well integrated. This leads to an accuracy of classification of 79.5%. The sensitivity was 80% and the specificity was 79.1%. PPV and NPV were 78% and 81%, respectively. CONCLUSION: The RTST provides a helpful tool in the clinical assessment of femoral stem fixation in THA with good accuracy and should be included in standard follow-up examinations and in the assessment of painful THAs.

Acute Myocardial Injury in Spontaneous Intracerebral Hemorrhage: A Secondary Observational Analysis of the FAST Trial.

BACKGROUND: Acute myocardial injury is associated with poor outcomes in patients with acute ischemic stroke, but its prognostic significance in patients with spontaneous intracerebral hemorrhage remains unclear. We investigated whether acute myocardial injury and the direction of the cardiac troponin I (cTnI) change (rising versus falling) affect post-intracerebral hemorrhage outcomes. METHODS AND RESULTS: We re-analyzed the FAST (Factor-Seven-for-Acute-Hemorrhagic-Stroke) trial. Acute myocardial injury was defined as at least 1 cTnI value above the upper reference limit with a rise/fall of >20%. Logistic regression tested for associations (1) between acute myocardial injury (presence versus absence) and poor outcome (modified Rankin Scale 4-6) and mortality at 15 and 90 days; (2) among 3 groups (rising versus falling versus no acute myocardial injury) and outcomes. Among the 841 FAST participants, 785 patients were included. Acute myocardial injury was detected in 29% (n=227); 170 had rising cTnI. At 15 and 90 days, respectively, those with acute myocardial injury had higher odds of poor outcome (adjusted odds ratio) ([aOR] 2.3 [95% CI, 1.3-3.9]); and adjusted odds ratio 2.5 [95% CI, 1.6-3.9];, and higher odds of mortality (adjusted odds ratio 2.4 [95% CI, 1.4-4.3]; and adjusted odds ratio 2.2 [CI, 1.3-3.6]) than patients without. There was no interaction between FAST group assignment and myocardial injury, and associations between myocardial injury and outcomes were consistent across group assignments. Rising cTnI was associated with the highest risk of poor outcomes and mortality. CONCLUSIONS: In this secondary analysis of the FAST trial, acute myocardial injury was common and associated with poor outcomes. The direction of the cTnI change might provide additional risk stratification after intracerebral hemorrhage.

Ventilation during cardiopulmonary resuscitation: A narrative review.

Ventilation during cardiopulmonary resuscitation is vital to achieve optimal oxygenation but continues to be a subject of ongoing debate. This narrative review aims to provide an overview of various components and challenges of ventilation during cardiopulmonary resuscitation, highlighting key areas of uncertainty in the current understanding of ventilation management. It addresses the pulmonary pathophysiology during cardiac arrest, the importance of adequate alveolar ventilation, recommendations concerning the maintenance of airway patency, tidal volumes and ventilation rates in both synchronous and asynchronous ventilation. Additionally, it discusses ventilation adjuncts such as the impedance threshold device, the role of positive end-expiratory pressure ventilation, and passive oxygenation. Finally, this review offers directions for future research.

Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes.

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.

REACT: a randomized trial to assess the efficacy and safety of clazosentan for preventing clinical deterioration due to delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Ischemic complications account for significant patient morbidity following aneurysmal subarachnoid hemorrhage (aSAH). The Prevention and Treatment of Vasospasm with Clazosentan (REACT) study was designed to assess the safety and efficacy of clazosentan, an endothelin receptor antagonist, in preventing clinical deterioration due to delayed cerebral ischemia (DCI) in patients with aSAH. METHODS: REACT was a prospective, multicenter, randomized, double-blind, phase 3 study. Eligible patients had aSAH secured by surgical clipping or endovascular coiling, and had presented with thick and diffuse clot on admission CT scan. Patients were randomized (1:1 ratio) to 15 mg/hour intravenous clazosentan or placebo within 96 hours of the aSAH for up to 14 days, in addition to standard of care treatment including oral or intravenous nimodipine. The primary efficacy endpoint was the occurrence of clinical deterioration due to DCI up to 14 days after initiation of the study drug. The main secondary endpoint was the occurrence of clinically relevant cerebral infarction at day 16 after study drug initiation. Other secondary endpoints included clinical outcome assessed on the modified Rankin Scale (mRS) and the Glasgow Outcome Scale-Extended (GOSE) at week 12 post-aSAH. Imaging and clinical endpoints were centrally adjudicated. RESULTS: A total of 409 patients were randomized between February 2019 and May 2022 across 74 international sites. Three patients did not start study treatment and were not included in the analysis set. The occurrence of clinical deterioration due to DCI was 15.8% (32/202 patients) in the clazosentan group and 17.2% (35/204 patients) in the placebo group, and the difference was not statistically significant (relative risk reduction [RRR] 7.2%, 95% CI -42.6% to 39.6%, p = 0.734). A nonsignificant RRR of 34.1% (95% CI -21.3% to 64.2%, p = 0.177) was observed in clinically relevant cerebral infarcts treated with clazosentan (7.4%, 15/202) versus placebo (11.3%, 23/204). Rescue therapy was less frequently needed for patients treated with clazosentan compared to placebo (10.4%, 21/202 vs 18.1%, 37/204; RRR 42.6%, 95% CI 5.4%-65.2%). A nonsignificant relative risk increase of 25.4% (95% CI -10.7% to 76.0%, p = 0.198) was reported in the risk of poor GOSE and mRS scores with clazosentan (24.8%, 50/202) versus placebo (20.1%, 41/204) at week 12 post-aSAH. Treatment-emergent adverse events were similar to those reported previously. CONCLUSIONS: Clazosentan administered for up to 14 days at 15 mg/hour had no significant effect on the occurrence of clinical deterioration due to DCI. Clinical trial registration no.: NCT03585270 (ClinicalTrials.gov) EU clinical trial registration no.: 2018-000241-39 (clinicaltrialsregister.eu).

In Situ Biofilm Affinity-Based Protein Profiling Identifies the Streptococcal Hydrolase GbpB as the Target of a Carolacton-Inspired Chemical Probe.

Natural products are important precursors for antibiotic drug design. These chemical scaffolds serve as synthetic inspiration for chemists who leverage their structures to develop novel antibacterials and chemical probes. We have previously studied carolacton, a natural product macrolactone fromSorangium cellulosum, and discovered a simplified derivative, A2, that maintained apparent biofilm inhibitory activity, although the biological target was unknown. Herein, we utilize affinity-based protein profiling (AfBPP) in situ during biofilm formation to identify the protein target using a photoexcitable cross-linking derivative of A2. From these studies, we identified glucan binding protein B (GbpB), a peptidoglycan hydrolase, as the primary target of A2. Further characterization of the interaction between A2 and GbpB, as well as PcsB, a closely related homologue from the more pathogenic S. pneumoniae, revealed binding to the catalytic CHAP (cysteine, histidine, aminopeptidase) domain. To the best of our knowledge, this is the first report of a small-molecule binder of a conserved and essential bacterial CHAP hydrolase, revealing its potential as an antibiotic target. This work also highlights A2 as a useful tool compound for streptococci and as an initial scaffold for the design of more potent CHAP binders.

Efficacy of subcutaneous sumatriptan in postcraniotomy pain and opioid consumption.

OBJECTIVE: Traditional pain management pathways following craniotomy are predicated on opioids. However, narcotics can confound critical neurological examination, contribute to respiratory depression, lower the seizure threshold, and lead to medication habituation, dependence, and/or abuse. Alternative medications to better address postoperative pain while mitigating opioid-related adverse effects remain insufficiently studied. Preliminary studies suggest sumatriptan, a 5-HT (1B/1D) receptor agonist known to regulate dural vasoactivity and inflammation, may moderate pain following trigeminal microvascular decompression and chronic postcraniotomy headache. In this study, the authors evaluated the efficacy of sumatriptan to modulate pain and opioid requirements following craniotomy surgery. METHODS: This was a single academic center, retrospective cohort study of 300 consecutive adult patients who underwent elective craniotomy surgery between 2015 and 2022. Patients were equally divided between a control and a sumatriptan cohort contingent upon administration of 6 mg of subcutaneous sumatriptan within 1 hour of surgery completion and prior to opioid administration. Postoperative opioid consumption at 6, 12, and 24 hours, as well as admission total, inpatient length of stay, and 30-day global reevaluation, were assessed. RESULTS: Three hundred patients were included for analysis. Significant differences were seen in baseline hypertension (p < 0.01), hyperlipemia (p < 0.01), anxiety (p = 0.04), and operative time (p = 0.02). A significant reduction of mean postoperative pain scores at 12 (p = 0.03) and 24 (p < 0.01) hours and total opioid consumption (p = 0.04) was observed in the sumatriptan cohort. Subgroup analysis revealed significantly lower postoperative pain scores at 6 (p = 0.05), 12 (p < 0.01), and 24 (p < 0.01) hours in patients who underwent burr hole placement in the sumatriptan cohort as compared with controls; however, no significant difference in opioid consumption was noted. No adverse events related to sumatriptan administration were noted throughout the study. CONCLUSIONS: Postoperative single-dose subcutaneous sumatriptan following elective craniotomy may reduce pain scores and opioid requirements. Additional studies are needed to better understand nuanced differences in opioid modulation and optimal patient selection.

Code-ICH: A New Paradigm for Emergency Intervention.

PURPOSE OF REVIEW: Intracerebral hemorrhage (ICH) is the most devastating type of stroke, causing widespread disability and mortality. Unfortunately, the acute care of ICH has lagged behind that of ischemic stroke. There is an increasing body of evidence supporting the importance of early interventions including aggressive control of blood pressure and reversal of anticoagulation in the initial minutes to hours of presentation. This review highlights scientific evidence behind a new paradigm to care for these patients called Code-ICH. RECENT FINDINGS: While numerous trials aimed at decreasing hematoma expansion through single interventions had failed to show statistically significant effects on primary outcomes, time-sensitive, multifaceted, bundled care approaches have recently shown substantial promise in improving functional outcomes in patients with ICH. The concept of Code-ICH can serve as a structural platform for the practice of acute care neurology to continuously measure its performance, reflect on best practices, advance care, and address disparities.

Screening Privileged Alkyl Guanidinium Motifs under Host-Mimicking Conditions Reveals a Novel Antibiotic with an Unconventional Mode of Action.

Screening large molecule libraries against pathogenic bacteria is often challenged by a low hit rate due to limited uptake, underrepresentation of antibiotic structural motifs, and assays that do not resemble the infection conditions. To address these limitations, we present a screen of a focused library of alkyl guanidinium compounds, a structural motif associated with antibiotic activity and enhanced uptake, under host-mimicking infection conditions against a panel of disease-associated bacteria. Several hit molecules were identified with activities against Gram-positive and Gram-negative bacteria, highlighting the fidelity of the general concept. We selected one compound (L15) for in-depth mode of action studies that exhibited bactericidal activity against methicillin-resistant Staphylococcus aureus USA300 with a minimum inhibitory concentration of 1.5 µM. Structure-activity relationship studies confirmed the necessity of the guanidinium motif for antibiotic activity. The mode of action was investigated using affinity-based protein profiling with an L15 probe and identified the signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification, docking, and molecular dynamics simulations demonstrated SpsB activation by L15, a recently described mechanism leading to the dysregulation of protein secretion and cell death. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.

Comparison of R2* and FerriScan liver iron concentration (LIC) quantification in the clinical classification of Iron overload states.

PURPOSE: This study assessed the clinical classification performance of an R2*-based MRI technique for LIC quantification relative to FerriScan, with intra-patient FerriScan LIC uncertainty taken into account. The variabilities of R2* and FerriScan LIC were also assessed. MATERIALS AND METHODS: This was an ethics approved retrospective study, performed on patients undergoing chelation treatment for iron overload. 126 patients (69 women, 57 men), with an age of 42 +/- 16 years (range 19-86 years) were included. FerriScan and R2* MRI at 1.5 T were performed as part of a routine liver iron assessment protocol. For R2* MRI, a commercially available pulse sequence and reconstruction implementation was used, together with a previously derived calibration curve to convert R2* to LIC. Clinical classifications arising from R2*-derived LIC estimates were compared to those based on FerriScan. The accuracy and precision of the R2* technique was calculated. The variabilities of FerriScan- and R2*-derived estimates of LIC were compared with a Wilcoxon Signed Rank test. Significance was set at the 95% confidence level. RESULTS: The precision of R2* ranged from 0.59 to 0.92, with an overall accuracy of 72%. When intra-patient FerriScan LIC uncertainty was considered, precision and accuracy increased to >94% and 97% respectively. The R2*-LIC variability (=17%) was significantly lower than the FerriScan-LIC variability (34%) at the 95% confidence level (p < 10-3). CONCLUSION: MRI R2*-based LIC estimates provided a similar clinical classification as FerriScan. The intra-patient uncertainty of R2*-based LIC estimates was significantly lower than FerriScan.

Unraveling the mechanism of small molecule induced activation of Staphylococcus aureus signal peptidase IB.

Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators.

Target identification of usnic acid in bacterial and human cells.

Usnic acid is a natural product with versatile biological activities against various organisms. Here, we utilise a chemical proteomic strategy to gain insights into its target scope in bacterial and human cells. First, we excluded DNA binding as a major reason for its antibacterial activity, and second, we commenced with target profiling, which unravelled several metal cofactor-dependent enzymes in both species indicating a polypharmacological mode of action. Interestingly, our synthetic studies revealed a selectivity switch at usnic acid, which maintains antibacterial activity but lacks strong cytotoxic effects.

Detection of colorectal-cancer-associated bacterial taxa in fecal samples using next-generation sequencing and 19 newly established qPCR assays.

We have previously identified increased levels of distinct bacterial taxa within mucosal biopsies from colorectal cancer (CRC) patients. Following prior research, the aim of this study was to investigate the detection of the same CRC-associated bacteria in fecal samples and to evaluate the suitability of fecal samples as a non-invasive material for the detection of CRC-associated bacteria. Next-generation sequencing (NGS) of the 16S ribosomal RNA (rRNA) V4 region was performed to evaluate the detection of the CRC-associated bacteria in the fecal microbiota of cancer patients, patients with adenomatous polyp and healthy controls. Furthermore, 19 novel species-specific quantitative PCR (qPCR) assays were established to detect the CRC-associated bacteria. Approximately, 75% of the bacterial taxa identified in biopsies were reflected in fecal samples. NGS failed to detect low-abundance CRC-associated taxa in fecal samples, whereas qPCR exhibited high sensitivity and specificity in identifying all targeted taxa. Comparison of fecal microbial composition between the different patient groups showed enrichment of Fusobacterium nucleatum, Parvimonas micra, and Gemella morbillorum in cancer patients. Our findings suggest that low-abundance mucosa-associated bacteria can be detected in fecal samples using sensitive qPCR assays.