Variants in the Kallikrein Gene Family and Hypermobile Ehlers-Danlos Syndrome.

Hypermobile Ehlers-Danlos syndrome (hEDS) is a common heritable connective tissue disorder that lacks a known genetic etiology. To identify genetic contributions to hEDS, whole exome sequencing was performed on families and a cohort of sporadic hEDS patients. A missense variant in Kallikrein-15 (KLK15 p. Gly226Asp ) , segregated with disease in two families and genetic burden analyses of 197 sporadic hEDS patients revealed enrichment of variants within the Kallikrein gene family. To validate pathogenicity, the variant identified in familial studies was used to generate knock-in mice. Consistent with our clinical cohort, Klk15 G224D/+ mice displayed structural and functional connective tissue defects within multiple organ systems. These findings support Kallikrein gene variants in the pathogenesis of hEDS and represent an important step towards earlier diagnosis and better clinical outcomes.

CRISPR-Cas9-mediated barcode insertion into Bacillus thuringiensis for surrogate tracking.

The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded B. thuringiensis strains for use in future studies on surrogate genomes. IMPORTANCE: The use of Bacillus anthracis as a biothreat agent poses significant challenges for public health and national security. Bacillus anthracis surrogates, like Bacillus thuringiensis, are invaluable tools for safely understanding Bacillus anthracis properties without the safety concerns that would arise from using a virulent strain of Bacillus anthracis. We report a simple method for barcode insertion into Bacillus thuringiensis using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in Bacillus thuringiensis suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded Bacillus thuringiensis surrogate strains, among other biotechnological applications in Bacillus species.

Extracorporeal Carbon Dioxide Removal With the Hemolung in Patients With Acute-on-Chronic Respiratory Failure: A Multicenter Retrospective Cohort Study.

Extracorporeal carbon dioxide removal (ECCO2R) devices are increasingly used in treating acute-on-chronic respiratory failure caused by chronic lung diseases. There are no large studies that investigated safety, efficacy, and the independent association of prognostic variables to survival that could define the role of ECCO2R devices in such patients. This multicenter, multinational, retrospective study investigated the efficacy, safety of a single ECCO2R device (Hemolung) in patients with acute on chronic respiratory failure and identified variables independently associated with intensive care unit (ICU) survival. The primary outcome was improvement in blood gasses with the use of Hemolung. Secondary outcomes included reduction in tidal volume, respiratory rate, minute ventilation, survival to ICU discharge, and complication profile. Multivariable regression analysis was used to identify variables that are independently associated with ICU survival. A total of 62 patients were included. There was a significant improvement in pH and partial pressure of carbon dioxide in arterial blood (PaCO2) along with a reduction in respiratory rate, tidal volume, and minute ventilation with Hemolung therapy. The complication profile did not differ between survivors and nonsurvivors. Multivariable analysis identified the duration of Hemolung therapy to be independently associated with survival to ICU discharge (adjusted odds ratio = 1.21; 95% confidence interval [CI] = 1.040-1.518; p = 0.01).

The choroid plexus maintains adult brain ventricles and subventricular zone neuroblast pool, which facilitates poststroke neurogenesis.

The brain's neuroreparative capacity after injuries such as ischemic stroke is partly contained in the brain's neurogenic niches, primarily the subventricular zone (SVZ), which lies in close contact with the cerebrospinal fluid (CSF) produced by the choroid plexus (ChP). Despite the wide range of their proposed functions, the ChP/CSF remain among the most understudied compartments of the central nervous system (CNS). Here, we report a mouse genetic tool (the ROSA26iDTR mouse line) for noninvasive, specific, and temporally controllable ablation of CSF-producing ChP epithelial cells to assess the roles of the ChP and CSF in brain homeostasis and injury. Using this model, we demonstrate that ChP ablation causes rapid and permanent CSF volume loss in both aged and young adult brains, accompanied by disruption of ependymal cilia bundles. Surprisingly, ChP ablation did not result in overt neurological deficits at 1 mo postablation. However, we observed a pronounced decrease in the pool of SVZ neuroblasts (NBs) following ChP ablation, which occurs due to their enhanced migration into the olfactory bulb. In the middle cerebral artery occlusion model of ischemic stroke, NB migration into the lesion site was also reduced in the CSF-depleted mice. Thus, our study establishes an important role of ChP/CSF in regulating the regenerative capacity of the adult brain under normal conditions and after ischemic stroke.

News Media Framing of Suicide Circumstances and Gender: Mixed Methods Analysis.

BACKGROUND: Suicide is a leading cause of death worldwide. Journalistic reporting guidelines were created to curb the impact of unsafe reporting; however, how suicide is framed in news reports may differ by important characteristics such as the circumstances and the decedent's gender. OBJECTIVE: This study aimed to examine the degree to which news media reports of suicides are framed using stigmatized or glorified language and differences in such framing by gender and circumstance of suicide. METHODS: We analyzed 200 news articles regarding suicides and applied the validated Stigma of Suicide Scale to identify stigmatized and glorified language. We assessed linguistic similarity with 2 widely used metrics, cosine similarity and mutual information scores, using a machine learning-based large language model. RESULTS: News reports of male suicides were framed more similarly to stigmatizing (P<.001) and glorifying (P=.005) language than reports of female suicides. Considering the circumstances of suicide, mutual information scores indicated that differences in the use of stigmatizing or glorifying language by gender were most pronounced for articles attributing legal (0.155), relationship (0.268), or mental health problems (0.251) as the cause. CONCLUSIONS: Linguistic differences, by gender, in stigmatizing or glorifying language when reporting suicide may exacerbate suicide disparities.

Publication Bias in Clinical Trials in Cataract Therapies: Implications for Evidence-Based Decision-Making: Publication Bias in Clinical Trials in Cataract Therapies.

ABSTRACT: Evidence-based decision-making is generally based on published evidence. Therefore, if the published evidence is biased, so will the decision-making. One possible bias is the "positive-results" publication bias. This study attempts to characterize this phenomenon in cataract therapy trials. Studies were categorized as "positive" if their results were congruent with the hypothesis and "negative" if not. Secondary outcomes included the influence of funding source and differences in publication metrics between "positive" and "negative" publications. The US NLM Clinical Trials database was reviewed for cataract trials, yielding 248 trials. Trials with less than 2 treatment arms, less than 5 participants, or insufficient reporting were excluded. Data was collected on intervention, treatment arms, funding type, publication rates, citation rate, and the impact factor/H-index of journals. Of the 132 trials included, there were 69 positive and 63 negative results. Publication rate for positive results (71%) was significantly greater than negative results (17%), (p<0.01), with no significant difference in the other publication metrics. In conclusion, "negative" result trials are published less frequently, but are equally valued, if published. There are implications for evidence-based medicine with these findings.

A Phase 1 Clinical Trial of NKTR-255 with CD19-22 CAR-T Cell Therapy for Refractory B-cell Acute Lymphoblastic Leukemia.

While chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies, many patients relapse and therefore strategies to improve antitumor immunity are needed. We previously designed a novel autologous bispecific CAR targeting CD19 and CD22 (CAR19-22), which was well tolerated and associated with high response rates but relapse was common. Interleukin-15 (IL15) induces proliferation of diverse immune cells and can augment lymphocyte trafficking. Here, we report the results of a phase 1 clinical trial of the first combination of a novel recombinant polymer-conjugated IL15 receptor agonist (NKTR-255), with CAR19-22, in adults with relapsed / refractory B-cell acute lymphoblastic leukemia. Eleven patients were enrolled, nine of whom successfully received CAR19-22 followed by NKTR-255. There were no dose limiting toxicities, with transient fever and myelosuppression as the most common possibly related toxicities. We observed favorable efficacy with eight out of nine patients (89%) achieving measurable residual disease negative remission. At 12 months, progression-free survival for NKTR-255 was double that of historical controls (67% vs 38%). We performed correlative analyses to investigate the effects of IL15 receptor agonism. Cytokine profiling showed significant increases in IL15 and the chemokines CXCL9 and CXCL10. The increase in chemokines was associated with decreases in absolute lymphocyte counts and CD8+ CAR T-cells in blood and ten-fold increases in CSF CAR-T cells, suggesting lymphocyte trafficking to tissue. Combining NKTR-255 with CAR19-22 was safe, feasible and associated with high rates of durable responses (NCT03233854).

Lymphocytes as a living drug for cancer.

Using a patient's lymphocytes is approved to treat melanoma and has wider applications.

Trigeminal ganglion neurons are directly activated by influx of CSF solutes in a migraine model.

Classical migraine patients experience aura, which is transient neurological deficits associated with cortical spreading depression (CSD), preceding headache attacks. It is not currently understood how a pathological event in cortex can affect peripheral sensory neurons. In this study, we show that cerebrospinal fluid (CSF) flows into the trigeminal ganglion, establishing nonsynaptic signaling between brain and trigeminal cells. After CSD, ~11% of the CSF proteome is altered, with up-regulation of proteins that directly activate receptors in the trigeminal ganglion. CSF collected from animals exposed to CSD activates trigeminal neurons in naïve mice in part by CSF-borne calcitonin gene-related peptide (CGRP). We identify a communication pathway between the central and peripheral nervous system that might explain the relationship between migrainous aura and headache.

ViViEchoformer: Deep Video Regressor Predicting Ejection Fraction.

Heart disease is the leading cause of death worldwide, and cardiac function as measured by ejection fraction (EF) is an important determinant of outcomes, making accurate measurement a critical parameter in PT evaluation. Echocardiograms are commonly used for measuring EF, but human interpretation has limitations in terms of intra- and inter-observer (or reader) variance. Deep learning (DL) has driven a resurgence in machine learning, leading to advancements in medical applications. We introduce the ViViEchoformer DL approach, which uses a video vision transformer to directly regress the left ventricular function (LVEF) from echocardiogram videos. The study used a dataset of 10,030 apical-4-chamber echocardiography videos from patients at Stanford University Hospital. The model accurately captures spatial information and preserves inter-frame relationships by extracting spatiotemporal tokens from video input, allowing for accurate, fully automatic EF predictions that aid human assessment and analysis. The ViViEchoformer's prediction of ejection fraction has a mean absolute error of 6.14%, a root mean squared error of 8.4%, a mean squared log error of 0.04, and an R 2 of 0.55. ViViEchoformer predicted heart failure with reduced ejection fraction (HFrEF) with an area under the curve of 0.83 and a classification accuracy of 87 using a standard threshold of less than 50% ejection fraction. Our video-based method provides precise left ventricular function quantification, offering a reliable alternative to human evaluation and establishing a fundamental basis for echocardiogram interpretation.

Macro and micro structural preservation of grey matter integrity after 24 weeks of rTMS in Alzheimer's disease patients: a pilot study.

Alzheimer's Disease (AD) is characterized by structural and functional dysfunction involving the Default Mode Network (DMN), for which the Precuneus (PC) is a key node. We proposed a randomized double-blind pilot study to determine neurobiological changes after 24 weeks of PC-rTMS in patients with mild-to-moderate AD. Sixteen patients were randomly assigned to SHAM or PC-rTMS, and received an intensive 2-weeks course with daily rTMS sessions, followed by a maintenance phase in which rTMS has been applied once a week. Before and after the treatment structural and functional MRIs were collected. Our results showed macro- and micro-structural preservation in PC-rTMS compared to SHAM-rTMS group after 24 weeks of treatment, correlated to an increase of functional connectivity (FC) within the PC in the PC-rTMS group. Even if preliminary, these results trigger the possibility of using PC-rTMS to arrest atrophy progression by manipulating distributed network connectivity patterns.

Domain 5 of Beta 2 glycoprotein I: Friend or foe in health? Context matters.

Beta 2 glycoprotein I (ß2GPI) is the major autoantigen in the antiphospholipid syndrome, an autoimmune disorder characterized by thrombotic and obstetric complications. The autoantibodies that target beta 2 glycoprotein I are pathogenic and contribute to disease pathogenesis. The ß2GPI molecule is composed of 5 domains that are numbered 1 through to 5. Autoantibodies bind mainly to domain 1 whereas the majority of the biological functions of the ß2GPI molecule in diverse processes such as apoptotic cell clearance, complement regulation, lipopolysaccharide clearance and anticoagulation have been localised to domain 5 and its unique biochemistry, reviewed in this article. The role of purified domain 5 peptide as a potential therapeutic agent in APS and ischemia reperfusion injury is discussed.

Topical Solution for Retinal Delivery: Bevacizumab and Ranibizumab Eye Drops in Anti-Aggregation Formula (AAF) in Rabbits.

PURPOSE: Wet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF). METHODS: Bevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent. RESULTS: Bevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies. CONCLUSIONS: Bevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.

Widespread detoxifying NO reductases impart a distinct isotopic fingerprint on N2O under anoxia.

Nitrous oxide (N2O), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce N2O with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O under anoxic conditions (~+10‰) that correlates with typical environmental N2O SP measurements. Using Pseudomonas aeruginosa as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo N2O SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net N2O SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar N2O SP biosignatures to those measured from our P. aeruginosa Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of N2O biological sources in nature and disease.

An Analysis of Tube Thoracostomy in Combat Implications for Improved Prehospital Recognition and Treatment.

BACKGROUND: Thoracic trauma occurs frequently in combat and is associated with high mortality. Tube thoracostomy (chest tube) is the treatment for pneumothorax resulting from thoracic trauma, but little data exist to characterize combat casualties undergoing this intervention. We sought to describe the incidence of these injuries and procedures to inform training and materiel development priorities. METHODS: This is a secondary analysis of a Department of Defense Trauma Registry (DoDTR) data set from 2007 to 2020 describing prehospital care within all theaters in the registry. We described all casualties who received a tube thoracostomy within 24 hours of admission to a military treatment facility. Variables described included casualty demographics; abbreviated injury scale (AIS) score by body region, presented as binary serious (=3) or not serious (<3); and prehospital interventions. RESULTS: The database identified 25,897 casualties, 2,178 (8.4%) of whom received a tube thoracostomy within 24 hours of admission. Of those casualties, the body regions with the highest proportions of common serious injury (AIS >3) were thorax 62% (1,351), extremities 29% (629), abdomen 22% (473), and head/neck 22% (473). Of those casualties, 13% (276) had prehospital needle thoracostomies performed, and 19% (416) had limb tourniquets placed. Most of the patients were male (97%), partner forces members or humanitarian casualties (70%), and survived to discharge (87%). CONCLUSIONS: Combat casualties with chest trauma often have multiple injuries complicating prehospital and hospital care. Explosions and gunshot wounds are common mechanisms of injury associated with the need for tube thoracostomy, and these interventions are often performed by enlisted medical personnel. Future efforts should be made to provide a correlation between chest interventions and pneumothorax management in prehospital thoracic trauma.

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.

Kynurenic acid inflammatory signaling expands in primates and impairs prefrontal cortical cognition.

Cognitive deficits from dorsolateral prefrontal cortex (dlPFC) dysfunction are common in neuroinflammatory disorders, including long-COVID, schizophrenia and Alzheimer's disease, and have been correlated with kynurenine inflammatory signaling. Kynurenine is further metabolized to kynurenic acid (KYNA) in brain, where it blocks NMDA and α7-nicotinic receptors (nic-α7Rs). These receptors are essential for neurotransmission in dlPFC, suggesting that KYNA may cause higher cognitive deficits in these disorders. The current study found that KYNA and its synthetic enzyme, KAT II, have greatly expanded expression in primate dlPFC in both glia and neurons. Local application of KYNA onto dlPFC neurons markedly reduced the delay-related firing needed for working memory via actions at NMDA and nic-α7Rs, while inhibition of KAT II enhanced neuronal firing in aged macaques. Systemic administration of agents that reduce KYNA production similarly improved cognitive performance in aged monkeys, suggesting a therapeutic avenue for the treatment of cognitive deficits in neuroinflammatory disorders.

Prospective Evaluation of Artificial Intelligence Triage of Incidental Pulmonary Emboli on Contrast-Enhanced CT Examinations of the Chest or Abdomen.

Background: Artificial intelligence (AI) algorithms improved detection of incidental pulmonary embolism (IPE) on contrast-enhanced CT (CECT) examinations in retrospective studies; however, prospective validation studies are lacking. Objective: To assess the effect on radiologists' real-world diagnostic performance and report turnaround times of a radiology department's clinical implementation of an AI triage system for detecting IPE on CECT examinations of the chest or abdomen. Methods: This prospective single-center study included consecutive adult patients who underwent CECT of the chest or abdomen for reasons other than PE detection from May 12, 2021 to June 30, 2021 (phase 1) or from July 1, 2021 to September 29, 2021 (phase 2). Before phase 1, the radiology department installed a commercially available AI triage algorithm for IPE detection that automatically processed CT examinations and notified radiologists of positive results through an interactive floating widget. In phase 1, the widget was inactive, and radiologists interpreted examinations without AI assistance. In phase 2, the widget was activated, and radiologists interpreted examinations with AI assistance. A review process involving a panel of radiologists was implemented to establish the reference standard for the presence of IPE. Diagnostic performance and report turnaround times were compared using Pearson Chi-square test and Wilcoxon rank-sum test, respectively. Results: Phase 1 included 1467 examinations in 1434 patients (mean age, 53.8±18.5 years; 753 male, 681 female); phase 2 included 3182 examinations in 2886 patients (mean age, 55.4±18.2 years; 1520 male, 1366 female). The frequency of IPE was 1.4% (20/1467) in phase 1 and 1.6% (52/3182) in phase 2. Radiologists without AI, in comparison with radiologists with AI, showed significantly lower sensitivity (80.0% vs 96.2%, P=.03), without a significant difference in specificity (99.1% vs 99.9%, P=.58), for detection of IPE. The mean report turnaround time for IPE-positive examinations was not significantly different between radiologists without AI and radiologists with AI (78.3 vs 64.6 min, P=.26). Conclusion: An AI triage system improved radiologists' sensitivity for IPE detection on CECT examinations of the chest or abdomen without significant change in report turnaround times. Clinical Impact: This prospective real-world study supports the use of AI assistance for maximizing IPE detection.

Navigating Barriers and Opportunities: Perceptions of the Non-Occupational Post-Exposure Prophylaxis Care Continuum Among Young Sexual Minority Men, Aged 17-24, in the United States.

Human immunodeficiency virus (HIV) is a public health concern among young sexual minority men (YSMM), ages 17 to 24, in the United States. Biomedical prevention methods, such as pre-exposure prophylaxis (PrEP) and non-occupational post-exposure prophylaxis (nPEP), can help reduce the risk of HIV transmission among this population. However, there is limited awareness and use of nPEP by YSMM. This study aims to explore the perceptions of YSMM regarding the nPEP care continuum, which consists of three areas of focus: awareness, uptake, and linkage to other HIV prevention services. This study draws on synchronous online focus groups with a sample of 41 YSMM in the United States. Transcripts from the focus groups were analyzed using reflexive thematic analysis. Participants reported limited nPEP awareness and prior use, a process of personal appraisal of nPEP need based on HIV risk and costs, and a preference for PrEP over PEP for long-term HIV prevention. Interventions should be tailored to increase awareness of nPEP among YSMM and reduce addressable barriers to nPEP use for YSMM, including cost and confidentiality concerns, in situations where nPEP is warranted. Finally, more research is needed on how nPEP use can act as a bridge to PrEP initiation for this population.