Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

Assessment of Survival Kinetics for Emergent Highly Pathogenic Clade 2.3.4.4 H5Nx Avian Influenza Viruses.

High pathogenicity avian influenza viruses (HPAIVs) cause high morbidity and mortality in poultry species. HPAIV prevalence means high numbers of infected wild birds could lead to spill over events for farmed poultry. How these pathogens survive in the environment is important for disease maintenance and potential dissemination. We evaluated the temperature-associated survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs (UK field strains between 2014 and 2021) incubated at up to three temperatures for up to ten weeks. The selected temperatures represented northern European winter (4 °C) and summer (20 °C); and a southern European summer temperature (30 °C). For each clade 2.3.4.4 HPAIV, the time in days to reduce the viral infectivity by 90% at temperature T was established (DT), showing that a lower incubation temperature prolonged virus survival (stability), where DT ranged from days to weeks. The fastest loss of viral infectivity was observed at 30 °C. Extrapolation of the graphical DT plots to the x-axis intercept provided the corresponding time to extinction for viral decay. Statistical tests of the difference between the DT values and extinction times of each clade 2.3.4.4 strain at each temperature indicated that the majority displayed different survival kinetics from the other strains at 4 °C and 20 °C.

Illustrated State-of-the-Art Capsules of the ISTH 2024 Congress.

Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis.

The survival benefit of adjuvant trastuzumab with or without chemotherapy in the management of small (T1mic, T1a, T1b, T1c), node negative HER2+ breast cancer.

There is limited data regarding the added benefit of adjuvant systemic therapy in the management of small, node-negative, HER2+ breast cancer. In a multi-institutional retrospective analysis using the American Society of Clinical Oncology CancerLinQ database, we compared survival outcomes among T1a-c N0 HER2+ patients diagnosed between 2010 to 2021 who received locoregional therapy alone or in combination with adjuvant trastuzumab (+/- chemotherapy). Primary outcomes were invasive disease-free survival (iDFS) and overall survival (OS). Of the 1,184 patients, 436 received locoregional therapy alone. We found a statistically significant improvement in iDFS (HR 0.73, P = 0.003) and OS (HR 0.63, P = 0.023) on univariate analysis with adjuvant trastuzumab with or without chemotherapy which remained statistically significant on multivariate analysis. Three-arm univariate analysis found that iDFS was significantly improved with trastuzumab monotherapy (P = 0.003) and combination therapy (P = 0.027) compared to observation. Subgroup data suggests that T1b/c tumors derive the greatest benefit.

Modulation of diabetic wound healing using carbon monoxide gas-entrapping materials.

Diabetic wound healing is uniquely challenging to manage due to chronic inflammation and heightened microbial growth from elevated interstitial glucose. Carbon monoxide (CO), widely acknowledged as a toxic gas, is also known to provide unique therapeutic immune modulating effects. To facilitate delivery of CO, we have designed hyaluronic acid-based CO-gas-entrapping materials (CO-GEMs) for topical and prolonged gas delivery to the wound bed. We demonstrate that CO-GEMs promote the healing response in murine diabetic wound models (full-thickness wounds and pressure ulcers) compared to N2-GEMs and untreated controls.

Cleavage of the syncytial protein of J paramyxovirus is required for its ability to promote cell-cell fusion.

Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the "LKTG" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.

Rapid mortality in captive bush dogs (Speothos venaticus) caused by influenza A of avian origin (H5N1) at a wildlife collection in the United Kingdom.

Europe has suffered unprecedented epizootics of high pathogenicity avian influenza (HPAI) clade 2.3.4.4b H5N1 since Autumn 2021. As well as impacting upon commercial and wild avian species, the virus has also infected mammalian species more than ever observed previously. Mammalian species involved in spill over events have primarily been scavenging terrestrial carnivores and farmed mammalian species although marine mammals have also been affected. Alongside reports of detections of mammalian species found dead through different surveillance schemes, several mass mortality events have been reported in farmed and wild animals. In November 2022, an unusual mortality event was reported in captive bush dogs (Speothos venaticus) with clade 2.3.4.4b H5N1 HPAIV of avian origin being the causative agent. The event involved an enclosure of 15 bush dogs, 10 of which succumbed during a nine-day period with some dogs exhibiting neurological disease. Ingestion of infected meat is proposed as the most likely infection route.

Adolescent-to-adult gains in cognitive flexibility are adaptively supported by reward sensitivity, exploration, and neural variability.

Cognitive flexibility exhibits dynamic changes throughout development, with different forms of flexibility showing dissociable developmental trajectories. In this review, we propose that an adolescent-specific mode of flexibility in the face of changing environmental contingencies supports the emergence of adolescent-to-adult gains in cognitive shifting efficiency. We first describe how cognitive shifting abilities monotonically improve from childhood to adulthood, accompanied by increases in brain state flexibility, neural variability, and excitatory/inhibitory balance. We next summarize evidence supporting the existence of a dopamine-driven, adolescent peak in flexible behavior that results in reward seeking, undirected exploration, and environmental sampling. We propose a neurodevelopmental framework that relates these adolescent behaviors to the refinement of neural phenotypes relevant to mature cognitive flexibility, and thus highlight the importance of the adolescent period in fostering healthy neurocognitive trajectories.

Fighting fibrin with fibrin: Vancomycin delivery into coagulase-mediated Staphylococcus aureus biofilms via fibrin-based nanoparticle binding.

Staphylococcus aureus skin and soft tissue infection is a common ailment placing a large burden upon global healthcare infrastructure. These bacteria are growing increasingly recalcitrant to frontline antimicrobial therapeutics like vancomycin due to the prevalence of variant populations such as methicillin-resistant and vancomycin-resistant strains, and there is currently a dearth of novel antibiotics in production. Additionally, S. aureus has the capacity to hijack the host clotting machinery to generate fibrin-based biofilms that confer protection from host antimicrobial mechanisms and antibiotic-based therapies, enabling immune system evasion and significantly reducing antimicrobial efficacy. Emphasis is being placed on improving the effectiveness of therapeutics that are already commercially available through various means. Fibrin-based nanoparticles (FBNs) were developed and found to interact with S. aureus through the clumping factor A (ClfA) fibrinogen receptor and directly integrate into the biofilm matrix. FBNs loaded with antimicrobials such as vancomycin enabled a targeted and sustained release of antibiotic that increased drug contact time and reduced the therapeutic dose required for eradicating the bacteria, both in vitro and in vivo. Collectively, these findings suggest that FBN-antibiotic delivery may be a novel and potent therapeutic tool for the treatment of S. aureus biofilm infections.

Biomaterial systems for evaluating the influence of ECM mechanics on anti-fibrotic therapeutic efficacy.

Cardiac fibrosis is characterized by excessive accumulation and deposition of ECM proteins. Cardiac fibrosis is commonly implicated in a variety of cardiovascular diseases, including post-myocardial infarction (MI). We have previously developed a dual-delivery nanogel therapeutic to deliver tissue plasminogen activator (tPA) and Y-27632 (a ROCK inhibitor) to address MI-associated coronary artery occlusion and downregulate cell-contractility mediated fibrotic responses. Initial in vitro studies were conducted on glass substrates. The study presented here employs the use of polyacrylamide (PA) gels and microgel thin films to mimic healthy and fibrotic cardiac tissue mechanics. Soft and stiff polyacrylamide substrates or high and low loss tangent microgel thin films were utilized to examine the influence of cell-substrate interactions on dual-loaded nanogel therapeutic efficacy. In the presence of Y-27632 containing nanogels, a reduction of fibrotic marker expression was noted on traditional PA gels mimicking healthy and fibrotic cardiac tissue mechanics. These findings differed on more physiologically relevant microgel thin films, where early treatment with the ROCK inhibitor intensified the fibrotic related responses.

Clot-Targeted Nanogels for Dual-Delivery of AntithrombinIII and Tissue Plasminogen Activator to Mitigate Disseminated Intravascular Coagulation Complications.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Fluid Biomarkers in Individuals at Risk for Genetic Prion Disease up to Disease Conversion.

OBJECTIVES: To longitudinally characterize disease-relevant CSF and plasma biomarkers in individuals at risk for genetic prion disease up to disease conversion. METHODS: This single-center longitudinal cohort study has followed known carriers of PRNP pathogenic variants at risk for prion disease, individuals with a close relative who died of genetic prion disease but who have not undergone predictive genetic testing, and controls. All participants were asymptomatic at first visit and returned roughly annually. We determined PRNP genotypes, measured NfL and GFAP in plasma, and RT-QuIC, total PrP, NfL, T-tau, and beta-synuclein in CSF. RESULTS: Among 41 carriers and 21 controls enrolled, 28 (68%) and 15 (71%) were female, and mean ages were 47.5 and 46.1. At baseline, all individuals were asymptomatic. We observed RT-QuIC seeding activity in the CSF of 3 asymptomatic E200K carriers who subsequently converted to symptomatic and died of prion disease. 1 P102L carrier remained RT-QuIC negative through symptom conversion. No other individuals developed symptoms. The prodromal window from detection of RT-QuIC positivity to disease onset was 1 year long in an E200K individual homozygous (V/V) at PRNP codon 129 and 2.5 and 3.1 years in 2 codon 129 heterozygotes (M/V). Changes in neurodegenerative and neuroinflammatory markers were variably observed prior to onset, with increases observed for plasma NfL in 4/4 converters, and plasma GFAP, CSF NfL, CSF T-tau, and CSF beta-synuclein each in 2/4 converters, although values relative to age and fold changes relative to individual baseline were not remarkable for any of these markers. CSF PrP was longitudinally stable with mean coefficient of variation 9.0% across all individuals over up to 6 years, including data from converting individuals at RT-QuIC-positive timepoints. DISCUSSION: CSF prion seeding activity may represent the earliest detectable prodromal sign in E200K carriers. Neuronal damage and neuroinflammation markers show limited sensitivity in the prodromal phase. CSF PrP levels remain stable even in the presence of RT-QuIC seeding activity. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov NCT05124392 posted 2017-12-01, updated 2023-01-27.

Effect of dietary macronutrients and immune challenge on gut microbiota, physiology and feeding behaviour in zebra finches.

Macronutrients play a vital role in host immunity and can influence host-pathogen dynamics, potentially through dietary effects on gut microbiota. To increase our understanding of how dietary macronutrients affect physiology and gut microbiota and investigate whether feeding behaviour is influenced by an immune threat, we conducted two experiments. First, we determined whether zebra finches (Taeniopygia guttata) exhibit shifts in physiology and gut microbiota when fed diets differing in macronutrient ratios. We found the type and amount of diet consumed affected gut microbiota alpha diversity, where microbial richness and Shannon diversity increased with caloric intake in birds fed a high-fat diet and decreased with caloric intake in birds fed a high protein diet. Diet macronutrient content did not affect physiological metrics, but lower caloric intake was associated with higher complement activity. In our second experiment, we simulated an infection in birds using the bacterial endotoxin lipopolysaccharide (LPS) and quantified feeding behaviour in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). We also examined whether social cues of infection alter physiological responses relevant to responding to an immune threat, an effect that could be mediated through shifts in feeding behaviour. LPS induced a reduction in caloric intake driven by a decrease in protein, but not fat consumption. No evidence was found for socially induced shifts in feeding behaviour, physiology or gut microbiota. Our findings carry implications for host health, as sickness-induced anorexia and diet-induced shifts in the microbiome could shape host-pathogen interactions.

Infectious bronchitis virus: an overview of the "chicken coronavirus".

Infectious bronchitis virus (IBV) is a highly contagious avian Gammacoronavirus that affects mainly chickens (Gallus gallus) but can circulate in other avian species. IBV constitutes a significant threat to the poultry industry, causing reduced egg yield, growth and mortality levels that can vary in impact. The virus can be transmitted horizontally by inhalation or direct/indirect contact with infected birds or contaminated fomites, vehicles, farm personnel and litter (Figure 1). The error-prone viral polymerase and recombination mechanisms mean diverse viral population results, with multiple genotypes, serotypes, pathotypes and protectotypes. This significantly complicates control and mitigation strategies based on vigilance in biosecurity and the deployment of vaccination.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

"You want people to listen to you": Patient experiences of women's healthcare within the Veterans Health Administration.

OBJECTIVE: To identify constructs that are critical in shaping Veterans' experiences with Veterans Health Administration (VA) women's healthcare, including any which have been underexplored or are not included in current VA surveys of patient experience. DATA SOURCES AND STUDY SETTING: From June 2022 to January 2023, we conducted 28 semi-structured interviews with a diverse, national sample of Veterans who use VA women's healthcare. STUDY DESIGN: Using VA data, we divided Veteran VA-users identified as female into four groups stratified by age (dichotomized at age 45) and race/ethnicity (non-Hispanic White vs. all other). We enrolled Veterans continuously from each recruitment strata until thematic saturation was reached. DATA COLLECTION/EXTRACTION METHODS: For this qualitative study, we asked Veterans about past VA healthcare experiences. Interview questions were guided by a priori domains identified from review of the literature, including trust, safety, respect, privacy, communication and discrimination. Analysis occurred concurrently with interviews, using inductive and deductive content analysis. PRINCIPAL FINDINGS: We identified five themes influencing Veterans' experiences of VA women's healthcare: feeling valued and supported, bodily autonomy, discrimination, past military experiences and trauma, and accessible care. Each emergent theme was associated with multiple of the a priori domains we asked about in the interview guide. CONCLUSIONS: Our findings underscore the need for a measure of patient experience tailored to VA women's healthcare. Existing patient experience measures used within VA fail to address several aspects of experience highlighted by our study, including bodily autonomy, the influence of past military experiences and trauma on healthcare, and discrimination. Understanding distinct factors that influence women and gender-diverse Veterans' experiences with VA care is critical to advance efforts by VA to measure and improve the quality and equity of care for all Veterans.

Records of fleas (Siphonaptera) from Delaware.

We present an annotated checklist of fleas (Siphonaptera) known to occur in the state of Delaware based on an examination of Siphonaptera collections at the University of Delaware and the Carnegie Museum of Natural History, as well as new specimens of fleas we collected from wildlife, other hosts, and tick flags. We review published records and compile them herein with our new records, which include 3 species previously unreported from Delaware. With these additions, there are now 18 flea species from 19 avian and mammalian hosts documented from Delaware.

Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation.

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.

Repeatability and reproducibility of hunter-harvest sampling for avian influenza virus surveillance in Great Britain.

Emerging pathogens can threaten human and animal health, necessitating reliable surveillance schemes to enable preparedness. We evaluated the repeatability and reproducibility of a method developed previously during a single year at one study site. Hunter-harvested ducks and geese were sampled for avian influenza virus at three discrete locations in the UK. H5N1 highly pathogenic avian influenza (HPAIV) was detected in four species (mallard [Anas platyrhynchos], Eurasian teal [Anas crecca], Eurasian wigeon [Mareca penelope] and pink-footed goose [Anser brachyrhynchus]) across all three locations and two non-HPAIV H5N1, influenza A positive detections were made from a mallard and Eurasian wigeon at two locations. Virus was detected within 1-to-4 days of sampling at every location. Application of rapid diagnostic methods to samples collected from hunter-harvested waterfowl offers potential as an early warning system for the surveillance and monitoring of emerging and existing strains of avian influenza A viruses in key avian species.