A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

FishTEDB 2.0: an update fish transposable element (TE) database with new functions to facilitate TE research.

We launched the initial version of FishTEDB in 2018, which aimed to establish an open-source, user-friendly, data-rich transposable element (TE) database. Over the past 5 years, FishTEDB 1.0 has gained approximately 10 000 users, accumulating more than 450 000 interactions. With the unveiling of extensive fish genome data and the increasing emphasis on TE research, FishTEDB needs to extend the richness of data and functions. To achieve the above goals, we introduced 33 new fish species to FishTEDB 2.0, encompassing a wide array of fish belonging to 48 orders. To make the updated database more functional, we added a genome browser to visualize the positional relationship between TEs and genes and the estimated TE insertion time in different species. In conclusion, we released a new version of the fish TE database, FishTEDB 2.0, designed to assist researchers in the future study of TE functions and promote the progress of biological theories related to TEs. Database URL: https://www.fishtedb.com/.

Heterologous expression of the insect SVWC peptide WHIS1 inhibits Candida albicans invasion into A549 and HeLa epithelial cells.

Candida albicans (C. albicans), a microbe commonly isolated from Candida vaginitis patients with vaginal tract infections, transforms from yeast to hyphae and produces many toxins, adhesins, and invasins, as well as C. albicans biofilms resistant to antifungal antibiotic treatment. Effective agents against this pathogen are urgently needed. Antimicrobial peptides (AMPs) have been used to cure inflammation and infectious diseases. In this study, we isolated whole housefly larvae insect SVWC peptide 1 (WHIS1), a novel insect single von Willebrand factor C-domain protein (SVWC) peptide from whole housefly larvae. The expression pattern of WHIS1 showed a response to the stimulation of C. albicans. In contrast to other SVWC members, which function as antiviral peptides, interferon (IFN) analogs or pathogen recognition receptors (PRRs), which are the prokaryotically expressed MdWHIS1 protein, inhibit the growth of C. albicans. Eukaryotic heterologous expression of WHIS1 inhibited C. albicans invasion into A549 and HeLa cells. The heterologous expression of WHIS1 clearly inhibited hyphal formation both extracellularly and intracellularly. Furthermore, the mechanism of WHIS1 has demonstrated that it downregulates all key hyphal formation factors (ALS1, ALS3, ALS5, ECE1, HWP1, HGC1, EFG1, and ZAP1) both extracellularly and intracellularly. These data showed that heterologously expressed WHIS1 inhibits C. albicans invasion into epithelial cells by affecting hyphal formation and adhesion factor-related gene expression. These findings provide new potential drug candidates for treating C. albicans infection.

KMT2A and chronic inflammation as potential drivers of sporadic parathyroid adenoma.

BACKGROUND: Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood. METHODS: Surgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10× Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations. RESULTS: There was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues. CONCLUSIONS: We revealed the previously underappreciated involvement of the KMT2A‒STAT3/GATA3‒CCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications. HIGHLIGHTS: Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis.

Prospects for the Protective Mechanisms and Compounds of Bufei Huoxue Capsule against COVID-19 Convalescence: Evaluation of Integrating UHPLC-HRMS Analysis and Network Pharmacology Strategy.

INTRODUCTION: Most COVID-19 survivors are troubled with chronic persistent symptoms, which have currently no definitive treatments. Bufei Huoxue (BFHX) capsule exerts clinical benefit, while the material basis and molecular mechanism remain unclear. AIM: The study aimed to elucidate the protective mechanisms of BFHX capsules against COVID-19 convalescence. UHPLC-HRMS and various databases were employed to explore potential compounds and targets. PPI, MCODE, transcription factor (TF), and miRNA analyses were conducted to receive hub targets and corresponding upstream regulators. METHOD: Molecular docking was applied to verify the binding activity of compound and target. Further, GO, KEGG, WIKI, and Reactome analyses were performed, and compound-targetsymptom and gene-disease networks were constructed. A total of 127 compounds and 313 targets were acquired. A sum of 10 hub targets were screened and showed good binding affinities with critical compounds. RESULT: MLLT1, CBFB, and EZH2 were identified as key TFs, and hsa-mir-146a-5p, hsa-mir- 26b-5p, and hsa-mir-24-3p were predicted to be important miRNAs. BFHX capsule may alleviate the symptoms by targeting TNF, IL-6, IFNG, and TGF-ß1. Besides, BFHX capsule may exert a therapeutic effect on respiratory disease (especially pulmonary fibrosis and lung infection) and multi-system damage during COVID-19 convalescence by regulating cytokine-cytokine receptor interaction, as well as TGF-ß, TNF, and Toll-like receptor signaling pathways. CONCLUSION: In summary, BFHX capsule may exert a therapeutic effect on multi-system damages during COVID-19 convalescence through multiple compounds (such as albiflorin, isopsoralen, and neobavaisoflavone), multiple targets (such as TNF, IL-6, and EGF) and multiple pathways (TGF-ß, TNF, and Toll-like receptor signaling pathways).

5(S)-5-carboxystrictosidine from the root of Mappianthus iodoides ameliorates H2O2-induced apoptosis in H9c2 cardiomyocytes via PI3K/AKT and ERK pathways.

5(S)-5-carboxystrictosidine (5-CS) is a compound found in the root of Mappianthus iodoides, a traditional Chinese medicine used for the treatment of coronary artery disease. The aim of the present study was to investigate the protective effect of 5-CS against oxidative stress-induced apoptosis in H9c2 cardiomyocytes and the underlying mechanisms. 5-CS pretreatment significantly protected against H2O2-induced cell death, LDH leakage and malondialdehyde (MDA) production which are indicators for oxidative stress injury. 5-CS also enhanced the activity of SOD and CAT. In addition, 5-CS pretreatment significantly inhibited H2O2-induced apoptosis, as determined by flow cytometer, suppressed the activity of caspase-3 and caspase-9 and attenuated the activation of cleaved caspase-3 and caspase-9. 5-CS also increased Akt and ERK activation altered by H2O2 using Western blot analysis. The PI3K-specific inhibitor LY294002 abolished 5-CS-induced Akt activation. The ERK-specific inhibitor PD98059 abolished 5-CS-induced ERK activation. Both LY294002 and PD98059 attenuated the protective effect of 5-CS on H9c2 cardiomyocytes against H2O2-induced apoptosis and cell death. Taken together, these results demonstrate that 5-CS prevents H2O2-induced oxidative stress injury in H9c2 cells by enhancing the activity of the endogenous antioxidant enzymes, inhibiting apoptosis, and modulating PI3K/Akt and ERK signaling pathways.

Clinical value of nano-carbon lymphatic tracer for regional lymph node dissections of rectal cancer after neoadjuvant chemoradiotherapy.

OBJECTIVES: Regional lymph node (LN) volume decreases after neoadjuvant therapy, requiring a tracer for more accurate detection. Nano-carbon tracer is a third-generation tracer with several advantages, but its use for LN detection after neoadjuvant chemoradiotherapy for middle and low rectal cancer remains unclear. Therefore, this study investigated the effects and safety of anoscope-guided subrectal injections of nano-carbon suspension in this patient population. METHODS: This study retrospectively reviewed the medical records of 45 patients with middle and low rectal cancer admitted to our institution from March 2019 to March 2022. All patients received preoperative neoadjuvant chemotherapy and radiotherapy and were divided into nano-carbon injection (n = 23; anoscope-guided injections of nano-carbon suspension in the rectal submucosa 2 cm above the dentate line 24 h preoperatively) and control (n = 22; directly underwent surgery) groups. The LN detection and complication rates were compared between the groups. RESULTS: The total and mean numbers of LNs and small LNs and the number of patients with > 12 LNs were significantly higher in the nano-carbon injection group than in the control group. The total number of positive LNs and LN metastasis did not differ between the groups, nor did the anastomotic leakage, bleeding, stenosis, and abscess occurrence rates. CONCLUSIONS: Anoscope-guided nano-carbon lymphatic tracing increased the LN detection rate, caused less trauma, and resulted in fewer postoperative complications than the direct surgical procedure. Thus, it is an effective, safe, and practical method that may improve dissections and the postoperative pathological staging accuracy.

Understanding the mechanism for sodium tripolyphosphate in improving the physicochemical properties of low-moisture extrusion textured protein from rapeseed protein and soybean protein blends.

Our previous experiments found that rapeseed protein (RP) has applicability in low-moisture textured proteins. The amount of RP added is limited to <20 %, but the addition of 20 % RP still brings some negative effects. Therefore, in order to improve the quality of 20%RP textured protein, this experiment added different proportions of sodium tripolyphosphate (STPP) to improve the quality of the product, and studied the physical-chemical properties and molecular structure changes of the product to explore the possible modification mechanism. The STPP not only improved the expansion characteristics of extrudates, but also increased the brightness of the extrudates, the rehydration rate. In addition, STPP increased the specific mechanical energy during extrusion, decreased the material mass flow rate. Furthermore, STPP decreased the starch digestibility, increased the content of slow-digesting starch and resistant starch. STPP increased the degree of denaturation of extrudate proteins, the proportion of ß-sheets in the secondary structure of proteins, as well as the intermolecular hydrogen bonding interactions. The gelatinization degradation degree of starch molecules also decreased with the addition of STPP. STPP also increased the protein-starch interactions and enhanced the thermal stability of the extrudate. All these indicate that STPP can improve the physical-chemical properties of extrudate.

Efficacy and safety of trifluridine/tipiracil plus bevacizumab versus trifluridine/tipiracil monotherapy for refractory metastatic colorectal cancer: a retrospective cohort study.

Background: Several studies demonstrated trifluridine/tipiracil (TAS-102) plus bevacizumab (BEV) had better efficacy than the monotherapy of TAS-102 in refractory metastatic colorectal cancer (mCRC). However, it remains unclear whether Chinese population can benefit from this combination or not. Hence, we conducted this retrospective cohort study to compare the efficacy and safety between TAS-102 plus BEV with TAS-102 monotherapy in refractory mCRC. Methods: This retrospective cohort study enrolled patients (any age) with refractory mCRC from Hunan Cancer Hospital. The main inclusion criteria were histopathologically and/or radiographically confirmed refractory mCRC, World Health Organization (WHO) performance status of 0 to 2, adequate organ function, and initial treatment of TAS-102 with or without BEV between November 2020 and October 2022. Previous therapy with fruquintinib or regorafenib was allowed but not mandatory. Baseline demographic and clinical characteristics were collected appropriately. Every 2 or 3 treatment cycles, the patients were assessed by computed tomography (CT) scans and clinical assessments until disease progression or loss to follow-up. The National Cancer Institute Common Terminology Criteria for Adverse Events 5.0 (NCI-CTCAE 5.0) were presented as n (%). The primary endpoint was investigator-evaluated overall survival (OS). As this is a retrospective cohort study, sample size calculation was not performed. Eligible patients would be enrolled as many as possible. Results: A total of 90 patients were enrolled, including 58 patients who received TAS-102 plus BEV and another 32 patients who received TAS-102 monotherapy. The known baseline characteristics were comparable (P<0.05). With a median follow-up of 4.60 months (range, 0.20-22.80), the median OS (mOS) time in the TAS-102 plus BEV group was longer than that in the TAS-102 monotherapy group (10.83 vs. 7.43 months), but the difference was not significant (P=0.79). The median progression-free survival (mPFS) time was comparable between the two groups (4.67 vs. 4.30 months, P=0.96). Multivariate Cox regression analysis demonstrated that undergoing therapy after TAS-102 either with or without BEV was an independent risk factor for OS [hazard ratio (HR) =0.25; 95% confidence interval (CI): 0.09-0.71, P<0.01], and previous treatment with cetuximab was an independent protective factor for PFS (HR =0.17; 95% CI: 0.03-0.91, P=0.04). Of the 70 patients who were evaluated, those receiving TAS-102 plus BEV showed trend of a higher objective response rate (ORR) and disease control rate (DCR) than those who received TAS-102 monotherapy (P=0.16 and P=0.29, respectively). Adverse events (AEs) were similar between the two groups, except that the incidence of platelet count decrease (grade ≥3) was significantly higher in the TAS-102 plus BEV group. Conclusions: There was a trend in favor of the combination of BEV plus TAS-102 regarding OS and DCR, without reaching statistical significance, and it means that there was no clear advantage of one over the other in terms of efficacy. Further prospective studies are still necessary to draw a definite conclusion.

Dissecting caspase-2-mediated cell death: from intrinsic PIDDosome activation to chemical modulation.

Caspase-2, a highly conserved member of the caspase family, is considered an initiator caspase that triggers apoptosis in response to some cellular stresses. Previous studies suggest that an intracellular multi-protein complex PIDDosome, induced by genotoxic stress, serves as a platform for caspase-2 activation. However, due to caspase-2's inability to process effector caspases, the mechanism underlying caspase-2-mediated cell death upon PIDDosome activation remains unclear. Here we conducted an unbiased genome-wide genetic screen and identified that the Bcl2 family protein BID is required for PIDDosome-induced, caspase-2-mediated apoptosis. PIDDosome-activated caspase-2 directly and functionally processes BID to signal the mitochondrial pathway for apoptosis induction. Additionally, a designed chemical screen identified a compound, HUHS015, that specifically activates caspase-2-mediated apoptosis. HUHS015-stimulated apoptosis also requires BID but is independent of the PIDDosome. Through extensive structure-activity relationship efforts, we identified a derivative with a potency of ~ 60 nmol/L in activating caspase-2-mediated apoptosis. The HUHS015-series of compounds act as efficient agonists that directly target the interdomain linker in caspase-2, representing a new mode of initiator caspase activation. Human and mouse caspase-2 differ in two crucial residues in the linker, rendering a selectivity of the agonists for human caspase-2. The caspase-2 agonists are valuable tools to explore the physiological roles of caspase-2-mediated cell death and a base for developing small-molecule drugs for relevant diseases.

Research on the outdoor thermal comfort of children in Hangzhou and Its influence on the underlying surface reflectance.

The outdoor thermal comfort (OTC) of children is more specific than that of adults, and the complex influence of outdoor activity spaces on children's thermal comfort warrants further investigation. To investigate the outdoor thermal comfort baseline (OTCB) of children in Hangzhou and explore the thermal impact of outdoor surfaces on children, a survey was conducted in six typical outdoor activity spaces in Hangzhou, China, during spring and summer utilizing physical measurements, questionnaire surveys, and the universal thermal climate index (UTCI). This study analyzed the differences in thermal perception among children in Hangzhou in different seasons, their OTCB, and the impact of surface reflectance (Rs) on children's OTC. The results indicated the following: 1) In spring, children in Hangzhou generally felt comfortable, but their discomfort with heat noticeably increased in summer. 2) The neutral UTCIs (NUTCIs) for Hangzhou children were 11.6 °C (spring) and 27.7 °C (summer), and the NUTCI ranges (NUTCIRs) were 9.7-17.5 °C (spring) and 25.7-30.0 °C (summer); additionally, the thermal acceptability ranges (TARs) were 13.2-25.2 °C (spring) and 11.8-34.8 °C (summer). 3) A high Rs made children feel more uncomfortable with heat, which was primarily due to the space's total shortwave and longwave radiation, which peaked between 14:00 and 15:00. 4) Based on the research findings, corresponding bioclimatic design strategies were proposed. Recommendations include using high Rs underlays with shading, composite underlays, or the future adoption of thermochromic coatings. Keeping permeable underlays moist is essential for activating their cooling mechanisms. Fundamental safety measures are imperative. This study provides valuable data for urban planners and landscape designers to create public spaces suitable for children's outdoor activities, contributing to a harmonious and unified living environment.

Preparation of a ß-cyclodextrin grafted magnetic biochar for efficient extraction of four antiepileptic drugs in plasma samples.

Simultaneous monitoring of plasma concentration levels of multiple antiepileptic drugs (AEDs) is essential for dose adjustment in comprehensive epilepsy treatment, necessitating a sensitive technique for accurate extraction and determination of AEDs. Herein, a magnetic solid-phase extraction (MSPE) technique on the basis of modified biochar (BC) is investigated to extract four AEDs from plasma, in conjunction with high performance liquid chromatography. BC derived from Zizyphus jujuba seed shells was activated by phosphoric acid (PBC) and magnetized via coprecipitation to produce MPBC. The MPBCCD obtained after modification with ß-cyclodextrin (CD) was characterized and evaluated for adsorption. It exhibited fast adsorption kinetics based on second-order kinetics and satisfactory adsorption capacity for AEDs. Then it was employed as the MSPE adsorbent and the influencing parameters were optimized. The enrichment factor was 18.75. The validation analysis revealed a favorable linearity that ranged from 0.04 to 20 µg·mL-1 along with a low limit of detection of 6.85 to 10.19 ng·mL-1. The recovery of the AEDs ranged from 78.7 to 109.2 %, with relative standard deviations below 6.7 %. Using quantum chemistry theory calculations and experimental results analysis, the adsorption mechanism was investigated. It disclosed that the suggested strategy built upon MPBCCD was appropriate for the assessment of AEDs in plasma and expanded the usage of BC as the environmentally favorable matrix for the analysis of biological samples.

A viral assembly inhibitor blocks SARS-CoV-2 replication in airway epithelial cells.

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

Improving the properties of whey protein isolate-zein nanogels with novel acidifiers: Re-dispersity, stability and quercetin bioavailability.

Low bioavailability of quercetin (Que) reduces its preclinical and clinical benefits. In order to improve Que bioavailability, a novel whey protein isolate (WPI)-zein nanogel was prepared by pH-driven self-assembly and heat-induced gelatinization. The results showed that hydrochloric acid can be substituted by both acetic acid and citric acid during the pH-driven process. After encapsulation, the bioavailability of Que in nanogels (composed of 70 % WPI) induced by different acidifiers increased to 19.89 % (citric acid), 21.65 % (hydrochloric acid) and 24.34 % (acetic acid), respectively. Comparatively, nanogels induced by acetic acid showed higher stability (pH and storage stability), re-dispersibility (75.62 %), Que bioavailability (24.34 %), and antioxidant capacity (36.78 % for DPPH scavenging rates). s improved performance of nanogels. In mechanism, acetic acid significantly balanced different intermolecular forces by weakening "acid-induced denaturation" effect. Moreover, the faster binding of Que and protein as well as higher protein molecular flexibility and randomness (higher ratio of random coil) was also observed in nanogels induced by acetic acid. All of these changes contributed to improve nanogels performances. Overall, WPI-zein nanogels induced by acetic acid might be a safe, efficiency and stable delivery system to improve the bioavailability of hydrophobic active ingredients.

Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity.

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Cleavage-independent activation of ancient eukaryotic gasdermins and structural mechanisms.

Gasdermins (GSDMs) are pore-forming proteins that execute pyroptosis for immune defense. GSDMs are two-domain proteins activated by proteolytic removal of the inhibitory domain. In this work, we report two types of cleavage-independent GSDM activation. First, TrichoGSDM, a pore-forming domain-only protein from the basal metazoan Trichoplax adhaerens, is a disulfides-linked autoinhibited dimer activated by reduction of the disulfides. The cryo-electron microscopy (cryo-EM) structure illustrates the assembly mechanism for the 44-mer TrichoGSDM pore. Second, RCD-1-1 and RCD-1-2, encoded by the polymorphic regulator of cell death-1 (rcd-1) gene in filamentous fungus Neurospora crassa, are also pore-forming domain-only GSDMs. RCD-1-1 and RCD-1-2, when encountering each other, form pores and cause pyroptosis, underlying allorecognition in Neurospora. The cryo-EM structure reveals a pore of 11 RCD-1-1/RCD-1-2 heterodimers and a heterodimerization-triggered pore assembly mechanism. This study shows mechanistic diversities in GSDM activation and indicates versatile functions of GSDMs.

[A study on the regulation of motor behavior in mouse based on temporal interference].

Temporal interference (TI) as a new neuromodulation technique can be applied to non-invasive deep brain stimulation. In order to verify its effectiveness in the regulation of motor behavior in animals, this paper uses the TI method to focus the envelope electric field to the ventral posterior lateral nucleus (VPL) of the thalamus in the deep brain of mouse to regulate left- and right-turning motor behavior. The focusability of TI in the mouse VPL was analyzed by finite element method, and the focus area and volume were obtained by numerical calculation. A stimulator was used to generate TI current to stimulate the mouse VPL to verify the effectiveness of the TI stimulation method, and the accuracy of the focus location was further determined by c-Fos immunofluorescence experiments. The results showed that the electric field generated by TI stimulation was able to focus on the VPL nuclei when the stimulation current reached 800 µA; the mouse were able to make corresponding left and right turns according to the stimulation position; and the c-Fos positive cell markers in the VPL nuclei increased significantly after stimulation. This study confirms the feasibility of TI in regulating animal motor behavior and provides a non-invasive stimulation method for brain tissue for animal robots.

Rapid identification of potential nonsteroidal anti-inflammatory drug overdose-induced liver toxicity and prediction of follow-up exposure: Integrating bioanalytical and population pharmacokinetic assay.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequently used drugs that can cause liver toxicity. The aim of this study was to integrate bioanalytical and population pharmacokinetic (PopPK) assay to rapidly screen and quantify the concentrations of NSAIDs in plasma and monitor clinical safety. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of acetaminophen (APAP), flurbiprofen (FLB), aspirin (ASP), and ibuprofen (IBP), four commonly used NSAIDs. The PopPK model of the signature toxicant was analyzed based on the published literature. The LC-MS/MS method was successfully validated and applied to determine NSAID concentrations in patient plasma samples. APAP, ASP, and IBP data were best fitted using a one-compartment model, and FLB data were best fitted using a two-compartment model. Bootstrapping and visual predictive checks suggested that a robust and reliable pharmacokinetic model was developed. A fast, simple, and sensitive LC-MS/MS method was developed and validated for determining APAP, FLB, ASP, and IBP in human plasma. Combined with the PopPK model, this method was applied to rapidly analyze the concentrations of NSAIDs in clinical samples from patients presenting to the emergency department with acute liver dysfunction and monitored NSAIDs clinical safety.