AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Non-canonical regulation of the reactivation of an oncogenic herpesvirus by the OTUD4-USP7 deubiquitinases.

Deubiquitinases (DUBs) remove ubiquitin from substrates and play crucial roles in diverse biological processes. However, our understanding of deubiquitination in viral replication remains limited. Employing an oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) to probe the role of protein deubiquitination, we found that Ovarian tumor family deubiquitinase 4 (OTUD4) promotes KSHV reactivation. OTUD4 interacts with the replication and transcription activator (K-RTA), a key transcription factor that controls KSHV reactivation, and enhances K-RTA stability by promoting its deubiquitination. Notably, the DUB activity of OTUD4 is not required for K-RTA stabilization; instead, OTUD4 functions as an adaptor protein to recruit another DUB, USP7, to deubiquitinate K-RTA and facilitate KSHV lytic reactivation. Our study has revealed a novel mechanism whereby KSHV hijacks OTUD4-USP7 deubiquitinases to promote lytic reactivation, which could be potentially harnessed for the development of new antiviral therapies.

Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

Daily glycome and transcriptome profiling reveals polysaccharide structures and correlated glycosyltransferases critical for cotton fiber growth.

Cotton fiber is the most valuable naturally available material for the textile industry and the fiber length and strength are key determinants of its quality. Dynamic changes in the pectin, xyloglucan, xylan, and cellulose polysaccharide epitope content during fiber growth contribute to complex remodeling of fiber cell wall (CW) and quality. Detailed knowledge about polysaccharide compositional and structural alteration in the fiber during fiber elongation and strengthening is important to understand the molecular dynamics of fiber development and improve its quality. Here, large-scale glycome profiling coupled with fiber phenotype and transcriptome profiling was conducted on fiber collected daily covering the most critical window of fiber development. The profiling studies with high temporal resolution allowed us to identify specific polysaccharide epitopes associated with distinct fiber phenotypes that might contribute to fiber quality. This study revealed the critical role of highly branched RG-I pectin epitopes such as ß-1,4-linked-galactans, ß-1,6-linked-galactans, and arabinogalactans, in addition to earlier reported homogalacturonans and xyloglucans in the formation of cotton fiber middle lamella and contributing to fiber plasticity and elongation. We also propose the essential role of heteroxylans (Xyl-MeGlcA and Xyl-3Ar), as a guiding factor for secondary CW cellulose microfibril arrangement, thus contributing to fiber strength. Correlation analysis of profiles of polysaccharide epitopes from glycome data and expression profiles of glycosyltransferase-encoding genes from transcriptome data identified several key putative glycosyltransferases that are potentially involved in synthesizing the critical polysaccharide epitopes. The findings of this study provide a foundation to identify molecular factors that dictate important fiber traits.

Mitochondrial Dysfunction of Peripheral Platelets as a Predictive Biomarker for Postoperative Delirium in Elderly Patients.

OBJECTIVE: To determine the association between the preoperative Bioenergetic Health Index (BHI) of platelets and the occurrence of postoperative delirium (POD) in elderly patients. METHODS: Elderly patients scheduled for major abdominal surgery under general anesthesia were included. The presence of POD was assessed within the 3 days after surgery. Seahorse XF analysis and transmission electron microscopy were utilized to evaluate the mitochondrial metabolism and morphology of platelets. RESULTS: A total of 20 out of 162 participants developed POD. Participants with POD showed lower preoperative Mini-Mental State Examination scores and total protein levels, fewer educational years, longer surgery duration, higher mean platelet volume, and lower platelet BHI compared with those without POD. Damaged mitochondria with swollen appearance and distorted cristae was detected in platelets from participants with POD. Preoperative platelet BHI was independently associated with the occurrence of POD after adjusting for age, education, preoperative Mini-Mental State Examination score, preoperative mean platelet volume and total protein levels, surgical type and duration, and lymphocyte counts on the first postoperative day (OR 0.11, 95% CI 0.03-0.37, p < 0.001). The areas under the receiver operating curves for predicting POD were 0.83 (95% CI 0.76-0.88) for platelet BHI. It showed a sensitivity of 85.00% and specificity of 73.24%, with an optimal cutoff value of 1.61. Using a serial combination (mean platelet volume followed by BHI) yielded a sensitivity of 80.00% and specificity of 82.39%. INTERPRETATION: Preoperative platelet BHI was independently associated with the occurrence of POD in elderly patients and has the potential as a screening biomarker for POD risk. ANN NEUROL 2024;96:74-86.

Uterine Immunoprivileged Cells Restore Cardiac Function of Male Recipients After Myocardial Infarction.

It has been documented that the uterus plays a key cardio-protective role in pre-menopausal women, which is supported by uterine cell therapy, to preserve cardiac functioning post-myocardial infarction, being effective among females. However, whether such therapies would also be beneficial among males is still largely unknown. In this study, we aimed to fill in this gap in knowledge by examining the effects of transplanted uterine cells on infarcted male hearts. We identified, based on major histocompatibility complex class I (MHC-I) expression levels, 3 uterine reparative cell populations: MHC-I(neg), MHC-I(mix), and MHC-I(pos). In vitro, MHC-I(neg) cells showed higher levels of pro-angiogenic, pro-survival, and anti-inflammatory factors, compared to MHC-I(mix) and MHC-I(pos). Furthermore, when cocultured with allogeneic mixed leukocytes, MHC-I(neg) had lower cytotoxicity and leukocyte proliferation. In particular, CD8+ cytotoxic T cells significantly decreased, while CD4+CD25+ Tregs and CD4-CD8- double-negative T cells significantly increased when cocultured with MHC-I(neg), compared to MHC-I(mix) and MHC-I(pos) cocultures. In vivo, MHC-I(neg) as well as MHC-I(mix) were found under both syngeneic and allogeneic transplantation in infarcted male hearts, to significantly improve cardiac function and reduce the scar size, via promoting angiogenesis in the infarcted area. All of these findings thus support the view that males could also benefit from the cardio-protective effects observed among females, via cell therapy approaches involving the transplantation of immuno-privileged uterine reparative cells in infarcted hearts.

Mitochondria transplantation alleviates cardiomyocytes apoptosis through inhibiting AMPKα-mTOR mediated excessive autophagy.

The disruption of mitochondria homeostasis can impair the contractile function of cardiomyocytes, leading to cardiac dysfunction and an increased risk of heart failure. This study introduces a pioneering therapeutic strategy employing mitochondria derived from human umbilical cord mesenchymal stem cells (hu-MSC) (MSC-Mito) for heart failure treatment. Initially, we isolated MSC-Mito, confirming their functionality. Subsequently, we monitored the process of single mitochondria transplantation into recipient cells and observed a time-dependent uptake of mitochondria in vivo. Evidence of human-specific mitochondrial DNA (mtDNA) in murine cardiomyocytes was observed after MSC-Mito transplantation. Employing a doxorubicin (DOX)-induced heart failure model, we demonstrated that MSC-Mito transplantation could safeguard cardiac function and avert cardiomyocyte apoptosis, indicating metabolic compatibility between hu-MSC-derived mitochondria and recipient mitochondria. Finally, through RNA sequencing and validation experiments, we discovered that MSC-Mito transplantation potentially exerted cardioprotection by reinstating ATP production and curtailing AMPKα-mTOR-mediated excessive autophagy.

α-ketoglutarate preconditioning extends the survival of engrafted adipose-derived mesenchymal stem cells to accelerate healing of burn wounds.

The application of adipose-derived stem cells (ADSCs) in treating hard-to-heal wounds has been widely accepted, while the short-term survival rate remains an obstacle in stem cell therapy. The aim of this study is to investigate the effect of preconditioning ADSCs with α-ketoglutarate (α-KG) on the healing of acid burn wounds and cell survival within wounds. Preconditioning of ADSCs was performed by treating cells at passage 3 with 3.5 mM DM-αKG for 24 h. Proliferation and migration of ADSCs was examined. An acid burn wound was created on the dorsal skin of mice. Cell suspension of ADSCs (2 × 106 cells/ml), either pre-treated with α-KG or not, was injected subcutaneously around the margin of wound. At 1,4,7,10,14 days after injection, the percentage of wound closure was evaluated. Expression of pro-angiogenic factors, matrix molecules and HIF1-α in pretreated ADSCs or in wounds was evaluated by qRT-PCR and immunohistochemistry staining, respectively. The survival rate of DiO-labelled ADSCs was determined with the in vivo bioluminescent imaging system. Treating with α-KG induced an enhancement in migration of ADSCs, while their proliferation was not affected. Expression of Vegf and Fgf-2 was significantly increased. With injection of pretreated ADSCs, healing of wounds was remarkably accelerated, along with increased ECM deposition and microvessel density. Moreover, pretreatment with α-KG resulted a prolonged survival of engrafted ADSCs was observed. Expression of HIF-1α was significantly increased in ADSCs treated with α-KG and in wounds injected with preconditioned ADSCs. Our results revealed that healing of acid burn wound was accelerated with administration of ADSCs pretreated with α-KG, which induced elevated expression of HIF-1α and prolonged survival of engrafted stem cells.

Engineering a targeted and safe bone anabolic gene therapy to treat osteoporosis in alveolar bone loss.

Alveolar bone loss in elderly populations is highly prevalent and increases the risk of tooth loss, gum disease susceptibility, and facial deformity. Unfortunately, there are very limited treatment options available. Here, we developed a bone-targeted gene therapy that reverses alveolar bone loss in patients with osteoporosis by targeting the adaptor protein Schnurri-3 (SHN3). SHN3 is a promising therapeutic target for alveolar bone regeneration, because SHN3 expression is elevated in the mandible tissues of humans and mice with osteoporosis while deletion of SHN3 in mice greatly increases alveolar bone and tooth dentin mass. We used a bone-targeted recombinant adeno-associated virus (rAAV) carrying an artificial microRNA (miRNA) that silences SHN3 expression to restore alveolar bone loss in mouse models of both postmenopausal and senile osteoporosis by enhancing WNT signaling and osteoblast function. In addition, rAAV-mediated silencing of SHN3 enhanced bone formation and collagen production of human skeletal organoids in xenograft mice. Finally, rAAV expression in the mandible was tightly controlled via liver- and heart-specific miRNA-mediated repression or via a vibration-inducible mechanism. Collectively, our results demonstrate that AAV-based bone anabolic gene therapy is a promising strategy to treat alveolar bone loss in osteoporosis.

Tpgen: a language model for stable protein design with a specific topology structure.

BACKGROUND: Natural proteins occupy a small portion of the protein sequence space, whereas artificial proteins can explore a wider range of possibilities within the sequence space. However, specific requirements may not be met when generating sequences blindly. Research indicates that small proteins have notable advantages, including high stability, accurate resolution prediction, and facile specificity modification. RESULTS: This study involves the construction of a neural network model named TopoProGenerator(TPGen) using a transformer decoder. The model is trained with sequences consisting of a maximum of 65 amino acids. The training process of TopoProGenerator incorporates reinforcement learning and adversarial learning, for fine-tuning. Additionally, it encompasses a stability predictive model trained with a dataset comprising over 200,000 sequences. The results demonstrate that TopoProGenerator is capable of designing stable small protein sequences with specified topology structures. CONCLUSION: TPGen has the ability to generate protein sequences that fold into the specified topology, and the pretraining and fine-tuning methods proposed in this study can serve as a framework for designing various types of proteins.

Protein Profiles and Novel Molecular Biomarkers of Schizophrenia Based on 4D-DIA Proteomics.

Schizophrenia is a severe psychological disorder. The current diagnosis mainly relies on clinical symptoms and lacks laboratory evidence, which makes it very difficult to make an accurate diagnosis especially at an early stage. Plasma protein profiles of schizophrenia patients were obtained and compared with healthy controls using 4D-DIA proteomics technology. Furthermore, 79 DEPs were identified between schizophrenia and healthy controls. GO functional analysis indicated that DEPs were predominantly associated with responses to toxic substances and platelet aggregation, suggesting the presence of metabolic and immune dysregulation in patients with schizophrenia. KEGG pathway enrichment analysis revealed that DEPs were primarily enriched in the chemokine signaling pathway and cytokine receptor interactions. A diagnostic model was ultimately established, comprising three proteins, namely, PFN1, GAPDH and ACTBL2. This model demonstrated an AUC value of 0.972, indicating its effectiveness in accurately identifying schizophrenia. PFN1, GAPDH and ACTBL2 exhibit potential as biomarkers for the early detection of schizophrenia. The findings of our studies provide novel insights into the laboratory-based diagnosis of schizophrenia.

Steering Photooxidation of Methane to Formic Acid over A Priori Screened Supported Catalysts.

Efficient methane photooxidation to formic acid (HCOOH) has emerged as a sustainable approach to simultaneously generate value-added chemicals and harness renewable energy. However, the persistent challenge lies in achieving a high yield and selectivity for HCOOH formation, primarily due to the complexities associated with modulating intermediate conversion and desorption after methane activation. In this study, we employ first-principles calculations as a comprehensive guiding tool and discover that by precisely controlling the O2 activation process on noble metal cocatalysts and the adsorption strength of carbon-containing intermediates on metal oxide supports, one can finely tune the selectivity of methane photooxidation products. Specifically, a bifunctional catalyst comprising Pd nanoparticles and monoclinic WO3 (Pd/WO3) would possess optimal O2 activation kinetics and an intermediate oxidation/desorption barrier, thereby promoting HCOOH formation. As evidenced by experiments, the Pd/WO3 catalyst achieves an exceptional HCOOH yield of 4.67 mmol gcat-1 h-1 with a high selectivity of 62% under full-spectrum light irradiation at room temperature using molecular O2. Notably, these results significantly outperform the state-of-the-art photocatalytic systems operated under identical condition.

Comprehensive analysis of the function of helicobacter-associated ferroptosis gene YWHAE in gastric cancer through multi-omics integration, molecular docking, and machine learning.

Gastric cancer is strongly associated with Helicobacter pylori (H. pylori) infection. However, the molecular mechanisms underlying the development of gastric cancer in the context of H. pylori infection, particularly in relation to ferroptosis, remain poorly understood. In this study, we investigated the role of the Helicobacter-associated ferroptosis gene YWHAE in gastric cancer. We analyzed multi-omics data, performed molecular docking, and employed machine learning to comprehensively evaluate the expression, function, and potential implications in gastric cancer, including its influence on drug sensitivity, mutation, immune microenvironment, immunotherapy, and prognosis. Our findings demonstrated that the YWHAE gene exhibits high expression in both H. pylori-associated gastritis and gastric cancer. Pan-cancer analysis revealed elevated expression of YWHAE in several cancer types compared to normal tissues. We also examined the methylation, single nucleotide variations (SNVs), and copy number variations (CNVs) associated with YWHAE. Single-cell analysis indicated that the YWHAE gene is expressed in various cell types, with its expression level potentially influenced by H. pylori infection. Functionally, we observed a positive correlation between YWHAE gene expression and ferroptosis in gastric cancer and associated with multiple cancer-related signaling pathways, including MAPK, NF-κB, and PI3K. Furthermore, we predicted five small molecule compounds that show promise for treating gastric cancer patients and screened five drugs with the highest correlation with YWHAE and validated them by molecular docking. Additionally, significant differences were observed in various immune cell types and immunotherapeutic response between the high and low YWHAE gene expression groups. Moreover, we found a positive correlation between YWHAE gene expression and the tumour mutation burden (TMB). By applying 10 machine learning algorithms and 101 integration combinations, we developed a prognostic model for YWHAE-related genes. Finally, qRT-PCR and immunohistochemistry (IHC) consistently demonstrated the upregulation of YWHAE in gastric cancer. In conclusion, we conducted a comprehensive analysis of YWHAE gene in gastric cancer. Our findings provided novel insights into the role of YWHAE as a gene associated with H. pylori infection and ferroptosis in gastric cancer and expanded our understanding of the molecular mechanisms underlying gastric carcinogenesis.

Development of an Interpretable Deep Learning Model for Pathological Tumor Response Assessment After Neoadjuvant Therapy.

BACKGROUND: Neoadjuvant therapy followed by surgery has become the standard of care for locally advanced esophageal squamous cell carcinoma (ESCC) and accurate pathological response assessment is critical to assess the therapeutic efficacy. However, it can be laborious and inconsistency between different observers may occur. Hence, we aim to develop an interpretable deep-learning model for efficient pathological response assessment following neoadjuvant therapy in ESCC. METHODS: This retrospective study analyzed 337 ESCC resection specimens from 2020-2021 at the Pudong-Branch (Cohort 1) and 114 from 2021-2022 at the Puxi-Branch (External Cohort 2) of Fudan University Shanghai Cancer Center. Whole slide images (WSIs) from these two cohorts were generated using different scanning machines to test the ability of the model in handling color variations. Four pathologists independently assessed the pathological response. The senior pathologists annotated tumor beds and residual tumor percentages on WSIs to determine consensus labels. Furthermore, 1850 image patches were randomly extracted from Cohort 1 WSIs and binarily classified for tumor viability. A deep-learning model employing knowledge distillation was developed to automatically classify positive patches for each WSI and estimate the viable residual tumor percentages. Spatial heatmaps were output for model explanations and visualizations. RESULTS: The approach achieved high concordance with pathologist consensus, with an R^2 of 0.8437, a RAcc_0.1 of 0.7586, a RAcc_0.3 of 0.9885, which were comparable to two senior pathologists (R^2 of 0.9202/0.9619, RAcc_0.1 of 8506/0.9425, RAcc_0.3 of 1.000/1.000) and surpassing two junior pathologists (R^2 of 0.5592/0.5474, RAcc_0.1 of 0.5287/0.5287, RAcc_0.3 of 0.9080/0.9310). Visualizations enabled the localization of residual viable tumor to augment microscopic assessment. CONCLUSION: This work illustrates deep learning's potential for assisting pathological response assessment. Spatial heatmaps and patch examples provide intuitive explanations of model predictions, engendering clinical trust and adoption (Code and data will be available at https://github.com/WinnieLaugh/ESCC_Percentage once the paper has been conditionally accepted). Integrating interpretable computational pathology could help enhance the efficiency and consistency of tumor response assessment and empower precise oncology treatment decisions.

Dapagliflozin attenuates LPS-induced myocardial injury by reducing ferroptosis.

Septic cardiomyopathy is a severe cardiovascular disease with a poor prognosis. Previous studies have reported the involvement of ferroptosis in the pathogenesis of septic cardiomyopathy. SGLT2 inhibitors such as dapagliflozin have been demonstrated to improve ischemia-reperfusion injury by alleviating ferroptosis in cardiomyocyte. However, the role of dapagliflozin in sepsis remains unclear. Therefore, our study aims to investigate the therapeutic effects of dapagliflozin on LPS-induced septic cardiomyopathy. Our results indicate that dapagliflozin improved cardiac function in septic cardiomyopathy experimental mice. Mechanistically, dapagliflozin works by inhibiting the translation of key proteins involved in ferroptosis, such as GPX4, FTH1, and SLC7A11. It also reduces the transcription of lipid peroxidation-related mRNAs, including PTGS2 and ACSL4, as well as iron metabolism genes TFRC and HMOX1.

IQ domain-containing protein ZmIQD27 modulates water transport in maize.

Plant metaxylem vessels provide physical support to promote upright growth and the transport of water and nutrients. A detailed characterization of the molecular network controlling metaxylem development is lacking. However, knowledge of the events that regulate metaxylem development could contribute to the development of germplasm with improved yield. In this paper, we screened an EMS-induced B73 mutant library, which covers 92% of maize (Zea mays) genes, to identify drought-sensitive phenotypes. Three mutants were identified, named iqd27-1, iqd27-2, and iqd27-3, and genetic crosses showed that they were allelic to each other. The causal gene in these 3 mutants encodes the IQ domain-containing protein ZmIQD27. Our study showed that defective metaxylem vessel development likely causes the drought sensitivity and abnormal water transport phenotypes in the iqd27 mutants. ZmIQD27 was expressed in the root meristematic zone where secondary cell wall deposition is initiated, and loss-of-function iqd27 mutants exhibited a microtubular arrangement disorder. We propose that association of functional ZmIQD27 with microtubules is essential for correct targeted deposition of the building blocks for secondary cell wall development in maize.

Antimicrobial potential of carvacrol against Edwardsiella piscicida in vitro.

With the alarming rise of antibiotic-resistant bacteria, novel antibacterial substances are urgently needed for controlling and treating multidrug-resistant bacterial infections. Edwardsiella piscicida is an important zoonotic enteric pathogen, that can cause systemic hemorrhagic septicemia in fish. Carvacrol, a major terpene of oregano essential oil, has a wide range of antibacterial activities. This study aimed to analyze the effect of carvacrol on the growth and virulence of E. piscicida in vitro. The minimum inhibitory concentration (MIC) of carvacrol against E. piscicida was 125 µg/mL. The sub-inhibitory concentrations of carvacrol significantly decreased the biofilm formation of E. piscicida in a dose dependent manner, whereas increased the hemolytic activity with a negative correlation. The quantitative real-time PCR results showed that carvacrol at sub-MICs downregulated the expression of related virulence genes, including flagellum (fimA, fliC, flgN), hemolysins (ethA, ethB), quorum sensing systems (luxR, qseB), T3SS (esrB, esrC) and T6SS (evpB, evpC). Moreover, carvacrol (≤1/8 MIC) reduced the cytotoxicity, adherence and internalization activities of E. piscicida to the EPC cells. In vivo trial, the diet mixed with carvacrol increased the survival of zebrafish infected with E. piscicida. Overall, these findings suggested that carvacrol might be a promising therapeutic agent against E. piscicida infection in aquaculture.

Automatic lung cancer subtyping using rapid on-site evaluation slides and serum biological markers.

BACKGROUND: Rapid on-site evaluation (ROSE) plays an important role during transbronchial sampling, providing an intraoperative cytopathologic evaluation. However, the shortage of cytopathologists limits its wide application. This study aims to develop a deep learning model to automatically analyze ROSE cytological images. METHODS: The hierarchical multi-label lung cancer subtyping (HMLCS) model that combines whole slide images of ROSE slides and serum biological markers was proposed to discriminate between benign and malignant lesions and recognize different subtypes of lung cancer. A dataset of 811 ROSE slides and paired serum biological markers was retrospectively collected between July 2019 and November 2020, and randomly divided to train, validate, and test the HMLCS model. The area under the curve (AUC) and accuracy were calculated to assess the performance of the model, and Cohen's kappa (κ) was calculated to measure the agreement between the model and the annotation. The HMLCS model was also compared with professional staff. RESULTS: The HMLCS model achieved AUC values of 0.9540 (95% confidence interval [CI]: 0.9257-0.9823) in malignant/benign classification, 0.9126 (95% CI: 0.8756-0.9365) in malignancy subtyping (non-small cell lung cancer [NSCLC], small cell lung cancer [SCLC], or other malignancies), and 0.9297 (95% CI: 0.9026-0.9603) in NSCLC subtyping (lung adenocarcinoma [LUAD], lung squamous cell carcinoma [LUSC], or NSCLC not otherwise specified [NSCLC-NOS]), respectively. In total, the model achieved an AUC of 0.8721 (95% CI: 0.7714-0.9258) and an accuracy of 0.7184 in the six-class classification task (benign, LUAD, LUSC, NSCLC-NOS, SCLC, or other malignancies). In addition, the model demonstrated a κ value of 0.6183 with the annotation, which was comparable to cytopathologists and superior to trained bronchoscopists and technicians. CONCLUSION: The HMLCS model showed promising performance in the multiclassification of lung lesions or intrathoracic lymphadenopathy, with potential application to provide real-time feedback regarding preliminary diagnoses of specimens during transbronchial sampling procedures. CLINICAL TRIAL NUMBER: Not applicable.

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway.

BACKGROUND: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI. METHODS AND RESULTS: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1ß production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis. CONCLUSION: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.

Molecular Dynamics Simulation of Dual Nanodroplet Impacts on a Cylindrical Surface.

Droplet impact behavior is ubiquitous in various fields. However, the dynamics and spreading mechanisms of micro- and nanoscale droplet impact on curved surfaces, particularly in the case of multiple droplets, have yet to be fully elucidated. In this study, molecular dynamics (MD) methods are employed to investigate the dynamic evolution of double nanodroplet impact on a nano cylindrical wall. The effects of droplet spacing, initial impact velocity, and wall wettability on droplet impact characteristics are analyzed. The results demonstrate that there are five impact modes of nanoscale double-droplet impacts with nanocylinders: spreading-partial wrapping-splitting-complete detachment (SPSC), spreading-complete wrapping-complete attachment (SCC), spreading-partial wrapping-complete attachment (SPC), spreading-partial wrapping-partial attachment (SPP), and spreading-partial wrapping-fragmentation-partial attachment (SPFP). The droplet spacing has an insignificant effect on the impact modes but affects the droplets' spreading shape in both the axial and radial directions. The initial velocity and wall wettability have significant impacts on the droplet impact modes and liquid film spreading characteristics. As the initial velocity increases, the liquid film's radial and axial spreading distances gradually increase. Under hydrophobic conditions, the spreading of the droplet is dominant in the radial direction, while under hydrophilic conditions, the spreading is dominant in the axial direction. Properly reducing the droplet spacing, increasing the impact velocity, and enhancing the wall hydrophobicity can promote detaching the droplet from the cylindrical wall.