Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system.

Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD+ and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD+-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5'-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD+ hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.

Anti-inflammatory bicyclic polyprenylated acylphloroglucinols with diverse architectures including an unprecedented 6/6/6 tricyclic core from Garcinia yunnanensis.

Garciyunnanol A (1), an unprecedented 1,2-seco-bicyclic polyprenylated acylphloroglucinol (BPAP) possessing a unique 6/6/6 tricyclic core, was characterized from Garcinia yunnanensis together with 16 BPAPs, including eight new compounds (garciyunnanols B-I, 2-9). Biogenetically, the bicyclo[3.3.1]nonane-2,4,9-trione moiety of 12 reconstructed the bicyclic δ-lactone core of 2 through Norrish type Ⅰ cleavage and cyclization, followed by a cyclization of two side chains to form an intriguing 6/6/6 tricyclic core of 1. Their structures were elucidated through analysis of spectroscopic data, calculation and comparison of ECD spectra. Bioactivity evaluation manifested that compounds 1, 2, 5, 6 and 14 demonstrated superior inhibition of NO production compared to the positive control dexamethasone. Notably, compound 5 exhibited a dose-dependent inhibitory effect on NO production, with an IC50 value of 0.25 ± 0.87 µM. Furthermore, experiments involving ELISA, Western blotting, and immunofluorescence staining revealed that 5 effectively reduced the secretion of interleukin-1ß in LPS plus nigericin-stimulated THP-1 macrophages by inhibiting the activation of the NLRP3 inflammasome.

Conformational and Solvent Effects on the Photoinduced Electron Transfer Dynamics of a Zinc Phthalocyanine-Benzoperylenetriimide Conjugate: A Nonadiabatic Dynamics Simulation.

Herein, we employed a combination of static electronic structure calculations and nonadiabatic dynamics simulations at linear-response time dependent density functional theory (LR-TDDFT) level with the optimally tuned range-separated hybrid (OT-RSH) functional to explore the ultrafast photoinduced dynamics of a zinc phthalocyanine-benzoperylenetriimide (ZnPc-BPTI) conjugate. Due to the flexibility of the linker, we identified two major conformations: the stacked conformation (ZnPc-BPTI-1) and the extended conformation (ZnPc-BPTI-2). Since the charge transfer states are much lower than the lowest local excitation in ZnPc-BPTI-1, which is contrary to ZnPc-BPTI-2, the ultrafast electron transfer (~3.6 ps) is only observed in the nonadiabatic simulations of ZnPc-BPTI-1 upon local excitation around the absorption maximum of ZnPc. However, when considering the solvent effects in benzonitrile: the lowest S1 states are both charge transfer states from ZnPc to BPTI for different conformers. Subsequent nonadiabatic dynamics simulations indicate that both conformers experience ultrafast electron transfer in benzonitrile with two time constants of 90 [100] fs and 1.40 [1.43] ps. Our present work not only agrees well with previous experimental study, but also points out the important role of conformational changes and solvent effects in regulating the photodynamics of organic donor-acceptor conjugates.

Lipoprotein(a) in atherosclerotic cardiovascular disease and proprotein convertase subtilisin/kexin-type 9 inhibitors.

High plasma lipoprotein(a) (Lp(a)) levels increase the cardiovascular risk in populations with atherosclerotic cardiovascular disease (ASCVD). Apolipoprotein (a) [apo(a)], a unique protein component of Lp(a), plays an important role in the pathogenesis of atherosclerosis. Statins, the primary medication in managing ASCVD, lower low-density lipoprotein cholesterol (LDL-C) but concurrently elevate plasma Lp(a) levels, contributing to an increased residual cardiovascular risk. In turn, proprotein convertase subtilisin/kexin-type 9 (PCSK9) inhibitors, a novel class of LDL-C lowering drugs, effectively reduce plasma Lp(a) levels, which is believed to decrease residual cardiovascular risk. However, the mechanism by which PCSK9 inhibitors reduce Lp(a) levels remains unknown. In addition, there are some clinical limitations of PCSK9 inhibitors. Here, we systematically review the past, present, and prospects of studies pertaining to Lp(a), PCSK9 inhibitors, and ASCVD.

Establishing M1 subdivision for de novo nasopharyngeal carcinoma patients receiving immuno-chemotherapy: A multicenter, retrospective cohort study.

OBJECTIVES: This study aims to better manage de novo metastatic nasopharyngeal carcinoma (NPC) patients receiving palliative immuno-chemotherapy (PICT), thereby easily determining individual survival outcomes. MATERIALS AND METHODS: Patients with de novo metastatic NPC from four centers who received first-line PICT were included. We developed a nomogram for the pretherapy overall survival (OS) prediction using a logistic regression model in the training cohort (n = 296). We assessed the performance of this nomogram in a validation cohort. RESULTS: A total of 592 patients were included. The median follow-up time was 29.83 months. Bone metastasis (HR, 2.46; 95 % CI, 1.01-6.21; p = 0.049) and the number of metastatic lesions > 3 (HR, 2.78; 95 % CI, 1.24-6.24; p = 0.013) were independent prognostic indicators. A new two-category M1 subdivision was generated: M1a, defined by the absence of co-existing bone metastasis and the presence of more than three metastatic lesions; and M1b, characterized by the presence of co-existing bone metastasis and the presence of more than three metastatic lesions. The 3-year OS rates of patients with M1a vs. M1b were 87.1 % vs. 60.3 % (p < 0.001). The C-indexes were 0.652 and 0.581 in the training and validation cohorts. The 1-, 2-, and 3-year areas under the curve (AUC) were 0.69, 0.68, 0.68 in the training cohort and 0.64, 0.6, 0.6 in the validation cohort. DCA curves also indicated that the nomogram has good clinical utility. CONCLUSION: The proposed M1 subdivision provides good OS segregation for patients receiving PICT.

Human umbilical cord mesenchymal stem cells derived extracellular vesicles ameliorate kidney ischemia-reperfusion injury by suppression of senescent tubular epithelial cells - Experimental Study.

BACKGROUND: Human umbilical cord mesenchymal stem cells derived extracellular vesicles (HUMSC-EVs) have drawn much interest in kidney transplantation, mainly because of their renoprotection by alleviating cell injury and stimulating tissue repair. Cellular senescence has been proven to play a dual regulatory role in kidney ischemia-reperfusion injury (IRI), and the regulation of HUMSC-EVs on tubular epithelial cell senescence may be a potential therapeutic target. MATERIALS AND METHODS: In vitro, the hypoxia-reoxygenation of human kidney-2 cells was used to simulate kidney IRI, and the regulation of HUMSC-EVs on human kidney-2 cells was detected. Transcriptome sequencing of human kidney-2 cells was used to explore the potential regulatory mechanism. In vivo, adult male mice were divided into five groups: control group, IRI group, HUMSC-EVs treatment group, senolytics treatment group (dasatinib + quercetin), and combined treatments group (HUMSC-EVs and senolytics). Kidney function, senescent features of tubular epithelial cells, acute kidney injury, and chronic interstitial fibrosis in mice were detected to explore the renoprotection effects of HUMSC-EVs. RESULTS: Kidney IRI significantly up-regulated expressions of LaminB1, p53, p21, p16, senescence-associated beta-galactosidase, and apoptosis of tubular epithelial cells. In the mouse kidney IRI model, kidney subcapsular injection of HUMSC-EVs significantly improved kidney function, reducing the senescent features of tubular epithelial cells and alleviating acute kidney injury and chronic interstitial fibrosis. HUMSC-EVs mainly achieved renoprotection by regulating Bax/Bcl-2-dependent apoptosis during acute kidney injury and mostly reduced tubular atrophy and kidney interstitial fibrosis by regulating Ras-pERK-Ets1-p53 pathway-dependent cell senescence. Oral administration of senolytics also alleviated kidney injury induced by IRI, while the combined treatments of HUMSC-EVs and senolytics had better renoprotection effects. CONCLUSIONS: The combination of HUMSC-EVs and senolytics alleviated acute kidney injury and chronic interstitial fibrosis by dynamic regulation of cell senescence and apoptosis, which provides a therapeutic potential strategy for organ preservation and tissue repair in kidney transplantation.

Burden of Voice Use and Voice Disorders in Early Childhood Educators in Singapore.

It is well established that teachers are prone to voice-related problems. Much of existing literature focuses on grade-school teachers, and early childhood educators are an understudied demographic. This study aims to determine the burden of voice use and prevalence of voice disorders in early childhood educators in Singapore. METHODS: A cross-sectional survey was performed in 2022. This was sent via email to all registered preschools/kindergartens in Singapore. RESULTS: There were a total of 413 responses. Mean age of respondents was 37.4years (range 28-46years) and 99.0% were female. 67.0% were Chinese, 14.8% Malay, 9.5% Indian, and 8.7% Others. Regarding burden of voice use, 81.4% of respondents reported talking for extended periods of time (>30 minutes) and 74.5% reported having to sing on >1 occasion a day. 321/413 respondents (77.9%) had to shout or raise their voice at least once a day, whereas 128 of out 413 respondents (31.1%) reported having to scream at least once a day. Point prevalence of voice disorder (defined by VHI-10 >11 was 37.5% and sVHI-10 >12) was 47.2%. Over the preceding 12months, 72.2% of respondents experienced hoarseness on at least 4-7 occasions, with 41.4% losing their voice. While 52.5% of respondents have taken at least 1 sick day off due to an issue with their voice, only 38% has consulted a GP and 13.6% consulted an ENT specialist on at least one occasion. 45.8% have considered leaving the profession on at least one occasion. Factors significantly associated with both point prevalence and year-long prevalence of voice disorders include large class size of >20 students and needing to shout and/or scream >1 time/day.

Preoperative gut microbiota of POCD patients induces pre- and postoperative cognitive impairment and systemic inflammation in rats.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is common following surgery in elderly patients. The role of the preoperative gut microbiota in POCD has attracted increasing attention, but the potential underlying mechanisms remain unclear. This research aimed to investigate the impact of the preoperative gut microbiota on POCD. METHODS: Herein, we analyzed the preoperative gut microbiota of POCD patients through a prospective specimen collection and retrospective blinded evaluation study. Then, we transferred the preoperative gut microbiota of POCD patients to antibiotic-treated rats and established POCD model by abdominal surgery to explore the impact of the preoperative gut microbiota on pre- and postoperative cognitive function and systemic inflammation. The gut microbiota was analyzed using 16S rRNA sequencing analysis. The Morris water maze test was performed to evaluate learning and memory abilities. The inflammatory cytokines TNF-α, IL-1ß and IL-6 in the serum and hippocampus were measured by ELISA. Microglia were examined by immunofluorescence staining for Iba-1. RESULTS: Based on the decrease in the postoperative MMSE score, 24 patients were identified as having POCD and were matched with 24 control patients. Compared with control patients, POCD patients exhibited higher BMI and lower preoperative MMSE score. The preoperative gut microbiota of POCD patients had lower bacterial richness but a larger distribution, decreased abundance of Firmicutes and increased abundance of Proteobacteria than did that of control patients. Compared with rats that received preoperative fecal samples of control patients, rats that received preoperative fecal samples of POCD patients presented an increased abundance of Desulfobacterota, decreased cognitive function, increased levels of TNF-α and IL-1ß in the serum, increased levels of TNF-α and greater microglial activation in the hippocampus. Additionally, correlation analysis revealed a positive association between the abundance of Desulfobacterota and the level of serum TNF-α in rats. Then, we performed abdominal surgery to investigate the impact of the preoperative gut microbiota on postoperative conditions, and the surgery did indeed cause POCD and inflammatory response. Notably, compared with rats that received preoperative fecal samples of control patients, rats that received preoperative fecal samples of POCD patients displayed exacerbated cognitive impairment; increased levels of TNF-α, IL-1ß and IL-6 in the serum and hippocampus; and increased activation of microglia in the hippocampus. CONCLUSIONS: Our findings suggest that the preoperative gut microbiota of POCD patients can induce preoperative and aggravate postoperative cognitive impairment and systemic inflammation in rats. Modulating inflammation by targeting the gut microbiota might be a promising approach for preventing POCD.

Baffled flow constructed wetland-microbial fuel cell coupling systems for combined secondary and tertiary wastewater treatment with simultaneous bioelectricity generation.

Baffled flow constructed wetland-microbial fuel cell (BFCW-MFC) coupling systems were constructed with baffles embedded in cathode chamber. The performance of BFCW-MFCs operated at different hydraulic retention times (HRTs) was evaluated. At the representative HRT of 48 h, embedding 1 or 2 baffles (i.e., BFCW-MFC1 and BFCW-MFC2) produced 32.9 % (29.5 mW/m3) and 53.2 % (34.0 mW/m3) more power density than control system (22.2 mW/m3), respectively. Comparable organics biodegradation efficiencies were observed in BFCW-MFCs at the same HRTs. BFCW-MFC1 and BFCW-MFC2 had higher ammonium and total nitrogen removal efficiency. All systems had decreased nitrogen removal performance as shortening HRT from 72 to 12 h. Multiple nitrogen removal processes were involved, including ammonium oxidation, anammox, and heterotrophic and autotrophic denitrification. The predominant bacteria on electrodes were identified for analyzing bioelectricity generation and wastewater treatment processes. Generally, simultaneous wastewater treatment and bioelectricity generation were obtained in BFCW-MFCs, and embedding 1 or 2 baffles was preferable.

The Association between Cardiac Arrest and Mortality in Patients with Acute Myocardial Infarction Complicated by Cardiogenic Shock.

Background: The impact of cardiac arrest (CA) at admission on the prognosis of patients with acute myocardial infarction (AMI) complicated by cardiogenic shock (CS) remains a subject of debate. Methods: We conducted a retrospective study at West China Hospital from 2018 to 2021, enrolling 247 patients with AMI complicated by CS (AMI-CS). Patients were categorized into CA and non-CA groups based on their admission status. Univariate and multivariate Cox regression analyses were performed, with 30-day and 1-year mortality as the primary endpoints. Kaplan-Meier plots were constructed, and concordance (C)-indices of the Global Registry of Acute Coronary Event (GRACE) score, Intra-aortic Balloon Pump in Cardiogenic Shock (IABP-SHOCK) II score, and IABP-SHOCK II score with CA were calculated. Results: Among the enrolled patients, 39 experienced CA and received cardiopulmonary resuscitation at admission. The 30-day and 1-year mortality rates were 40.9% and 47.0%, respectively. Neither univariate nor multivariate Cox regression analyses identified CA as a significant risk factor for 30-day and 1-year mortality. In C-statistics, the GRACE score exhibited a moderate effect (C-indices were 0.69 and 0.67, respectively), while the IABP-SHOCK II score had a better predictive performance (C-indices were 0.79 and 0.76, respectively) for the 30-day and 1-year mortality. Furthermore, CA did not enhance the predictive value of the IABP-SHOCK II score for 30-day (p = 0.864) and 1-year mortality (p = 0.888). Conclusions: Cardiac arrest at admission did not influence the survival of patients with AMI-CS. Active resuscitation should be prioritized for patients with AMI-CS, regardless of the presence of cardiac arrest.

Epigenetic modification of Castor zinc finger 1 (CASZ1) is associated with tumor microenvironments and prognosis of clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) represents the predominant and remarkably diverse form of renal cell carcinoma. The involvement of the Castor zinc finger 1 (CASZ1) gene in adverse prognostic outcomes has been observed across different cancer types. Nevertheless, the specific altered activities and associated multi-omics characteristics of CASZ1 in ccRCC remain unelucidated. METHOD: In order to explore the expression of CASZ1, evaluate its prognostic significance, and aid in the therapeutic decision-making process for patients with ccRCC, the The Cancer Genome Atlas(TCGA), Gene expression omnibus (GEO), and The Human Protein Atlas (HPA) databases were utilized to gather data on clinicopathological data, prognostic information, genomic, methylomic and immunomic data. Additionally, the Genomics of drug sensitivity in cancer (GDSC) database provided information on drug sensitivity. RESULTS: CASZ1 expression was found to be significantly reduced in ccRCC and was associated with unfavorable pathological characteristics and a bleak prognosis. Diminished CASZ1 mRNA levels were notably correlated with heightened cytosine-phosphate-guanine (CpG) methylation , indicating a poorer prognosis for patients with increased methylation. Examination of RNA-seq data from TCGA indicated that the CASZ1-high expression subgroup displayed heightened immune cell infiltration and increased expression of immune checkpoint markers, potentially suggesting a more favorable response to immunotherapy. Furthermore, data from the GDSC database indicated that the CASZ1-low expression subgroup might exhibit greater sensitivity to anti-angiogenetic treatments, such as Sunitinib and Axitinib. CONCLUSIONS: These results indicate that CASZ1 may function as a biomarker for distinguishing various tumor microenvironment phenotypes, predicting prognosis, and assisting in treatment decisions for individuals with ccRCC.

Association between Healthy Eating Index-2015 and prostate enlargement: A cross-sectional study of the National and Nutrition Examination Survey 2001-2008.

Background: Benign prostate hyperplasia (BPH) occurs in elder men globally with high prevalence. Human diet and lifestyle aroused great attention in the prevalence of BPH. Prostate enlargement (PE) is a major symptom of BPH. Objectives: To elaborate the effect of total diet quality for adults from the United States, we investigated the association between Health Eating Index (HEI)-2015 and the risk of PE in adults from the National Health and Nutrition Examination Survey (NHANES). Methods: This cross-sectional study was conducted based on NHANES 2001-2008. Participants who reported a PE history were included. We conducted a logistic regression analysis to investigate the association between HEI-2015 and PE. Results: A total of 4,866 male participants aged 40 and above were enrolled. Compared with Q1 of HEI-2015, no significant differences were found in adjusted models. Higher vegetables intake (Odds ratio [OR] = 1.073; 95% confidence interval [95%CI] 1.015 to 1.134, P = 0.02) and higher total dairy intake (OR = 1.034; 95%CI 1.009 to 1.061, P = 0.01) were significantly related with higher risk of PE. Conclusions: There was no significant difference between HEI-2015 and PE after full adjustment. Total vegetables and dairy product might be associated with higher risk of PE and needed further validation.

Unraveling how hydrogen-bonding networks affect the capture of amphetamine-type stimulants by polymerized deep eutectic solvent modified magnetic biochar: Coupling quantum chemical calculations with experiment.

The abuse of amphetamine-type stimulants (ATSs) has caused irreversible harm to public safety and ecosystems. A novel polymerized deep eutectic solvent modified magnetic pomelo peel biochar (PMBC) was prepared, and the differences in adsorption of four abused amphetamine-type stimulants (ATSs: AMP, MAMP, MDA and MDMA) were due to varying hydrogen bonds quantities and strengths. PMBC showed excellent chemical reactivity to MDMA, with a maximum adsorption capacity of 926.13 µg g-1, which was 3.25, 2.52 and 1.15 times higher than that of AMP, MAMP and MDA, respectively. Modern spectral analysis showed that there were a series of active centers (-COOH, -NH2 and -OH) on the PMBC, which could form hydrogen bond networks with the nitrogen and oxygen functional groups of ATSs. In various chemical environments: pH level (4-11), inorganic ion and organic matter (humic acid), PMBC maintained high activity towards four ATSs. Additionally, the quantum chemical calculations revealed that the methylenedioxy bridge of ATSs can increase the active sites, and the -NH- and -NH2 groups had different hydrogen bond formation capabilities, which together resulted in the adsorption order of PMBC on the four ATSs: MDMA > MDA > MAMP > AMP. Moreover, the hydrogen-bonding binding energies of several common hydrogen-bonding types were compared, including O-H····O, N-H····O/O-H····N and N-H···N. This study laid an empirical and theoretical foundation for the efficient capture of ATSs in water and contributed to the innovative design of materials.

Label-Aware Dual Graph Neural Networks for Multi-Label Fundus Image Classification.

Fundus disease is a complex and universal disease involving a variety of pathologies. Its early diagnosis using fundus images can effectively prevent further diseases and provide targeted treatment plans for patients. Recent deep learning models for classification of this disease are gradually emerging as a critical research field, which is attracting widespread attention. However, in practice, most of the existing methods only focus on local visual cues of a single image, and ignore the underlying explicit interaction similarity between subjects and correlation information among pathologies in fundus diseases. In this paper, we propose a novel label-aware dual graph neural networks for multi-label fundus image classification that consists of population-based graph representation learning and pathology-based graph representation learning modules. Specifically, we first construct a population-based graph by integrating image features and non-image information to learn patient's representations by incorporating associations between subjects. Then, we represent pathologies as a sparse graph where its nodes are associated with pathology-based feature vectors and the edges correspond to probability of the co-occurrence of labels to generate a set of classifier scores by the propagation of multi-layer graph information. Finally, our model can adaptively recalibrate multi-label outputs. Detailed experiments and analysis of our results show the effectiveness of our method compared with state-of-the-art multi-label fundus image classification methods.

Exploration on cold adaptation of Antarctic lichen via detection of positive selection genes.

Lichen as mutualistic symbiosis is the dominant organism in various extreme terrestrial environment on Earth, however, the mechanisms of their adaptation to extreme habitats have not been fully elucidated. In this study, we chose the Antarctic dominant lichen species Usnea aurantiacoatra to generate a high-quality genome, carried out phylogenetic analysis using maximum likelihood and identify genes under positive selection. We performed functional enrichment analysis on the positively selected genes (PSGs) and found that most of the PSGs focused on transmembrane transporter activity and vacuole components. This suggest that the genes related to energy storage and transport in Antarctic U. aurantiacoatra were affected by environmental pressure. Inside of the 86 PSGs screened, two protein interaction networks were identified, which were RNA helicase related proteins and regulator of G-protein signaling related proteins. The regulator of the G-protein signaling gene (UaRGS1) was chosen to perform further verification by the lichen genetic manipulation system Umbilicaria muhlenbergii. Given that the absence of UmRgs1 resulted in elevated lethality to cold shock, the role for UaRgs1 in Antarctic U. aurantiacoatra resistance to cold can be inferred. The investigation of lichen adaptation to extreme environments at the molecular level will be opened up.

Application of real-time shear wave elastography to Achilles tendon hardness evaluation in older adults.

BACKGROUND: Real-time shear wave elastography (SWE) is a non-invasive imaging technique used to measure tissue stiffness by generating and tracking shear waves in real time. This advanced ultrasound-based method provides quantitative information regarding tissue elasticity, offering valuable insights into the mechanical properties of biological tissues. However, the application of real-time SWE in the musculoskeletal system and sports medicine has not been extensively studied. AIM: To explore the practical value of real-time SWE for assessing Achilles tendon hardness in older adults. METHODS: A total of 60 participants were enrolled in the present study, and differences in the elastic moduli of the bilateral Achilles tendons were compared among the following categories: (1) Age: 55-60, 60-65, and 65-70-years-old; (2) Sex: Male and female; (3) Laterality: Left and right sides; (4) Tendon state: Relaxed and tense state; and (5) Tendon segment: Proximal, middle, and distal. RESULTS: There were no significant differences in the elastic moduli of the bilateral Achilles tendons when comparing by age or sex (P > 0.05). There were, however, significant differences when comparing by tendon side, state, or segment (P < 0.05). CONCLUSION: Real-time SWE plays a significant role compared to other examination methods in the evaluation of Achilles tendon hardness in older adults.

A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion.

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25+ TIGIT+ double-positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explore the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25+ TIGIT+ double-positive cells over single-positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

Deep longitudinal lower respiratory tract microbiome profiling reveals genome-resolved functional and evolutionary dynamics in critical illness.

The lower respiratory tract (LRT) microbiome impacts human health, especially among critically ill patients. However, comprehensive characterizations of the LRT microbiome remain challenging due to low microbial mass and host contamination. We develop a chelex100-based low-biomass microbial-enrichment method (CMEM) that enables deep metagenomic profiling of LRT samples to recover near-complete microbial genomes. We apply the method to 453 longitudinal LRT samples from 157 intensive care unit (ICU) patients in three geographically distant hospitals. We recover 120 high-quality metagenome-assembled genomes (MAGs) and associated plasmids without culturing. We detect divergent longitudinal microbiome dynamics and hospital-specific dominant opportunistic pathogens and resistomes in pneumonia patients. Diagnosed pneumonia and the ICU stay duration were associated with the abundance of specific antibiotic-resistance genes (ARGs). Moreover, CMEM can serve as a robust tool for genome-resolved analyses. MAG-based analyses reveal strain-specific resistome and virulome among opportunistic pathogen strains. Evolutionary analyses discover increased mobilome in prevailing opportunistic pathogens, highly conserved plasmids, and new recombination hotspots associated with conjugative elements and prophages. Integrative analysis with epidemiological data reveals frequent putative inter-patient strain transmissions in ICUs. In summary, we present a genome-resolved functional, transmission, and evolutionary landscape of the LRT microbiota in critically ill patients.