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Abstract Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges in modulating their protein interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of traditional tools. Aptamers possess large molecular weights, expansive blocking surfaces and efficient cellular internalization, making them compelling tools for modulating TF interactions. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamers) that selectively block TF interactions. Our approach leads to the discovery of iAptamers that cooperatively disrupt SCAF4/SCAF8-RNAP2 interactions, dysregulating RNAP2-dependent gene expression, which impairs cell proliferation. This approach is further applied to develop iAptamers blocking WDR5-MYC interactions. Overall, our study highlights the potential of iAptamers in disrupting pathogenic TF interactions, implicating their potential utility in studying the biological functions of TF interactions and in nucleic acids drug discovery.

Abstract The conversion of farmland to forest in China has been recognized for its positive impact on above-ground vegetation and carbon sequestration. However, the impact on soil quality during land conversion, particularly in vulnerable karst areas, has received less attention. In this study conducted in a karst area of southwest China, eight different farmland conversion strategies were investigated to assess improvements in surface soil carbon, nitrogen, and ecosystem multi-functionality (EMF). Our results showed that farmland converted to afforestation areas or farmland that was abandoned contained higher amounts of carbon (total, organic, active) and ammonium nitrogen (NH4 +-N) in the soil compared to farmland converted to grassland or maize crop. Soluble organic carbon levels were higher in afforestation and grassland areas compared to maize crop controls. By contrast, soil from grassland and abandoned land exhibited higher levels of nitrate nitrogen (NO3 --N) compared to afforestation land or maize crop controls. There were no differences in NH4 +-N content between any condition, except for afforestation land that specifically contained the Zenia insignis plant species. Afforestation land consistently exhibited higher EMF values than grassland. Pearson correlation analysis revealed positive relationships between soil indices and EMF scores, except for NO3 --N.Random forest analysis explained 95% of the variation in soil EMF and identified specific soil factors: total carbon, organic carbon, active labile organic carbon, total nitrogen, and ammonium nitrogen, as the main drivers of soil multi-functionality. Our studies show how various reforestation strategies can enhance soil nutrient sequestration and improve soil multi-functionality of farmland in the karst areas.These findings provide insight into sustainable soil management practices for converting farmland into natural areas.

Abstract The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Abstract Background Diabetic cardiomyopathy (DCM), a serious complication of diabetes, leads to structural and functional abnormalities of the heart and ultimately evolves to heart failure. IL-37 exerts a substantial influence on the regulation of inflammation and metabolism. Whether IL-37 is involved in DCM is unknown. Methods The plasma samples were collected from healthy controls, diabetic patients and DCM patients, and the level of IL-37 and its relationship with heart function were observed. The changes in cardiac function, myocardial fibrosis and mitochondrial injury in DCM mice with or without IL-37 intervention were investigated in vivo. By an in vitro co-culture approach involving HG challenge of cardiomyocytes and fibroblasts, the interaction carried out by cardiomyocytes on fibroblast profibrotic activation was studied. Finally, the possible interactive mediator between cardiomyocytes and fibroblasts was explored, and the intervention role of IL-37 and its relevant molecular mechanisms. Results We showed that the level of plasma IL-37 in DCM patients was upregulated compared to that in healthy controls and diabetic patients. Both recombinant IL-37 administration or inducing IL-37 expression alleviated cardiac dysfunction and myocardial fibrosis in DCM mice. Mechanically, hyperglycemia impaired mitochondria through SIRT1/AMPK/PGC1α signaling, resulting in significant cardiomyocyte apoptosis and the release of extracellular vesicles containing mtDNA. Fibroblasts then engulfed these mtDNA-enriched vesicles, thereby activating TLR9 signaling and the cGAS-STING pathway to initiate pro-fibrotic process and adverse remodeling. However, the presence of IL-37 ameliorated mitochondrial injury by preserving the activity of SIRT1-AMPK-PGC1α axis, resulting in a reduction in release of mtDNA-enriched vesicle and ultimately attenuating the progression of DCM. Conclusions Collectively, our study demonstrates a protective role of IL-37 in DCM, offering a promising therapeutic agent for this disease. Graphical abstract Hyperglycemia aggravates mitochondrial injury through SIRT1/AMPK/PGC1α signaling, resulting in significant cardiomyocyte apoptosis and the release of extracellular vesicles containing mtDNA in DCM mice. Fibroblasts then engulf these mtDNA-enriched vesicles, activating TLR9 signaling and the cGAS-STING pathway to initiate profibrotic process and adverse remodeling. However, both exogenous and endogenous IL-37 ameliorate mitochondrial injury by preserving the activity of SIRT1-AMPK-PGC1α axis, and reducing the release of mtDNA-enriched vesicles, which attenuates the progression of DCM

Abstract Objective To comprehensively evaluate the effect of low birth weight on premature ovarian insufficiency. Methods We performed a systematic review of the literature by searching MEDLINE, EMBASE, Web of Science, Scopus, Wanfang and CNKI up to August 2023. All cohort and case-control studies that included birth weight as an exposure and premature ovarian insufficiency as an outcome were included in the analysis. Data were combined using inverse-variance weighted meta-analysis with fixed and random effects models and between-study heterogeneity evaluated. We evaluated risk of bias using the Newcastle Ottawa Scale and using Egger’s method to test publication bias. All statistical analyses were performed with the use of R software. Results Five articles were included in the review. A total of 2,248,594 women were included, including 21,813 (1%) cases of premature ovarian insufficiency, 150,743 cases of low birth weight, and 220,703 cases of macrosomia. We found strong evidence that changed the results of the previous review that low birth weight is associated with an increased risk of premature ovarian insufficiency (OR = 1.15, 95%CI 1.09–1.22) in adulthood compared with normal birth weight. No effect of macrosomia on premature ovarian insufficiency was found. Conclusions Our meta-analysis showed strong evidence of an association between low birth weight and premature ovarian insufficiency. We should reduce the occurrence of low birth weight by various methods to avoid the occurrence of premature ovarian insufficiency.

Virus-like particles (VLPs) are used as nanocontainers for targeted drug, protein, and vaccine delivery. The phage P22 VLP is an ideal macromolecule delivery vehicle, as it has a large exterior surface area, which facilitates multivalent genetic and chemical modifications for cell recognition and penetration. Arginine-rich cell-penetrating peptides (CPPs) can increase cargo transport efficiency in vivo. However, studies on the tissue distribution and retention of P22 VLPs mediated by TAT and 8R are lacking. This study aimed to analyze the TAT and 8R effects on the P22 VLPs transport efficiency and tissue distribution both in vitro and in vivo. We used a prokaryotic system to prepare P22 VLP self-assembled particles and expressed TAT-or 8R-conjugated mCherry on the VLP capsid protein as model cargoes and revealed that the level of P22 VLP-mCherry penetrating the cell membrane was low. However, both TAT and 8R significantly promoted the cellular uptake efficiency of P22 VLPs in vitro, as well as enhanced the tissue accumulation and retention of P22 VLPs in vivo. At 24 h postinjection, TAT enhanced the tissue distribution and retention in the lung, whereas 8R could be better accumulation in brain. Thus, TAT was superior in terms of cellular uptake and tissue accumulation in the P22 VLPs delivery system. Understanding CPP biocompatibility and tissue retention will expand their potential applications in macromolecular cargo delivery.

ABSTRACT Central venous catheters (CVCs) are widely used for intravenous medication administration. However, biofilm formation along the catheter surface is the main most important cause of catheter-related bloodstream infections. Nowadays, several antimicrobial-coated catheters are available to prevent biofilm development. In this study, we introduced a new dynamic in vitro model to evaluate the antimicrobial activity against bacterial biofilms on CVCs. Rifampicin–minocycline-coated catheters and control catheters without antimicrobial component were assembled into the model to test the antimicrobial activity on external surface and internal surface. After 1 h irrigation of Staphylococcus epidermidis or Staphylococcus aureus preculture and 23 h irrigation of Trypticase Soy Broth, the viable adherent organism was collected and counted. The enumeration results showed that the number of bacteria attached to antibacterial catheter was significantly less than that of the control catheter, both on external surface (P < 0.05) and internal surface (P < 0.05). The results were further confirmed by the scanning electron microscopy. In conclusion, the dynamic in vitro model can be applied to evaluate the antimicrobial activity against bacterial biofilms grown on the external and internal surfaces of CVCs used in clinical practice.IMPORTANCEFor the first time, a new dynamic in vitro model was constructed to evaluate the antimicrobial activity against bacterial biofilms on central venous catheters (CVCs) on both external surface and internal surface. This model could be applied to evaluate the antimicrobial activity against bacterial biofilms not only on CVCs but also other types of catheters.

ABSTRACT Companion animals such as cats and dogs harbor diverse microbial communities that can potentially impact human health due to close and frequent contact. To better characterize their total infectomes and assess zoonotic risks, we characterized the overall infectomes of companion animals (cats and dogs) and evaluated their potential zoonotic risks. Meta-transcriptomic analyses were performed on 239 samples from cats and dogs collected across China, identifying 24 viral species, 270 bacterial genera, and two fungal genera. Differences in the overall microbiome and infectome composition were compared across different animal species (cats or dogs), sampling sites (rectal or oropharyngeal), and health status (healthy or diseased). Diversity analyses revealed that viral abundance was generally higher in diseased animals compared to healthy ones, while differences in microbial composition were mainly driven by sampling site, followed by animal species and health status. Disease association analyses validated the pathogenicity of known pathogens and suggested potential pathogenic roles of previously undescribed bacteria and newly discovered viruses. Cross-species transmission analyses identified seven pathogens shared between cats and dogs, such as alphacoronavirus 1, which was detected in both oropharyngeal and rectal swabs albeit with differential pathogenicity. Further analyses showed that some viruses, like alphacoronavirus 1, harbored multiple lineages exhibiting distinct pathogenicity, tissue, or host preferences. Ultimately, a systematic evolutionary screening identified 27 potential zoonotic pathogens in this sample set, with far more bacterial than viral species, implying potential health threats to humans. Overall, our meta-transcriptomic analysis reveals a landscape of actively transcribing microorganisms in major companion animals, highlighting key pathogens, those with the potential for cross-species transmission, and possible zoonotic threats.IMPORTANCEThis study provides a comprehensive characterization of the entire community of infectious microbes (viruses, bacteria, and fungi) in companion animals like cats and dogs, termed the “infectome.” By analyzing hundreds of samples from across China, the researchers identified numerous known and novel pathogens, including 27 potential zoonotic agents that could pose health risks to both animals and humans. Notably, some of these zoonotic pathogens were detected even in apparently healthy pets, highlighting the importance of surveillance. The study also revealed key microbial factors associated with respiratory and gastrointestinal diseases in pets, as well as potential cross-species transmission events between cats and dogs. Overall, this work sheds light on the complex microbial landscapes of companion animals and their potential impacts on animal and human health, underscoring the need for monitoring and management of these infectious agents.

IntroductionMuscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry.MethodsThe present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. ResultsThe results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%.DiscussionThe developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing.

ABSTRACT Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies.IMPORTANCEPorcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.

Abstract Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV‐1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion‐stabilized HIV‐1 envelope (Env) trimer, BG505 DS‐SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4‐binding site (CD4bs) of vulnerability. Two of the vaccine‐elicited CD4bs‐targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a‐hinge region and human IgG1‐constant region (G36×3‐IgG2a and R27×3‐IgG2a), neutralized 96% of a multiclade 208‐strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL−1, respectively. Cryo‐EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV‐1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2‐apex‐targeting broadly neutralizing antibody, CAP256V2LS. The resultant human‐llama bispecific antibody CAP256L‐R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV‐1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn‐Fc mice similar to the parent CAP256V2LS. Vaccine‐elicited llama nanobodies, when combined with V2‐apex broadly neutralizing antibodies, may therefore be able to fulfill anti‐HIV‐1 therapeutic and prophylactic clinical goals.

The spatial structure evolution of forests in karst areas is a key scientific issue in global forest research. In order to explore the dynamics of spatial structure in karst evergreen and deciduous broad-leaved forests, a study was conducted in Mulun National Nature Reserve, focusing on a fixed monitoring plot of 2 hm2. Based on data from three surveys conducted in 2007, 2012, and 2017, the dynamics of community spatial structure were analyzed using the spatial structure parameters, i. e., angular scale (Wi), mixing degree (Mi), and size ratio (Ui). The results were as follows: (1) The univariate distribution results of spatial structure parameters revealed that the forest structure gradually shifted towards a slightly clumped distribution state as the stand age increased between 2007 and 2017. The mixing degree was at a level of strong mixing and showed a tendency towards extreme mixing. In terms of the size ratio, the stand exhibited a moderate state, shifting towards subdominance as the stand grew. (2) The study of spatial structure for different diameter classes of trees showed that the anglular scale, size ratio, and mixing degree of trees in diameter classes 1-15 cm gradually increased with stand growth. As the diameter class increased, the values of the size ratio showed a decreasing trend while the mixing degree showed an increasing trend. (3) The study of spatial structure for dominant tree species revealed that, during the period from 2007 to 2017, all five dominant tree species groups exhibited a slightly clumped distribution. The size ratio showed a tendency towards subdominance and a moderate distribution state, while the mixing level was at a high degree and gradually shifted towards extreme mixing. In conclusion, the karst evergreen and deciduous broad-leaved forest in Mulun currently exhibits a slightly clumped distribution, a strong degree of mixing, and a tendency towards subdominance. The community is relatively stable, the stand regeneration is in good condition, and the forest is gradually transitioning towards a climax community. The analysis of the dynamic changes in vegetation structure in karst evergreen and deciduous broad-leaved forests is of great significance for predicting the future development and changes of forests.

Abstract Background In this study, we investigated the effects of alpine meadow in different phenological periods on ruminal fermentation, serum biochemical indices, and gastrointestinal tract microbes in grazing yak on the Qinghai-Tibetan Plateau. A total of eighteen female freely grazing yaks with an average age of 3 years old and a body weight of 130 ± 19 kg were selected. According to the plant phenological periods, yaks were randomly allocated to one of three treatments: (1) regreen periods group (RP, n = 6); (2) grassy periods group (GP, n = 6); and (3) hay periods group (HP, n = 6). At the end of the experiment, the blood, rumen fluids, and rectal contents were collected to perform further analysis. Results The concentrations of total volatile fatty acid (TVFA), acetate, glucose (GLU), triglyceride (TG), cholesterol (CHO), high density lipoprotein (HDL), and low density lipoprotein (LDL) were higher in the GP group than in the HP group (P

Abstract Purpose This research aimed to develop a machine learning model to predict the potential risk of prolonged length of stay in hospital before operation, which can be used to strengthen patient management. Methods Patients who underwent posterior spinal deformity surgery (PSDS) from eleven medical institutions in China between 2015 and 2022 were included. Detailed preoperative patient data, including demographics, medical history, comorbidities, preoperative laboratory results, and surgery details, were collected from their electronic medical records. The cohort was randomly divided into a training dataset and a validation dataset with a ratio of 70:30. Based on Boruta algorithm, nine different machine learning algorithms and a stack ensemble model were trained after hyperparameters tuning visualization and evaluated on the area under the receiver operating characteristic curve (AUROC), precision-recall curve, calibration, and decision curve analysis. Visualization of Shapley Additive exPlanations method finally contributed to explaining model prediction. Results Of the 162 included patients, the K Nearest Neighbors algorithm performed the best in the validation group compared with other machine learning models (yielding an AUROC of 0.8191 and PRAUC of 0.6175). The top five contributing variables were the preoperative hemoglobin, height, body mass index, age, and preoperative white blood cells. A web-based calculator was further developed to improve the predictive model's clinical operability. Conclusions Our study established and validated a clinical predictive model for prolonged postoperative hospitalization duration in patients who underwent PSDS, which offered valuable prognostic information for preoperative planning and postoperative care for clinicians. Trial registration ClinicalTrials.gov identifier NCT05867732, retrospectively registered May 22, 2023, https://classic.clinicaltrials.gov/ct2/show/NCT05867732 .

Abstract Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Abstract Background To investigate the prevalence and evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) at commercial fattening pig farms, a total of 1397 clinical samples were collected from a single fattening cycle at seven pig farms in five provinces of China from 2020 to 2021. Results The RT‒PCR results revealed that PRRSV was present on all seven farms, and the percentage of PRRSV-positive individuals was 17.54–53.33%. A total of 344 partial NSP2 gene sequences and 334 complete ORF5 gene sequences were obtained from the positive samples. The statistical results showed that PRRSV-2 was present on all seven commercial fattening farms, and PRRSV-1 was present on only one commercial fattening farm. A total of six PRRSV-2 subtypes were detected, and five of the seven farms had two or more PRRSV-2 subtypes. L1.8 (L1C) PRRSV was the dominant epidemic strain on five of the seven pig farms. Sequence analysis of L1.8 (L1C) PRRSV from different commercial fattening pig farms revealed that its consistency across farms varied substantially. The amino acid alignment results demonstrated that there were 131 aa discontinuous deletions in NSP2 between different L1.8 (L1C) PRRSV strains and that the GP5 mutation in L1.8 (L1C) PRRSV was mainly concentrated in the peptide signal region and T-cell epitopes. Selection pressure analysis of GP5 revealed that the use of the PRRSV MLV vaccine had no significant episodic diversifying effect on L1.8 (L1C) PRRSV. Conclusion PRRSV infection is common at commercial fattening pig farms in China, and the percentage of positive individuals is high. There are multiple PRRSV subtypes of infection at commercial fattening pig farms in China. L1.8 (L1C) is the main circulating PRRSV strain on commercial fattening pig farms. L1.8 (L1C) PRRSV detected at different commercial fattening pig farms exhibited substantial differences in consistency but similar molecular characteristics. The pressure on the GP5 of L1.8 (L1C) PRRSV may not be directly related to the use of the vaccines.