Multiplexed CRISPR-Cas system targeting ASFV genes in vivo: solution lies within.

The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo. Application of this system showed a significant reduction of viral replication in vitro, while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology.

[Progress in research and application of the biotin ligase BirA and its mutants in pathogen-host interaction].

Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.

Resilience mediates the prediction of alienation towards parents on subjective well-being in rural left-behind children: a 12-month longitudinal study.

Objective: Parent-child relationship is critical for children's well-being. In China, the large number of left-behind children (LBC, one or both parents leaving for work for at least 6 months) raises public concern. Although LBC often report poor mental health status and higher alienation towards parents, the dynamic trend of subjective well-being in this population, as well as the prediction of alienation towards parents on LBC's subjective well-being, remain unrevealed. This study aimed to examine the dynamic trend of subjective well-being in Chinese LBC and further explore the predictional influence of alienation towards parents, with resilience as a potential mediator. Methods: We recruited 916 rural LBC in China and investigated them at five waves (baseline, and 1, 3, 6, and 12 months later) using Inventory of Alienation towards Parents (IAP), Resilience Scale for Chinese Adolescents (RSCA) and Subjective Happiness Scale (SHS). We used hierarchical linear modeling (HLM) for analysis. Results: At baseline, no significant differences were found in the scores of alienation towards parents, resilience, and subjective well-being on gender, grade, or type of LBC. A significant correlation existed between the scores of alienation towards parents, resilience, and subjective well-being. HLM showed a linear increase in the subjective well-being of rural LBC. Alienation toward both mother and father negatively predicted the developmental trajectory of children's subjective well-being over 12 months. Moreover, resilience partially mediated this prediction. Conclusion: This study is among the first to reveal that alienation towards parents predicts the developmental trajectory of later LBC's subjective well-being, with resilience as a mediator. These findings warrant the necessity of paying attention to alienation toward parents to ensure the mental health of LBC, giving valuable guidance to parents, schools and governments.

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target.

Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

Molecular Research on Coronavirus: Pathogenic Mechanisms, Antiviral Drugs, and New Vaccines.

Since the COVID-19 outbreak in 2019, five coronaviruses have been found to infect humans, including SARS-CoV (severe acute respiratory syndrome coronavirus) [...].

A portable transistor immunosensor for fast identification of porcine epidemic diarrhea virus.

Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Anti-Coronavirus Potential of Polyether Ionophores: The New Application of Veterinary Antibiotics in Livestock.

Coronaviruses have consistently posed a major global concern in the field of livestock industry and public health. However, there is currently a lack of efficient drugs with broad-spectrum antiviral activity to address the challenges presented by emerging mutated strains or drug resistance. Additionally, the method for identifying multitarget drugs is also insufficient. Aminopeptidase N (APN) and 3C-like proteinase (3CLpro) represent promising targets for host-directed and virus-directed strategies, respectively, in the development of effective drugs against various coronaviruses. In this study, maduramycin ammonium demonstrated a broad-spectrum antiviral effect by targeting both of the proteins. The binding domains 4 Å from the ligand of both target proteins shared a structural similarity, suggesting that screening and designing drugs based on these domains might exhibit broad-spectrum and highly effective antiviral activity. Furthermore, it was identified that the polyether ionophores' ability to carry zinc ion might be one of the reasons why they were able to target APN and exhibit antiviral effect. The findings of this experiment provide novel perspectives for future drug screening and design, while also offering valuable references for the utilization of polyether ionophores in the management of livestock health.

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus.

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Hydrothermal Hydrolyzation-Driven Topological Transformation of Ni-Co Bimetallic Compounds with Hollow Nanoflower Structure for Optimizing Hydrogen Evolution Catalysis.

Composition screening and structure optimization are two critical factors in improving the electrocatalytic performance of hybrid materials. Herein, we present a straightforward hydrothermal hydrolyzation-topological transformation strategy for the synthesis of a range of Ni-Co bimetallic compounds with a hollow nanoflower structure. Among these Ni-Co compounds, Ni2P/Co2P hollow nanoflowers (HNFs) exhibit the most impressive electrocatalytic activity for the hydrogen evolution reaction (HER), necessitating only an 153 mV overpotential to achieve a current density of 10 mA cm-2 under alkaline conditions. Importantly, this performance remains stable for over 48 h, indicating exceptional durability. The exceptional catalytic performance of Ni2P/Co2P HNFs arises from the synergy between the hybrid Ni2P/Co2P components and the hollow nanoflower structure. The former provides abundant catalytic sites, while electron rearrangement at the heterointerfaces enhances the adsorption/desorption of active species and facilitates electron transfer. The latter contributes to the exposure of catalytic sites, shortening mass and charge transfer routes, and bolstering structural stability during prolonged electrocatalysis. This research offers valuable insights into the screening and optimization of advanced hybrid electrocatalysts, holding significant promise for applications in the emerging field of new energy technologies.

Pharmacological effects and target analysis of Guipi wan in the treatment of cerebral ischemia-reperfusion injury.

Guipi wan (GPW) is a traditional Chinese medicine commonly used in clinical practice, typically to treat neurological diseases such as neurasthenia and traumatic brain injury. It may have positive effects on cerebral ischemia‒reperfusion injury (cI/R). This study aimed to assess the effects of GPW in a mouse model of cI/R and find its possible targets. C57BL/6J mice were used to establish the cI/R model, and the laser speckle doppler was used to determine the success of the model. GPW was administered intragastrically for 7 days, brain tissue sections were stained with TTC, HE, and TUNEL, Western blot assay was performed to detect the effect of apoptosis-related proteins. Furthermore, we screened active ingredients from the TCM Database and constructed a compound‒target network using the Cytoscape 3.8.0 software. Moreover, we employed protein‒protein interaction and component‒target‒pathway network analyses to determine the potential components of GPW and its target genes, the key target was verified through molecular docking. Finally, we detected the influence of the downstream signaling pathway of the target through Western blot. The results showed that GPW decreased the cerebral infarction area, neurological function scores, and neuronal apoptosis in mice by regulating PI3K/AKT signaling pathway. Network analysis indicated that gamma-aminobutyric acid B receptor 1 (GABBR1) might be a potential target for the treatment of cI/R. Molecular docking indicated that 9 active components in GPW could bind to GABBR1 with desirable binding energy. This study represented the demonstratable effect of GPW in the treatment of cI/R injury and suggested GABBR1 as a potential target using network analysis.

Design and synthesis of APN and 3CLpro dual-target inhibitors based on STSBPT with anticoronavirus activity.

Coronaviruses (CoVs) have continuously posed a threat to human and animal health. However, existing antiviral drugs are still insufficient in overcoming the challenges caused by multiple strains of CoVs. And methods for developing multi-target drugs are limited in terms of exploring drug targets with similar functions or structures. In this study, four rounds of structural design and modification on salinomycin were performed for novel antiviral compounds. It was based on the strategy of similar topological structure binding properties of protein targets (STSBPT), resulting in the high-efficient synthesis of the optimal compound M1, which could bind to aminopeptidase N and 3C-like protease from hosts and viruses, respectively, and exhibit a broad-spectrum antiviral effect against severe acute respiratory syndrome CoV 2 pseudovirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, feline infectious peritonitis virus and mouse hepatitis virus. Furthermore, the drug-binding domains of these proteins were found to be structurally similar based on the STSBPT strategy. The compounds screened and designed based on this region were expected to have broad-spectrum and strong antiviral activities. The STSBPT strategy is expected to be a fundamental tool in accelerating the discovery of multiple targets with similar effects and drugs.

Humic acid-mediated transport of a typical soil passivation remediation product (chloropyromorphite) in saturated porous media.

Conversion of labile Pb species into chloropyromorphite (CPY) using phosphorus-bearing amendments was considered to be an ideal strategy in soil passivation remediation. However, the fate and transport of CPY in the soil are poorly understood. This study aims to fill the knowledge gap by evaluating the fate and transport of CPY under environmentally relevant conditions of humic acid (HA), pH, electrolyte concentration, and species through the saturated sandy medium. Results showed that bare CPY colloids are basically immobile in sandy porous media while the co-existence of HA made the transport of CPY improved by 30%-93.5%. Facilitated transport of CPY was attributed to the increased stability of CPY and the repulsive interaction between CPY particles and sands due to HA adsorption. The mobility of CPY was also increased with increasing pH from 5.0 to 9.0. When the pH was 9 with a 10 mmol/L NaCl background solution, the stronger energy barrier between CPY and sand led to enhanced transport behavior. The divalent Ca2+ had a more dramatic effect than monovalent Na+ on the aggregation and sedimentation of CPY colloids due to its effectivescreening of the surface charge of CPY and bridging interaction with CPY particles. Derjaguin-Landau-Verwey-Overbeek theory and attachment efficiency calculation indicated that high energy barriers were responsible for the high mobility of CPY colloids, while the retention of CPY in sands was mainly caused by secondary energy minimum and physically straining. The findings of this work can help to evaluate the fate of soil passivation remediation products in natural water and soil.

Inhibition of porcine deltacoronavirus entry and replication by Cepharanthine.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that mainly causes acute diarrhea/vomiting, dehydration, and mortality in piglets, possessing economic losses and public health concerns. However, there are currently no proven effective antiviral agents against PDCoV. Cepharanthine (CEP) is a naturally occurring alkaloid used as a traditional remedy for radiation-induced symptoms, but its underlying mechanism of CEP against PDCoV has remained elusive. The aim of this study was to investigate the anti-PDCoV effects and mechanisms of CEP in LLC-PK1 cells. The results showed that the antiviral activity of CEP was based on direct action on cells, preventing the virus from attaching to host cells and virus replication. Importantly, Surface Plasmon Resonance (SPR) results showed that CEP has a moderate affinity to PDCoV receptor, porcine aminopeptidase N (pAPN) protein. AutoDock predicted that CEP can form hydrogen bonds with amino acid residues (R740, N783, and R790) in the binding regions of PDCoV and pAPN. In addition, RT-PCR results showed that CEP treatment could significantly reduce the transcription of ZBP1, cytokine (IL-1ß and IFN-α) and chemokine genes (CCL-2, CCL-4, CCL-5, CXCL-2, CXCL-8, and CXCL-10) induced by PDCoV. Western blot analysis revealed that CEP could inhibit viral replication by inducing autophagy. In conclusion, our results suggest that the anti-PDCoV activity of CEP is not only relies on competing the virus binding with pAPN, but also affects the proliferation of the virus in vitro by downregulating the excessive immune response caused by the virus and inducing autophagy. CEP emerges as a promising candidate for potential anti-PDCoV therapeutic development.

Generation and Characterization of Monoclonal Antibodies against Swine Acute Diarrhea Syndrome Coronavirus Spike Protein.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a member of the family Coronaviridae and the genus Alphacoronavirus, primarily affects piglets under 7 days old, causing symptoms such as diarrhea, vomiting, and dehydration. It has the potential to infect human primary and passaged cells in vitro, indicating a potential risk of zoonotic transmission. In this study, we successfully generated and purified six monoclonal antibodies (mAbs) specifically targeting the spike protein of SADS-CoV, whose epitope were demonstrated specificity to the S1A or S1B region by immunofluorescence assay and enzyme-linked immunosorbent assay. Three of these mAbs were capable of neutralizing SADS-CoV infection on HeLa-R19 and A549. Furthermore, we observed that SADS-CoV induced the agglutination of erythrocytes from both humans and rats, and the hemagglutination inhibition capacity and antigen-antibody binding capacity of the antibodies were assessed. Our study reveals that mAbs specifically targeting the S1A domain demonstrated notable efficacy in suppressing the hemagglutination phenomenon induced by SADS-CoV. This finding represents the first instance of narrowing down the protein region responsible for SADS-CoV-mediated hemagglutination to the S1A domain, and reveals that the cell attachment domains S1A and S1B are the main targets of neutralizing antibodies.

A novel Fc-enhanced humanized monoclonal antibody targeting B7-H3 suppresses the growth of ESCC.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor of the digestive tract with a low 5-year survival rate due to the lack of effective treatment methods. Although therapeutic monoclonal antibodies (mAbs) now play an important role in cancer therapy, effective targeted mAbs are still lacking for ESCC. B7-H3 is highly expressed in a variety of tumors and has emerged as a promising therapeutic target. Several mAbs against B7-H3 have advanced to clinical trials, but their development has not yet been pursued for ESCC. Here, we developed a humanized and Fc-engineered anti-B7H3 mAb 24F-Hu-mut2 and systematically evaluated its anti-tumor activity in vitro and in vivo. The 24F-Hu-mut2 was humanized and modified in Fc fragment to obtain stronger antibody-dependent cell-mediated cytotoxicity(ADCC) activity and nanomolar affinity. Furthermore, both of ESCC cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models indicated that 24F-Hu-mut2 displayed potent in vivo anti-tumor activity. In addition, a computational docking model showed that the mAb bound to IgC1 and IgC2 domain of B7-H3, which is closer to the cell membrane. Consistently, our ELISA results verified the binding of 24F-Hu-WT and IgC1 and IgC2. Our results indicate that 24F-Hu-mut2 has significant anti-ESCC activity both in vitro and in vivo, and this monoclonal antibody may be a promising antibody against ESCC and other B7-H3 overexpressing tumors.

Effects of Cr(VI) oxyanion, humic acid and solution chemistry on the aggregation and colloidal stability of green synthesized chlorapatite nanoparticles.

Aggregation is a crucial process determining the fate, mobility and ecological risks of nanomaterials. Chlorapatite nanoparticles (nClAP) exhibit widely applications in environmental remediation and consequently will inevitably enter aquatic systems. However, the aggregation characteristics of nClAP are still mostly uncovered. This study investigated the aggregation kinetics and colloidal stability of nClAP as a function of pH, humic acid (HA), Cr(VI) oxyanions, monovalent and divalent electrolytes. Results showed that pH values from 5 to 9 had a notable impact on the aqueous behaviors of nClAP. The addition of HA made the zeta potential (ZP) of nClAP more negative and thus enhanced nClAP stability through electrostatic and steric effects. Similarly, the adsorption of Cr(VI) on the surface of nClAP created a physical barrier and negative charge, improving the stability of nClAP by inducing steric force. Lower ZP and hydrodynamic diameter (HDD) reflected that the enhanced stability of nClAP by HA was more significant than Cr(VI). In comparison, the presence of Ca2+ ions were more effective than monovalent Na + ions in promoting the aggregation of nClAP. The classical DLVO theory incorporating the steric repulsion were used to interpret the aggregation and dispersion of nClAP, making it was easier to overcome energy barriers and agglomerate. This study provides new mechanistic insights which could help better understand the effects of Cr(VI) oxyanions and HA on nClAP's colloidal stability.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Luteolin in the Qi Bi Anshen decoction improves propionic acid-induced autism-like behavior in rats by inhibiting LRP1/MMP9.

BACKGROUND: A neurodevelopmental illness with a high frequency and unidentified pathophysiology is known as autism spectrum disorder (ASD). A research hotspot in this field is the identification of disease-specific biomarkers and drug intervention targets. Traditional Chinese medicine (TCM) can eliminate the symptoms of autism by precisely regulating human physiology. The Qi Bi Anshen decoction (QAT) is a commonly used TCM clinical drug commonly-used to treat for treating ASD. However, the primary active ingredients and underlying mechanisms of action of this decoction remain unknown. PURPOSE: This study aimed to investigate the active ingredients and pharmacodynamics of QAT in the treatment of ASD using a Sprague-Dawley rat model that resembled autism. METHODS: Autism-like rat models were established through intracerebroventricular injections of propionic acid (PPA). Subsequently, the rats were treated with QAT, and their efficacy was evaluated using the three-chamber method to analyze social interactions and grooming behavior. Additionally, open-field tests, elevated cross-maze tests, hematoxylin and eosin staining, Nissl staining, and enzyme-linked immunosorbent assays were performed; Western blot analysis was employed to determine the expression of synaptic plasticity-related proteins. Utilizing ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS), the effectiveness of active QAT components was assessed, and potential QAT targets were screened through molecular docking, surface plasmon resonance, and thermal migration experiments. To better understand the precise processes involved in treating ASD with active QAT components, in vivo and in vitro knockdown tests were also performed. RESULTS: QATexhibited a significant improvement in autism-like behavior and a notable increase in the production of proteins associated with synaptic plasticity. Furthermore, luteolin (LUT), identified as a potentially important active ingredient in QAT for treating ASD, reduced matrix metallopeptidase-9 (MMP9) expression. However, this effect was attenuated by the knockdown of low-density lipoprotein receptor-associated protein 1 (LRP1), which is the target binding site for LUT. CONCLUSIONS: LUT emerges as a potentially crucial active component of QAT in the treatment of ASD, with the ability to antagonize LRP1 and subsequently reduce MMP9 expression.