A full-length cDNA infectious clone, pKS15-01-Clone, was constructed from an emerging Senecavirus A (SVA; strain KS15-01). To explore the potential use as a viral backbone for expressing marker genes, the enhanced green fluorescent protein (EGFP)-tagged reporter virus (vKS15-01-EGFP) was generated using reverse genetics. Compared to the parental virus, the pKS15-01-Clone derived virus (vKS15-01-Clone) replicated efficiently in vitro and in vivo, and induced similar levels of neutralizing antibody and cytokine responses in infected animals. In contrast, the vKS15-01-EGFP virus showed impaired growth ability and induced lower level of immune response in infected animals. Lesions on the dorsal snout and coronary bands were observed in all pigs infected by parental virus KS15-01, but not in pigs infected with vKS15-01-Clone or vKS15-01-EGFP viruses. These results demonstrated that the infectious clone and EGFP reporter virus could be used as important tools in further elucidating the SVA pathogenesis and development of control measures.

Haemophilus parasuis (H. parasuis) is the etiological agent of Glässer's disease in pigs. Currently, the molecular basis of this infection is largely unknown. The innate immune response is the first line of defense against the infectious disease. Systematical analysis on host innate immune response to the infection is important for understanding the pathogenesis of the infectious microorganisms.

IL-1α in vitro promotes immunoglobulin secretion by inducing proliferation of mature B cells, whereas IL-1α deficiency has no effect on in vivo antibody production. However, the reason IL-1α deficiency does not reduce in vivo antibody production is still unclear. In this study, we found that similar as in vivo data, IL-1α deficiency did not affect antibody production in in vitro LPS-stimulated B cells. Surprisingly, LPS-stimulated IL-1α-/- B cells reduced a key antibody production-related transcription factor X-box binding protein 1 (Xbp-1) expression. Furthermore, we found that IL-1α deficiency up-regulated mTOR expression, which bypassed Xbp-1 for immunoglobulin secretion. Finally, we showed that Xbp-1 suppressed mTOR expression, whereas mTOR suppressed the activation of Xbp-1 promoter via JunB. Together, these data suggest that IL-1a deficiency reduced Xbp-1 and up-regulated mTOR. This may explain why IL-1α deficiency has no effect on antibody production.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus (CoV) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. Unlike most CoVs that antagonize type I interferon (IFN) production, previous studies showed that TGEV infection induces IFN-I production both in vivo and in vitro. However, the underlying mechanism(s) remain largely unknown. In this study, we found that TGEV infection significantly facilitated IFN-β production as well as activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor-kappaB (NF-κB) in porcine kidney (PK-15) cells. Screening of TGEV-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent IFN-β inducer and induced IFN-β production mainly by activating NF-κB but not IRF3. Further analysis showed that nsp14 interacted with DDX1, a member of the DExD/H helicase family. Knockdown of DDX1 by specific small interfering RNA (siRNA) significantly decreased nsp14-induced IFN-β production and NF-κB activation. Furthermore, TGEV-induced IFN-β production and IFN-stimulated gene (ISG) expression were decreased in cells transfected with DDX1-specific siRNA, indicating the vital role of DDX1 to TGEV-induced IFN-β responses. In summary, our data revealed a potential coactivator role of host RNA helicase DDX1 to the induction of IFN-β response initiated by TGEV and demonstrated that nsp14 is an important IFN inducer among the TGEV-encoded proteins.

Notch1 signaling regulates B and T lymphocyte development and also in vitro promotes antibody secretion upon B cell activation. However, it is still unclear about the role of Notch1 in antibody production upon in vitro and in vivo mixture lymphocytes activation. We first showed that Notch1 expressed in LPS-activated CD19hi B cells and CD19cre mediated Notch1 knock-down in LPS-activated B cells. Furthermore, we demonstrated that Notch1 knock-down in B cells reduced antibody production in LPS-stimulated B cells but did not affect antibody production in LPS-stimulated splenocytes and in experimental allergic encephalomyelitis (EAE) mice. Importantly, Notch1 ligands Dll1 and Jag1 expressed in B cells and pre-coated Notch1 protein promotes Notch1-knocked down B cells to produce antibody in LPS-stimulated B cells suggesting that Notch1 in other cells may promote antibody production by binding its ligands Dll1 and Jag1 in B cells. Together, our results suggest that both Notch1 and its ligands in B cells play an important role in antibody production.

Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators.

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow.

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.

Cardiac side effects of doxorubicin (Dox) have limited its clinical application. The aim of this study was to explore new Dox-loaded dextran-based nano-carriers (NCs) in efficiently targeting tumor growth with less cardiac toxicity.

Elderly patients with diffuse large B-cell lymphoma (DLBCL) present with poor clinical outcome and intolerance to intensive chemotherapy. Histone deacetylase inhibitors (HDACIs) show anti-lymphoma activities and can be applied to treat DLBCL. This study aimed to evaluate efficacy and safety of oral HDACI tucidinostat (formerly known as chidamide) plus R-CHOP (CR-CHOP) in elderly patients with newly diagnosed DLBCL (International Prognostic Index ≥ 2).

Since first identified in December of 2019, COVID-19 has been quickly spreading to the world in few months and COVID-19 cases are still undergoing rapid surge in most countries worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adapts and evolves rapidly in nature. With the availability of 16,092 SARS-CoV-2 full genomes in GISAID as of 13 May, we removed the poor-quality genomes and performed mutational profiling analysis for the remaining 11,183 viral genomes. Global analysis of all sequences identified all single nucleotide polymorphisms (SNPs) across the whole genome and critical SNPs with high mutation frequency that contributes to five-clade classification of global strains. A total of 119 SNPs were found with 74 non-synonymous mutations, 43 synonymous mutations and two mutations in intergenic regions. Analysis of geographic pattern of mutational profiling for the whole genome reveals differences between each continent. A transition mutation from C to T represents the most mutation types across the genome, suggesting rapid evolution and adaptation of the virus in host. Amino acid (AA) deletions and insertions found across the genome results in changes in viral protein length and potential function alteration. Mutational profiling for each gene was analysed, and results show that nucleocapsid gene demonstrates the highest mutational frequency, followed by Nsp2, Nsp3 and Spike gene. We further focused on non-synonymous mutational distributions on four key viral proteins, spike with 75 mutations, RNA-dependent-RNA-polymerase with 41 mutations, 3C-like protease with 22 mutations and Papain-like protease with 10 mutations. Results show that non-synonymous mutations on critical sites of these four proteins pose great challenge for development of anti-viral drugs and other countering measures. Overall, this study provides more understanding of genetic diversity/variability of SARS-CoV-2 and insights for development of anti-viral therapeutics.

Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission.

The clinical importance of aberrantly expressed microRNAs (miRNAs) in diagnosing inflammatory bowel disease (IBD) has not been well established, so was investigated in this systematic review and meta-analysis.

Excessive proliferation and migration of fibroblasts in the lumbar laminectomy area can lead to epidural fibrosis, eventually resulting in failed back surgery syndrome. It has been reported that laminin α1, a significant biofunctional glycoprotein in the extracellular matrix, is involved in several fibrosis‑related diseases, such as pulmonary, liver and keloid fibrosis. However, the underlying mechanism of laminin α1 in epidural fibrosis remains unknown. The present study aimed to explore the effect and mechanism of laminin α1 in fibroblast proliferation, apoptosis and migration, and epidural fibrosis. Following the establishment of a laminectomy model, hematoxylin and eosin, Masson's trichrome and immunohistochemical staining were performed to determine the degree of epidural fibrosis, the number of fibroblasts, collagen content and the epidural expression levels of laminin α1, respectively. Furthermore, a stable small interfering RNA system was used to knock down the expression of laminin α1 in fibroblasts. The transfection efficiency was confirmed by reverse transcription‑quantitative PCR and immunofluorescence staining. Western blot analysis, scratch wound assay, EdU incorporation assay, flow cytometric analysis and Cell Counting Kit 8 assay were performed to assess the proliferation, apoptosis, migration and viability of fibroblasts, as well as the expression levels of the AKT/mechanistic target of rapamycin (mTOR) signaling‑related proteins. In vivo experiments revealed that laminin α1 was positively and time‑dependently associated with epidural fibrosis. In addition, laminin α1 knockdown attenuated cell proliferation, viability and migration, and promoted apoptosis. Furthermore, the results revealed that the activation of the AKT/mTOR signaling pathway was involved in the aforementioned processes. Overall, the current study illustrated the positive association between laminin α1 and epidural fibrosis, and also verified the effect of laminin α1 on fibroblast proliferation, apoptosis and migration. Furthermore, the results suggested that the AKT/mTOR signaling pathway may serve a significant role in regulating the behavior of laminin α1‑induced fibroblasts.

Most people with cerebral palsy (CP) suffer from impaired walking ability and pathological gait patterns. Seeking to improve the effectiveness of gait training in this patient population, this study developed and assessed the feasibility of a real-time biofeedback mechanism to augment untethered ankle exoskeleton-assisted walking performance in individuals with CP. We selected step length as a clinically-relevant gait performance target and utilized a visual interface with live performance scores. An adaptive ankle exoskeleton control algorithm provided assistance proportional to the real-time ankle moment. We assessed lower-extremity gait mechanics and muscle activity in seven ambulatory individuals with CP as they walked with adaptive ankle assistance alone and with ankle assistance plus step-length biofeedback. We achieved our technical validation goal by demonstrating a strong correlation between estimated step length and real step length (R = 0.771, p < 0.001). We achieved our clinical feasibility goal by demonstrating that biofeedback-plus-assistance resulted in a 14% increase in step length relative to baseline (p ≤ 0.05), while no difference in step length was observed for assistance alone. Additionally, we observed near immediate improvements in lower-extremity posture, moments, and positive power relative to baseline for biofeedback-plus-assistance (p < 0.05), with none, or more-limited improvements observed for assistance alone. Our findings suggest that providing real-time biofeedback and using step length as the target can be effective for increasing the rate at which individuals with CP improve their gait mechanics when walking with wearable ankle assistance.

Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-β signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.

Periodontitis is a chronic inflammatory oral disease that affects almost half of the adult population. NF-κB activator 1 (Act1) is mainly expressed in immune cells, including macrophages, and modulates immune cells' function to regulate inflammation in inflammatory diseases. Macrophages play a vital role in the pathophysiology of periodontitis. However, the effect of macrophage-specific Act1 on periodontitis has not been investigated yet. This study aims to unravel the role of macrophage-specific Act1 on the pathophysiology of periodontitis. The expression of Act1 in healthy and periodontitis periodontal tissue was confirmed by immunohistochemistry. Macrophage-specific Act1 expression downregulated (anti-Act1) mice were developed by inserting anti-Act1 antisense oligonucleotides after the CD68 promoter of C57BL/6 mice. Ligature-induced periodontitis (LIP) was induced in anti-Act1 mice and wildtype mice. Micro-CT, histology, and TRAP staining analyzed the periodontal tissue status, alveolar bone loss, and osteoclast numbers. Immunohistochemistry, RT-qPCR, and ELISA analyzed the inflammatory cells infiltration, expression of inflammatory cytokines, and M1/M2 macrophage polarization. mRNA sequencing of in vitro bacterial lipopolysaccharide (LPS)-treated peritoneal macrophages analyzed the differentially expressed genes in anti-Act1 mice during inflammation. Anti-Act1 mice showed aggravated periodontitis and alveolar bone loss compared to wildtype. Periodontitis-affected periodontal tissue (PAPT) of anti-Act1 mice showed a higher degree of macrophage infiltration, and M1 macrophage polarization compared to wildtype. Levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα), and macrophage activity-related factors (CCL2, CCL3, and CCL4) were robustly high in PAPT of anti-Act1 mice compared to wildtype. mRNA sequencing and KEGG analysis showed activated TNF/NF-κB signaling in LPS-treated macrophages from anti-Act1 mice. In vitro studies on LPS-treated peritoneal macrophages from anti-act1 mice showed a higher degree of cell migration and expression of inflammatory cytokines, macrophage activity-related factors, M1 macrophage-related factors, and TNF/NF-κB signaling related P-p65 protein. In conclusion, downregulation of macrophage-specific Act1 aggravated periodontitis, alveolar bone loss, macrophage infiltration, inflammation, and M1 macrophage polarization. Furthermore, LPS-treated macrophages from anti-Act1 mice activated TNF/NF-κB signaling. These results indicate the distinct role of macrophage-specific Act1 on the pathophysiology of periodontitis possibly via TNF/NF-κB signaling.

The -2/-1 programmed ribosomal frameshifting (-2/-1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This -2/-1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for -2/-1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate -2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both -2 and -1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus -2/-1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in -2/-1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of -2/-1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, -2/-1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that -2/-1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication.IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved ribosomal frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.

Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD.IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.