Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P<0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and (1) H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post-natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild-type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N-acetylaspartate (NAA), myo-inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long-lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.

Recent works have reported that long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and prognosis of cancers, suggesting the potential utility of lncRNAs as cancer prognostic markers. However, lncRNA signatures in predicting the survival of patients with clear cell renal cell carcinoma (ccRCC) remain unknown. In this study, we attempted to identify lncRNA signatures and their prognostic values in ccRCC. Using lncRNA expression profiling data in 440 ccRCC tumors from The Cancer Genome Atlas (TCGA) data, a five-lncRNA signature (AC069513.4, AC003092.1, CTC-205M6.2, RP11-507K2.3, U91328.21) has been identified to be significantly associated with ccRCC patients' overall survival in both training set and testing set. Based on the lncRNA signature, ccRCC patients could be divided into high-risk and low-risk group with significantly different survival rate. Further multivariable Cox regression analysis suggested that the prognostic value of this signature was independent of clinical factors. Functional enrichment analyses showed the potential functional roles of the five prognostic lncRNAs in ccRCC oncogenesis. These results indicated that this five-lncRNA signature could be used as an independent prognostic biomarker in the prediction of ccRCC patients' survival.

Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-SIgA secretory component (SC) mAb probe for the detection of anti-PEDV-specific SIgA in swine. On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Results showed that the immunochromatographic test strip had high sensitivity and specificity. When compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect PEDV specific SIgA in colostrum samples. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. We found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of PEDV specific SIgA, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying PEDV.

Integration of multi-omics data with molecular interaction networks enables elucidation of the pathophysiology of Alzheimer's disease (AD). Using the latest genome-wide association studies (GWAS) including proxy cases and the STRING interactome, we identified an AD network of 142 risk genes and 646 network-proximal genes, many of which were linked to synaptic functions annotated by mouse knockout data. The proximal genes were confirmed to be enriched in a replication GWAS of autopsy-documented cases. By integrating the AD gene network with transcriptomic data of AD and healthy temporal cortices, we identified 17 gene clusters of pathways, such as up-regulated complement activation and lipid metabolism, down-regulated cholinergic activity, and dysregulated RNA metabolism and proteostasis. The relationships among these pathways were further organized by a hierarchy of the AD network pinpointing major parent nodes in graph structure including endocytosis and immune reaction. Control analyses were performed using transcriptomics from cerebellum and a brain-specific interactome. Further integration with cell-specific RNA sequencing data demonstrated genes in our clusters of immunoregulation and complement activation were highly expressed in microglia.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and interleukin-6 (IL-6). Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present study, we found that interleukin-8 (IL-8) was upregulated by PDCoV infection. We then demonstrated that PDCoV E protein induced IL-8 production and that the TM domain and the C-terminal domain of the E protein were important for IL-8 production. Subsequently, we showed here that deleting the AP-1 and NF-κB binding motif in porcine IL-8 promoter abrogated its activation, suggesting that IL-8 expression was dependent on AP-1 and NF-κB. Furthermore, PDCoV E induced IL-8 production, which was also dependent on the NF-κB pathway through activating nuclear factor p65 phosphorylation and NF-κB inhibitor alpha (IκBα) protein phosphorylation, as well as inducing the nuclear translocation of p65, eventually resulting in the promotion of IL-8 production. PDCoV E also activated c-fos and c-jun, both of which are members of the AP-1 family. These findings provide new insights into the molecular mechanisms of PDCoV-induced IL-8 production and help us further understand the pathogenesis of PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes vomiting, diarrhea, dehydration, and even death of piglets, resulting in significant losses to the pig industry worldwide. However, the epitopes of PDCoV remain largely unknown. In this study, a monoclonal antibody (mAb) against the PDCoV nucleocapsid (N) protein, termed 9G1, was prepared using the lymphocyte hybridoma technique, and was identified as a type IgG1 with a κ light chain and reacted with the native N protein of PDCoV. Furthermore, the epitope recognized by the 9G1 mAb was subjected to western blot and an ELISA using truncated recombinant proteins and synthetic polypeptides of the PDCoV N protein. The results indicate that 9G1 mAb recognized the epitope, G59TPIPPSYAFYY70 (EP-9G1), a novel linear B cell epitope of the PDCoV N protein. A comparison analysis revealed that the EP-9G1 epitope was highly conserved among PDCoV strains, in which four residues (G59-F68YY70) were observed among different coronavirus genera. These data demonstrate that the EP-9G1 epitope identified in this study provides some basic information for further characterization of the antigenic structure of the PDCoV N protein and has potential use for developing diagnostic reagents for PDCoV.

A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as "drug" transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric coronavirus that causes vomiting, severe diarrhea, dehydration and death in suckling piglets. NS7a is putative accessory protein that is predicted to be encoded by SADS-CoV, but still to be confirmed experimentally. In the present study, recombinant NS7a protein was expressed in a prokaryotic expression system and used as an antigen to prepare monoclonal antibodies (mAbs) specific to NS7a protein. We obtained two anti-NS7a mAbs, termed AH5 and EH3, that were shown by western blotting to react with the natural NS7a protein in Vero E6 cells infected with SADS-CoV. Using the produced mAbs, we observed by confocal microscopy that NS7a protein was expressed in the cytoplasm. Further studies revealed that the motif 31VNTWQEFA38 was the minimal unit of the linear B-cell epitope recognized by mAb AH5, and the motif 82FDLFERF88 was the minimal unit of the linear B-cell epitope recognized by mAb EH3. Alignment of amino acids showed that these two epitopes were highly conserved among different SADS-CoV strains and SADS-related coronaviruses from bats, but with one substitution in these two motifs in bat coronavirus HKU2. In summary, we generated and characterized two mAbs against SADS-CoV NS7a protein, and demonstrated NS7a expression in SADS-CoV-infected cells for the first time.

In patients with liver or kidney disease, it is especially important to consider the routes of metabolism and elimination of small-molecule pharmaceuticals. Once in the blood, numerous drugs are taken up by the liver for metabolism and/or biliary elimination, or by the kidney for renal elimination. Many common drugs are organic anions. The major liver uptake transporters for organic anion drugs are organic anion transporter polypeptides (OATP1B1 or SLCO1B1; OATP1B3 or SLCO1B3), whereas in the kidney they are organic anion transporters (OAT1 or SLC22A6; OAT3 or SLC22A8). Since these particular OATPs are overwhelmingly found in the liver but not the kidney, and these OATs are overwhelmingly found in the kidney but not liver, it is possible to use chemoinformatics, machine learning (ML) and deep learning to analyze liver OATP-transported drugs versus kidney OAT-transported drugs. Our analysis of >30 quantitative physicochemical properties of OATP- and OAT-interacting drugs revealed eight properties that in combination, indicate a high propensity for interaction with "liver" transporters versus "kidney" ones based on machine learning (e.g., random forest, k-nearest neighbors) and deep-learning classification algorithms. Liver OATPs preferred drugs with greater hydrophobicity, higher complexity, and more ringed structures whereas kidney OATs preferred more polar drugs with more carboxyl groups. The results provide a strong molecular basis for tissue-specific targeting strategies, understanding drug-drug interactions as well as drug-metabolite interactions, and suggest a strategy for how drugs with comparable efficacy might be chosen in chronic liver or kidney disease (CKD) to minimize toxicity.

Endoplasmic reticulum (ER) stress is associated with numerous mammalian diseases, especially viral diseases. Porcine parvovirus (PPV) is the causative agent of reproductive failure in swine. Here, we observed that the PPV infection of porcine kidney 15 and porcine testis cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 and activating transcription factor 6 (ATF6). ER stress activation obviously blocked PPV replication. Depletion of proteins, such as PERK, eukaryotic initiation factor 2, and ATF4, by small interfering RNA significantly enhanced PPV replication. Moreover, the pro-apoptotic factor C/EBP homologous protein was identified a key factor in the inhibition of PPV replication. These data demonstrate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress inhibits further PPV replication by promoting apoptosis.

Porcine parvovirus (PPV) is a pathogen of infectious reproductive disease, which can cause stillbirth, mummification, embryo death, and infertility (SMEDI) syndrome in pigs. The objective of this study was to gain new insights into the evolution and phylogeny of the PPV1 genome. In this study, we isolated two new PPV1 (HLJ202108-Y and SDLC202109) from northern China and sequenced their whole genomes. The new isolates were found to have three amino acid substitutions (K195R, K562R, and S578P) in nonstructural protein 1. The VP2 amino acid site contained nine nonsynonymous substitutions, including six substitutions of the Kresse strain corresponding to the NADL-2 strain and three substitutions of A414S, S436T, and N555K. Genetic evolution analysis was conducted on 107 reference sequences available in the GenBank database, and 4-5 PPV1 taxa were defined. The new isolates were in the same phylogenetic cluster as strain 27a. The changes in the cluster, specifically marker amino acids, and their potential role in enhancing pathogenicity are discussed in this study. Furthermore, the evolutionary tree map results showed that the strains in China were evolving in two directions: one was becoming increasingly similar to early NADL-2 strains, while the other was evolving toward 27a-like strains. We also compared the proliferation ability of the isolated strains in susceptible cells by analyzing the multistep growth curves. The results showed that the virulence titer of the mutant strain was high. In summary, this study introduced the latest changes in PPV and discussed the virus characteristics that were considered to affect virulence.

The purpose of this study was to determine if variables calculated from diffusion tensor imaging (DTI) would serve as a reliable marker of damage after a muscle strain injury in dystrophic (mdx) and wild type (WT) mice. Unilateral injury to the tibialis anterior muscle (TA) was induced in vivo by 10 maximal lengthening contractions. High resolution T1- and T2-weighted structural MRI, including T2 mapping and spin echo DTI was acquired on a 7T small animal MRI system. Injury was confirmed by a significant loss of isometric torque (85% in mdx versus 42% in WT). Greater increases in apparent diffusion coefficient (ADC), axial, and radial diffusivity (AD and RD) of the injured muscle were present in the mdx mice versus controls. These changes were paralleled by decreases in fractional anisotropy (FA). Additionally, T2 was increased in the mdx mice, but the spatial extent of the changes was less than those in the DTI parameters. The data suggest that DTI is an accurate indicator of muscle injury, even at early time points where the MR signal changes are dominated by local edema.

The major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV). Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein.

Porcine epidemic diarrhea virus (PEDV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleolus. The mechanism of nuclear translocation, and whether N protein associates with particular nucleolar components, is unknown. In this study, we confirm that a nucleolar phosphoprotein nucleophosmin (NPM1) interacts and co-localizes with the N protein in the nucleolus. In vitro binding studies indicated that aa 148-294 of N and aa 118-188 of NPM1 were required for binding. Interestingly, N protein importation into the nucleolus is independent of the ability of NPM1 to shuttle between the nucleus and the cytoplasm. Furthermore, overexpression of NPM1 promoted PEDV growth, while knockdown of NPM1 suppressed PEDV growth. In addition, binding of N protein to NPM1 protects it from proteolytic degradation by caspase-3, leading to increased cell survival. Taken together, our studies demonstrate a specific interaction of the N protein with the host cell protein NPM1 in the nucleolus. The results suggest potential linkages among viral strategies for the regulation of cell survival activities, possibly through an interaction of N protein with NPM1 which prevents its proteolytic cleavage and enhances cell survival, thus ultimately promoting the replication of PEDV.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that can cause vomiting and watery diarrhea in pigs and death in piglets. Since PDCoV was first detected in 2009 in Hong Kong, the prevalence of PDCoV has increased in recent years, resulting in serious economic losses to the swine industry. The coronavirus spike (S) protein is an antigen that has been demonstrated to contain epitopes that induce neutralizing antibodies. The presence of serum and milk IgA antibodies against pathogens that replicate primarily on mucosal surfaces is important for mucosal immunity. Here, an indirect anti-PDCoV IgA antibody enzyme-linked immunosorbent assay (PDCoV S1 IgA ELISA) using the purified S1 portion of S protein as the coating antigen was developed to detect PDCoV IgA antibodies in serum and sow's milk. A receiver operating characteristic (ROC) curve analysis showed high specificity and sensitivity of the PDCoV-S1-IgA-ELISA based on samples confirmed by IFA. Anti-PDCoV IgA antibodies in 152 serum samples and 65 milk samples collected from six farms that had experienced diarrhea outbreaks within previous last two years were detected by this assay, and 62.5% of the serum samples and 100% of the milk samples were positive for PDCoV. The indirect ELISA method established in this study will provide a convenient tool for measurement of serum and milk IgA levels against PDCoV in pig herds, rapid detection of PDCoV infection in pigs, and evaluation of the immunogenicity of vaccines.

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.

The human kinome plays a crucial role in several pathways. Its dysregulation has been linked to diverse central nervous system (CNS)-related disorders with a drastic impact on the aging population. Among them, tauopathies, such as Alzheimer's Disease (AD) and Frontotemporal Lobar degeneration (FTLD-tau), are neurodegenerative disorders pathologically defined by the presence of hyperphosphorylated tau-positive intracellular inclusions known as neurofibrillary tangles (NFTs). Compelling evidence has reported the great potential of the simultaneous modulation of multiple protein kinases (PKs) involved in abnormal tau phosphorylation through a concerted pharmacological approach to achieve a superior therapeutic effect relative to classic "one target, one drug" approaches. Here, we report on the identification and characterization of ARN25068 (4), a low nanomolar and well-balanced dual GSK-3β and FYN inhibitor, which also shows inhibitory activity against DYRK1A, an emerging target in AD and tauopathies. Computational and X-Ray studies highlight compound 4's typical H-bonding pattern of ATP-competitive inhibitors at the binding sites of all three PKs. In a tau phosphorylation assay on Tau0N4R-TM-tGFP U2OS cell line, 4 reduces the extent of tau phosphorylation, promoting tau-stabilized microtubule bundles. In conclusion, this compound emerges as a promising prototype for further SAR explorations to develop potent and well-balanced triple GSK-3β/FYN/DYRK1A inhibitors to tackle tau hyperphosphorylation.

Exposure to acrolein was reported to be related with adverse health effects. However, the associations between acrolein exposure and blood lipids remain largely unknown. We assessed the associations of urinary acrolein metabolites with blood lipids using data from the National Health and Nutrition Examination Survey (NHANES) and further investigated the existence of mediation by systemic inflammation in the associations. Urinary acrolein metabolites, N-acetyl-S-(carboxyethyl)-l-cysteine (CEMA) and N-acetyl-S-(3-hydroxypropyl)-l-cysteine (3-HPMA), blood lipids, and serum high sensitivity C-reactive protein (hs-CRP) were measured in the NHANES. The associations of urinary acrolein metabolites with blood lipids and dyslipidemia and hs-CRP were estimated by multiple linear and logistic regression models. Mediation analysis was conducted to evaluate the mediating effects of hs-CRP on the associations between urinary acrolein metabolites and blood lipids. We found urinary CEMA+3-HPMA (∑acrolein) was significantly associated with higher levels of serum triglycerides (TG), hs-CRP, and lower levels of high-density lipoprotein cholesterol (HDL-C). Each 1-unit increment in ln-transformed level of ∑acrolein was associated with a 0.06 mmol/L increment in TG and 0.02 mmol/L decrement in HDL-C (all P <0.05). A positive dose-response relationship was observed between urinary ∑acrolein and dyslipidemia risk. In addition, hs-CRP significantly mediated the associations of urinary ∑acrolein with serum TG and HDL-C, with mediated proportions of 22.12% and 41.41%, respectively. In conclusion, acrolein exposure is associated with the levels of serum TG, HDL-C, and hs-CRP. Hs-CRP may mediate acrolein-associated alterations of blood lipids. Our results indicated that decreased exposure to acrolein may reduce systemic inflammation and dyslipidemia risk.