Antigenicity, epitope mapping, and intracellular distribution of the NSP7α protein of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the global pig industry. Nonstructural protein 7α (NSP7α) of PRRSV is highly conserved among different lineages of PRRSV and could be a potential target for the development of detection methods. In this study, NSP7α was expressed in prokaryote (Escherichia coli) and purified. An NSP7α-ab-ELISA detection method was established, the NSP7α-ab-ELISA has 93.1 % coincidence rate with IDEXX PRRS X3 ab test kit. NSP7α antibody was detected in pig serum by ELISA 14 days following PRRSV infection. Three monoclonal antibodies (4H9, 3F2, and C10) against NSP7α prepared by a hybridoma technique were used for epitope mapping by indirect immunofluorescence. The 4H9, 3F2, and C10 antibodies all recognized the C-terminal 72-149 amino acid region of NSP7α. 4H9 reacted with amino acids 135-143, but 3F2 and C10 did not react with any truncated polypeptide. In addition, by using the monoclonal antibodies, NSP7α was localized solely in the cytoplasm, while the N protein was distributed in the cytoplasm and nucleus. The collective findings of the antigenicity and epitope of NSP7α will be helpful for understanding the antigenicity of NSP7α and developing PRRSV diagnostic methods.

Genomic surveillance and evolutionary dynamics of type 2 porcine reproductive and respiratory syndrome virus in China spanning the African swine fever outbreak.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a serious threat to the pig industry in China. Our previous study demonstrated that PRRSV persists with local circulations and overseas imports in China and has formed a relatively stable epidemic pattern. However, the sudden African swine fever (ASF) outbreak in 2018 caused serious damage to China's pig industry structure, which resulted in about 40 per cent of pigs being slaughtered. The pig yields recovered by the end of 2019. Thus, whether the ASF outbreak reframed PRRSV evolution with changes in pig populations and further posed new threats to the pig industry becomes a matter of concern. For this purpose, we conducted genomic surveillance and recombination, NSP2 polymorphism, population dynamics, and geographical spread analysis of PRRSV-2, which is dominant in China. The results showed that the prevalence of ASF had no significant effects on genetic diversities like lineage composition, recombination patterns, and NSP2 insertion and deletion patterns but was likely to lead to changes in PRRSV-2 recombination frequency. As for circulation of the two major sub-lineages of Lineage 1, there was no apparent transmission of NADC30-like among provinces, while NADC34-like had obvious signs of inter-provincial transmission and foreign importation during the ASF epidemic. In addition, two suspected vaccine recombinant epidemic strains suggest a slight safety issue of vaccine use. Herein, the interference of ASF to the PRRSV-2 evolutionary pattern was evaluated and vaccine safety was analyzed, in order to monitor the potential threat of PRRSV-2 to China's pig industry in the post-epidemic era of ASF.

Application and Comparison of Different Regression Models in Iodine Balance Experiment on Women of Childbearing Age and Pregnant Women.

The iodine balance experiment is a traditional approach to evaluate the physiological requirement for iodine, while the simple linear regression model (SLM) and the mixed effects model (MEM) are two primary methods used to analyze iodine balance experiments. In the present study, we aimed to compare the effects of these two regression models on the evaluation of iodine balance experiments to investigate appropriate valuation methods. By constructing SLM and MEM, zero iodine balance values (IBV) were determined, and the evaluation effects were compared. No changes were made to the experimental data for women of childbearing age, and cutoff values of 600 µg/day and 1000 µg/day, respectively, were chosen for further processing of the experimental data for pregnant women. Equation combinations 1-3 (EC1-3) were obtained by fitting SLM, and zero IBV were calculated as 110.26 µg/day, 333.06 µg/day, and 434.84 µg/day, respectively. EC4-6 were obtained by fitting MEM, and zero IBV were calculated as 110.44 µg/day, 335.79 µg/day, and 418.06 µg/day, respectively. The inclusion of inter-measurement variation as a random factor in the MEM yielded EC7-8, which reduced the test power of the iodine balance experiment on women of childbearing age. Our study suggested that when experimental conditions were tightly controlled, with fewer uncertainties or significant influences, computationally straightforward and well-understood SLM was preferred. If some uncertain factors might cause large changes in the experimental results, it was advised to use a more "conservative" MEM to calculate the zero IBV. ClinicalTrials.gov Identifier: Registered at Clinicaltrials.gov, NCT03279315 (17th September 2017, retrospectively registered), NCT03710148 (18th October 2018, retrospectively registered).

T817MA Regulates Mitochondrial Dynamics via Sirt1 and Arc Following Subarachnoid Hemorrhage.

Spontaneous subarachnoid hemorrhage (SAH) is an acute neurologic emergency with poor outcomes, and mitochondrial dysfunction is known as one of the key pathological mechanisms underlying the SAH-induced early brain injury (EBI). 1-{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl} azetidin-3-ol maleate (T817MA) is a newly synthesized neurotrophic compound that has been demonstrated to exert protective effects against brain injury. Here, we investigated the effect of T817MA in neuronal injury following experimental SAH both in vitro and in vivo. Primary cultured cortical neurons were treated with oxyhemoglobin (OxyHb) to mimic SAH in vitro, and T817MA at concentrations higher than 0.1 µM reduced OxyHb-induced neuronal injury. T817MA treatment significantly inhibited lipid peroxidation, reduced neuronal apoptosis and attenuated mitochondrial fragmentation. The results of western blot showed that T817MA markedly reduced the expression of mitochondrial fission proteins, fission protein 1 (Fis-1) and dynamin-related GTPase-1 (Drp-1), but prolonged the expression of the postsynaptic protein activity-regulated cytoskeleton-associated protein (Arc). In addition, T817MA significantly increased the expression of sirtuin 1 (Sirt1), which was accompanied by preserved enzymatic of isocitrate dehydrogenase (IDH2) and superoxide dismutase (SOD). Knockdown of Sirt1 and Arc via small interfere RNA (siRNA) transfection partially prevented the T817MA-induced protection in cortical neurons. Furthermore, treatment with T817MA in vivo significantly reduced brain damage and preserved neurological function in rats. The decreased expression of Fis-1 and Drp-1, as well as the increased expression of Arc and Sirt1 were also observed in vivo. Taken together, these data indicate that the neuroprotective agent T817MA protects against SAH-induced brain injury via Sirt1- and Arc-mediated regulation of mitochondrial dynamics.

Genomic Analysis of Porcine Reproductive and Respiratory Syndrome Virus 1 Revealed Extensive Recombination and Potential Introduction Events in China.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is considered one of the most devastating swine diseases worldwide. PRRSV-1 was first isolated in China in 2006. However, there were few reports concerning the genetic characteristics of PRRSV-1 in China. In this study, three PRRSV-1 strains (HL85, HeB3, and HeB47) were detected by a general RT-qPCR method from clinical samples in 2018. HeB47 was identified as a recombinant between the BJEU06-1 and CReSA228-like strains. To further analyze the recombination and deletion features of PRRSV-1, all the available 88 complete genome sequences (isolated in 19 countries) from 1991 to 2018 in GenBank were analyzed. The high-frequency recombination regions were concentrated in NSP2 and GP2 to GP4. More importantly, phylogenetic analysis of PRRSV-1 revealed four independent introductions in China. Therefore, it is necessary to strengthen the important monitoring of breeding pigs and pork products and epidemiological surveys on pig farms to prevent the further spread of PRRSV-1.

CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA.

Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA. After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment, direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.

WTAP mediates FOXP3 mRNA stability to promote SMARCE1 expression and augment glycolysis in colon adenocarcinoma.

N6-methyladenosine (m6A) is the most abundant mRNA internal modification and has reportedly been linked to aerobic glycolysis, a hallmark event in tumor development. This work focuses on the role of the m6A methyltransferase WT1-associated protein (WTAP) in metabolic reprogramming and development of colon adenocarcinoma (COAD) and the molecules involved. The WTAP expression in COAD tissues and cells was detected. WTAP was knocked down in two COAD cell lines to figure out its role in the glycolytic activity and malignant phenotype of cancer cells. Cancer cells were further injected into nude mice subcutaneously or via tail vein to evaluate tumor growth and metastasis. The downstream molecules involved were explored using bioinformatics tools, and the molecular interactions were confirmed by immunoprecipitation, luciferase assays, and rescue experiments. WTAP was abundantly expressed in COAD samples. Knockdown of WTAP suppressed glucose consumption, lactate production, and glycolysis, which consequently suppressed cancer cell growth and dissemination in vitro and in vivo. WTAP promoted m6A methylation and stabilized forkhead box P3 (FOXP3) mRNA with the participation of the m6A "reader" YTHDF1. FOXP3 could further bind to SMARCE1 promoter for transcriptional activation. Rescue experiments showed that upregulation of FOXP3 or SMARCE1 restored the glycolytic activity in COAD cells and augmented the growth and mobility of cells both in vitro and in vivo. This study demonstrates that WTAP grants glycolytic activity to COAD and promotes tumor malignant development via the m6A modification of FOXP3 mRNA and the upregulation of SMARCE1.

Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration.

Colon adenocarcinoma is one of the tumors with the highest mortality rate, and tumorigenesis or development of colon adenocarcinoma is the major reason leading to patient death. However, the molecular mechanism and biomarker to predict tumor progression are currently unclear. With the goal of understanding the molecular mechanism and tumor progression, we utilized the TCGA database to identify differentially expressed genes. After identifying the differentially expressed genes among colon adenocarcinoma tissues with different expression levels of LGR4 and normal tissue, protein-protein interaction, gene ontology, pathway enrichment, gene set enrichment analysis, and immune cell infiltration analysis were conducted. Here, the top 10 hub genes, i.e., ALB, F2, APOA2, CYP1A1, SPRR2B, APOA1, APOB, CYP3A4, SST, and GCG, were identified, and relative correlation analysis was conducted. Kaplan-Meier analysis revealed that higher expression of LGR4 correlates with overall survival of colon adenocarcinoma patients, although expression levels of LGR4 in normal tissues are higher than in tumor tissues. Further functional analysis demonstrated that higher expression of LGR4 in colon adenocarcinoma may be linked to up-regulate metabolism-related pathways, for example, the cholesterol biosynthesis pathway. These results were confirmed by gene set enrichment analysis. Immune cell infiltration analysis clearly showed that the infiltration percentage of T cells was significantly higher than other immune cells, and TIMER analysis revealed a positive correlation between T-cell infiltration and LGR4 expression. Finally, COAD cancer cells, Caco-2, were employed to be incubated with squalene and 25-hydroxycholesterol-3-sulfate, and relative experimental results confirmed that the cholesterol biosynthesis pathway involved in modulating the proliferation of COAD tumorigenesis. Our investigation revealed that LGR4 can be an emerging diagnostic and prognostic biomarker for colon adenocarcinoma by affecting metabolism-related pathways.

Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease with great economic importance in the pig industry. Although vaccines against the PRRS virus (PRRSV) have been employed for more than 20 years, differentiating infected from vaccinated animals remains challenging. In this study, all 907 non-structural protein 2 (NSP2) full-length sequences of PRRSV-2 available from GenBank were aligned. Two peptides, at positions 562-627 (m1B) and 749-813 (m2B) of NSP2, were selected, and their potential for use in differential diagnosis was assessed. Both m1B and m2B were recognized by PRRSV-positive pig serum in peptide-coated enzyme-linked immunosorbent assays. Further epitope identification yielded five overlapping short peptides for the immunodominant regions of m1B and m2B. Using the infectious clone of PRRSV HuN4-F112 as a template, the deletion mutants, rHuN4-F112-m1B, rHuN4-F112-m2B, and rHuN4-F112-C5-m1B-m2B, were generated and successfully rescued in Marc-145 cells. Growth kinetics revealed that the deletion of m1B and m2B did not significantly affect virus replication. Hence, m1B and m2B show potential as molecular markers for developing a PRRSV vaccine.

Recombinant characteristics, pathogenicity, and viral shedding of a novel PRRSV variant derived from twice inter-lineage recombination.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a significant threat to the global pig industry. In this study, a novel recombinant PRRSV, SD043, was isolated from a pig farm experiencing disease in 2019. Phylogenetic analysis revealed that SD043 belonged to lineage 1 of PRRSV-2 while recombination analyses revealed that it is a recombinant virus from lineage 1 and lineage 8 strains. Based on further analysis, SD043 underwent recombination twice. Pathogenicity studies revealed that SD043 causes mild clinical symptoms, thymus atrophy, and severe histopathological lesions in the lungs. Notably, virus shedding in SD043-infected piglets was detectable at 10 days post-inoculation with a high viral load in the respiratory or digestive tract, indicating that the recombinant PRRSV appears to shed higher numbers of virus. Furthermore, genomic surveillance based on all available PRRSVs circulating in Shandong province revealed an increasing increase in recombinant PRRSV since 2015, with the recombinant pattern (between lineages 1 and 8) being the same as that of SD043. These findings enable a better understanding of the process of twice recombination and virus shedding of recombinant PRRSV and can strengthen the prevention and control of the PRRSV epidemic.

Monkfish Peptides Mitigate High Fat Diet-Induced Hepatic Steatosis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic syndrome usually accompanied by fatty degeneration and functional impairment. The aim of the study was to determine whether monkfish peptides (LPs) could ameliorate high-fat diet (HFD)-induced NAFLD and its underlying mechanisms. NAFLD was induced in mice by giving them an HFD for eight weeks, after which LPs were administered in various dosages. In comparison to the HFD control group: body weight in the LP-treated groups decreased by 23-28%; triacylglycerol levels in the blood decreased by 16-35%; and low-density lipoproteins levels in the blood decreased by 23-51%. Additionally, we found that LPs elevated the activity of hepatic antioxidant enzymes and reduced the inflammatory reactions within fatty liver tissue. Investigating the effect on metabolic pathways, we found that in LP-treated mice: the levels of phospho-AMP-activated protein kinase (p-AMPK), and phospho-acetyl CoA carboxylase (p-ACC) in the AMP-activated protein kinase (AMPK) pathway were up-regulated and the levels of downstream sterol regulatory element-binding transcription factor 1 (SREBP-1) were down-regulated; lipid oxidation increased and free fatty acid (FFA) accumulation decreased (revealed by the increased carnitine palmitoyltransferase-1 (CPT-1) and the decreased fatty acid synthase (FASN) expression, respectively); the nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant pathway was activated; and the levels of heme oxygenase-1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) were increased. Overall, all these findings demonstrated that LPs can improve the antioxidant capacity of liver to alleviate NAFLD progression mainly through modulating the AMPK and Nrf2 pathways, and thus it could be considered as an effective candidate in the treatment of human NAFLD.

A total infectome approach to understand the etiology of infectious disease in pigs.

BACKGROUND: The global pork industry is continuously affected by infectious diseases that can result in large-scale mortality, trade restrictions, and major reductions in production. Nevertheless, the cause of many infectious diseases in pigs remains unclear, largely because commonly used diagnostic tools fail to capture the full diversity of potential pathogens and because pathogen co-infection is common. RESULTS: We used a meta-transcriptomic approach to systematically characterize the pathogens in 136 clinical cases representing different disease syndromes in pigs, as well as in 12 non-diseased controls. This enabled us to simultaneously determine the diversity, abundance, genomic information, and detailed epidemiological history of a wide range of potential pathogens. We identified 34 species of RNA viruses, nine species of DNA viruses, seven species of bacteria, and three species of fungi, including two novel divergent members of the genus Pneumocystis. While most of these pathogens were only apparent in diseased animals or were at higher abundance in diseased animals than in healthy animals, others were present in healthy controls, suggesting opportunistic infections. Importantly, most of the cases examined here were characterized by co-infection with more than two species of viral, bacterial, or fungal pathogens, some with highly correlated occurrence and abundance levels. Examination of clinical signs and necropsy results in the context of relevant pathogens revealed that a multiple-pathogen model was better associated with the data than a single-pathogen model was. CONCLUSIONS: Our data demonstrate that most of the pig diseases examined were better explained by the presence of multiple rather than single pathogens and that infection with one pathogen can facilitate infection or increase the prevalence/abundance of another. Consequently, it is generally preferable to consider the cause of a disease based on a panel of co-infecting pathogens rather than on individual infectious agents. Video abstract.

Protective mechanism of traditional Chinese medicine guizhi fuling pills against carbon tetrachloride-induced kidney damage is through inhibiting oxidative stress, inflammation and regulating the intestinal flora.

BACKGROUND: Chemical or drug-induced kidney damage has been recognized as a critical cause of kidney failure. The oxidative stress, inflammation, and imbalance of intestinal flora caused by carbon tetrachloride (CCl4) play a fundamental role in chronic kidney damage. Guizhi Fuling pills (GZFL) is a traditional formula consisting of five traditional Chinese medicinal herbs, which can promote blood circulation and improve kidney function. The underlying mechanisms of GZFL improving kidney damage are not fully understood yet. AIM: The current study aimed to explore the effects of GZFL on CCl4-induced kidney damage and intestinal microbiota in mice. METHODS: Male ICR mice were intraperitoneally administered with 20% CCl4 (mixed in a ratio of 1:4 in soybean oil) twice a week, for 4 weeks to induce kidney damage. Creatinine (CRE), urea nitrogen, antioxidant enzymes, and inflammatory cytokines were measured and the histology of the kidney, jejunum, and colon examination to assess kidney and intestinal damage. The expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) family members, nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome in kidney tissues, and the tight junction proteins in colonic tissues were detected by Western blot. The gut microbiota was analyzed through 16S rRNA gene sequencing. RESULTS: GZFL treatment decreased the serum CRE and urea nitrogen levels. Moreover, GZFL reduced the levels of pro-inflammatory cytokines and increased antioxidant enzyme activities in kidney and colonic tissues. GZFL improved the kidney, jejunum, and colon histology. Furthermore, GZFL inhibited the expressions of NLRP3, ASC, and cleaved-Caspase-1, while Nrf2, HO-1, NQO1, GCLM, and tight junction proteins were increased. The dysbiosis of intestinal microbiota improved after GZFL treatment. CONCLUSIONS: This study showed that GZFL could improve kidney damage, which might be mainly via the integrated regulations of the Nrf2 pathway, NLRP3 inflammasome, and composition of intestinal microbiota.

Fucoxanthin Attenuates Free Fatty Acid-Induced Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism/Oxidative Stress/Inflammation via the AMPK/Nrf2/TLR4 Signaling Pathway.

Fucoxanthin, a xanthophyll carotenoid abundant in brown algae, is reported to have several biological functions, such as antioxidant, anti-inflammatory, and anti-tumor activities, in mice. We investigated the effects and mechanisms of fucoxanthin in the mixture oleate/palmitate = 2/1(FFA)-induced nonalcoholic fatty liver disease (NAFLD) cell model in this study. The results showed that the content of superoxide dismutase in the FFA group was 9.8 ± 1.0 U/mgprot, while that in the fucoxanthin high-dose (H-Fx) group (2 µg/mL) increased to 22.9 ± 0.6 U/mgprot. The content of interleukin-1ß in the FFA group was 89.3 ± 3.6 ng/mL, while that in the H-Fx group was reduced to 53.8 ± 2.8 ng/mL. The above results indicate that fucoxanthin could alleviate the FFA-induced oxidative stress and inflammatory levels in the liver cells. Oil red-O staining revealed visible protrusions and a significant decrease in the number of lipid droplets in the cytoplasm of cells in the fucoxanthin group. These findings on the mechanisms of action suggest that fucoxanthin can repair FFA-induced NAFLD via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway and nuclear factor erythroid-2-related factor 2-mediated (Nrf2) signaling pathway, as well as by downregulating the expression of the Toll-like receptor 4-mediated (TLR4) signaling pathway. Fucoxanthin exhibited alleviating effects in the FFA-induced NAFLD model and could be explored as a potential anti-NAFLD substance.

Using Collagen Peptides From the Skin of Monkfish (Lophius litulon) to Ameliorate Kidney Damage in High-Fat Diet Fed Mice by Regulating the Nrf2 Pathway and NLRP3 Signaling.

BACKGROUND: Oxidative stress and inflammation play important roles in high-fat diet (HFD) induced kidney damage. Previous studies show that the collagen extracted from the skin of monkfish (Lophius litulon) with pepsin (pepsin-solubilized collagen, PSC) exhibits good biological activities. This study investigates the protective effect of PSCP against chronic kidney injury in HFD-fed mice. METHODS: Pepsin-solubilized collagen was further hydrolyzed into collagen peptides, and the compound with the best 2,2-diphenyl-1-picrylhydrazyl (DPPH) clearance rate was named pepsin-solubilized collagen peptide (PSCP). A group of mice were fed an HFD for 4 weeks, and then for another 6 weeks PSCP was added to their diet at the amount of either 100 or 200 mg/kg. RESULTS: Pepsin-solubilized collagen peptide treatment (200 mg/kg) reduced the mice's serum levels of uric acid (UA), creatinine (CRE), and blood urea nitrogen (BUN) by 27, 20, and 37%, respectively. This treatment also remarkably improved renal histopathology. Moreover, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were increased by 96, 52, and 74%, respectively, and decreased the malondialdehyde (MDA) level by 36%. Additionally, PSCP activated the Nrf2 pathway and inhibited NLRP3 signaling to significantly reduce the levels of inflammatory cytokines IL-1ß, IL-6, and TNF-α. CONCLUSIONS: Our results indicate that compound PSCP has the potential to prevent or control chronic kidney damage.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

Monkfish (Lophius litulon) Peptides Ameliorate High-Fat-Diet-Induced Nephrotoxicity by Reducing Oxidative Stress and Inflammation via Regulation of Intestinal Flora.

BACKGROUND: Renal damage and intestinal flora imbalance due to lipotoxicity are particularly significant in terms of oxidative stress and inflammation, which can be alleviated with bioactive peptides. The monkfish (Lophius litulon) is rich in proteins, which can be used as a source of quality bioactive peptides. This study aimed to examine the protective effect of monkfish peptides on renal injury and their potential role in regulating gut microbiota. METHODS: Monkfish meat was hydrolyzed using neutral protease and filtered, and the component with the highest elimination rate of 2,2-diphenyl-1-picrylhydrazyl was named lophius litulon peptides (LPs). Lipid nephrotoxicity was induced via high-fat diet (HFD) feeding for 8 weeks and then treated with LPs. Oxidative stress, inflammatory factors, and intestinal flora were evaluated. RESULTS: LP (200 mg/kg) therapy reduced serum creatinine, uric acid, and blood urea nitrogen levels by 49.5%, 31.6%, and 31.6%, respectively. Renal vesicles and tubules were considerably improved with this treatment. Moreover, the activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity increased significantly by 198.7%, 167.9%, 61.5%, and 89.4%, respectively. LPs attenuated the upregulation of HFD-induced Toll-like receptor 4 and phospho-nuclear factor-kappa B and increased the protein levels of heme oxygenase 1, nicotinamide quinone oxidoreductase 1, and nuclear factor erythroid 2-related factor 2. The dysbiosis of intestinal microbiota improved after LP treatment. CONCLUSIONS: LPs significantly improve antioxidant activity, reduce inflammatory cytokine levels, and regulate intestinal dysbiosis. Thus, LPs are potential compounds that can alleviate HFD-induced renal lipotoxicity.

Optimization of Extraction of Bioactive Peptides from Monkfish (Lophius litulon) and Characterization of Their Role in H2O2-Induced Lesion.

BACKGROUND: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs. METHODS: Peptides were prepared from the muscles of monkfish (Lophius litulon), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities. RESULTS: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H2O2-related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels. CONCLUSION: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.

Collagen Peptides from Swim Bladders of Giant Croaker (Nibea japonica) and Their Protective Effects against H2O2-Induced Oxidative Damage toward Human Umbilical Vein Endothelial Cells.

Five different proteases were used to hydrolyze the swim bladders of Nibea japonica and the hydrolysate treated by neutrase (collagen peptide named SNNHs) showed the highest DPPH radical scavenging activity. The extraction process of SNNHs was optimized by response surface methodology, and the optimal conditions were as follows: a temperature of 47.2 °C, a pH of 7.3 and an enzyme concentration of 1100 U/g, which resulted in the maximum DPPH clearance rate of 95.44%. Peptides with a Mw of less than 1 kDa (SNNH-1) were obtained by ultrafiltration, and exhibited good scavenging activity for hydroxyl radicals, ABTS radicals and superoxide anion radicals. Furthermore, SNNH-1 significantly promoted the proliferation of HUVECs, and the protective effect of SNNH-1 against oxidative damage of H2O2-induced HUVECs was investigated. The results indicated that all groups receiving SNNH-1 pretreatment showed an increase in GSH-Px, SOD, and CAT activities compared with the model group. In addition, SNNH-1 pretreatment reduced the levels of ROS and MDA in HUVECs with H2O2-induced oxidative damage. These results indicate that collagen peptides from swim bladders of Nibea japonica can significantly reduce the oxidative stress damage caused by H2O2 in HUVECs and provides a basis for the application of collagen peptides in the food industry, pharmaceuticals, and cosmetics.

Glycine- and Proline-Rich Protein OsGPRP3 Regulates Grain Size and Quality in Rice.

The grain size and shape of rice are limited by the growth of the spikelet hulls and are important selective target during domestication and breeding. In this study, we identified a glycine- and proline-rich protein (OsGPRP3), which belongs to a conserved family rarely studied. We found that OsGPRP3 was highly expressed in the seed at 10 days after pollination (DAP) using qRT-PCR, pOsGPRP3::GUS and in situ hybridization. Knockout and knockdown of OsGPRP3 led to significant decrease of 1000-grain weight, grain width, and grain thickness. We further found that the content of storage protein and total lipid were decreased in osgprp3 lines. In particular, the contents of C14:0 (myristic acid), C16:0 (palmitic acid), C18:1 (oleic acid), and C18:2 (linoleic acid) were reduced in osgprp3 lines. Cytological experiments revealed that the cell width of spikelet hull in osgprp3 lines was significantly reduced than that in WT. Taken together, our results reveal that OsGPRP3 regulates the grain size and shape of rice by influencing the cell width of spikelet hulls and the accumulation of storage protein and lipids.