CancerMHL: the database of integrating key DNA methylation, histone modifications and lncRNAs in cancer.

The discovery of key epigenetic modifications in cancer is of great significance for the study of disease biomarkers. Through the mining of epigenetic modification data relevant to cancer, some researches on epigenetic modifications are accumulating. In order to make it easier to integrate the effects of key epigenetic modifications on the related cancers, we established CancerMHL (http://www.positionprediction.cn/), which provide key DNA methylation, histone modifications and lncRNAs as well as the effect of these key epigenetic modifications on gene expression in several cancers. To facilitate data retrieval, CancerMHL offers flexible query options and filters, allowing users to access specific key epigenetic modifications according to their own needs. In addition, based on the epigenetic modification data, three online prediction tools had been offered in CancerMHL for users. CancerMHL will be a useful resource platform for further exploring novel and potential biomarkers and therapeutic targets in cancer. Database URL: http://www.positionprediction.cn/.

Metabolic and Bioprocess Engineering of Clostridium tyrobutyricum for Butyl Butyrate Production on Xylose and Shrimp Shell Waste.

Microbial conversion of agri-food waste to valuable compounds offers a sustainable route to develop the bioeconomy and contribute to sustainable biorefinery. Clostridium tyrobutyricum displays a series of native traits suitable for high productivity conversion of agri-food waste, which make it a promising host for the production of various compounds, such as the short-chain fatty acids and their derivative esters products. In this study, a butanol synthetic pathway was constructed in C. tyrobutyricum, and then efficient butyl butyrate production through in situ esterification was achieved by the supplementation of lipase into the fermentation. The butyryl-CoA/acyl-CoA transferase (cat1) was overexpressed to balance the ratio between precursors butyrate and butanol. Then, a suitable fermentation medium for butyl butyrate production was obtained with xylose as the sole carbon source and shrimp shell waste as the sole nitrogen source. Ultimately, 5.9 g/L of butyl butyrate with a selectivity of 100%, and a productivity of 0.03 g/L·h was achieved under xylose and shrimp shell waste with batch fermentation in a 5 L bioreactor. Transcriptome analyses exhibited an increase in the expression of genes related to the xylose metabolism, nitrogen metabolism, and amino acid metabolism and transport, which reveal the mechanism for the synergistic utilization of xylose and shrimp shell waste. This study presents a novel approach for utilizing xylose and shrimp shell waste to produce butyl butyrate by using an anaerobic fermentative platform based on C. tyrobutyricum. This innovative fermentation medium could save the cost of nitrogen sources (~97%) and open up possibilities for converting agri-food waste into other high-value products.

Presenilin 1 Is a Therapeutic Target in Pulmonary Hypertension and Promotes Vascular Remodeling.

Small muscular pulmonary artery remodeling is a dominant feature of PAH. PSEN1 affects angiogenesis, cancer and Alzheimer's disease. We aimed to determine the role of PSEN1 in the pathogenesis of vascular remodeling in PH. Haemodynamics and vascular remodeling in the Psen1-knockin and smooth muscle-specific Psen1-knockout mice were assessed. The functional partners of PSEN1 were predicted by bioinformatics analysis and biochemical experiments. The therapeutic effect of PH was evaluated by administration of the PSEN1-specific inhibitor ELN318463. We discovered that both the mRNA and protein levels of PSEN1 were increased over time in hypoxic rats, monocrotaline (MCT) rats and Su5416/hypoxia (SuHx) mice. Psen1 transgenic mice were highly susceptible to PH, whereas smooth muscle-specific Psen1-knockout mice were resistant to hypoxic PH. STRING analysis showed that the Notch1/2/3, ß-catenin, Cadherin-1, DNER, TMP10 and ERBB4 appeared to be highly correlated with PSEN1. Immunoprecipitation confirmed that PSEN1 interacts with ß-catenin and DNER, and these interactions were suppressed by the catalytic PSEN1 mutations D257A, D385A and C410Y. PSEN1 was found to mediate the nuclear translocation of the Notch1 intracellular domains and activated RBP-Jκ. Octaarginine-coated liposome-mediated pharmacological inhibition of PSEN1 significantly prevented and reversed the pathological process of in hypoxic and MCT induced PH. PSEN1 essentially drives the pathogenesis of PAH and interacted with the non-canonical Notch ligand DNER. PSEN1 can be used as a promising molecular target for treating PAH. PSEN1 inhibitor ELN318463 can prevent and reverse the progression of PH and be developed as a potential anti-PAH drug.

Therapeutic effect of long-acting FGF21 with controlled site-specific modification on nonalcoholic steatohepatitis.

FGF21 plays an active role in the treatment of type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). However, the short half-life and poor stability of wild-type FGF21 limit its clinical application. Previous studies found that PEGylation can significantly increase the stability of FGF21. However, the uneven distribution of PEGylation sites in FGF21 makes it difficult to purify PEG-FGF21, thereby affecting its yield, purity, and activity. To obtain long-acting FGF21 with controlled site-specific modification, we mutated lysine residues in FGF21, resulting in PEGylation only at the N-terminus of FGF21 (mFGF21). In addition, we modified mFGF21 molecules with different PEG molecules and selected the PEG-mFGF21 moiety with the highest activity. The yield of PEG-mFGF21 in this study reached 1 g/L (purity >99 %), and the purification process was simple and efficient with strong quality controllability. The half-life of PEG-mFGF21 in rats reached 40.5-67.4 h. Pharmacodynamic evaluation in mice with high-fat, high-cholesterol- and methionine and choline deficiency-induced NASH illustrated that PEG-mFGF21 exhibited long-term efficacy in improving liver steatosis and reducing liver cell damage, inflammation, and fibrosis. Taken together, PEG-mFGF21 could represent a potential therapeutic drug for the treatment of NASH.

Potential biomarkers: The hypomethylation of cg18949415 and cg22193385 sites in colon adenocarcinoma.

Overall cancer hypomethylation had been identified in the past, but it is not clear exactly which hypomethylation site is the more important for the occurrence of cancer. To identify key hypomethylation sites, we studied the effect of hypomethylation in twelve regions on gene expression in colon adenocarcinoma (COAD). The key DNA methylation sites of cg18949415, cg22193385 and important genes of C6orf223, KRT7 were found by constructing a prognostic model, survival analysis and random combination prediction a series of in-depth systematic calculations and analyses, and the results were validated by GEO database, immune microenvironment, drug and functional enrichment analysis. Based on the expression values of C6orf223, KRT7 genes and the DNA methylation values of cg18949415, cg22193385 sites, the least diversity increment algorithm were used to predict COAD and normal sample. The 100 % reliability and 97.12 % correctness of predicting tumor samples were obtained in jackknife test. Moreover, we found that C6orf223 gene, cg18949415 site play a more important role than KRT7 gene, cg22193385 site in COAD. In addition, we investigate the impact of key methylation sites on three-dimensional chromatin structure. Our results will be help for experimental studies and may be an epigenetic biomarker for COAD.

Development and validation of CT-based radiomics deep learning signatures to predict lymph node metastasis in non-functional pancreatic neuroendocrine tumors: a multicohort study.

Background: Lymph node status is an important factor for the patients with non-functional pancreatic neuroendocrine tumors (NF-PanNETs) with respect to the surgical methods, prognosis, recurrence. Our aim is to develop and validate a combination model based on contrast-enhanced CT images to predict the lymph node metastasis (LNM) in NF-PanNETs. Methods: Retrospective data were gathered for 320 patients with NF-PanNETs who underwent curative pancreatic resection and CT imaging at two institutions (Center 1, n = 236 and Center 2, n = 84) between January 2010 and March 2022. RDPs (Radiomics deep learning signature) were developed based on ten machine-learning techniques. These signatures were integrated with the clinicopathological factors into a nomogram for clinical applications. The evaluation of the model's performance was conducted through the metrics of the area under the curve (AUC). Findings: The RDPs showed excellent performance in both centers with a high AUC for predicting LNM and disease-free survival (DFS) in Center 1 (AUC, 0.88; 95% CI: 0.84-0.92; DFS, p < 0.05) and Center 2 (AUC, 0.91; 95% CI: 0.85-0.97; DFS, p < 0.05). The clinical factors of vascular invasion, perineural invasion, and tumor grade were associated with LNM (p < 0.05). The combination nomogram showed better prediction capability for LNM (AUC, 0.93; 95% CI: 0.89-0.96). Notably, our model maintained a satisfactory predictive ability for tumors at the 2-cm threshold, demonstrating its effectiveness across different tumor sizes in Center 1 (≤2 cm: AUC, 0.90 and >2 cm: AUC, 0.86) and Center 2 (≤2 cm: AUC, 0.93 and >2 cm: AUC, 0.91). Interpretation: Our RDPs may have the potential to preoperatively predict LNM in NF-PanNETs, address the insufficiency of clinical guidelines concerning the 2-cm threshold for tumor lymph node dissection, and provide precise therapeutic strategies. Funding: This work was supported by JSPS KAKENHI Grant Number JP22K20814; the Rare Tumor Research Special Project of the National Natural Science Foundation of China (82141104) and Clinical Research Special Project of Shanghai Municipal Health Commission (202340123).

Personalized glucose-lowering effect of chiglitazar in type 2 diabetes.

Chiglitazar (carfloglitazar) is a peroxisome proliferator-activated receptor pan-agonist presenting non-inferior glucose-lowering efficacy with sitagliptin in patients with type 2 diabetes. To delineate the subgroup of patients with greater benefit from chiglitazar, we conducted a machine learning-based post-hoc analysis in two randomized controlled trials. We established a character phenomap based on 13 variables and estimated HbA1c decline to the effects of chiglitazar in reference to sitagliptin. Out of 1,069 patients, 63.3% were found to have greater reduction in HbA1c levels with chiglitazar, while 36.7% showed greater reduction with sitagliptin. This distinction in treatment response was statistically significant between groups (pinteraction<0.001). To identify patients who would gain the most glycemic control benefit from chiglitazar, we developed a machine learning model, ML-PANPPAR, which demonstrated robust performance using sex, BMI, HbA1c, HDL, and fasting insulin. The phenomapping-derived tool successfully identified chiglitazar responders and enabled personalized drug allocation in patients with drug-naïve diabetes.

Large-scale pancreatic cancer detection via non-contrast CT and deep learning.

Pancreatic ductal adenocarcinoma (PDAC), the most deadly solid malignancy, is typically detected late and at an inoperable stage. Early or incidental detection is associated with prolonged survival, but screening asymptomatic individuals for PDAC using a single test remains unfeasible due to the low prevalence and potential harms of false positives. Non-contrast computed tomography (CT), routinely performed for clinical indications, offers the potential for large-scale screening, however, identification of PDAC using non-contrast CT has long been considered impossible. Here, we develop a deep learning approach, pancreatic cancer detection with artificial intelligence (PANDA), that can detect and classify pancreatic lesions with high accuracy via non-contrast CT. PANDA is trained on a dataset of 3,208 patients from a single center. PANDA achieves an area under the receiver operating characteristic curve (AUC) of 0.986-0.996 for lesion detection in a multicenter validation involving 6,239 patients across 10 centers, outperforms the mean radiologist performance by 34.1% in sensitivity and 6.3% in specificity for PDAC identification, and achieves a sensitivity of 92.9% and specificity of 99.9% for lesion detection in a real-world multi-scenario validation consisting of 20,530 consecutive patients. Notably, PANDA utilized with non-contrast CT shows non-inferiority to radiology reports (using contrast-enhanced CT) in the differentiation of common pancreatic lesion subtypes. PANDA could potentially serve as a new tool for large-scale pancreatic cancer screening.

Construction of a Collagen-like Protein Based on Elastin-like Polypeptide Fusion and Evaluation of Its Performance in Promoting Wound Healing.

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.

Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase.

Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.

Alcoholic Setdb1 suppression promotes hepatosteatosis in mice by strengthening Plin2.

BACKGROUND AND AIMS: Hepatosteatosis is one of the early features of alcoholic liver disease (ALD) and pharmaceutical or genetic interfering of the development of hepatosteatosis will efficiently alleviate the progression of ALD. Currently, the role of histone methyltransferase Setdb1 in ALD is not yet well understood. METHOD: Lieber-De Carli diet mice model and NIAAA mice model were constructed to confirm the expression of Setdb1. The hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice was established to determine the effects of Setdb1 in vivo. Adenovirus-Setdb1 were produced to rescue the hepatic steatosis in both Setdb1-HKO and Lieber-De Carli mice. The enrichment of H3k9me3 in the upstream sequence of Plin2 and the chaperone-mediated autophagy (CMA) of Plin2 were identified by ChIP and co-IP. Dual-luciferase reporter assay was used to detect the interaction of Setdb1 3'UTR and miR216b-5p in AML12 or HEK 293 T cells. RESULTS: We found that Setdb1 was downregulated in the liver of alcohol-fed mice. Setdb1 knockdown promoted lipid accumulation in AML12 hepatocytes. Meanwhile, hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice exhibited significant lipid accumulation in the liver. Overexpression of Setdb1 was performed with an adenoviral vector through tail vein injection, which ameliorated hepatosteatosis in both Setdb1-HKO and alcoholic diet-fed mice. Mechanistically, downregulated Setdb1 promoted the mRNA expression of Plin2 by desuppressing H3K9me3-mediated chromatin silencing in its upstream sequence. Pin2 acts as a critical membrane surface-associated protein to maintain lipid droplet stability and inhibit lipase degradation. The downregulation of Setdb1 also maintained the stability of Plin2 protein through inhibiting Plin2-recruited chaperone-mediated autophagy (CMA). To explore the reasons for Setdb1 suppression in ALD, we found that upregulated miR-216b-5p bound to the 3'UTR of Setdb1 mRNA, disturbed its mRNA stability, and eventually aggravated hepatic steatosis. CONCLUSIONS: Setdb1 suppression plays an important role in the progression of alcoholic hepatosteatosis via elevating the expression of Plin2 mRNA and maintaining the stability of Plin2 protein. Targeting hepatic Setdb1 might be a promising diagnostic or therapeutic strategy for ALD.

Design and pharmaceutical evaluation of bifunctional fusion protein of FGF21 and GLP-1 in the treatment of nonalcoholic steatohepatitis.

Fibroblast growth factor 21 (FGF21) and glucagon-like peptide-1 (GLP-1) may be useful for the treatment of type 2 diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). Previous studies have shown that GLP-1 may synergize with FGF21 in the regulation of glucose and lipid metabolism. Currently, no approved drug therapy is available for non-alcoholic steatohepatitis (NASH). Here, we constructed and screened dual-targeting fusion proteins of GLP-1 and FGF21, connected by elastin-like polypeptides (ELPs), to investigate whether a combination of these two hormones would have therapeutic effects in models of NASH. The temperature phase transition and release of the hormones under physiological conditions were studied to identify a bifunctional fusion protein of FGF21 and GLP-1 (GEF) that was highly stable and showed sustained release. We further evaluated the quality and therapeutic efficacy of GEF in three mouse models of NASH. We successfully synthesized a novel recombinant bifunctional fusion protein with high stability and low immunogenicity. The GEF protein synthesized ameliorated hepatic lipid accumulation, hepatocyte damage, and inflammation; prevented the progression of NASH in the three models; reduced glycemia; and caused weight loss. This novel GEF molecule may be suitable for clinical use for the treatment of NAFLD/NASH and related metabolic diseases.

mTORC1 inhibition uncouples lipolysis and thermogenesis in white adipose tissue to contribute to alcoholic liver disease.

BACKGROUND: Adipose tissue thermogenic activities use fatty acids from lipolysis for heat generation. Therefore, a tight coupling between lipolysis and thermogenesis is physiologically imperative in maintaining not only body temperature but also lipids homeostasis. Adipose tissue dysfunction contributes to alcoholic liver disease (ALD). Here, studies were conducted to examine how alcohol intake affects adipose tissue thermogenic activities and whether altered adipose tissue thermogenesis contributes to ALD. METHODS: Both the Lieber-DeCarli and the NIAAA mouse models of ALD were used. Denervation surgery in epididymal fat pads was performed. CL316,243, a selective ß3-adrenoceptor agonist, SR59230A, a selective ß3 adrenoceptor (ADRB3) antagonist, and rapamycin, a selective mechanistic target of rapamycin complex 1 (mTORC1) inhibitor, were administrated through i.p. injection. Adipocyte-specific Prdm16 knockout mice were subjected to alcohol-containing diet chronically. RESULTS: Chronic alcohol consumption, which enhances adipose tissue lipolysis, inhibits thermogenic activities of beige adipocytes in inguinal white adipose tissue (WAT), leading to an uncoupling status between lipolysis and thermogenesis in WAT at both basal and ADRB3 stimulation states. CL316,243 administration exacerbates liver pathologies of ALD. Alcohol intake inhibits mTORC1 activities in WAT. In mice, mTORC1 inhibition by rapamycin inhibits the thermogenesis of iWAT, whereas enhancing WAT lipolysis. Further investigations using adipocyte-specific Prdm16 knockout mice revealed that functional deficiency of beige adipocytes aggravates liver pathologies of ALD, suggesting that the inhibitory effect of alcohol on WAT browning/thermogenesis contributes to ALD pathogenesis. CONCLUSION: Chronic alcohol consumption induces an "uncoupling status" between lipolysis and browning/thermogenesis in WAT by inhibiting mTORC1 activation. Diminished WAT browning/thermogenesis, concomitant with enhanced lipolysis, contributes to ALD pathogenesis.

Sodium Danshensu protects against oxygen glucose deprivation/reoxygenation-induced astrocytes injury through regulating NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome and tuberous sclerosis complex-2 (TSC2)/mammalian target of rapamycin (mTOR) pathways.

Background: Cerebral ischemic stroke is a serious condition with high incidence, mortality, and associated disability. Currently, effective therapeutic options are available for ischemic stroke are limited. Accumulating evidence indicates that sodium Danshensu, mono sodium compound derived from Salvia miltiorrhiza, plays protective roles in ischemic stroke. However, the underlying protective mechanism of sodium Danshensu in cerebral ischemic stroke remains unknown. Methods: In the current study, we explored the role and mechanism of sodium Danshensu on astrocytes exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), which mimics the process of ischemia-reperfusion. The impact of sodium Danshensu on cell viability and apoptosis after OGD/R were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-dophenyl tetrazolium bromide (MTT) assay and flow cytometry. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were used to detect the expression of target messenger RNA (mRNA) and proteins associated with apoptosis and autophagy. The release of lactate dehydrogenase (LDH) was determined, and the production of proinflammatory cytokines were detected using enzyme-linked immunosorbent assay (ELISA) kits. Results: It was found that sodium Danshensu could significantly increase cell viability and decrease LDH release and apoptosis. Besides, it inhibited the production of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. Sodium Danshensu also dose-dependently decreased protein and mRNA levels of nucleotide binding oligomerization NOD-like receptor pyrin domain containing 3 (NLRP3) and high mobility group box 1 (HMGB1), which play a crucial role in promoting ischemic stroke-induced cell injury. Moreover, sodium Danshensu dose-dependently upregulated Beclin 1 expression, downregulated P62 protein expression, and further increased LC3B-II/LC3B-I ratio through inducing autophagy in astrocytes. Additionally, we noticed that sodium Danshensu dose-dependently increased tuberous sclerosis complex-2 (TSC2) protein expression, while significantly reduced the levels of mammalian target of rapamycin (mTOR) in the presence of OGD/R insult. Conclusions: These findings suggest that sodium Danshensu protects against OGD/R-induced injury by modulating the NLRP3 inflammasome and TSC2/mTOR pathways.

Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses in pigs and cattle.

Foot-and-mouth disease (FMD) remains a very serious barrier to agricultural development and the international trade of animals and animal products. Recently, serotype O has been the most prevalent FMDV serotype in China, and it has evolved into four different lineages: O/SEA/Mya-98, O/ME-SA/PanAsia, O/ME-SA/Ind-2001 and O/Cathay. PanAsia-2, belonging to the O/ME-SA topotype, is prevalent in neighbouring countries and poses the risk of cross-border spread in China. This study aimed to develop a promising vaccine candidate strain that can not only provide the best protection against all serotype O FMDVs circulating in China but also be used as an emergency vaccine for the prevention and control of transboundary incursion of PanAsia-2. Here, two chimeric FMDVs (rHN/TURVP1 and rHN/NXVP1) featuring substitution of VP1 genes of the O/TUR/5/2009 vaccine strain (PanAsia-2) and O/NXYCh/CHA/2018 epidemic strain (Mya98) were constructed and evaluated. The biological properties of the two chimeric FMDVs were similar to those of the wild-type (wt) virus despite slight differences in plaque sizes observed in BHK-21 cells. The structural protein-specific antibody titres induced by the rHN/TURVP1 and wt virus vaccines in pigs and cows were higher than those induced by the rHN/NXVP1 vaccine at 28-56 dpv. The vaccines prepared from the two chimeric viruses and wt virus all induced the production of protective cross-neutralizing antibodies against the viruses of the Mya-98, PanAsia and Ind-2001 lineages in pigs and cattle at 28 dpv; however, only the animals vaccinated with the rHN/TURVP1 vaccine produced a protective immune response to the field isolate of the Cathay lineage at 28 dpv, whereas the animals receiving the wt virus and the rHN/NXVP1 vaccines did not, although the wt virus and O/GXCX/CHA/2018 both belong to the Cathay topotype. This study will provide very useful information to help develop a potential vaccine candidate for the prevention and control of serotype O FMD in China.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia.

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

The long non-coding RNA LNC_000397 negatively regulates PRRSV replication through induction of interferon-stimulated genes.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant threats to the global swine industry. It is of great importance to understand viral-host interactions to develop novel antiviral strategies. Long non-coding RNAs (lncRNAs) have emerged as critical factors regulating host antiviral immune responses. However, lncRNAs participating in virus-host interactions during PRRSV infection remain largely unexplored. METHOD: RNA transcripts of porcine alveolar macrophages (PAMs) infected with two different PRRSV strains, GSWW/2015 and VR2332, at 24 h post-infection were sequenced by high-throughput sequencing. Four programs namely, CNCI, CPC, PFAM, and phyloCSF, were utilized to predict the coding potential of transcripts. mRNAs co-localized or co-expressed with differentially expressed lncRNAs were considered as their targets. Fuction of lncRNAs was predicted by GO and KEGG analysis of their target mRNAs. The effect of LNC_000397 on PRRSV replication was validated by knockdown its expression using siRNA. Target genes of LNC_000397 were identified by RNA-Sequencing and validated by RT-qPCR. RESULT: In this study, we analyzed lncRNA and mRNA expression profiles of PRRSV GSWW/2015 and VR2332 infected porcine alveolar macrophages. A total of 1,147 novel lncRNAs were characterized, and 293 lncRNAs were differentially expressed. mRNAs co-localized and co-expressed with lncRNAs were enriched in pathogen-infection-related biological processes such as Influenza A and Herpes simplex infection. Functional analysis revealed the lncRNA, LNC_000397, which was up-regulated by PRRSV infection, negatively regulated PRRSV replication. Knockdown of LNC_000397 significantly impaired expression of antiviral ISGs such as MX dynamin-like GTPase 1 (MX1), ISG15 Ubiquitin-like modifier (ISG15), and radical S-adenosyl methionine domain containing 2 (RSAD2). CONCLUSIONS: LNC_000397 negatively regulated PRRSV replication by inducing interferon-stimulated genes (ISGs) expression. Our study is the first report unveiling the role of host lncRNA in regulating PRRSV replication, which might be beneficial for the development of novel antiviral therapeutics.

Two Cross-Protective Antigen Sites on Foot-and-Mouth Disease Virus Serotype O Structurally Revealed by Broadly Neutralizing Antibodies from Cattle.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that infects cloven-hoofed animals. Neutralizing antibodies play critical roles in antiviral infection. Although five known antigen sites that induce neutralizing antibodies have been defined, studies on cross-protective antigen sites are still scarce. We mapped two cross-protective antigen sites using 13 bovine-derived broadly neutralizing monoclonal antibodies (bnAbs) capable of neutralizing 4 lineages within 3 topotypes of FMDV serotype O. One antigen site was formed by a novel cluster of VP3-focused epitopes recognized by bnAb C4 and C4-like antibodies. The cryo-electron microscopy (cryo-EM) structure of the FMDV-OTi (O/Tibet/99)-C4 complex showed close contact with VP3 and a novel interprotomer antigen epitope around the icosahedral 3-fold axis of the FMDV particle, which is far beyond the known antigen site 4. The key determinants of the neutralizing function of C4 and C4-like antibodies on the capsid were ßB (T65), the B-C loop (T68), the E-F loop (E131 and K134), and the H-I loop (G196), revealing a novel antigen site on VP3. The other antigen site comprised two group epitopes on VP2 recognized by 9 bnAbs (B57, B73, B77, B82, F28, F145, F150, E46, and E54), which belong to the known antigen site 2 of FMDV serotype O. Notably, bnAb C4 potently promoted FMDV RNA release in response to damage to viral particles, suggesting that the targeted epitope contains a trigger mechanism for particle disassembly. This study revealed two cross-protective antigen sites that can elicit cross-reactive neutralizing antibodies in cattle and provided new structural information for the design of a broad-spectrum molecular vaccine against FMDV serotype O. IMPORTANCE FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response. Monoclonal neutralization antibodies provide both crucial defense components against FMDV infection and valuable tools for fine analysis of the antigenic structure. In this study, we found a cluster of novel VP3-focused epitopes using 13 bnAbs against FMDV serotype O from natural host cattle, which revealed two cross-protective antigen sites on VP2 and VP3. Antibody C4 targeting this novel epitope potently promoted viral particle disassembly and RNA release before infection, which may indicate a vulnerable region of FMDV. This study reveals new structural information about cross-protective antigen sites of FMDV serotype O, providing valuable and strong support for future research on broad-spectrum vaccines against FMD.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

The rescue and selection of thermally stable type O vaccine candidate strains of foot-and-mouth disease virus.

Inactivated foot-and-mouth disease virus (FMDV) vaccines have been used widely to control foot-and-mouth disease (FMD). However, the virions (146S) of this virus are easily dissociated into pentamer subunits (12S), which limits the immune protective efficacy of inactivated vaccines when the temperature is higher than 30 °C. A cold-chain system can maintain the quality of the vaccines, but such systems are usually not reliable in limited-resource settings. Thus, it is imperative to improve the thermostability of vaccine strains to guarantee the quality of the vaccines. In this study, four recombinant FMDV strains containing single or multiple amino acid substitutions in the structural proteins were rescued using a previously constructed FMDV type O full-length infectious clone (pO/DY-VP1). We found that single or multiple amino acid substitutions in the structural proteins affected viral replication to different degrees. Furthermore, the heat and acid stability of the recombinant viruses was significantly increased when compared with the parental virus. Three thermally stable recombinant viruses (rHN/DY-VP1Y2098F, rHN/DY-VP1V2090A-S2093H, and rHN/DY-VP1V2090A-S2093H-Y2098F) were prepared as inactivated vaccines to immunize pigs. Blood samples were collected every week to prepare sera, and a virus neutralization test showed that the substitutions S2093H and Y2098F, separately or in combination, did not affect the immunogenicity of the virus, but the Y2098F mutation increased the thermostability significantly (p < 0.05). Therefore, the rHN/DY-VP1Y2098F mutant should be considered for use in future vaccines.