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List of 40 genes in next-generation sequencing (NGS) panel

PCR-based assays to detect human circulating DNA in mice demonstrate that human PTEGR2 assays are specific (A) and sensitive (B). Human-derived DNA could be detected in plasma of PDX (C).

Porcine epidemic diarrhea virus (PEDV) is the main cause of diarrhea, vomiting, and mortality in pigs, which results in devastating economic loss to the pig industry around the globe. In recent years, the advent of RNA-sequencing technologies has led to delineate host responses at late stages of PEDV infection; however, the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown. Here, using the BGI DNBSEQ RNA-sequencing, we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent (GDS01) or avirulent (HX) PEDV strains for 3, 6, and 12 ​h. It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern. Functional enrichment analyses indicated that the differentially expressed genes (DEGs) in the GDS01 group were predominantly related to autophagy and apoptosis, whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation. Among the DEGs, the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells. TLR3 expression was found to inhibit HX strain, but not GDS01 strain, replication by enhancing the IFIT2 expression in infected cells. In conclusion, our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains. These findings may provide a foundation for establishing novel therapies to control PEDV infection.

With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.

With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.

Acute kidney injury (AKI) is a common clinical disease with high morbidity and mortality, and with a lack of effective drugs for treatment. Oxidative stress is very important in the occurrence and progression of AKI, and antioxidants use is one of the promising treatments. Rosmarinic acid (RA) is a ubiquitous natural polyphenol with powerful antioxidant and anti-inflammatory activities. Due to its inherent characteristic with poor water solubility and inferior bioavailability, its clinical application is impeded. Hence, the authors design a nanoparticle for effectively delivering RA, which is a chemical complex of RA and fourth-generation poly-amidoamine-based amphiphilic polymer (G4-PAMAM). The nanoparticle is modified with l-serine due to the specific interaction between kidney injury molecule-1 (Kim-1) and serine, which eventually generates a promising AKI kidney-targeting nanoparticle (S-G-R). The S-G-R is rapidly cumulated and long-term retained in ischemia-reperfusion-induced AKI kidneys, especially in the damaged renal tubular cells. The S-G-R exhibits more excellent antioxidative and antiapoptotic effects in vitro and has a more outstanding ability to improve the renal function, repair damaged renal tissue, and decrease oxidative stress, inflammatory response and apoptosis of tubular cells in vivo. Overall, this study might develop a safe and effective targeting strategy for the therapy of AKI.

BackgroundRenin-angiotensin system inhibitors (RASi) are the first choice and basic therapy for the treatment of IgA nephropathy (IgAN) with proteinuria. However, approximately 40% of patients have no response to RASi treatment. The aim of this study was to screen potential biomarkers for predicting the treatment response of RASi in patients with IgAN.MethodsWe included IgAN patients who were treatment-naive. They received supportive treatment, including a maximum tolerant dose of RASi for 3 months. According to the degree of decrease in proteinuria after 3 months of follow-up, these patients were divided into a sensitive group and a resistant group. The plasma of the two groups of patients was collected, and the exosomes were extracted for high-throughput sequencing. The screening of hub genes was performed using a weighted gene co-expression network (WGCNA) and filtering differentially expressed genes (DEGs). We randomly selected 20 patients in the sensitive group and 20 patients in the resistant group for hub gene validation by real-time quantitative polymerase chain reaction (qRT−PCR). A receiver operating characteristic (ROC) curve was used to evaluate hub genes that predicted the efficacy of the RASi response among the 40 validation patients.ResultsAfter screening 370 IgAN patients according to the inclusion and exclusion criteria and the RASi treatment response evaluation, there were 38 patients in the sensitive group and 32 patients in the resistant group. IRAK1, ABCD1 and PLXNB3 were identified as hub genes by analyzing the high-throughput sequencing of the plasma exosomes of the two groups through WGCNA and DEGs screening. The sequencing data were consistent with the validation data showing that these three hub genes were upregulated in the resistant group compared with the sensitive group. The ROC curve indicated that IRAK1 was a good biomarker to predict the therapeutic response of RASi in patients with IgAN.ConclusionsPlasma exosomal IRAK1 can be a potential biomarker for predicting the treatment response of RASi in patients with IgAN.

A two-step process of carbonization coupled with steam activation was proposed for wooden activated carbon production from four kinds of biomass waste materials. The TG-FTIR results show that the carbonization process started at around 250 °C and finished at 500 °C for the coconut shell, pinewood, and plywood. The carbonization temperature of corn straw was lower than those of the other three samples, which was attributed to the higher concentration of ash content. FTIR results for the volatile compounds during carbonization show that CH4, CO, CO2, and hydrocarbons are the main detected gaseous species. The CH4 and C m H n yields of pinewood and plywood are higher than those of the coconut shell and corn straw. The carbonization results on the tubular furnace reactor show that furfural and phenol and its derivatives are the main tar compounds in waste carbonization. Carbonization experiments show that a temperature of 500 °C and residence time of 30 min are the optimized parameters for the three biomass wastes. The char yields are 26.4, 25.73, and 30.38% for pinewood, plywood, and coconut shell, respectively. CFD modeling has proven that using 20% of the volatiles could achieve lowest pollution and provide heat for carbonization of biomass waste. The steam activation results show that an activation temperature of 800 °C and activation time of 30 min are suitable for all three biomass samples, which could obtain optimized AC yields and adsorption quality for dioxin.

Fibronectin glomerulopathy (FNG) is a rare inherited kidney disease characterized by extensive deposition of fibronectin in the glomeruli, especially in the mesangial and subendothelial regions. The disease progresses slowly and eventually leads to kidney failure in 15–20 years. Here, we report an interesting case. The patient presented with proteinuria and was diagnosed with immune complex–mediated glomerulonephritis, and lupus nephritis was suspected. This patient progressed to end-stage renal disease after 18 years and received an allogeneic kidney transplant. However, proteinuria recurred 27 months after kidney transplantation. The renal biopsy found extensive deposition in glomeruli, and the patient was diagnosed with FNG using mass spectrometry analysis and confirmed by immunohistochemistry in both the native and transplanted kidneys. Gene sequencing revealed that a missense mutation in the fibronectin 1 (FN1) gene caused reduced binding to heparin, endothelial cells, and podocytes and impaired stress fiber formation. The patient had stable renal function but persistent nephrotic proteinuria after 6 months of follow-up. Given the persistence of abnormal circulating fibronectin levels, FNG can relapse following renal transplantation. The circulating fibronectin deposits on grafts, and renal function progressively deteriorates after recurrence. Therefore, whether renal transplantation is an acceptable treatment for FNG is still debatable.

Since September 2020, the clinical symptoms of Muscovy duck spleen spots have appeared in Guangdong, Guangxi, Jiangxi, Hunan, Hubei, and other provinces, resulting in a large number of Muscovy duck deaths and great economic losses. The absence of the typical clinical symptoms caused by pathogenic microorganisms makes the cause of the spotted spleen a mystery. High-throughput sequencing results suggested that Riemerella anatipestifer (R. anatipestifer) may be the pathogen. Then, R. anatipestifer was regarded as the research target for isolation, identification, and pathogenicity assessment. After biochemical test, PCR amplification, and serotype determination, it was confirmed that the isolated strain CZG-1 was serotype 15 R. anatipestifer. Typical spotted spleen symptoms were observed after CZG-1 infection. Furthermore, drug sensitivity assays showed the similar drug-resistant spectrum of R. anatipestifer serotype 15 to other serotypes; for example, all test strains were resistant to polymyxin, gentamicin, and neomycin. The CZG-1 strain has high pathogenicity, and its lethal dose of 50% (LD50) is 35.122 CFU/ml. Virulence gene determination showed that the CZG-1 strain had at least five virulence genes, bioF, TSS9-1, TSS9-2, PncA, and 0373Right. Above all, this study identified and proved that the pathogen of spotted spleen in ducks was R. anatipestifer serotype 15, which caused death of ducks without the typical symptoms of bacterial infection. The results of this study enriched the knowledge of symptom after R. anatipestifer infection, provided a reference to the identification of the pathogen of spotted spleen, and provided theoretical basis for prevention and control of spotted spleen.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. mRNA optimization is indispensable for reducing immunogenicity, ensuring stability, and maximizing protein output. We present mRNAid, an experimentally validated software for mRNA optimization and visualization that generates mRNA sequences with comparable if not superior characteristics to commercially optimized sequences. To encompass all aspects of mRNA design, we also interrogated the impact of uridine content, nucleoside analogs and UTRs on expression and immunogenicity.

Riemerella anatipestifer is an important bacterial pathogen associated with epizootic infections in waterfowl and various other birds. The complete genome sequence of Riemerella anatipestifer xi1, isolated in China, was sequenced. The genome consisted of 2,062,911 bp carrying 1,879 predicted genes.

Background The association between homocysteine (Hcy) and IgA nephropathy (IgAN) is not well understood. We aimed to investigate the relationship between Hcy and clinicopathologic features in IgAN patients. Methods A total of 337 IgAN patients and 150 sex- and age- matched healthy controls were enrolled in this single-center retrospective study. According to Hcy ≤ 10 μmol/L or > 10 μmol/L, patients were divided into low and high Hcy groups. Multivariate logistic regression was performed to explore the risk factors for elevated Hcy. Results Serum Hcy was higher in IgAN patients than in healthy controls [11.6 (9.1,15.3) vs. 8.8 (7.5,10.6) μmol/L, P 2) [9.9 (7.6,12.4) vs. 8.8 (7.5,10.6) μmol/L, P Conclusions Pathologic T was an independent risk factor associated with elevated Hcy, especially at the early stage of IgAN.

Targeted protein degradation has emerged as a powerful technology – both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced ‘biodegraders’ — targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.

BackgroundIgA nephropathy (IgAN) is the most common primary glomerulonephritis globally. Increasing evidence suggests the importance of host immunity in the development of IgAN, but its dynamics during the early stage of IgAN are still largely unclear.ResultsHere we successfully resolved the early transcriptomic changes in immune cells of IgAN by conducting single-cell RNA-sequencing (scRNA-seq) with peripheral blood mononuclear cells. The differentially expressed genes (DEGs) between control and IgAN were predominantly enriched in NK cell-mediated cytotoxicity and cell killing pathways. Interestingly, we discovered that the number and cytotoxicity of NK cells are significantly reduced in IgAN patients, where both the number and marker genes of NK cells were negatively associated with the clinical parameters, including the levels of urine protein creatinine ratio (UPCR), serum galactose-deficient IgA1 and IgA. A distinctive B cell subset, which had suppressed NFκB signaling was predominantly in IgAN and positively associated with disease progression. Moreover, the DEGs of B cells were enriched in different viral infection pathways. Classical monocytes also significantly changed in IgAN and a monocyte subset expressing interferon-induced genes was positively associated with the clinical severity of IgAN. Finally, we identified vast dynamics in intercellular communications in IgAN.ConclusionsWe dissected the immune landscape of IgAN at the single-cell resolution, which provides new insights in developing novel biomarkers and immunotherapy against glomerulonephritis.

Avian nephritis virus (ANV) infections of chicken flocks cause enteric and kidney disease, uneven growth, and runting stunting syndrome, leading to economic losses in the poultry industry. In this study, one ANV strain, designated as AH202017, was isolated from a diseased broiler flock in Anhui province, China, in 2020. Virus production in LMH cell culture was confirmed by real-time RT-PCR and immunofluorescence assay. The complete genome sequencing analysis indicated that AH202017 shares 77.5%-85.5% identity with 12 reference strains in GenBank. Phylogenetic analysis of the capsid protein revealed that AH202017 is more closely related to VIC-6a/Australia/2014 belonging to ANV genotype 2. However, the phylogenetic tree, based on the ORF1a protein and ORF1b protein, indicated that AH202017 manifests a close relationship with GXJL815/China/2017 belonging to genotype 8. In infection experiments, four infected chickens showed depression and one chicken died at 6 days post-infection, corresponding to 5% mortality. The virus was shed daily in the feces of infected chickens, and was found distributed in multiple organs. Macroscopic and microscopic lesions in the kidneys were observed. This is the first paper that describes the genomic characteristics and pathogenicity of a novel ANV strain in China.

Mycoplasma synoviae (MS), an important avian pathogen, can cause chronic respiratory disease, eggshell apex abnormalities, infectious synovitis, and arthritis in avian species, leading serious economic losses in the global poultry industry. To date, studies have shown significant different transcript profiles using various chicken cells after MS infection. However, in vitro cell models cannot fully represent the complex in vivo regulations after adventitious infection. The objective of this study was to explore the nature of the host-pathogen interaction during MS infection. The tracheal and spleen tissues of chickens were collected at d 0, 1, 3, and 5 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A lot of significantly differentially expressed genes (DEGs) were observed in this analysis, and 861 DEGs were observed in trachea tissues and 753 DEGs were observed in spleen samples. Many of DEGs in trachea tissues participate in a variety of cellular activities, especially cellular metabolism. Immune-related DEGs were mainly enriched at d 3, and 5 postinfection in trachea tissues. While, DEGs in spleen tissues were significantly and mainly enriched into immune-related pathways. The results of this study show the direct interactions between MS and the chicken trachea and spleen for the first time. Early dysregulation of tissue-wide gene expression as observed here set the stage for persistent infection of MS.

Graphical abstract, Infectious disease is a great enemy of humankind. The ravages of COVID-19 are leading to profound crises across the world. There is an urgent requirement for analyzing the current pandemic situation, predicting trends over time, and assessing the effectiveness of containment measures. Thus, numerous statistical models, primarily based on the susceptible–exposed–infected–recovered or removed (SEIR) model, have been established. However, these models are highly technical, which are difficult for the public and governing bodies to understand and use. To address this issue, we developed a simple operating software based on our improved K-SEIR model termed as the kernelkernel SEIR simulator (K-SEIR-Sim). This software includes natural propagation parameters, containment measure parameters, and certain characteristic parameters that can deduce the effects of natural propagation and containment measures. Further, the applicability of the proposed software was demonstrated using the example of the COVID-19 outbreak in the United States and the city of Wuhan, China. Operating results verified the potency of the proposed software in evaluating the epidemic situation and human intervention during COVID-19. Importantly, the software can perform real-time, backward-looking, and forward-looking analysis by functioning in data-driven and model-driven ways. All of them have considerable practical values in their applications according to the actual needs of personal use. Conclusively, K-SEIR-Sim is the first simple customized operating software that is highly valuable for the global fight against COVID-19 and other infectious diseases.

Pathological angiogenesis is fundamental to progression of cancerous tumors and blinding eye diseases. Anti-angiogenic receptor tyrosine kinase inhibitors (TKIs) are in broad use for the treatment of these diseases. With more and more TKIs available, it is a challenge to make an optimal choice. It remains unclear whether TKIs demonstrate similar anti-angiogenesis activities in different tissues. Many TKIs have shown varying degrees of toxic effects that should also be considered in clinical use. This study investigates the anti-angiogenic effects of 13 FDA-approved TKIs on the intersegmental vessels (ISVs), subintestinal vessels (SIVs) and retinal vasculature in zebrafish embryos. The results show that vascular endothelial growth factor receptor TKIs (VEGFR-TKIs) exhibit anti-angiogenic abilities similarly on ISVs and SIVs, and their efficacy is consistent with their IC

Cisplatin-induced acute kidney injury (AKI), which is accompanied by a rapid decline in renal function and a high risk of death, is a complex critical illness with no effective or specific treatment. Polo-like kinase 2 (PLK2), a serine/threonine kinase, is involved in the progression of multiple diseases, including cancers, cardiac fibrosis, diabetic nephropathy, etc. Here, by integrating two Gene Expression Omnibus (GEO) datasets of cisplatin-induced AKI animal models, we identified PLK2 as a significantly up-regulated gene in AKI renal tissues, which was then verified in different AKI animal models and cell models. Suppressing PLK2 using siRNAs or inhibitors could enhance cisplatin-induced AKI by inducing severe apoptosis and oxidative stress damage, while enforced PLK2 expression could prevent renal dysfunction induced by cisplatin. We further discovered that PLK2 might phosphorylate glycogen synthase kinase 3β (GSK3β) in the pathogenesis of AKI. In conclusion, our results show that PLK2 play a protective role in cisplatin-induced AKI and may be a new protective target of cisplatin nephrotoxicity.

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen (DO) to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum (ER) over culture time, disrupting cellular homeostasis. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions and could serve as a baseline for enabling the quality optimization and control of mAb production.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia with osteolysis at the carpal and tarsal bones. Heterozygous missense mutations in the transcription factor MAFB are found in patients with MCTO. MAFB is reported to negatively regulate osteoclastogenesis in vitro. However, the in vivo function of MAFB and its relation to MCTO remains unknown. In this study, we generated zebrafish MAFB homolog mafbb mutant utilizing CRISPR/Cas9 technology. Mafbb deficient zebrafish demonstrated enhanced osteoclast cell differentiation and abnormal cartilage and bone development resembling MCTO patients. It is known that osteoclasts are hematopoietic cells derived from macrophages. Loss of mafbb caused selective expansion of definitive macrophages and myeloid cells, supporting that mafbb restricts myeloid differentiation in vivo. We also demonstrate that MAFB MCTO mutations failed to rescue the defective osteoclastogenesis in mafbb−/− embryos, but did not affect osteoclast cells in wild type embryos. The mechanism of MCTO mutations is likely haploinsufficiency. Zebrafish mafbb mutant provides a useful model to study the function of MAFB in osteoclastogenesis and the related MCTO disease.

Objective: To explore the application value of goal-directed fluid therapy (GDFT) in the enhanced recovery after surgery (ERAS) of patients undergoing radical lung cancer surgery (RLCS). Methods: A total of 74 patients undergoing elective RLCS based on the enhance recovery after surgery (ERAS) concept in the HanDan Central Hospital between December 2016 and December 2019 were enrolled and assigned to a group treated by regular conventional liquids (regular group, n=34) and a group treated by goal-directed fluid (GDFT group, n=40) according to the fluid infusion scheme. The two groups were compared in intraoperative fluid inflow and outflow, hemodynamic indexes at 30 min (T0) before operation, 4 h (T1) and 24 h (T2) after operation, postoperative complications, postoperative recovery, inflammatory factors at 1 day (d 0) before operation, and at 1 day (d 1) and 7 days (d 3) after operation, as well as for postoperative life quality. Results: Crystalloid fluid input, fluid infusion, and urine output of the GDFT group were all significantly less than those of the regular group (all P

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Epilepsy was characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. Previous studies suggested that microRNA (miR)-139-5p and the Notch pathway were implicated in Epilepsy; however, their interaction remained vague. Rat primary hippocampal neurons were isolated and identified by immunofluorescence staining. The cells were then used for SREDs model construction and further subjected to flow cytometry for apoptosis detection. Contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), super oxidase dismutase (SOD) contents, and reactive oxygen species (ROS) and the level of mitochondrial membrane potential (MMP) were determined using commercial kits. Target gene and potential binding sites of miR-139-5p were predicted with TargetScan and confirmed by dual-luciferase reporter assay. Expressions of miR-139-5p, Notch pathway-related proteins and apoptosis-related proteins were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. The results showed that the hippocampal neurons were microtubule-associated protein 2 (MAP2)-positive. MiR-139-5p was downregulated in SREDs model cells. SREDs promoted apoptosis and increased the contents of LDH, MDA, and ROS and the level of MMP while reducing miR-139-5p expression and SOD content in cells, which was reversed by miR-139-5p overexpression. Notch-1 was recognized as the target gene of miR-139-5p, and its expression was negatively regulated by miR-139-5p. Besides, Notch-1 overexpression reversed the effects of miR-139-5p upregulation on the expressions of Notch pathway-related proteins and apoptosis-related proteins, cell apoptosis, oxidative stress and MMP in SREDs-treated cells. Our results indicated that miR-139-5p upregulation alleviated SREDs-induced oxidative stress and cell apoptosis via regulating the Notch pathway, which provides new insights into the role of miRNA in the occurrence and development of epilepsy. This article is protected by copyright. All rights reserved.

Liver macrophages, Kupffer cells (KCs), play a critical role in drug-induced liver injury (DILI) and liver diseases including cholestasis, liver fibrosis and viral hepatitis. Application of KCs in in vitro models of DILI and liver diseases is hindered due to limited source of human KCs. In vivo, KCs originate from MYB-independent macrophage progenitors, which differentiate into liver-specific macrophages in response to hepatic cues in the liver. Here, we recapitulated KCs ontogeny by differentiation of MYB-independent iPSCs to macrophage-precursors and exposing them to hepatic cues to generate iPSC-derived KCs (iKCs). iKCs expressed macrophage markers (CD11/CD14/CD68/CD163/CD32) at 0.3-5 folds of primary adult human KCs (pKCs) and KC-specific CLEC-4F, ID1 and ID3. iKCs phagocytosed and secreted IL-6 and TNFα upon stimulation at levels similar to pKCs but different from non-liver macrophages. Hepatocyte-iKCs co-culture model was more sensitive in detecting hepatotoxicity induced by inflammation-associated drugs, Acetaminophen and Trovafloxacin, and Chlorpromazine-induced cholestasis when compared to hepatocytes alone. Overall, iKCs were mature, liver-specific and functional. Furthermore, donor-matched iKCs and iPSC-hepatocyte co-culture exhibited minimal non-specific background response compared to donor-mismatched counterpart. iKCs offer a mature renewable human cell source for liver-specific macrophages, useful in developing in vitro model to study DILI and liver diseases such as cholestasis.

Pancreatic ductal adenocarcinoma (PDAC) has up to half the tumor mass of tumor-associated myeloid cells. Myeloid innate immune cells play important roles in regulating cancer cell recognition and tumor growth. PDAC cells often mold myeloid cells into pro-tumoral state to fuel cancer growth and induce immune suppression. However, how tumor cells educate the innate immune responses remains largely unknown. In this study, we used four different human PDAC cell lines (PANC1, BxPC3, AsPC1, and CFPAC1) to establish the zebrafish xenograft model and investigated the interaction between pancreatic cancer and innate immune cells. The primary tumor-derived cancer cells PANC1 and BxPC3 activated innate immune anti-tumoral responses efficiently, while cancer cells from metastatic tissues AsPC1 and CFPAC1 induced an innate immune suppression and educated innate immune cells towards pro-tumoral state. Chemical conversion of innate immune cells to anti-tumoral state inhibited tumor growth for AsPC1 and CFPAC1. Moreover, genetic and pharmacological inhibition of macrophages also significantly reduced tumor growth, supporting the important roles of macrophages in innate immune suppression. REG4 expression is high in AsPC1 and CFPAC1. Knockdown of REG4 induced innate immune activation and reduced tumor growth in the xenografts, indicating that REG4 is a beneficial target for PDAC therapy. Our study provides a fast in-vivo model to study PDAC-innate immune interaction and their plasticity that could be used to study the related mechanism as well as identify new drugs to enhance immunotherapy.

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) characterized by vomit, watery diarrhea, dehydration and high mortality. Outbreaks of highly pathogenic variant strains of PEDV have resulted in extreme economic losses to the swine industry all over the world. The study of host–virus interaction can help to better understand the viral pathogenicity. Many studies have shown that poly(A)-binding proteins are involved in the replication process of various viruses. Here, we found that the infection of PEDV downregulated the expression of poly(A)-binding protein cytoplasmic 1 (PABPC1) at the later infection stage in Vero cells. The overexpression of PABPC1 inhibited the proliferation of PEDV at transcription and translation level, and siRNA-mediated depletion of PABPC1 promoted the replication of PEDV. Furthermore, mass spectrometry analysis and immunoprecipitation assay confirmed that PABPC1 interacted with the nucleocapsid (N) protein of PEDV. Confocal microscopy revealed the co-localizations of PABPC1 with N protein in the cytoplasm. Taken together, these results demonstrate the antiviral effect of PABPC1 against PEDV replication by interacting with N protein, which increases understanding of the interaction between PEDV and host.

The demand for Filipino teachers to teach in other countries provides boundless opportunities, including monetary reward, despite challenges concerning differences on curricula and cultures when teaching abroad. One thing that facilitates their adjustment is by embracing the Vision, Mission and Objectives (VMO) of the school where they teach. The focus of this inquiry is to gather teaching experiences of Filipino teachers in foreign schools, to share their perceptions and understanding of their present VMO’s and how they managed to embrace all these through paradigm shifting. Purposive sampling was used to conduct interview to 6 Filipino teachers abroad through email, messenger, Skype application and data were analyzed through HyperResearch 4.0.0. The highest level of achievement is in improving the lives of the children towards success of schools and its student development. Communication barriers in understanding the native language and in producing appropriate accent, cultural differences and individual preferences hinder harmonious relationship; noted were minimal prevalence of discrimination.

This article proposes a novel collaborative optimization (NCO) framework that uses a persuasive multi-agent negotiation method for satellite system design. Satellite system design is a typical multi-disciplinary design optimization problem. The traditional collaborative optimization (CO) is a competitive method but has the disadvantage of inefficient convergence caused by the interdisciplinary compatibility constraints in system-level optimization. By introducing the multi-agent negotiation method, an NCO framework is proposed, in which the system-level and subsystem-level variable negotiations replace the system-level compatibility constraints in traditional CO. The negotiation introduces a modification for candidate values that do not satisfy the compatibility constraints, ensures that an alternative compatible candidate will be obtained and has high convergence efficiency. Two numerical benchmark functions and a multi-disciplinary satellite system design are used to analyse the performance of t...

There is increasing preclinical evidence suggesting that metformin, an antidiabetic drug, has anticancer properties against various malignancies, including colorectal cancer. However, the majority of evidence, which was derived from cancer cell lines and xenografts, was likely to overestimate the benefit of metformin because these models are inadequate and require supraphysiologic levels of metformin. Here, we generated patient-derived xenograft (PDX) lines from 2 colorectal cancer patients to assess the properties of metformin and 5-fluorouracil (5-FU), the first-line drug treatment for colorectal cancer. Metformin (150 mg/kg) as a single agent inhibits the growth of both PDX tumors by at least 50% (P < 0.05) when administered orally for 24 days. In one of the PDX models, metformin given concurrently with 5-FU (25 mg/kg) leads to an 85% (P = 0.054) growth inhibition. Ex vivo culture of organoids generated from PDX demonstrates that metformin inhibits growth by executing metabolic changes to decrease oxygen consumption and activating AMPK-mediated pathways. In addition, we also performed genetic characterizations of serial PDX samples with corresponding parental tissues from patients using next-generation sequencing (NGS). Our pilot NGS study demonstrates that PDX represents a useful platform for analysis in cancer research because it demonstrates high fidelity with parental tumor. Furthermore, NGS analysis of PDX may be useful to determine genetic identifiers of drug response. This is the first preclinical study using PDX and PDX-derived organoids to investigate the efficacy of metformin in colorectal cancer. Mol Cancer Ther; 16(9); 2035–44. ©2017 AACR.

PURPOSEEvaluation of 12 ccRCC publicly-available ccRCC gene expression datasets showed that previously proposed discrete molecular subtypes are unstable. To reflect the continuous nature of gene expression observed, we developed a quantitative score (Continuous Linear Enhanced Assessment of Renal cell carcinoma, or CLEAR) using expression analysis founded on pathologic parameters.MATERIALS AND METHODS265 ccRCC gene expression profiles were used to develop the CLEAR score, representing a genetic correlate of the continuum of morphological tumor grade. A signature derivation method based on correlation of CLEAR score with gene expression ranking was used to derive an 18-transcript signature. External validation was conducted in multiple public expression datasets.ResultsAs a measure of intertumoral gene expression heterogeneity, the CLEAR score demonstrated both superior prognostic estimates (94% vs 83% adequacy index in TCGA dataset) and inverse correlation with anti-angiogenic tyrosine-kinase inhibition (65% vs 55% adequacy index) in comparison to previously proposed discrete subtyping classifications. Inverse correlation with high-dose interleukin-2 outcomes was also observed for the CLEAR score (p=0.05). Multiple somatic mutations (VHL, PBRM1, SETD2, KDM5C, TP53, BAP1, PTEN, MTOR) were associated with the CLEAR score. Application of the CLEAR score to independent expression profiling of intratumoral ccRCC regions demonstrated its ability to reflect intratumoral expression heterogeneity and further analysis showed average intertumoral heterogeneity exceeded intratumoral heterogeneity.ConclusionsThe CLEAR score, a gene expression signature developed on histopathology, outperformed discrete subtype-classification in prognostic estimates and correlated better with treatment outcomes. Recognizing cancer as a continuum has important implications for laboratory and clinical research.

Calcium bisphosphonate/calcium polyacrylate spheres were synthesized by a facile method and applied for the first time as gene vectors for transfection. The colloidal spheres of the PAA–Ca2+–H2O complex, formed by sodium polyacrylate and calcium ions in the solution, were used as template to synthesize a spherical PAA–Ca2+–BPMP composite (CaBPMP/CaPAA) in the presence of 1,4-bis(phosphomethyl)piperazine (BPMP). The CaBPMP/CaPAA composite exhibits uniform and well-dispersed spheres with a particle size of about 200 nm as expected. The cytotoxicity assays confirm that CaBPMP/CaPAA spheres are quite safe for different cells even at a high concentration of 500 μg/mL. In vitro transfection results show that CaBPMP/CaPAA spheres serving as gene vectors are capable of transferring exogenous genes into different cells with about 25% of transfection efficiency and good reproducibility. The transfection capacity of CaBPMP/CaPAA spheres may be attributed to the controllable sphere morphology, low cytotoxicity, moderate DNA loading capacity, and bioresorbable property. The application of calcium phosphonates with adjustable surface properties derived from the different organic groups of phosphonic acid in gene delivery provides a new design idea for gene vectors.

Chicken astrovirus (CAstV) is associated with kidney disease and visceral gout, runting and stunting syndrome, and white chick hatchery disease, causing economic losses to the poultry industry worldwide. In this study, 55.6% of 36 clinical samples from Guangdong province in China were positive for CAstV, but negative for other common enteric viruses, including avian nephritis virus, infectious bronchitis virus, fowl adenovirus Group I, Newcastle disease virus, chicken parvovirus, reovirus, and rotavirus by PCRs and RT-PCRs. A CAstV strain, named GD202013, was isolated from Guangdong province in south China, and was identified by CAstV RT-PCR. A whole genome sequence analysis demonstrated that GD202013 shares 76.0 to 88.1% identity with 24 reference strains in GenBank. Phylogenetic analysis, based on whole genome and capsid protein, showed that GD202013 is more closely related to 2 US strains (GA2011/US/2011 and 4175/US/2011) belonging to subgroup Bii. Recombination analysis indicated that GD202013 is a recombinant strain formed by 3 strains: a major parent strain CkP5/US/2016, and 2 minor parent strains (GA2011/US/2011 and G059/PL/2014). In addition, the chicken embryo infection experiment demonstrated that GD202013 causes hatchability reduction, growth depression, and death of embryos. Macroscopic and microscopic lesions in the liver, kidney and small intestine were observed in the dead-in-shell embryos. This is the first report of the novel CAstV infection in China.

First, for a processU(t,τ)∣t≥τ, we introduce a new concept, called the weakD-pullback exponential attractor, which is a family of setsM(t)∣t≤T, for anyT∈R, satisfying the following: (i)M(t)is compact, (ii)M(t)is positively invariant, that is,U(t,τ)M(τ)⊂M(t), and (iii) there existk,l>0such thatdist(U(t,τ)B(τ),M(t))≤ke-(t-τ); that is,M(t)pullback exponential attractsB(τ). Then we give a method to obtain the existence of weakD-pullback exponential attractors for a process. As an application, we obtain the existence of weakD-pullback exponential attractor for reaction diffusion equation inH01with exponential growth of the external force.

Under the assumption that g(t) is translation bounded in , and using the method developed in [3], we prove the existence of pullback exponential attractors in for nonlinear reaction diffusion equation with polynomial growth nonlinearity( is arbitrary).

The nonaxisymmetric transverse free vibrations of radially inhomogeneous circular Mindlin plates with variable thickness are governed by three coupled differential equations with variable coefficients, which are quite difficult to solve analytically in general. In this paper, we discover that if the geometrical and material properties of the plates vary in generalized power form along the radial direction, then the complicated governing differential equations can be reduced into three uncoupled second-order ordinary differential equations which are very easy to solve analytically. Most strikingly, for a class of solid circular Mindlin plates with absolutely sharp edge, the natural frequencies can be expressed explicitly in terms of elementary functions, with the corresponding mode shapes given in terms of Jacobi polynomials. These analytical expressions can serve as benchmark solutions for various numerical methods.

Mesoporous zirconium phenylphosphonates were synthesized by one-pot co-condensation of ZrCl4 and phenylphosphonic acid (PPA) under weak acidic medium using sodium dodecyl sulfate (SDS) as a template. The structure and chemical states of the synthesized materials were characterized by N2 sorption, powder XRD, TEM and FT-IR, ICP, and solid-state NMR, indicating that the hybrids possess mesoporous structure and the frameworks are constructed by the coordination of zirconium(IV) with phenylphosphonates. These mesoporous materials present a considerable amount of uniformly distributed zirconium(IV) throughout the frameworks available for the enrichment of phosphopeptides, which causes them to circumvent the complex preloading of metal ions required for traditional immobilized metal affinity chromatographic (IMAC) adsorbents. Thus, the materials are first applied as IMAC adsorbents to enrich phosphopeptides from the tryptic digests of both standard phosphoproteins and a mixture of proteins. MALDI-TOF MS analysi...

Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has been a potential pandemic threat. The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses; thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed. Our previous studies demonstrated that the HA-based structural design approach by introducing a CFLLC minidomain into transmembrane domain (TM) of H1, H5 or H9 hemagglutinin (HA) proteins by replacing with H3 subtype HA TM could enhance their cross-protection. In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM containing CFLLC minidomian by replacing its TM domain with H3 HA TM. We investigated whether introduction of CFLLC minidomain into H7 HA (H7-53TM) could increase its cross-reactivity and cross-protection against different genotypes of H7N9 viruses. The results showed that the H7-53TM either with or without squalene adjuvant induced increased HI antibodies, serum IgG antibodies, and IFN-γ production to a panel of 7 H7N9 viruses in mice. Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection (80%). Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and less pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT. In summary, this study presents a better subunit vaccine candidate (H7-53TM) against potential H7N9 pandemic.

The weighted sum and genetic algorithm-based hybrid method (WSGA-based HM), which has been applied to multiobjective orbit optimizations, is negatively influenced by human factors through the artificial choice of the weight coefficients in weighted sum method and the slow convergence of GA. To address these two problems, a cluster and principal component analysis-based optimization method (CPC-based OM) is proposed, in which many candidate orbits are gradually randomly generated until the optimal orbit is obtained using a data mining method, that is, cluster analysis based on principal components. Then, the second cluster analysis of the orbital elements is introduced into CPC-based OM to improve the convergence, developing a novel double cluster and principal component analysis-based optimization method (DCPC-based OM). In DCPC-based OM, the cluster analysis based on principal components has the advantage of reducing the human influences, and the cluster analysis based on six orbital elements can reduce the search space to effectively accelerate convergence. The test results from a multiobjective numerical benchmark function and the orbit design results of an Earth observation satellite show that DCPC-based OM converges more efficiently than WSGA-based HM. And DCPC-based OM, to some degree, reduces the influence of human factors presented in WSGA-based HM.

Influenza A H7N9 virus is the latest emerging pandemic threat, and has rapidly diverged into three clades, demanding a H7N9 virus vaccine with broadened protection against unmatched strains. Hemagglutinin (HA)-based structural design approaches for stabilizing HA proteins have provided excitingly promising results. However, none of the HA-based structural design approaches has been applied to a recombinant replicative influenza virus. Here we report that our HA-based structural design approach is a first in the field to generate a recombinant replicative H7N9 virus (H7N9-53TM) showing broadened protection. The H7N9-53TM contains a replaced H3 HA transmembrane domain (TM) in its HA protein. In mice, the inactivated H7N9-53TM vaccine induced significantly higher HI titers, HA-specific IgG titers, and IFN-γ production than the corresponding H7N9-53WT inactivated virus vaccine containing wild-type HA. More excitingly, mice immunized with the H7N9-53TM showed full protection against homologous (H7N9-53) and interclade (H7N9-MCX) challenges with minimal weight loss, no detectable lung viral loads, and no apparent pulmonary lesions and inflammation, while mice immunized with the H7N9-53WT showed partial protection (only 60% against H7N9-MCX) with severe weight loss, detectable lung viral loads, and severe pulmonary lesions and inflammation. In summary, this study presents a better vaccine candidate (H7N9-53TM) against H7N9 pandemics. Furthermore, our HA-based structural design approach would be conceivably applicable to other subtype influenza viruses, especially the viruses from emerging pandemic and epidemic influenza viruses such as H5N1 and H1N1.

Vibrio vulnificus is an opportunistic pathogen widely distributed in estuarine and coastal seawaters. In this study, a culture-free method was developed to rapid detection of V. vulnificus in all seasons, based on loop-mediated isothermal amplification (LAMP) targeting virulent-correlated gene (vcg). The new assay method allows differentiation between the virulent and non-virulent strain of V. vulnificus accurately. This method also allows effective detection of the pathogeny in winter when the bacterium lives in the viable but non-culturable (VBNC) state. A total of 30 costal seawater samples collected in all seasons were used for the evaluation of this method. The results show that the method is sensitive, accurate and convenient.

Increasing numbers of proteins, nucleic acids and other molecular entities have been explored as therapeutic targets, hundreds of which are targets of approved and clinical trial drugs. Knowledge of these targets and corresponding drugs, particularly those in clinical uses and trials, is highly useful for facilitating drug discovery. Therapeutic Target Database (TTD) has been developed to provide information about therapeutic targets and corresponding drugs. In order to accommodate increasing demand for comprehensive knowledge about the primary targets of the approved, clinical trial and experimental drugs, numerous improvements and updates have been made to TTD. These updates include information about 348 successful, 292 clinical trial and 1254 research targets, 1514 approved, 1212 clinical trial and 2302 experimental drugs linked to their primary targets (3382 small molecule and 649 antisense drugs with available structure and sequence), new ways to access data by drug mode of action, recursive search of related targets or drugs, similarity target and drug searching, customized and whole data download, standardized target ID, and significant increase of data (1894 targets, 560 diseases and 5028 drugs compared with the 433 targets, 125 diseases and 809 drugs in the original release described in previous paper). This database can be accessed at http://bidd.nus.edu.sg/group/cjttd/TTD.asp.

In this study, a loop-mediated isothermal amplification assay targeting the 16S-23S intergenic spacer regions (internal transcribed spacer) of Aeromonas caviae was developed. Eighteen reference strains and 109 clinical samples were analyzed. The results showed this detection technique is more reliable and convenient compared with common polymerase chain reaction and biochemical culture methods.

Background Breast fibroepithelial lesions are biphasic tumors and include fibroadenomas and phyllodes tumors. Preoperative distinction between fibroadenomas and phyllodes tumors is pivotal to clinical management. Fibroadenomas are clinically benign while phyllodes tumors are more unpredictable in biological behavior, with potential for recurrence. Differentiating the tumors may be challenging when they have overlapping clinical and histological features especially on core biopsies. Current molecular and immunohistochemical techniques have a limited role in the diagnosis of breast fibroepithelial lesions. We aimed to develop a practical molecular test to aid in distinguishing fibroadenomas from phyllodes tumors in the pre-operative setting.