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The current coronavirus disease 2019 (COVID-19) pandemic has caused significant challenges throughout the world and a rapid, reliable diagnostic test is in high demand. Real-time reverse transcription polymerase chain reaction (RT-PCR) was one of the most quickly established methods of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and is considered to be the gold standard. In this report, we share our experience of using two different testing platforms: the cobas 6800 SARS-CoV-2 test, an automated system that was recently granted Emergency Use Authorization by the FDA, and a laboratory-developed test based on the protocol from the Taiwan Centers for Disease Control (CDC). There was an overall 96.2% agreement between the two platforms. However, the positive agreement between the two platforms was only 80.0%. We found 3 instances of discordance between the two systems and this emphasized the need for timely diagnosis with a reliable testing platform.

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Ferritin is a ubiquitous and conserved iron storage protein that plays a significant role in host detoxification, iron storage, and immune response. Although ferritin has been studied in many species, little is known about its role in the Eri-silkworm (Samia cynthia ricini). In this study, the ferritin light-chain subunit gene, named ScFerLCH, was identified from S. c. ricini. The full-length gene, ScFerLCH, was 1,155 bp and encoded a protein consisting of 231 amino acids with a deduced molecular weight of 26.38 kDa. Higher ScFerLCH expression levels were found in the midgut, silk gland, and fat body by quantitative reverse-transcription polymerase chain reaction and western blot analysis. Injection of Staphylococcus aureus and Pseudomonas aeruginosa could induce upregulation of ScFerLCH in the hemolymph, fat body, and midgut, indicating that ScFerLCH may contribute to the host defense against invading pathogens. In addition, the native ferritin protein was isolated from S. c. ricini by native polyacrylamide gel electrophoresis and its two subunits, ferritin heavy-chain subunit (ScFerHCH) and ferritin light-chain subunit (ScFerLCH), were identified by mass spectrometry. Specifically, we found that recombinant ferritin subunits could self-assemble into a protein complex in vitro; moreover, both recombinant subunits and the protein complex were found to bind different bacteria, including Escherichia coli, P. aeruginosa, S. aureus, and Bacillus subtilis. However, bactericidal tests showed that the protein complex could not inhibit the growth of bacteria directly. Taken together, our results suggest that ScFerritin might play an important role in mediating molecular interaction with pathogens.

The protracted maturational process of temporal processing in layer 4 (L4) of primary auditory cortex (A1) has been extensively studied. Accumulating evidences show that layer 5 (L5) receives direct thalamic inputs as well. How the temporal responses in L5 may developmentally emerge remains unclear. Using in vivo loose-patch recordings in rat A1, we found that putative pyramidal (Pyr) neurons in developing L5 exhibited adult-like stimulus-following ability but less bursting shortly after hearing onset. L5 Pyr neurons in adult A1 exhibited phase-locking similar to L4 neurons, while L5 fast-spiking (FS) neurons showed greater phase-locking at 7 and 12.5 pps. In developing L5, whole-cell recordings revealed inhibition with decay constant comparable to that in adult L5, thereby avoiding the summation of inhibition that contributed to the strong adaptation in L4. Given the targets of L5 outputs, the relatively precocious temporal processing in L5 might contribute to temporal response maturation in connected cortical and subcortical areas. Our findings were in agreement with the idea that L5 may be a "hub" for processing cortical inputs and outputs that can operate independently of L4.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

Background CUL4A has been known for its oncogenic properties in various human cancers. However, its role in intrahepatic cholangiocarcinoma (iCCA) has not been explored. Methods We retrospectively investigated 105 iCCA cases from a single medical institution. Tissue microarrays were used for immunohistochemical analysis of CUL4A expression. CUL4A expression vectors were introduced in cell lines. Cell migration and invasion assays were used to compare the mobility potential of iCCA cells under basal conditions and after manipulation. Then we evaluated the effects of CUL4A on the cell growth by proliferation assay, and further checked the susceptibility to cisplatin in iCCA cells with or without CUL4A overexpression. Results CUL4A overexpression was detected in 34 cases (32.4%). Patients with CUL4A-overexpressing tumors exhibited shortened disease-free survival (mean, 27.7 versus 90.4 months; P = 0.011). In the multivariate analysis model, CUL4A overexpression was shown to be an independent unfavorable predictor for disease-free survival (P = 0.045). Moreover, stably transfected CUL4A-overexpressing iCCA cell lines displayed an increased mobility potential and enhanced cell growth without impact on susceptibility to cisplatin. Conclusions Our data demonstrate that overexpression of CUL4A plays an oncogenic role in iCCA and adversely affects disease-free survival. Thus, it may prove to be a powerful prognostic factor and a potential therapeutic target.

Atrial fibrillation (AF) is a commonly encountered heart arrhythmia and a risk factor for cardiovascular system. The purpose of the present study was to explore the role of long non-coding RNA myocardial infarction-associated transcript (MIAT) in AF and AF-induced myocardial fibrosis and the possible mechanisms involved in this process. We successfully induced an AF rat model. Expression of MIAT presented a dramatic increase, while microRNA (miR)-133a-3p presented a dramatic decrease in atrium tissues of rats with AF induction. In addition, we also found that MIAT was highly expressed and miR-133a-3p was significantly reduced in peripheral blood leukocyte of AF patients. For biological function exploration of MIAT/miR-133a-3p axis, MIAT was knocked down using small hairpin RNA (shRNA) lentivirus injection and the rescue experiments were performed simultaneously by inhibiting miR-133a-3p using anti-miR-133a-3p lentivirus injection in rats with AF. MIAT downregulation significantly alleviated AF, increased atrial effective refractory period (AERP), and reduced the duration of AF as well as cardiomyocytes apoptosis. Whereas these effects of MIAT downregulation on AF were reversed by anti-miR-133a-3p administration. Luciferase reporter revealed that miR-133a-3p was directly regulated by MIAT. Moreover, MIAT knockdown effectively reduced AF-induced atrial fibrosis by detecting reduced collagen in the right atria and inhibited expression of fibrosis-related gene expression of collagen I, collagen III, connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGF-β1) in rats with AF, these findings were in contrast with the findings for rats with inhibition of miR-133a-3p. In conclusion, our study demonstrated the role of MIAT downregulation in alleviating AF and AF-induced myocardial fibrosis, and the functional regulatory pathway of MIAT targeting miR-133a-3p.

Fungal keratitis (FK) is one of the most severe corneal infectious diseases. FK often leads to poor visual prognosis and thus requires accurate diagnosis. Conventional approaches, including clinical diagnoses, smears, and cultures, often fail to provide reliable diagnostic value. Omics approaches, such as those using genomic, metagenomic, and tear proteomic data sources, provide promising features for improving the diagnosis and monitoring the progression of FK. Genomic approaches are based mainly on detecting amplicons of ribosomal RNA genes, and internal transcribed spacers are gradually gaining popularity in clinical practices. A metagenomic approach based on 16S rRNA genes may help monitor the dynamic change of conjunctival microbiota associated with an FK event, whereas that based on shot-gun and 18S rRNA target enrichment sequencing could have the potential to diagnose FK using clinical samples. A tear proteomic approach may provide comprehensive information about ocular surface defense and injury during FK. Representative up- and down-regulated proteins during FK could also be used as biomarkers to determine the clinical course and develop a treatment strategy in different stages of FK. Consequently, a personalized tear proteomic approach will soon play a key role in FK management.

Restoration of sinus rhythm in atrial fibrillation (AF) by radiofrequency catheter ablation (RFCA) is associated with a transient stunning of left atrial (LA) function. However, the long-term effects of different ablation strategies on LA function remain undetermined. We performed randomized controlled trial to evaluate the effects of RFCA, cryoablation, and 3D mapping-guided cryoablation on LA function of proximal AF patients within 1 year. The 3D mapping-guided cryoablation was defined as a maximum of two cryoablation procedures for each pulmonary vein accompanied by RFCA for additional points until complete pulmonary vein isolation was achieved. Conventional and speckle tracking echocardiographic analyses were performed to evaluate LA function. Among the 210 patients (70 in each group) included, a trend of decreasing LA systolic and diastolic function was observed in all groups, as evidenced by decreases in peak A-wave velocity, the global LA peak systolic strain, the peak strain rate, the peak early diastolic strain rate, and the peak late diastolic strain rate within 7 days to 3 months after ablation followed by gradual recovery thereafter. However, the temporal changes in the above four strain parameters among the three groups did not differ significantly within 1 year after ablation (all p > 0.05). Parameters of the LA emptying fraction and LA dimensions were not significantly affected. These results suggested that stunning of LA function occurred within 7 days to 3 months after ablation, and different strategies of AF ablation did not differentially affect the temporal changes in LA function up to 1 year after ablation.

Extranodal nasal-type natural killer (NK)/T-cell lymphoma (NKTCL) is an aggressive lymphoma that is prevalent among East Asian and South American populations. Although Epstein-Barr virus (EBV) is commonly detected in NKTCL, there are limited studies that have analyzed the EBV genomic variations in NKTCL. In this study, 8 EBV latent genes were analyzed using targeted gene sequencing in 23 formalin-fixed paraffin-embedded tissues derived from 18 patients with NKTCL. Five cases with paired samples were comparatively analyzed. The consistency of EBV sequencing data between tissue samples was high (96.3%–98.7%), whereas that of variant calling among the tissue samples and plasma samples (74.3%–79.2%) was low. The highest densities of non-synonymous variants were detected in the EBNA3B gene. Among the 74 known T-cell epitopes, 363 non-synonymous variants were identified in 32 (43.2%) epitopes. Additionally, the AVFDRKSDAK (A1S/P and V2F/M/L) and YHLIVDTDSL (I4L and L10R/V/G/H) epitopes were associated with 5 patterns of amino acid changes in EBNA3B and EBNA-2, respectively. The frequency of variation in the human leukocyte antigen (HLA)-restricted epitopes with corresponding HLA types common among Taiwanese population was significantly low (P = 0.011), whereas that in anchor residues was significantly high (P = 0.012). In conclusion, this study demonstrated the genomic diversity of EBV in NKTCL and its correlation with the HLA-restricted epitope variations in Taiwanese population. The findings of this study provide useful insights for the development of novel therapeutic strategies for NKTCL.

Genome-wide association studies (GWAS) have identified Aldehyde dehydrogenase 2 (ALDH2) as a susceptibility locus for coronary artery disease (CAD) previously. However, the impacts of common variants in this gene on CAD and its outcomes have not been extensively studied. This study explored the association between the Tagging SNPs in ALDH2 and CAD as well as its main outcomes. Six common variants in ALDH2 were selected as tagging SNPs and two cohorts containing 7296 individuals were genotyped to investigate the impacts of ALDH2 on CAD and its main outcomes. The results show that the variant rs671 in ALDH2 is associated with an increased risk of CAD in southern Chinese (OR=1.26, 95%CI: 1.07-1.48, p=0.004), while not in northern Chinese (OR=1.00, 95%CI: 0.86-1.50, p=0.94). Meanwhile, we find that rs671 genotypes may not influence the outcomes of CAD (HR=1.11, 95%CI: 0.892-1.38, p=0.346). Additionally, we also tested the effect of rs671 genotype on CAD severity, while no significant association was found between them. In the subgroup analysis, the results revealed that rs671 were significantly associated with CAD (OR=1.24, 95%CI: 1.11-1.38, p

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection. Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori., Graphical abstract Unlabelled Image, Highlights • The digestive juice antiviral activity of resistant strain was higher than susceptible strain. • Silkworm digestive juice was analyzed by label-free quantitative proteomics. • Eighty DEPs were identified in different resistant strains following BmNPV infection. • Some DEPs were validated by RT-qPCR and western blot. • These results provide insights into the potential mechanisms for B. mori resistant to BmNPV infection.

Background and Purpose: Drynaria fortunei J. Sm (D. fortunei), known as Gu-Sui-Bu, is used in traditional Chinese medicine to treat common injuries, including bone fractures and bruising. The specific functional mechanisms of the angiogenic and endothelial cell migration properties of D. fortunei are currently unclear. Thus, the purpose of this study is to validate the potential angiogenic and cellular migration properties and related mechanisms by D. fortunei both in vivo and in vitro. Experimental Approach: The present study investigates, both in vivo and in vitro, the wound healing effects of D. fortunei as associated with angiogenesis, specifically by the modulation of matrix metalloproteinases (MMPs) and upregulation of vascular endothelial growth factor (VEGF) ligand/receptors. In order to determine the potential angiogenic effects of D. fortunei, in vivo neovascularization of chick chorioallantoic membranes (CAMs) assay, and directed in vivo angiogenesis assay (DIVVA) were performed, while in vitro scratch wound healing, migration, and matrix-induced tube formation assays were performed by using human umbilical vascular endothelial cells (HUVECs). Furthermore, we used qPCR to analyze the gene expressions and Western blot to observe protein expressions of MMP-2, MMP-14, TIMP-2, RECK, and VEGF/VEGFRs. Results: This study identified five major compounds from the water extract of D. fortunei: protocatechuic acid, caffeic acid 4-O-β-D-glucopyranoside, 5,7-dihydroxychromone-7-O-rutinoside, neoeriocitrin, and naringin. D. fortunei was confirmed to activate in vivo angiogenesis by CAM and DIVVA assays. D. fortunei further exhibited in vitro angiogenic effects associated with cell migration, as demonstrated by the tube formation assay, transwell migration assay, and scratch wound healing assay. The extracellular MMP-2 activity was found to be dose-dependently augmented both in vitro and in vivo by D. fortunei. The mRNA and protein expressions of MMP-2, and MMP-14 were increased; while the tissue inhibitor metalloproteinase-2 (TIMP-2), and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were both decreased. Furthermore, D. fortunei activated the gene and protein expressions of VEGF-A, -B, and VEGFR-2, -3. Conclusion: D. fortunei increased MMP-2 activity, thereby stimulating angiogenesis and cell migration, both in vivo and in vitro, as a result of MMP-2 and TIMP-2 balance modulation and the activation of VEGF/VEGFRs expression.

Carnosic acid (CA), a major polyphenolic diterpene present in Rosmarinus officinalis, has been reported to have multiple functions, including antitumor activity. The MTT assay, BrdU incorporation, wound healing, and colony formation were used to detect melanoma B16F10 cell growth and proliferation. Flow cytometry was used for cell cycle detection. p21 and p27 expression was detected by Western blotting. B16F10 cell xenograft model was established, and treated with CA, carmustine (BCNU), or lomustine (CCNU). The present study found that CA exhibits significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggers cell cycle arrest at G0/G1 phase, and enhances p21 expression. Additionally, CA can enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibits tumor growth, and reduces the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo. The present study study concluded that CA may be safe and useful as a novel chemotherapeutic agent.

We sought to investigate the association of single nucleotide polymorphisms (SNPs) of the genes involved in βAR signaling with the response of patients to βAR blockers. A total of 2403 hospitalized patients with chronic heart failure (HF) were enrolled in a multicenter observational study as the first cohort and followed up for a mean period of 20 months. Genes for β1AR, β2AR, and the major cardiac G-protein-coupled receptor kinases (GRKs) GRK2 and GRK5 were analyzed to identify SNPs, and patients were stratified according to genotypes. A second independent cohort enrolling 919 patients with chronic HF was applied to validate the observed associations. The signaling properties of the key identified SNPs were assessed in vitro. Our data showed that HF patients harboring the Gly16 allele in the gene for β2AR (ADRB2) had an increased risk of the composite end point relative to patients who were homozygous for Arg16. Notably, these patients showed a beneficial response to βAR-blocker treatment in a G allele-dose-dependent manner, whereas Arg16 homozygotes had no response to βAR-blocker therapy. This Arg16Gly genotype-dependent heterogeneity in clinical outcomes of HF was successfully validated in the second independent population. Besides, the in vitro experiments revealed that G allele carriers were defective in β2AR-coupled inhibitory adenylate cyclase g (Gi) protein signaling.

The World Health Organization, in a 2016 revision, introduced recurrent genetic abnormalities for classifying mature T- and NK-cell neoplasms. However, the role of genetic analyses from lymph node aspiration cytology is still not elucidated. We hypothesize that the use of genetic analyses may increase the accuracy of diagnosis from cytological preparations.Fifty-seven formalin-fixed paraffin-embedded (FFPE) samples were collected for next-generation sequencing (NGS) targeting potential driver mutations including TET2, DMN3TA, IDH2, RHOA, STAT3, and STAT5B. Competitive allele-specific TaqMan polymerase chain reaction (cast-PCR) was performed to validate the mutation status by using FFPE and preoperative fine needle aspiration cytology (FNAC) samples.Among these six candidate genes, only IDH2 and RHOA mutations were significantly more frequent in nodal subtypes, angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) (P = .002 and0.001, respectively). All genes exhibited different mutation patterns except RHOA with a hotspot mutation involving the Gly17 residue. The RHOA G17V mutation was found in 15 (75%) of 20 AITL and two (22%) of nine PTCL, NOS. Cast-PCR using FFPE samples showed 100% concordance with NGS. Among 12 lymph node aspirates, the preliminary diagnoses were suspicious for lymphoma (3, 25%), atypical lymphoid cells (3, 25%) and benign/negative (6, 50%). Cast-PCR detected the RHOA G17V mutation in six (75%) of eight RHOA-mutated aspirates and revealed negative results in all (100%) of four wild-type aspirates, with an 83.3% (10/12) concordance comparing to FFPE samples.The RHOA G17V mutation serves as a useful biomarker for cytological assessment in AITL. The use of cast-PCR is valuable in the diagnosis of malignant lymphomas from cytological preparations, and thus avoiding the potential risks of invasive procedures.

Faithful representation of sound envelopes in primary auditory cortex (A1) is vital for temporal processing and perception of natural sounds. However, the emergence of cortical temporal processing mechanisms during development remains poorly understood. Although cortical inhibition has been proposed to play an important role in this process, direct in-vivo evidence has been lacking. Using loose-patch recordings in rat A1 immediately after hearing onset, we found that stimulus-following ability in fast-spiking neurons was significantly better than in regular-spiking (RS) neurons. In-vivo whole-cell recordings of RS neurons revealed that inhibition in the developing A1 demonstrated much weaker adaptation to repetitive stimuli than in adult A1. Furthermore, inhibitory synaptic inputs were of longer duration than observed in vitro and in adults. Early in development, overlap of the prolonged inhibition evoked by 2 closely following stimuli disrupted the classical temporal sequence between excitation and inhibition, resulting in slower following capacity. During maturation, inhibitory duration gradually shortened accompanied by an improving temporal following ability of RS neurons. Both inhibitory duration and stimulus-following ability demonstrated exposure-based plasticity. These results demonstrate the role of inhibition in setting the pace for experience-dependent maturation of temporal processing in the auditory cortex.

Limited data are available about the role of common variants at the aldehyde dehydrogenase 2 gene (ALDH2) on the clinical outcome in Chinese patients with coronary heart disease (CHD) undergoing percutaneous coronary intervention (PCI). In the present study, a total of 1089 patients were consecutively enrolled from January 2012 and July 2013. Six common variants at ALDH2 gene, including rs2339840, rs4648328, rs4767939, rs11066028, rs16941669, and rs671, were selected to test the associations of those polymorphisms with the cardiovascular outcome in patients with CHD after PCI. The clinical endpoints included cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke. The composite of clinical endpoints was defined as the primary endpoint, and every endpoint alone was considered as the secondary endpoints. The median follow-up time was 38.27 months. Our results showed that the common variant rs2339840 was independently associated with a lower risk of stroke in patients with CHD after PCI (codominant model, HR = 0.32, 95% CI, 0.11–0.91, P = .074 for heterozygotes; HR = 0.25, 95% CI, 0.06–1.14, P = .033 for homozygotes; dominant model, HR = 0.32, 95% CI, 0.14–0.74, P = .007). However, no significant associations were found between other 5 single nucleotide polymorphisms (SNPs) and the clinical endpoints. For the first time, the common variant rs2339840 was reported to be a protective factor against stroke in CHD patients with PCI.

Peripheral mature T and NK cell lymphomas consist of a heterogeneous group of neoplasms with cytogenetic and molecular diversities. TP53 mutation is involved in the events of tumorigenesis and present in a variety of cancer subtypes. However, TP53 mutation in peripheral mature T and NK cell lymphomas has not been extensively investigated. In this study, 57 formalin-fixed paraffin-embedded samples were collected for whole-exome sequencing and immunohistochemical study. Ten cases had TP53 mutation variant allele frequency (VAF) of more than 5%, predominantly in intestinal T-cell lymphoma (60%), peripheral T-cell lymphoma, not otherwise specified (22.2%), and extranodal nasal-type NK/T-cell lymphoma (21.7%). Six of 16 (37.5%) cases with 72Pro homozygous genotype showed higher frequency of TP53 mutation VAF > 5% than did those with 72Arg homozygous genotype (P = .044). To a variable extent, overexpression of p53 was observed in 11 of 56 (19.6%) tumors. The percentage of tumor cells with strong p53 staining was positively correlated with TP53 mutation VAF (R2 = 0.95, P < .001). TP53 mutation and p53 expression tended to indicate poor prognosis (P = .054 and P = .009, respectively). In conclusion, we demonstrated TP53 mutation and p53 expression in subtypes of peripheral mature T and NK cell lymphomas and found positive correlation of this immunostaing pattern with the status of TP53 mutation.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

Insulin receptor substrate 2 (IRS2) is a candidate driver oncogene frequently amplified in cancer and is positively associated with IRS2 expression. The overexpression of IRS2 has been suggested to promote tumor metastasis. However, its function in intrahepatic cholangiocarcinoma (iCCA) has not been investigated extensively. The present study examined 86 cases of iCCA to analyze IRS2 expression and its correlation with clinicopathological characteristics using immunohistochemical assays. Three stable cell lines overexpressing IRS2 were established. The mobility potential of cells was compared in the basal condition and following manipulation using cell migration and invasion assays. Epithelial-mesenchymal transition (EMT)-associated proteins were assessed by western blotting. IRS2 was overexpressed in 29 iCCA cases (33.7%) and was significantly more frequent in cases with large tumor size (P=0.033), classified as an advanced stage by the American Joint Committee on Cancer (P=0.046). In comparison with the control cells, the three IRS2-overexpressing iCCA cell lines exhibited a statistically significant increase in mobility potential. Expression analysis of EMT markers demonstrated decreased epithelial marker levels and increased mesenchymal marker levels in IRS2-overexpressing cells compared with their corresponding control cells. The results of the present study indicate that IRS2 overexpression is characterized by a large tumor size and advanced tumor stage in iCCA, and that it may increase tumor mobility potential by regulating EMT pathways. Therefore, it is a valuable predictive indicator of metastasis and may provide a novel direction for targeted therapy in iCCA.

This study presented an integrated microfluidic device which can perform rapid antimicrobial susceptibility tests (AST) of antibiotics combination against clinical bacteria. A new approach to measure either drug interactions of three antibiotics or minimum inhibitory concentration (MIC) of antibiotics by performing bacteria dispensation and antibiotics dilution automatically was proposed and it substantially reduced detection time (from 24 hr to 4 hr) and bacteria consumption (from 100 μL to 3 μL) when compared to the broth microdilution method. A new membrane-type micromixer composed of two circular micropumps was integrated in order to achieve on-chip pumping/mixing with high accuracy and efficiency. Experimental results revealed that the proposed device can produce accurate liquid transportation and chaotic mixing and visually interpret AST results including individual MIC determination and drug interactions. Synergistic and antagonistic effects of pairwise and triple interactions among three common antibiotics (vancomycin, gentamicin and ceftazidime) with ten different combinations were demonstrated on one clinical isolate of vancomycin-intermediate staphylococcus aureus. It is the first time that a microfluidic device was demonstrated for drug screening to inhibit bacteria growth with three antibiotics by performing AST automatically. The developed device could be utilized as a robust tool to rapidly explore drug screening for drug cocktail therapy.

Background Platelets play an important role in the pathogenesis of acute coronary syndrome (ACS). Patients with ACS have an increased mean platelet volume (MPV) and immature platelet fraction (IPF) resulting in elevation of thrombotic ability. In this study, we evaluated the diagnostic performance of MPV and IPF in identifying suspected ACS patients at emergency department. Moreover, we investigated the correlation between MPV or IPF with initial troponin I (TnI), one of the current ACS biomarkers. Methods This was a single-center study recruiting suspected ACS patients who had acute chest pain at the emergency department. Whole blood samples were obtained from all participants and MPV and IPF were measured by Sysmex XE-5000 hematology analyzer within 20 min of blood sampling. The diagnostic values of MPV and IPF in identifying ACS were analyzed retrospectively. Result In this study, 63 in 104 suspected ACS patients were diagnosed as ACS (65.3%). MPV and IPF were higher in ACS patients compared to non-ACS patients (MPV: 10.7 ± 0.80 fL vs 10.0 ± 0.64 fL, p

The genetic status of candidate tumor suppressor genes (TSGs) at chromosome 3p has not yet been elucidated in intrahepatic cholangiocarcinoma (iCCA). Herein, we retrospectively investigated 32 fresh iCCA case samples from a single medical institution to clarify mutations of 11 TSGs by next-generation sequencing. Validation of the mutations was performed on the MassARRAY platform or by high-resolution melting curve analysis. We then integrated the gene mutations into copy number alterations at chromosome 3p that had been generated in a previous study using the same fresh iCCA samples, and correlated the integration results with the clinicopathologic features. Nine of the 32 (28.1%) iCCA patients had gene mutations at chromosome 3p, totaling 11 mutations across five genes. Those included five (15.6%) BAP1 mutations, two each (6.3%) of CACNA2D3 and RASSF1 mutations, and one each (3.1%) of ATG7 and PLCD1 mutations. Six (18.8%) cases had concurrent loss of chromosome 3p and gene mutations. Patients with TSG mutations had shorter disease-free and survival times than those without the mutations. Our data showed that iCCA patients with TSG mutations at chromosome 3p faced an adverse prognosis. BAP1 was the common target of mutational inactivation and may be a principal driver of 3p21 losses.

Background Patients with arsenic-induced Bowen’s disease (As-BD) are at risk of developing invasive cancers in the skin, lung, and urinary bladder. However, a longitudinal follow-up study on the association between As-BD and invasive cancers is still lacking. Objectives This study aims to investigate the underlying molecular mechanisms of this malignant progression in the skin and internal organs. Methods This is a biopsy-based follow-up study. We tested the DNA histograms, Cyclin D1 (CCND1) protein expression and CCND1 promoter DNA methylation in 40 pathologically confirmed specimens from As-BD patients to correlate with individual’s invasive cancer occurrence in the 5-year follow-up. Results Flow cytometric DNA histogram analysis of skin specimens showed aneuploid (n = 15), G2/M arrest (n = 22), and normal (n = 3) DNA histograms. No patients with normal DNA histograms developed invasive cancers, whereas 13 developed invasive cancers in the aneuploid group and 2 developed invasive cancers in the G2/M arrest group. The aneuploid group showed a high risk of invasive cancer development. In all assessed aneuploid specimens, the CCND1 promoter hypomethylation was observed. Statistically, percentage of un-methylation more than 55.85% among 17 detected CpG sites showed extremely high predictive power in the occurrence of invasive arsenical cancers. Furthermore, the un-methylation at -56 and -54 bp CpG sites was statistically significantly associated with invasive arsenical cancer development (p = 1.29 × 10−5). Conclusions As-BD lesions showing an aneuploid DNA histogram had a high risk of invasive cancer development. Un-methyaltion at -56 and -54 bp CpG in the CCND1 promoter serves as a predictor for invasive progression in As-BD patients.

Digital loop-mediated amplification (LAMP) is an isothermal, quantitative DNA amplification approach which has high sensitivity and specificity and could be used to analyze extremely low amount of DNA within a short period of time. In this study, an integrated self-driven microfluidic chip was proposed herein for performing a digital LAMP process for vancomycin-resistant Enterococcus (VRE) diagnosis. The entire bacterial diagnosis could be automatically performed by capillary forces through the new polydimethylsiloxane (PDMS) surface treatment without using external pumps. Moreover, digitized droplets could be separated from each other by normally-close valves such that it could be quantitated. The contact angle of the treated PDMS surface was measured to be only 24°, which was the lowest value in literatures. This is the first time that a rapid (within 1 hour), simple, and hydrophilic PDMS surface modification method has been reported, which could be used in self-driven microfluidic devices for digital LAMP. This device may become a promising tool for clinical diagnosis and point-of-care applications.

Acetylcholine receptors (AChRs) serve as connections between motor neurons and skeletal muscle and are essential for recovery from spinal cord transection (SCT). Recently, microRNAs have emerged as important potential biotherapeutics for several diseases; however, whether miRNAs operate in the modulation of AChRs remains unknown. We found increased AChRs numbers and function scores in rats with SCT; these increases were reduced following the injection of a eukaryotic translation initiation factor 5A1 (eIF5A1) shRNA lentivirus into the hindlimb muscle. Then, high-throughput screening for microRNAs targeting eIF5A1 was performed, and miR-434-3p was found to be robustly depleted in SCT rat skeletal muscle. Furthermore, a highly conserved miR-434-3p binding site was identified within the mRNA encoding eIF5A1 through bioinformatics analysis and dual-luciferase assay. Overexpression or knockdown of miR-434-3p in vivo demonstrated it was a negative post-transcriptional regulator of eIF5A1 expression and influenced AChRs expression. The microarray-enriched Gene Ontology (GO) terms regulated by miR-434-3p were muscle development terms. Using a lentivirus, one functional gene (map2k6) was confirmed to have a similar function to that of miR-434-3p in GO terms. Finally, HRM and MeDIP-PCR analyses revealed that DNA demethylation also up-regulated eIF5A1 after SCT. Consequently, miR-434-3p/eIF5A1 in muscle is a promising potential biotherapy for SCI repair.

Vancomycin-resistant Enterococcus (VRE) is a kind of enterococci, which shows resistance toward antibiotics. It may last for a long period of time and meanwhile transmit the vancomycin-resistant gene (vanA) to other bacteria. In the United States alone, the resistant rate of Enterococcus to vancomycin increased from a mere 0.3% to a whopping 40% in the past two decades. Therefore, timely diagnosis and control of VRE is of great need so that clinicians can prevent patients from becoming infected. Nowadays, VRE is diagnosed by antibiotic susceptibility test or molecular diagnosis assays such as matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and polymerase chain reaction. However, the existing diagnostic methods have some drawbacks, for example, time-consumption, no genetic information, or high false-positive rate. This study reports an integrated microfluidic system, which can automatically identify the vancomycin resistant gene (vanA) from live bacteria in clinical samples. A new approach using ethidium monoazide, nucleic acid specific probes, low temperature chemical lysis, and loop-mediated isothermal amplification (LAMP) has been presented. The experimental results showed that the developed system can detect the vanA gene from live Enterococcus in joint fluid samples with detection limit as low as 10 colony formation units/reaction within 1 h. This is the first time that an integrated microfluidic system has been demonstrated to detect vanA gene from live bacteria by using the LAMP approach. With its high sensitivity and accuracy, the proposed system may be useful to monitor antibiotic resistance genes from live bacteria in clinical samples in the near future.

Background Both respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance. Methods In order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay. Results Our one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously. Conclusions In summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.