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We report a rapid and accurate quantitative detection method using droplet digital PCR (ddPCR) technology to identify cassava adulteration in starch products. The ddPCR analysis showed that the weight of cassava (M) and cassava-extracted DNA content had a significant linear relationship-the correlation coefficient was R2 = 0.995, and the maximum coefficient of variation of replicates was 7.48%. The DNA content and DNA copy number (C) measured by ddPCR also had a linear relationship with R2 = 0.992; the maximum coefficient of variation of replicates was 8.85%. The range of cassava ddPCR DNA content was 25 ng/μL, and the formula M = (C + 32.409)/350.579 was obtained by converting DNA content into the median signal. The accuracy and application potential of the method were verified using the constructed adulteration model., Competing Interests: The authors have declared that no competing interests exist.

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic studies using this strategy.

Current study aims to investigate the biological function of circular RNA (circRNA, circ_0000337) in cervical cancer (CC). Bioinformatic analyses were used to predict targets for circ_0000337 and miR-155-5p, and analyze the gene expression differences between cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tissues and normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to assess mRNA and protein expressions of circ_0000337, microRNA-155-5p (miR-155-5p) and member RAS oncogene family (RAB3B), respectively. Following the establishment of gain/loss-of-function models, CCK-8 was performed to evaluate cell proliferation. Bioinformatics analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were used to identify the interaction in circ_0000337, miR-155-5p, and RAB3B. Circ_0000337 and RAB3B were upregulated, while miR-155-5p was downregulated in CC tissues and cell lines. circ_0000337 overexpression promoted cell proliferation, circ_0000337 knock down inhibited cell proliferation by sponging miR-155-5p. RAB3B was a target of miR-155-5p which was positively regulated by circ_0000337. In the collected CC tissues, there was a negative correlation between miR-155-5p and circ_0000337 or RAB3B, and a positive correlation between circ_0000337 and RAB3B. miR-155-5p was positively, while RAB3B was negatively correlated with OS in patients with CC, and they were negatively correlated. In conclusion, circ_0000337 upregulates RAB3B by sponging miR-155-5p to promote CC cell proliferation. [ABSTRACT FROM AUTHOR]

We present the development of a novel point-of-care electrochemical immunosensor for the detection of the Epstein-Barr virus capsid antigen (EBVCA-IgA) using a biocarbon-modified screen-printed electrode platform. The biosensor achieves sensitive and rapid detection within a broad linear range of 0.1 pM to 10 nM of EBVCA-IgA, with an impressive limit of detection of 33 fM. The key to its practical utility is that the sensor operates efficiently, providing results in under one hour, which is a significant improvement over traditional methods such as enzymelinked immunosorbent assays and polymerase chain reaction that typically exceed two hours. We also emphasize the sensor's high selectivity by demonstrating minimal cross-reactivity with other common respiratory viruses. With its combination of rapidity, sensitivity, and simplicity, this biosensor represents a significant advancement in Epstein-Barr virus (EBA) diagnostics, which makes it particularly beneficial in clinical and low-resource settings where rapid and reliable testing is paramount. [ABSTRACT FROM AUTHOR]

The melon fly, Bactrocera cucurbitae (Coquillett) (Tephritidae: Diptera), is an invasive pest that poses a significant threat to agriculture in Africa and other regions. Flies are known to use their olfactory systems to recognise environmental chemical cues. However, the molecular components of the chemosensory system of B. cucurbitae are poorly characterised. To address this knowledge gap, we have used nextgeneration sequencing to analyse the antenna transcriptomes of sexually immature B. cucurbitae adults. The results have identified 160 potential chemosensory genes, including 35 odourant-binding proteins (OBPs), one chemosensory protein (CSP), three sensory neuron membrane proteins (SNMPs), 70 odourant receptors (ORs), 30 ionotropic receptors (IRs), and 21 gustatory receptors (GRs). Quantitative real-time polymerase chain reaction quantitative polymerase chain reaction was used to validate the results by assessing the expression profiles of 25 ORs and 15 OBPs. Notably, high expression levels for BcucOBP5/9/10/18/21/23/26 were observed in both the female andmale antennae. Furthermore, BcucOROrco/6/7/9/13/15/25/27/28/42/62 exhibited biased expression in the male antennae, whereas BcucOR55 showed biased expression in the female antennae. This comprehensive investigation provides valuable insights into insect olfaction at the molecular level and will, thus, help to facilitate the development of enhanced pest management strategies in the future. [ABSTRACT FROM AUTHOR]

Lung cancer accounts for the vast majority of cancer-related deaths worldwide, and aberrant miRNA expression is commonly observed as the disease progresses. The current study aimed to determine the role of miR-4757-3p in the development of lung cancer. The real-time PCR test was performed to determine the expression of miR-4757-3p in lung cancer cell lines. miR-4757-3p was downregulated in A549 cells. CCK8 and transwell assays demonstrated that overexpression of miR-4757-3p significantly reduced A549 cell invasion and migration. Bioinformatic analysis by the TargetScan database predicted the possible targets of miR-4757-3p. A luciferase activity test was used to determine the direct relationship between miR-4757-3p, Wnt5a, and Wnt8b. The overexpression of miR-4757-3p drastically inhibited the expression of Wnt5a and Wnt8b. Furthermore, we discovered that silencing Wnt5a and Wnt8b significantly lowered β-catenin expression and hampered invasion and migration. Finally, miR-4757-3p inhibited lung cancer cell migration and invasion by inhibiting the activation of the Wnt signaling pathway. Our study provided evidence that miR-4757-3p could be developed as an indicator or an anticancer target in the clinical application. [ABSTRACT FROM AUTHOR]

Background: With the development of metagenomic sequencing technologies, more and more cases of pneumonia caused with Chlamydia psittaci (C. psittaci) have been reported. However, it remains unknown about the characteristics of patients with pneumonia caused by different strains of C. psittaci. Here, we shared the clinical characteristics of two cases of pneumonia caused with C. psittaci strains SZ18-2 and SZ15 which were rarely identified in humans. Case presentation: Case 1: A 69-year-old male farmer who fed ducks presented to hospital for cough, diarrhea and lethargy with the temperature of 39.8°C. Case 2: A 48-year-old male worker who slaughtered ducks was transferred to hospital for high fever, cough, myalgia, diarrhea and loss of appetite. Both patients did not take any protective measures (wearing face masks or gloves) while processing ducks. C. psittaci pneumonia was diagnosed by metagenomic next-generation sequencing and polymerase chain reaction. After treatment with doxycycline and azithromycin individually, they recovered well and discharged from hospital. Through OmpA sequencing, two different strains of SZ18-2 and SZ15 were identified in case 1 and case 2, respectively. Conclusions: Patients infected with different strains of C. psittaci may own different clinical manifestations. C. psittaci infection should be suspected when pneumonia appears, accompanied by digestive symptoms and multiple organ dysfunction, especially under the exposure of specific birds. [ABSTRACT FROM AUTHOR]

Background: Dysregulated activation of the inflammasome is involved in various human diseases including acute cerebral ischemia, multiple sclerosis and sepsis. Though many inflammasome inhibitors targeting NOD-like receptor protein 3 (NLRP3) have been designed and developed, none of the inhibitors are clinically available. Growing evidence suggests that targeting apoptosis-associated speck-like protein containing a CARD (ASC), the oligomerization of which is the key event for the assembly of inflammasome, may be another promising therapeutic strategy. Lonidamine (LND), a small-molecule inhibitor of glycolysis used as an antineoplastic drug, has been evidenced to have anti-inflammation effects. However, its anti-inflammatory mechanism is still largely unknown. Methods: Middle cerebral artery occlusion (MCAO), experimental autoimmune encephalomyelitis (EAE) and LPS-induced sepsis mice models were constructed to investigate the therapeutic and anti-inflammasome effects of LND. The inhibition of inflammasome activation and ASC oligomerization by LND was evaluated using western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) in murine bone marrow-derived macrophages (BMDMs). Direct binding of LND with ASC was assessed using molecular mock docking, surface plasmon resonance (SPR), and drug affinity responsive target stability (DARTS). Results: Here, we find that LND strongly attenuates the inflammatory injury in experimental models of inflammasome-associated diseases including autoimmune disease-multiple sclerosis (MS), ischemic stroke and sepsis. Moreover, LND blocks diverse types of inflammasome activation independent of its known targets including hexokinase 2 (HK2). We further reveal that LND directly binds to the inflammasome ligand ASC and inhibits its oligomerization. Conclusions: Taken together, our results identify LND as a broad-spectrum inflammasome inhibitor by directly targeting ASC, providing a novel candidate drug for the treatment of inflammasome-driven diseases in clinic. [ABSTRACT FROM AUTHOR]

The renal ischemia/reperfusion (I/R)-induced acute kidney injury incidence after nephron-sparing surgery for localized renal tumors is 20%, but the biological determinant process of postoperative acute kidney injury remains unclear. Using Gene Expression Omnibus database (GSE192883) and several bioinformatics analyses (discrete time points analysis, gene set enrichment analysis, dynamic network biomarker analysis, etc), combined with the establishment of the I/R model for verification, we identified three progressive patterns involving five core pathways confirmed using gene set enrichment analysis and six key genes (S100a10, Pcna, Abat, Kmo, Acadm, and Adhfe1) verified using quantitative polymerase chain reaction The dynamic network biomarker (DNB) subnetwork composite index value is the highest in the 22-min ischemia group, suggesting the transcriptome expression level fluctuated sharply in this group, which means 22-min ischemia is an critical warning point. This study illustrates the core molecular progressive patterns from mild to severe I/R kidney injury, laying the foundation for precautionary biomarkers and molecular intervention targets for exploration. In addition, the safe renal artery blocking time of nephron-sparing surgery that we currently accept may not be safe anymore. [ABSTRACT FROM AUTHOR]

Background. Colorectal cancer (CRC), one of the main causes of death, remains a leading cause of mortality in gastrointestinal cancer and tends to affect the younger generation. However, the pathological process of colorectal cancer is unclear. Exploring potential pathogenesis and therapeutic targets of CRC is significant as its high prevalence and high mortality. Nowadays, the rapid development of bioinformatics provides us an opportunity to explore potential molecular markers of CRC. Materials and Methods. First, three CRC gene chips with paracancerous controls were downloaded from the Gene Expression Omnibus (GEO) database. Second, after combining and batch correcting the three chips using the R language and Perl language, the differentially expressed genes (DEGs) were selected to investigate how they affect the CRC occurrence and development by GO and KEGG enrichment analysis. Third, based on the STRING website and the Cytoscape software, the protein-protein interaction (PPI) network was constructed and the core genes were screened out. Finally, through polymerase chain reaction (PCR) and immunohistochemistry (IHC), the expression and function of the core gene CXCL8 in CRC were explored. Results. GSE10950, GSE44076, and GSE75970, including 126 intestinal cancer samples and 126 paracancer samples, were screened as the datasets. 192 DEGs were screened, including 43 upregulated genes and 149 downregulated genes. Through the DEGs screened out, GO enrichment analysis, KEGG enrichment analysis, and the construction of PPI interaction network were carried out. Finally, according to the nodes and edges in the PPI network, the DEGs were sorted and the core genes were selected. Through basic experiments, the first ranked CXCL8 was further studied, and the results suggest that the expression of CXCL8 is related to the proliferation, migration, invasion, and even distant metastasis of CRC. Conclusion. The present study showed that DEGs of CRC are associated with multiple tumor-related biological processes and signaling pathways. The core gene CXCL8 has the potential to be a new therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

Background: The incidence of atrial fibrillation (AF)-related stroke increases with aging. Natriuretic peptides (NPs) family, including Corin-B type natriuretic peptide (BNP)-neprilysin (NEP) protein levels increased with age and are risk markers of cardiovascular and cerebrovascular diseases, such as AF and cardioembolic stroke. Aging is also linked to epigenetics, specifically DNA methylation. However, only a few studies have investigated the effect of DNA methylation on the NP system. Thus, the present study aimed to investigate whether the Corin-BNP-NEP protein pathway is involved in the pathogenesis of AF-stroke and CpG methylation in the promoter region of the Corin protein gene has an effect on AF-related ischemic stroke. Methods: A total of 82 patients hospitalized with acute ischemic strokes were enrolled in this study. The differences in clinical information were compared between the AF-stroke (n = 37) and no AF-stroke groups (n = 45). Plasma-soluble Corin and NEP were detected using an ELISA kit. CpG methylation in the promoter region of the gene was assessed by a next-generation sequencing-based bisulfite sequencing polymerase chain reaction (BSP). Results: (1) Patients in AF-stroke were older, had higher initial NIHSS score, 90-day mRs, higher D2-dimer, INR, and APTT, and low TG, TC, and HbA1c (all p < 0.05). (2) Serum levels of Corin and BNP in the AF-stroke group were significantly higher than that in the no AF-stroke group (p < 0.05). No significant difference was detected in the serum levels of NEP between the two groups. (3) The levels of CpG methylation in the promoter region of the Corin protein gene in the AF-stroke group was significantly lower than that in the no AF-stroke group (p < 0.05). The CpG sites with maximal methylation differences between the two groups were CORIN:678, CORIN:682, CORIN:694, and CORIN:700. Conclusion: The current findings raise the possibility that the Corin–BNP–NEP protein pathway may be involved in the pathogenesis of AF-related ischemic stroke. Deficient CpG methylation in the promoter region of the Corin protein gene is associated with AF-related ischemic stroke. [ABSTRACT FROM AUTHOR]

Sepsis can easily cause acute kidney injury (AKI) and seriously endanger human health. This article aims to investigate and study the role of microRNA-665 (miR-665) in septic AKI and the underlying molecular mechanism. Lipopolysaccharide (LPS) was used to construct cell and animal models of septic AKI. The expression of miR-665 in cells and kidney tissues was detected by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The contents of inflammatory factors (TNF-α, IL-1β, and IL-6) in the cell supernatant were detected using commercial kits. Renal tissue damage was observed by hematoxylin-eosin (HE) staining. Kidney function was assessed by serum Cr, serum BUN, and urine NAG levels. The apoptosis of HK-2 cells was analyzed by flow cytometry and TUNEL staining. Luciferase activity assay was performed for the verification of the target of miR-665. The expression of miR-665 was increased in the cell model and animal model of septic AKI constructed by LPS. By transfecting miR-665 inhibitor in HK-2 cells and injecting miR-665 antagomir (antagomiR-665) through the tail vein of rats, the expression of miR-665 in HK-2 cells and rat kidneys was remarkably reduced. Silencing miR-665 dramatically inhibited the expression of inflammatory factors (TNF-α, IL-1β, and IL-6) in LPS-induced HK-2 cells and reduced LPS-induced apoptosis in HK-2 cells. At the same time, the levels of serum Cr, serum BUN, and urine NAG decreased markedly, and the damage of the kidney was also alleviated. Finally, luciferase reporter experiments demonstrated that miR-665 directly targets Bcl-2. We revealed that miR-665 expression was increased in septic AKI, and silencing miR-665 could inhibit LPS-induced inflammation and apoptosis of the kidney by targeting Bcl-2, thereby improving renal function. [ABSTRACT FROM AUTHOR]

[Display omitted] • Circ-G6PC3 is highly expressed in LUAD tissues and cells. • Circ-G6PC3 promotes cell proliferation and suppresses cell apoptosis in LUAD. • Circ-G6PC3 promotes LUAD cell proliferation through regulating autophagy. • Circ-G6PC3 could bind to HMGB1 and Beclin 1. • Circ-G6PC3 overexpression promotes the binding of HMGB1 with Beclin 1. Circular RNAs (circRNAs) have been corroborated to be vital modulators in multiple cancers, including lung adenocarcinoma (LUAD). Cellular autophagy is the way in which cells utilize lysosomes to degrade the damaged or senescent macromolecules and organelles through self-digestion. Reportedly, autophagy is closely associated with tumor progression. The current study targeted to probe circ-G6PC3 function in LUAD progression as well as its regulatory mechanism concerning autophagy. Through ISH, this research identified a high expression pattern of circ-G6PC3 in LUAD tissues compared to adjacent normal tissues. Consistently, high level of circ-G6PC3 was also determined in LUAD cells through quantitative real-time polymerase chain reaction (qRT-PCR). Through functional assays in vitro and in vivo , we found circ-G6PC3 augmentation promoted LUAD cell growth. Moreover, circ-G6PC3 facilitated LUAD cell proliferation via inducing autophagy. Mechanistically, circ-G6PC3 could bind with both HMGB1 and Beclin 1 and facilitate the binding between HMGB1 and Beclin 1, forming the circ-G6PC3-HMGB1-Beclin 1 complex. This study then ascertained that circ-G6PC3 boosted LUAD cell proliferation and suppressed the cell apoptosis through Beclin 1-mediated autophagy. In conclusion, circ-G6PC3 promotes LUAD progression through HMGB1-Beclin 1 complex-mediated autophagy. [ABSTRACT FROM AUTHOR]

Background: Patients with non‐small cell lung cancer (NSCLC) are diagnosed in advanced stages and with a poor 5‐year survival rate. There is a critical need to identify novel biomarkers to improve the therapy and overall prognosis of this disease. Methods: Differentially expressed genes (DEGs) were identified from three profiles of GSE101586, GSE101684 and GSE112214 using Venn diagrams. hsa_circ_0043256 were validated using quantitative real‐time polymerase chain reaction (RT‐qPCR). The circular RNA–microRNA–messenger RNA (circRNA–miRNA–mRNA) regulatory network was constructed with Cytoscape 3.7.0. Hub genes were identified with protein interaction (PPI) and validated with the Gene Expression Profiling Interactive Analysis (GEPIA), Human Protein Atlas (HPA) databases, and immunohistochemistry. Survival analyses were also performed using a Kaplan–Meier (KM) plotter. The effects of hsa_circ_0043256 on cell proliferation and cell cycles were evaluated by EdU staining and flow cytometry, respectively. Results: hsa_circ_0043256, hsa_circ_0029426 and hsa_circ_0049271 were obtained. Following RT‐qPCR validation, hsa_circ_0043256 was selected for further analysis. In addition, functional experiment results indicated that hsa_circ_0043256 could inhibit cell proliferation and cell‐cycle progression of NSCLC cells in vitro. Prediction by three online databases and combining with DEGs identified from The Cancer Genome Atlas (TCGA), a network containing one circRNAs, three miRNAs, and 209 mRNAs was developed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated DEGs might be associated with lung cancer onset and progression. A PPI network based on the 209 genes was established, and five hub genes (BIRC5, SHCBP1, CCNA2, SKA3, and GINS1) were determined. Following verification of five hub genes using GEPIA database, HPA database, and immunohistochemistry. High expression of all five hub genes led to poor overall survival. Conclusion: Our study constructed a circRNA–miRNA–mRNA network of hsa_circ_0043256. hsa_circ_0043256 may be a potential therapeutic target for lung cancer. [ABSTRACT FROM AUTHOR]

Introduction: Circ_HECW2 plays a key role in lipopolysaccharide (LPS)‐induced signal transduction, which is critical in osteoarthritis (OA). Thus, we analyzed the role of Circ_HECW2 in osteoarthritis. Methods: The expression of Circ_HECW2 and miR‐93 was examined using reverse‐transcription polymerase chain reaction. Cell apoptosis was evaluated using Annexin V‐FITC Apoptosis Detection Kit. Results: Circ_HECW2 and miR‐93 were inversely correlated, with Circ_HECW2 upregulated and miR‐93 downregulated in OA and LPS‐induced chondrocytes. Circ_HECW2 overexpression inhibited miR‐93 expression and increased methylation of miR‐93 coding gene. Cell apoptosis analysis showed that Circ_HECW2 overexpression increased LPS‐induced chondrocyte apoptosis, while MiR‐93 overexpression reversed the effects of Circ_HECW2 on chondrocyte apoptosis. Conclusion: In summary, our data revealed that the Circ_HECW2 is highly expressed in OA and might inhibit miR‐93 expression through methylation to affect LPS‐induced chondrocyte apoptosis. [ABSTRACT FROM AUTHOR]

Aims: Long non-coding RNAs (lncRNAs) are critical regulators of viral infection and inflammatory responses. However, the roles of lncRNAs in acute myocarditis (AM), especially fulminant myocarditis (FM), remain unclear. Methods: FM and non-fulminant myocarditis (NFM) were induced by coxsackie B3 virus (CVB3) in different mouse strains. Then, the expression profiles of the lncRNAs in the heart tissues were detected by sequencing. Finally, the patterns were analyzed by Pearson/Spearman rank correlation, Kyoto Encyclopedia of Genes and Genomes, and Cytoscape 3.7. Results: First, 1,216, 983, 1,606, and 2,459 differentially expressed lncRNAs were identified in CVB3-treated A/J, C57BL/6, BALB/c, and C3H mice with myocarditis, respectively. Among them, 88 lncRNAs were commonly dysregulated in all four models. Quantitative real-time polymerase chain reaction analyses further confirmed that four out of the top six commonly dysregulated lncRNAs were upregulated in all four models. Moreover, the levels of ENSMUST00000188819, ENSMUST00000199139, and ENSMUST00000222401 were significantly elevated in the heart and spleen and correlated with the severity of cardiac inflammatory infiltration. Meanwhile, 923 FM-specific dysregulated lncRNAs were detected, among which the levels of MSTRG.26098.49, MSTRG.31307.11, MSTRG.31357.2, and MSTRG.32881.28 were highly correlated with LVEF. Conclusion: Expression of lncRNAs is significantly dysregulated in acute myocarditis, which may play different roles in the progression of AM. [ABSTRACT FROM AUTHOR]

Postoperative cognitive dysfunction (POCD) is a common complication in elderly patients. Circular RNAs (circRNAs) may contribute to neurodegenerative diseases. However, the role of circRNAs in POCD in aged mice has not yet been reported. This study aimed to explore the potential circRNAs in a POCD model. First, a circRNA microarray was used to analyze the expression profiles. Differentially expressed circRNAs were validated using quantitative real-time polymerase chain reaction. A bioinformatics analysis was then used to construct a competing endogenous RNA (ceRNA) network. The database for annotation, visualization, and integrated discovery was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of circRNA-related genes. Moreover, protein-protein interactions were analyzed to predict the circRNA-regulated hub genes using the STRING and molecular complex detection plug-in of Cytoscape. Microarray screen 124 predicted circRNAs in the POCD of aged mice. We found that the up/downregulated circRNAs were involved in multiple signaling pathways. Hub genes, including Egfr and Prkacb , were identified and may be regulated by ceRNA networks. These results suggest that circRNAs are dysexpressed in the hippocampus and may contribute to POCD in aged mice. [ABSTRACT FROM AUTHOR]

Objective. To reveal the molecular mechanism of the antagonistic effect of traditional Chinese medicine Tianma formula (TF) on dementia including vascular dementia (VaD) and Alzheimer's disease (AD) and to provide a scientific basis for the study of traditional Chinese medicine for prevention and treatment of dementia. Method. The TF was derived from the concerted application of traditional Chinese medicine. We detected the pharmacological effect of TF in VaD rats. The molecular mechanism of TF was examined by APP/PS1 mice in vivo, Caenorhabditis elegans (C. elegans) in vitro, ELISA, pathological assay via HE staining, and transcriptome. Based on RNA-seq analysis in VaD rats, the differentially expressed genes (DEGs) were identified and then verified by quantitative PCR (qPCR) and ELISA. The molecular mechanisms of TF on dementia were further confirmed by network pharmacology and molecular docking finally. Results. The Morris water maze showed that TF could improve the cognitive memory function of the VaD rats. The ELISA and histological analysis suggested that TF could protect the hippocampus via reducing tau and IL-6 levels and increasing SYN expression. Meanwhile, it could protect the neurological function by alleviating Aβ deposition in APP/PS1 mice and C. elegans. In the RNA-seq analysis, 3 sphingolipid metabolism pathway-related genes, ADORA3, FCER1G, and ACER2, and another 5 nerve-related genes in 45 key DEGs were identified, so it indicated that the protection mechanism of TF was mainly associated with the sphingolipid metabolism pathway. In the qPCR assay, compared with the model group, the mRNA expressions of the 8 genes mentioned above were upregulated, and these results were consistent with RNA-seq. The protein and mRNA levels of ACER2 were also upregulated. Also, the results of network pharmacology analysis and molecular docking were consistent with those of RNA-seq analysis. Conclusion. TF alleviates dementia by reducing Aβ deposition via the ACER2-mediated sphingolipid signaling pathway. [ABSTRACT FROM AUTHOR]

A single-copy specific primer was designed based on beef and duck samples and through drop digital polymerase chain reaction (ddPCR) for the quantitative analysis. Results revealed that the primers had no specific amplification with sheep, chicken, pork, or other species. Both the relationships between meat weight and DNA weight and between DNA weight and DNA copy number (C) were nearly linear within the dynamic range. To calculate the original meat weight from the DNA copy number, the DNA weight was used as the intermediate value to establish the following formulae: Mbeef = 0.058C − 1.86; Mduck = 0.0268C − 7.78. To achieve a good quantitative analysis, all species used in the experiment were made of lean meat. The accuracy of the method was verified by artificial adulteration of different proportions. Testing of the commercial samples indicated that adulteration is present in the market. The established digital PCR method provided an effective tool for monitoring the adulterated meat products and reducing the adulteration in the market. [ABSTRACT FROM AUTHOR]

Neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS) are inflammatory demyelinating diseases of the central nervous system. Exosomal microRNAs (miRNAs) are emerging biomarkers for demyelinating diseases. In this study, 52 aquaporin-4 antibody serum-positive NMOSD patients, 18 relapsing-remitting multiple sclerosis (RRMS) patients and 17 healthy controls (HCs) were included for the next-generation sequencing (NGS). To validate the NGS results, the valuable miRNAs were selected for validation by real-time quantitative polymerase chain reaction in another cohort of patients, comprising 31 NMOSD patients and 14 HCs. In addition, these miRNAs were also validated in a longitudinal study. NGS data revealed the exosomal miRNAs profile in NMOSD patients was different from HCs. Among those potential exosomal miRNAs which can distinguish NMOSD status, hsa-miR-122-3p and hsa-miR-200a-5p were the most abundant miRNAs. In addition, hsa-miR-122-3p and hsa-miR-200a-5p were significantly upregulated in the serum exosome of relapsing NMOSD compared with that in remitting NMOSD. Hsa-miR-122-3p and hsa-miR-200a-5p had positive correlations with disease severity in NMOSD patients. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the MAPK, Wnt and Ras signaling pathways were enriched. Further biological function analysis demonstrated that these two miRNAs might be involved in the immunoregulation of NMOSD pathogenesis. Our results indicated that miRNAs delivered by exosomes could be applied as potential biomarkers for NMOSD. [ABSTRACT FROM AUTHOR]

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. The current study explored the effect of mmLDL on the endothelin type A (ETA) receptor in mouse mesenteric arteries in vivo, as well as the role of autophagy in this process. mmLDL was injected via the caudal vein, and the Class III PI3K autophagic pathway inhibitor 3-methyladenine (3-MA) was injected intraperitoneally. The animals were divided into physiological saline (NS), mmLDL, and mmLDL + 3-MA groups. The dose-effect curve of endothelin-1- (ET-1-) induced mesenteric artery contraction was measured using myography, while ETA receptor mRNA expression was detected using real-time polymerase chain reactions, and the protein levels of the ETA receptor, class III PI3K, Beclin-1, LC3 II/I, p62, NF-κB, and p-NF-κB were observed using Western blot analysis. mmLDL significantly strengthened ET-1-induced contraction (the Emax value increased from 184.87 ± 7.46% in the NS group to 319.91 ± 20.31% in the mmLDL group (P < 0.001), and the pEC50 value increased from 8.05 ± 0.05 to 9.11 ± 0.09 (P < 0.01). In addition to upregulating the protein levels of Class III PI3K, Beclin-1, and LC3 II/I and downregulating that of p62, mmLDL significantly increased the mRNA expression and protein level of the ETA receptor and increased the protein level of p-NF-κB. However, these effects were significantly inhibited by 3-MA. mmLDL activates autophagy via the Class III PI3K/Beclin-1 pathway and upregulates the ETA receptor via the downstream NF-κB pathway. Understanding the effect of mmLDL on the ETA receptor and the underlying mechanisms may provide a new idea for the prevention and treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Angelica dahurica is a widely grown plant species with multiple uses, especially in the medical field. However, the frequent introduction of A. dahurica to new areas has made it difficult to distinguish between varieties. Simple sequence repeats (SSRs) detected based on transcriptome analyses are very useful for constructing genetic maps and analyzing genetic diversity. They are also relevant for the molecular marker-assisted breeding of A. dahurica. We identified 33,724 genic SSR loci based on transcriptome sequencing data. A total of 114 primer pairs were designed for the SSR loci and were tested for their specificity and diversity. Ten SSR loci in untranslated regions were ultimately selected. Subsequently, 56 A. dahurica ecotypes collected from different regions were analyzed. The SSR loci comprised 2–8 alleles, with a mean of 5.2 alleles per locus. The polymorphic information content value and Shannon’s information index were 0.6274–0.2702 (average of 0.4091) and 1.3040–0.5618 (average of 0.8475), respectively. Thus, the 10 novel SSRs identified in this study were almost in accordance with Harvey-Weinberg equilibrium and will be useful for analyzing A. dahurica genetic relationships. The results of this study confirm the potential value of transcriptome databases for the development of new SSR markers. [ABSTRACT FROM AUTHOR]

The purpose of this experiment is to establish a rat model of thin endometrium and to explore the effect of ligustrazine on the thin endometrium of rats. The thin endometrium model was made by using infusing absolute ethyl alcohol into the uterine cavity. The thickness of endometrium was measured. Hematoxylin-Eosin (HE) staining was used to observe the histopathological changes of endometrium. The mRNA levels of VEGF, VEGFR-2, PI3K, and AKT were detected by RT-PCR. Western blotting was used to detect the levels of VEGF, VEGFR-2, PI3K, and AKT in endometrial tissue. The thickness of endometrium in the model group was significantly thinner than that in the control group. Compared with the model group, the thickness of endometrium in ligustrazine group was increased. HE staining shown that ligustrazine restored the histopathological changes of endometrium. RT-PCR and Western Blotting results showed that the mRNA and protein levels of VEGF, VEGFR-2, PI3K, and AKT in the model group were significantly decreased compared with the control group, while ligustrazine restored the changes. Ligustrazine can improve the morphology of endometrium, can promote the growth of endometrium, and has obvious therapeutic effect. Its mechanism is related to the activation of PI3K/Akt signaling pathway through upregulation of VEGF and VEGFR-2 expression to induce the repair of thin endometrium in rats. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 (CYP3A4) enzyme activity is known to show considerable ethnic heterogeneity and inter-individual differences, affecting the outcome of drug treatment. CYP3A4 genetic polymorphisms are believed to be one of the important causes, leading to inter-individual variability in drug metabolism. Quinine is an antipyretic drug with antimalarial properties that is metabolized primarily by CYP3A4. Quinine 3-hydroxylation has been proven as a biomarker reaction for evaluating CYP3A4 ability. Quinine has frequent adverse effects and there are distinct inter-individual differences in quinine sensitivity. The open reading frame for 30 CYP3A4 allelic variants were constructed from wild-type CYP3A4*1A by an overlap extension polymerase chain reaction. Recombinant CYP3A4 variants were expressed using baculovirus-insect cell expression system, and their catalytic activities towards quinine hydroxylation were determined and evaluated. Of the 30 CYP3A4 allelic variants, 23 variants exhibited significantly reduced intrinsic clearance towards quinine, 2 variants showed increased intrinsic clearance for quinine, 2 variants possessed no significant differences towards quinine, compared with CYP3A4*1A, and 3 variants had no detected expression and enzyme activity. Our assessment on the enzymatic activities of CYP3A4 variants towards quinine may contribute to laying an experimental foundation for further clinical studies so as to accelerate the process of determining the associations between genetic variations and clinical phenotypes. [ABSTRACT FROM AUTHOR]

Silymarin (SM), a standardized extract derived from Silybum marianum (L.) Gaertn, is primarily composed of flavonolignans, with silibinin (SB) as its major active constituent. The present study aimed to evaluate the antigenotoxic activities of SM and SB using the alkaline comet assay in whole blood cells and to assess their effects on the expression of genes associated with carcinogenesis and chemopreventive processes. Different concentrations of SM or SB (1.0, 2.5, 5.0, and 7.5 mg/ml) were used in combination with the DNA damage-inducing agent methyl methanesulfonate (MMS, 800 μM) to evaluate their genoprotective potential. To investigate the role of SM and SB in modulating gene expression, we performed quantitative real-time PCR (qRT-PCR) analysis of five genes that are known to be involved in DNA damage, carcinogenesis, and/or chemopreventive mechanisms. Treatment with SM or SB was found to significantly reduce the genotoxicity of MMS, upregulate the expression of PTEN and BCL2, and downregulate the expression of BAX and ABL1. We observed no significant changes in ETV6 expression levels following treatment with SM or SB. In conclusion, both SM and SB exerted antigenotoxic activities and modulated the expression of genes related to cell protection against DNA damage. [ABSTRACT FROM AUTHOR]

Background/Aims: Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy. Methods: We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability, cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in in vivo studies. Results: The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our in vitro findings were supported by our in vivo evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. Conclusion: LPALPAR1- Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy. [ABSTRACT FROM AUTHOR]

Background: miR-124-3p has been reported to be involved in the pathogenesis of many diseases by modulating a variety of signaling pathways. In this study, we aimed to understand the impact of miR-124-3p expression level on the fracture healing in the patients of metaphyseal fracture of distal tibia, who received minimal invasive percutaneous plate osteosynthesis. Methods: We firstly collected 195 patients of metaphyseal fracture of distal tibia, and the genotype of rs531564 was determined: GG (n=124) and GC+CC (n=71). We collected information of the participants including age, gender, total in-hospital time, smoking and alcohol consumption. Subsequently, we searched the miRNA database online to identify the possible binding sequence of miR-124-3p located within the 3'-UTR of the target gene. We did correlation analysis and luciferase to understand the regulatory relationship between miR- 124-3p and BMP6. Meanwhile, we also conducted real time PCR and western blotting analysis to study the mRNA and protein expression level of BMP6 in different genotype groups. We then treated the cells with scramble control, miR-124-3p mimics, BMP6 siRNA and miR-124- 3p inhibitors to investigate the influence of miR-124-3p on the expression of BMP6, viability and apoptosis of cells. Results: Total in-hospital time was significantly longer in GC+CC group than GG group. MiR-124-3p was up-regulated in GG group than GC and CC groups. BMP6 was virtual target of miR-124-3p. There existed negative regulatory relationship betweenmiR- 124-3p and BMP6. The mRNA and protein expression level of BMP6 decreased in GG group. MiR-124-3p decreased the expression of BMP6. MiR-124-3p negatively interfered with the viability of cells and BMP6 positively interfered with the viability of cells. MiR-124-3p reduced apoptosis and BMP6 promoted apoptosis. Conclusion: These data proved the expression of miR-124-3p was associated with the healing of metaphyseal fracture of distal tibia, and could be recognized as a biomarker to predict the healing of metaphyseal fracture of distal tibia. [ABSTRACT FROM AUTHOR]

This study investigated the immunomodulatory effects of ferulic acid (FA) on antigen-presenting dendritic cells (DCs) in vitro and its antiallergic effects against ovalbumin- (OVA-) induced Th2-mediated allergic asthma in mice. The activation of FA-treated bone marrow-derived DCs by lipopolysaccharide (LPS) stimulation induced a high level of interleukin- (IL-) 12 but reduced the expression levels of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor- (TNF-) α. Compared to control-treated DCs, FA significantly enhanced the expressions of Notch ligand Delta-like 4 (Dll4), MHC class II, and CD40 molecules by these DCs. Furthermore, these FA-treated DCs enhanced T-cell proliferation and Th1 cell polarization. In animal experiments, oral administration of FA reduced the levels of OVA-specific immunoglobulin E (IgE) and IgG1 and enhanced IgG2a antibody production in serum. It also ameliorated airway hyperresponsiveness and attenuated eosinophilic pulmonary infiltration in dose-dependent manners. In addition, FA treatment inhibited the production of eotaxin, Th2 cytokines (IL-4, IL-5, and IL-13), and proinflammatory cytokines but promoted the Th1 cytokine interferon- (IFN-) γ production in bronchoalveolar lavage fluid (BALF) and the culture supernatant of spleen cells. These findings suggest that FA exhibits an antiallergic effect via restoring Th1/Th2 imbalance by modulating DCs function in an asthmatic mouse model. [ABSTRACT FROM AUTHOR]

Oxidative stress-induced mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in peripheral neurons is considered to be important in the development of diabetic neuropathy. Mitochondrial transcription factor A (TFAM) wraps mtDNA and promotes mtDNA replication and transcription. We studied whether overexpression of TFAM reverses experimental peripheral diabetic neuropathy using TFAM transgenic mice (TFAM Tg) that express human TFAM (hTFAM). Levels of mouse mtDNA and the total TFAM (mouse TFAM + hTFAM) in the dorsal root ganglion (DRG) increased by approximately twofold in the TFAM Tg mice compared with control (WT) mice. WT and TFAM Tg mice were made diabetic by the administration of streptozotocin. Neuropathy end points were motor and sensory nerve conduction velocities, mechanical allodynia, thermal nociception, and intraepidermal nerve fiber density (IENFD). In the DRG neurons, mtDNA copy number and damage to mtDNA were quantified by qPCR, and TFAM levels were measured by Western blot. Mice with 16-wk duration of diabetes developed motor and sensory nerve conduction deficits, behavioral deficits, and intraepidermal nerve fiber loss. All of these changes were mostly prevented in diabetic TFAM Tg mice and were independent of changes in blood parameters. Mice with 16 wk of diabetes had a 40% decrease in mtDNA copy number compared with nondiabetic mice (P < 0.01). Importantly, the mtDNA copy number in diabetic TFAM Tg mice reached the same level as that of WT nondiabetic mice. In comparison, there was upregulation of mtDNA and TFAM in 6-wk diabetic mice, suggesting that TFAM activation could be a therapeutic strategy to treat peripheral neuropathy. [ABSTRACT FROM AUTHOR]

Atherosclerosis progress is regulated by a variety of factors. Here, we show that miR-320a, an intergenic miRNA, is markedly elevated in the peripheral blood of coronary heart disease patients and high-risk patients. Microarray analysis and qRT- PCR assays showed that circulating miRNA-320a was highly expressed in coronary artery disease patients. In vivo study showed that overexpression of miR-320a resulted in significant increase in levels of plasma lipid ( total cholesterol, Triglyceride and low-density lipoprotein) and serum inflammatory cytokines ( IL-6, MCP-1, sICAM, pSelectin, TNF-α and fibrinogen). In ApoE−/− mice, miR-320a expression attenuates endothelium cell function and promotes atherogenesis. Bioinformatics analysis identified serum response factor as a potential target for miR-320a, which was validated by luciferase reporter activity assay and western-blot in vitro and in vivo. Moreover, miR-320a expression inhibits human-derived endothelium cell proliferation and induces apoptosis. We also found that SP1 transcriptionally up-regulates hsa-miR-320a expression. Our observations indicate that miR-320a is a key regulator contributing to multiple aspects of atherogenesis. [ABSTRACT FROM AUTHOR]

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices.Methods: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D).Results: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818.Conclusions: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing. [ABSTRACT FROM AUTHOR]

Background: The consumption of omega 3 polyunsaturated fatty acids (PUFAs) is important for human health and is closely associated with cell proliferation and differentiation. This study aimed to investigate the influence of omega 3 PUFAs on embryonic stem cell (ESC) proliferation and explore potential mechanisms that mediate these effects. Methods: In this study, we isolated ESCs from fad3b-expressing transgenic mice. We detected the fatty-acid composition of ESCs using gas chromatography-mass spectroscopy, analyzed cell-cycle phases using flow cytometry, and detected gene expression using real-time polymerase chain reaction (PCR) and western blots. Results: The amount of omega 3 PUFAs significantly increased in fad3b versus control ESCs. However, the growth of fad3b ESCs was slower than that of control cells, and most fad3b ESCs were in a prolonged G0/G1 phase after being passaged for 18 h. Therefore, we hypothesized that fad3b expression inhibited the cell cycle in ESCs by increasing the expression of P21, which then decreased the expression of cyclin-dependent kinase 4 (Cdk4). We found that pretreatment of fad3b ESCs with PD0325901, a P21 inhibitor, clearly attenuated the inhibitory effects of P21 on Cdk4, and resumed the cell cycle. Conclusions: Expression of the fad3b gene in ESCs increased the omega 3 PUFA content, which inhibited cell proliferation by prolonging the G1 phase but did not arrest the G0-to-G1 or G1-to-S transitions. The prolonged G1 phase in fad3b ESCs was probably induced by downregulation of Cdk4 expression via p21 upregulation. These results suggest that accumulation of omega 3 PUFAs in vivo may beneficially affect ESC differentiation and that fad3b ESCs may be a useful tool for investigating related mechanisms. [ABSTRACT FROM AUTHOR]

Background: Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. Methods: In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. Results: The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). Conclusion: The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with the second highest mortality rate in all cancer. Energy reprogramming is one of the hallmarks of cancer, and emerging evidence showed that targeting glycolysis is a promising strategy for HCC treatment. Cryptocaryone has been shown to display promising anti-cancer activity against numerous types of cancer. Previous study also indicated that cryptocaryone induces cytotoxicity by inhibiting glucose transport in cancer cells, but the detailed mechanism still needs to be elucidated. Therefore, this study aimed to investigate the relationship between the anti-cancer effect and glycolytic metabolism of cryptocaryone in human HCC cells. In this study, we found that cryptocaryone potently induced growth inhibition by apoptotic cell death in HCC cells. Cryptocaryone also suppressed the ATP synthesis, lactate production and glycolytic capacity of HCC cells. Mechanistic investigations showed that phosphorylation of Akt and c-Src, as well as the expression of HK1 were impeded by cryptocaryone. Moreover, cryptocaryone markedly increased the expression level of transcription factor FoxO1. Importantly, clinical database analysis confirmed the negative correlation between HK1 and FoxO1. High expression levels of HK-1 were positively correlated with poorer survival in patients with HCCs. These results suggest that cryptocaryone may promote cell apoptosis by inhibiting FoxO1-mediated aerobic glycolysis through Akt and c-Src signaling cascades in human HCC cells. This is the first study to indicate that cryptocaryone exerts anti-cancer property against human HCC cells. Cryptocaryone is a potential natural product worthy of further development into a promising candidate for HCC treatment. [ABSTRACT FROM AUTHOR]

Abstract: Renal hypouricemia (RHUC), as an infrequent hereditary disease, is associated with severe complications such as exercise-induced acute renal failure (EIARF). Loss-of-function mutations in urate transporter gene URAT1 (Type 1) and in glucose transporter gene GLUT9 (Type 2) are major causes of this disorder. In this study, URAT1 and GLUT9 were screened in two uncorrelated families from mainland China and a total of five mutations were identified in exons, including two novel heterozygous URAT1 mutations. In four members of the first family, c.151delG (p.A51fsX64) in exon 1 was detected, which resulted in a frameshift and truncated the original 553-residue-protein to 63 amino acid protein. A missense mutation c.C1546A (p.P516T) in exon 9 in GLUT9 was revealed in the second family, which caused a functional protein substitution at codon 516. These two novel mutations were neither identified in the subsequent scanning of 200 ethnically matched healthy control subjects with normal serum UA level nor in a 1000 genome project database. Thus our report identifies two novel loss-of-function mutations (c.151delG in URAT1 and p.P516T in GLUT9) which cause RHUC and renal dysfunction in two independent RHUC pedigrees. [Copyright &y& Elsevier]

BackgroundUnbiased metagenomic next-generation sequencing (mNGS) detects pathogens in a target-independent manner. It is not well-understood whether mNGS has comparable sensitivity to target-dependent nucleic acid test for pathogen identification.MethodsThis study included 31 patients with chickenpox and neurological symptoms for screening of possible varicella-zoster virus (VZV) central nervous system (CNS) infection. Microbiological diagnosing of VZV cerebrospinal fluid (CSF) infection was performed on stored CSF samples using mNGS, quantitative and qualitative VZV-specific PCR assays, and VZV IgM antibodies test.ResultsThe median age was 30.0 [interquartile range (IQR), 24.3–33.3] years. 51.6% of the patients were men. About 80.6% of the patients had normal CSF white blood cell counts (≤ 5 × 106/L). VZV IgM antibodies presented in 16.1% of the CSF samples, and nucleic acids were detectable in 16.1 and 9.7% using two different VZV-specific real-time PCR protocols. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS (p = 0.001 and p = 007; compared with qualitative PCR and VZV IgM antibody test, respectively), with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples. All VZV PCR positive samples were positive when analyzed by mNGS. Of note, human betaherpesvirus 6A with clinical significance was unexpectedly detected in one CSF sample.ConclusionsOur study suggests that CSF mNGS may have higher sensitivity for VZV detection than CSF VZV PCR and antibody tests, and has the advantage of identifying unexpected pathogens.

Carbendazim. a widely used fungicide, exhibits reproductive and developmental toxicity. The objective of this study was to investigate the expression of androgen receptor induced by carbendazim and the antagonistic effect of flutamide. Rats were treated with carbendazim, flutamide or a combination of both to measure androgen receptor mRNA, immune activity and protein expression. Carbendazim treatment increased androgen receptor mRNA in a dose-dependent manner, while flutamide, an androgen receptor antagonist, blocked the androgen receptor mRNA. Co-treatment with carbendazim and various flutamide doses decreased the androgen receptor mRNA in a dose-dependent manner. Conversely, co-treatment with flutamide and various carbendazim doses increased the androgen receptor mRNA in a dose-dependent manner. Immunohistochemistry and Western blot analyses showed that carbendazim increased androgen receptor activity (especially in rat testes) in a dose-dependent manner, while flutamide decreased it. Furthermore, carbendazim or flutamide treatment for 7 days increased testosterone and follicular stimulating hormone concentrations in the serum of male rats in a dose-dependent manner, which might involve the disruption of the androgen receptor. Although we need to examine the exact mechanism of androgen receptor involved in the reproductive toxicity and endocrine-disrupting activity induced by carbendazim and its parent, benomyl, we should first discuss how to take advantage of flutamide antagonism on carbendazim-induced reproductive and endocrine- disrupting activity possibly in human. In summary, carbendazim induced androgen receptor expression in mRNA and protein levels, while flutamide antagonized this effect. This is the first report on the antagonistic effect of flutamide on the carbendazim-androgenic effect on mRNA and protein levels. The results would help to clarify the mechanism of carbendazim- and chemical-induced developmental toxicity and endocrine-disrupting activity. [ABSTRACT FROM AUTHOR]

Background. This study was aimed at exploring the effects of miR-215 and its target gene stearoyl-CoA desaturase (SCD) on colorectal cancer (CRC) cell migration and invasion. Methods. Here, we analyzed the relationship between miR-215 and SCD, as well as the regulation of miR-215 on CRC cells. We constructed wild-type and mutant plasmids of SCD to identify whether SCD was a target gene of miR-215 by using a luciferase reporter assay. The expression of miR-215 and SCD was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. MTT, wound healing, and Transwell assays were applied to determine the effect of miR-215 on CRC cell proliferation, migration, and invasion. Results. It was found that miR-215 expression was significantly decreased in CRC tissue while SCD was highly expressed compared with those in adjacent normal tissue. The luciferase reporter assay indicated that SCD was a direct target gene of miR-215. Functional analysis revealed that miR-215 overexpression significantly inhibited CRC cell proliferation, migration, and invasion in vitro. In addition, the result of rescue experiments showed that overexpression of SCD could promote the proliferation, migration, and invasion of CRC cells, and the carcinogenic effect of SCD could be inhibited by miR-215. Conclusions. Taken together, our findings suggested that miR-215 could inhibit CRC cell migration and invasion via targeting SCD. The result could eventually contribute to the treatment for CRC. [ABSTRACT FROM AUTHOR]

Background: Excessive reactive oxygen species generated in mitochondria has been implicated as a causal event in hypertensive cardiomyopathy. Multiple recent studies suggest that microRNAs (miRNAs) are able to translocate to mitochondria to modulate mitochondrial activities, but the medical significance of such a new miRNA function has remained unclear. Here, we characterized spontaneous hypertensive rats (SHRs) in comparison with Wistar rats, finding that micro RNA-21 (miR-21) was dramatically induced in SHRs relative to Wistar rats. We designed a series of experiments to determine whether miR-21 is involved in regulating reactive oxygen species generation in mitochondria, and if so, how induced miR-21 may either contribute to hypertensive cardiomyopathy or represent a compensatory response.Methods: Western blotting was used to compare the expression of key nuclear genome (nDNA)-encoded and mitochondrial genome (mtDNA)-encoded genes involved in reactive oxygen species production in SHRs and Wistar rats. Bioinformatics was used to predict miRNA targets followed by biochemical validation using quantitative real-time polymerase chain reaction and Ago2 immunoprecipitation. The direct role of miRNA in mitochondria was determined by GW182 dependence, which is required for miRNA to function in the cytoplasm, but not in mitochondria. Recombinant adeno-associated virus (type 9) was used to deliver miRNA mimic to rats via tail vein, and blood pressure was monitored with a photoelectric tail-cuff system. Cardiac structure and functions were assessed by echocardiography and catheter manometer system.Results: We observed a marked reduction of mtDNA-encoded cytochrome b (mt-Cytb) in the heart of SHRs. Downregulation of mt-Cytb by small interfering RNA in mitochondria recapitulates some key disease features, including elevated reactive oxygen species production. Computational prediction coupled with biochemical analysis revealed that miR-21 directly targeted mt-Cytb to positively modulate mt-Cytb translation in mitochondria. Circulating miR-21 levels in hypertensive patients were significantly higher than those in controls, showing a positive correlation between miR-21 expression and blood pressure. Remarkably, recombinant adeno-associated virus-mediated delivery of miR-21 was sufficient to reduce blood pressure and attenuate cardiac hypertrophy in SHRs.Conclusions: Our findings reveal a positive function of miR-21 in mitochondrial translation, which is sufficient to reduce blood pressure and alleviate cardiac hypertrophy in SHRs. This observation indicates that induced miR-21 is part of the compensatory program and suggests a novel theoretical ground for developing miRNA-based therapeutics against hypertension. [ABSTRACT FROM AUTHOR]

Abstract: Acetyl-CoA carboxylase α (ACC1), the major regulatory enzyme of fatty acid biosynthesis, catalyzes the conversion of acetyl-CoA to malonyl-CoA. The full-length cDNA coding ACC1 isoform was cloned from liver of grass carp. The cDNA obtained was 7515bp with a 7173bp open reading frame encoding 2389 amino acids. The ACC1 protein has a calculated molecular weight of 269.2kDa and isoelectric point of 6.23. Tissue distribution of ACC1 mRNA in brain, mesenteric adipose, spleen, white muscle and liver of grass carp was analyzed by real-time PCR method using β-actin as an internal control for cDNA normalization. The results showed that the expressions of ACC1 mRNA were detected in all examined tissues. Relative expression profile of ACC1 mRNA in liver normalized with β-actin level was 15, 92, 135 and 165-fold compared with the level in brain, white muscle, mesenteric adipose and spleen, respectively. In addition, we present evidence for the presence of two isoforms of ACC1 (265.7kDa and 267.2kDa) in grass carp liver that differ from the 269.2kDa ACC1 by the absence of 34 and 15 amino acids. In conclusion, the liver is one of the main ACC1 producing tissues in grass carp and ACC1 gene was highly homologous to that of mammals. [Copyright &y& Elsevier]