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He-Xi Du,1–3 Xian-Guo Chen,1–3 Li Zhang,1–3 Yi Liu,1–3 Chang-Sheng Zhan,1–3 Jing Chen,1–3 Yong Zhang,1–3 Zi-Qiang Yu,1–3 Jin Zhang,4,5 Hong-Yi Yang,4,5 Kai Zhong,4,5 Chao-Zhao Liang1–31Department of Urology, The First Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei 230022, People’s Republic of China; 2Institute of Urology, Anhui Medical University, Hefei 230022, People’s Republic of China; 3Anhui Province Key Laboratory of Genitourinary Diseases, Anhui Medical University, Hefei 230022, People’s Republic of China; 4High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China; 5Key Laboratory of Anhui Province for High Magnetic Resonance Imaging, Hefei 230031, People’s Republic of ChinaBackground: Patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) frequently show depressive symptoms clinically and increasing evidence indicates a correlation between CP/CPPS and depression. However, the underlying mechanisms of CP/CPPS-related depression remain poorly understood. Here, we sought to determine the role of hippocampal microglial activation and neurobiological changes in a mouse model of experimental autoimmune prostatitis (EAP)-induced depression and the treatment efficacy of Chinese herb extract baicalein.Methods: EAP was induced through intradermal injection of prostate antigen and adjuvant twice. Then, mice were assessed for affective behaviors in the open field test, elevated plus maze, forced swim test, and tail suspension test. The morphology and function of microglia and astrocytes were detected by immunofluorescence, Western blotting, and transmission electron microscopy. Proinflammatory mediators along with serotonin transporter (SLC6A4/SERT) and indoleamine 2,3-dioxygenase (IDO) were quantified with reverse transcription-polymerase chain reaction (RT‑PCR), and serum serotonin concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Proton magnetic resonance spectroscopy (1H-MRS) was performed to measure hippocampal glutamate levels. In addition, baicalein was used in a subset of EAP mice to test its anti-depressant action.Results: EAP was successfully established and induced depressive- and anxiety-like behavior in mice. Increasing levels of co-expressed ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) and ultrastructural observations suggested microglial activation and reactive astrocytosis in the hippocampus. These activated microglia resulted in increased expressions of multiple proinflammatory cytokines. Simultaneously, EAP mice showed higher gene expressions of SLC6A4 and IDO and lower concentrations of serotonin. 1H-MRS indicated a decrease in the glutamate + glutamine (Glx)/total creatine (tCr) ratio in EAP mice. Furthermore, baicalein treatment alleviated the depressive-like behavior and neuroinflammation by suppressing the nuclear factor-kappa B (NF-κB) pathway.Conclusion: Our data indicate that EAP-induced depressive-like behavior is linked to microglia activation and subsequent neurotransmitter metabolism. Moreover, baicalein attenuates behavioral changes by inhibiting neuroinflammation via downregulation of the NF-κB pathway.Keywords: EAP, microglial activation, serotonin, neurochemical changes, baicalein

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is very common worldwide, and alcohol consumption is a notable contributing factor. Researches have shown that gut microbiota can be influenced by alcohol consumption and is an important mediator in regulating Th17 cell immunity. However, it is still unclear the exact mechanism by which alcohol exacerbates the CP/CPPS and the role of gut microbiota in this process., Method: We first constructed the most-commonly used animal model for CP/CPPS, the experimental autoimmune prostatitis (EAP) model, through immunoassay. Based on this, mice were divided into EAP group and alcohol-consuming EAP group. By 16S rRNA sequencing and non-targeted metabolomics analysis, differential gut microbiota and their metabolites between the two groups were identified. Subsequently, metabolomics detection targeting cholesterols was carried out to identify the exact difference in cholesterol. Furthermore, multiple methods such as flow cytometry and immunohistochemistry were used to detect the differentiation status of Th17 cells and severity of prostatitis treated with 27-hydroxycholesterol (the differential cholesterol) and its upstream regulatory factor-sterol regulatory element-binding protein 2 (SREBP2). Lastly, fecal transplantation was conducted to preliminary study on whether alcohol intake exacerbates EAP in immune receptor mice., Results: Alcohol intake increased the proportion of Th17 cells and levels of related inflammatory factors. It also led to an altered gut bacterial richness and increased gut permeability. Further metabolomic analysis showed that there were significant differences in a variety of metabolites between EAP and alcohol-fed EAP mice. Metabolic pathway enrichment analysis showed that the pathways related to cholesterol synthesis and metabolism were significantly enriched, which was subsequently confirmed by detecting the expression of metabolic enzymes. By targeting cholesterol synthesis, 27-hydroxycholesterol was significantly increased in alcohol-fed EAP mice. Subsequent mechanistic research showed that supplementation with 27-hydroxycholesterol could aggravate EAP and promote Th17 cell differentiation both in vivo and in vitro, which is regulated by SREBP2. In addition, we observed that fecal transplantation from mice with alcohol intake aggravated EAP in immunized recipient mice fed a normal diet., Conclusion: Our study is the first to show that alcohol intake promotes Th17 cell differentiation and exacerbates EAP through microbiota-derived cholesterol biosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To investigate the mechanism of the CXCL10/CXCR3 axis regulating Th1 cell differentiation and migration through the PI3K/AKT pathway in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS)., Methods: Experimental autoimmune prostatitis (EAP) model, a well-described and validated animal model of CP/CPPS, was used in our study. After treatment with CXCL10, the severity of EAP and Th1 cell proportion were respectively measured by HE stains, immunohistochemistry, and flow cytometry. Then, the protein expression of the PI3K/AKT pathway in CXCL10/CXCR3-regulated Th1 cell differentiation and migration was evaluated by western blotting. Additionally, by the CXCR3 antagonist AMG487 and the PI3K inhibitor LY294002 applications, the effects of CXCL10/CXCR3 through PI3K/AKT pathway on the Th1 cell differentiation and migration were further assessed., Results: The EAP model was successfully built. CXCL10 increased the proportion of Th1 cells in EAP mice, accompanied by upregulation of the PI3K/AKT pathway. Additionally, the PI3K/AKT pathway was found to be involved in CXCL10/CXCR3 axis-mediated Th1 cell differentiation and migration., Conclusions: Our investigations indicate that the CXCL10/CXCR3 axis regulates Th1 cell differentiation and migration in EAP through the PI3K/AKT pathway, which provides a new perspective on the immunological mechanisms of CP/CPPS., (© 2023 American Society of Andrology and European Academy of Andrology.)

Wheatgrass is recognized for its nutritional and medicinal properties, partly attributed to its flavonoid content. The objective of this study was to assess the flavonoid content and antioxidant properties of wheatgrass obtained from a wide range of 145 wheat cultivars, which included Chinese landraces (CL), modern Chinese cultivars (MCC), and introduced modern cultivars (IMC). The flavonoids were extracted using a solution of 80% methanol, and their content was evaluated using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). The results revealed the assessed cultivars showed significant variation in their total flavonoid content (TFC), with MCCs generally having higher amounts compared to CLs. PCA analysis demonstrated clear variations in flavonoid profiles between different cultivar groups, emphasizing the evolutionary inconsistencies in wheat breeding. The antioxidant assays, ABTS, DPPH, and FRAP, exhibited robust abilities for eliminating radicals, which were found to be directly associated with the amounts of flavonoids. In addition, this study investigated the correlation between the content of flavonoids and the ability to resist powdery mildew in a collection of mutated wheat plants. Mutants exhibiting heightened flavonoid accumulation demonstrated a decreased severity of powdery mildew, suggesting that flavonoids play a protective role against fungal infections. The results highlight the potential of wheatgrass as a valuable source of flavonoids that have antioxidant and protective effects. This potential is influenced by the genetic diversity and breeding history of wheatgrass. Gaining insight into these connections can guide future wheat breeding endeavors aimed at improving nutritional value and in strengthening disease resistance. The current finding provides critical information for developing wheatgrass with high flavonoid content and antioxidant activity.

Aims/hypothesis: The relationship between metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes mellitus, insulin resistance and the metabolic syndrome is well established. While zinc finger BED-type containing 3 (ZBED3) has been linked to type 2 diabetes mellitus and the metabolic syndrome, its role in MASLD remains unclear. In this study, we aimed to investigate the function of ZBED3 in the context of MASLD., Methods: Expression levels of ZBED3 were assessed in individuals with MASLD, as well as in cellular and animal models of MASLD. In vitro and in vivo analyses were conducted using a cellular model of MASLD induced by NEFA and an animal model of MASLD induced by a high-fat diet (HFD), respectively, to investigate the role of ZBED3 in MASLD. ZBED3 expression was increased by lentiviral infection or tail-vein injection of adeno-associated virus. RNA-seq and bioinformatics analysis were employed to examine the pathways through which ZBED3 modulates lipid accumulation. Findings from these next-generation transcriptome sequencing studies indicated that ZBED3 controls SREBP1c (also known as SREBF1; a gene involved in fatty acid de novo synthesis); thus, co-immunoprecipitation and LC-MS/MS were utilised to investigate the molecular mechanisms by which ZBED3 regulates the sterol regulatory element binding protein 1c (SREBP1c)., Results: In this study, we found that ZBED3 was significantly upregulated in the liver of individuals with MASLD and in MASLD animal models. ZBED3 overexpression promoted NEFA-induced triglyceride accumulation in hepatocytes in vitro. Furthermore, the hepatocyte-specific overexpression of Zbed3 promoted hepatic steatosis. Conversely, the hepatocyte-specific knockout of Zbed3 resulted in resistance of HFD-induced hepatic steatosis. Mechanistically, ZBED3 interacts directly with polypyrimidine tract-binding protein 1 (PTBP1) and affects its binding to the SREBP1c mRNA precursor to regulate SREBP1c mRNA stability and alternative splicing., Conclusions/interpretation: This study indicates that ZBED3 promotes hepatic steatosis and serves as a critical regulator of the progression of MASLD., Data Availability: RNA-seq data have been deposited in the NCBI Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231875 ). MS proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository ( https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD041743 )., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common inflammatory immune disease of the urogenital system. High glucose intake is considered to be a potential promoter of autoimmune diseases. However, the influence of high glucose intake on CP/CPPS is unknown. This research aimed to explore the influences of high glucose intake on experimental autoimmune prostatitis (EAP), a valid animal model of CP/CPPS, and the underlying mechanism. NOD mice received 20% glucose water or normal water treatment during EAP induction. EAP severity and Th17 cell responses were evaluated. Then, we explored the effects of an IL-17A neutralizing antibody, an inhibitor of TGF-β, the reactive oxygen species (ROS) inhibitor NAC, and the mitochondrial ROS (mtROS) antioxidant MitoQ on glucose-fed EAP mice. The results demonstrated that high glucose intake aggravated EAP severity and promoted Th17 cell generation, which could be ameliorated by the neutralization of IL-17A. In vitro experiments showed that high dextrose concentrations promoted Th17 cell differentiation through mtROS-dependent TGF-β activation. Treatment with TGF-β blockade, NAC, or MitoQ suppressed Th17 cell generation both in vivo and in vitro, resulting in the amelioration of EAP manifestations caused by high glucose intake. This study revealed that high glucose intake exacerbates EAP through mtROS-dependent TGF-β activation-mediated Th17 differentiation. Our results may provide insights into the molecular mechanisms underlying the detrimental effects of an environmental factor, such as high glucose intake, on CP/CPPS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Atmospheric PM 2.5 samples were collected in Heze, Shandong Province, from a total of three sampling sites at Heze College, Huarun Pharmacy, and a wastewater treatment plant between October 15, 2017 and January 31, 2018, to determine the concentrations of 21 metal elements in PM 2.5 using inductively coupled plasma mass spectrometry (ICP-MS). The degree of elemental enrichment was also discussed, the health risks and potential heavy metal ecological risks were assessed. The results showed that ρ (PM 2.5 ) ranged from 26.7 to 284.1 μg·m -3 at the three sampling sites during the sampling period, and the concentration values did not differ significantly, all of which were at high pollution levels. The highest concentrations of K were found in the three sampling sites, accounting for 31.03%, 39.47%, and 38.43% of the total, respectively, mainly due to the high contribution of biomass burning in autumn and winter in Heze, a large agricultural city. The highest concentrations of Zn, 89.70, 84.21, and 67.68 ng·m -3 , were found in the trace elements at the three sampling sites, respectively. The enrichment factor results showed that the enrichment factor values of Zn, Pb, Sn, Sb, Cd, and Se were higher than 100, among which the enrichment factors of Cd and Se were higher than 2 000 and 4 000, respectively, which were significantly influenced by anthropogenic activities and might have been related to industrial production, metal smelting, road sources, and coal combustion emissions. The health risk results showed that there was some potential non-carcinogenic risk (HQ>0.1 for children and adults) for As and a combined potential non-carcinogenic risk (HI>0.1) and some potential carcinogenic risk (CRT>1×10 -6 ) for both children and adults at the three sampling sites. There was a more significant carcinogenic risk (CRT>1×10 -4 ) for adults at the wastewater treatment plant, and the slightly higher carcinogenic risk for adults than that for children may have been related to the longer outdoor activity and higher PM 2.5 exposure for adults. The elements with the highest potential ecological risk values were Cd, As, and Pb, with Cd exhibiting a very high potential ecological risk that should be taken seriously. All three sampling sites showed a very high combined potential ecological risk, with the intensity spatially expressed as Heze College>Huarun Pharmacy>wastewater treatment plant.

Background: Bladder cancer (BLCA) is a prevalent urinary system malignancy. Understanding the interplay of immunological and metabolic genes in BLCA is crucial for prognosis and treatment., Methods: Immune/metabolism genes were extracted, their expression profiles analyzed. NMF clustering found prognostic genes. Immunocyte infiltration and tumor microenvironment were examined. Risk prognostic signature using Cox/LASSO methods was developed. Immunological Microenvironment and functional enrichment analysis explored. Immunotherapy response and somatic mutations evaluated. RT-qPCR validated gene expression., Results: We investigated these genes in 614 BLCA samples, identifying relevant prognostic genes. We developed a predictive feature and signature comprising 7 genes (POLE2, AHNAK, SHMT2, NR2F1, TFRC, OAS1, CHKB). This immune and metabolism-related gene (IMRG) signature showed superior predictive performance across multiple datasets and was independent of clinical indicators. Immunotherapy response and immune cell infiltration correlated with the risk score. Functional enrichment analysis revealed distinct biological pathways between low- and high-risk groups. The signature demonstrated higher prediction accuracy than other signatures. qRT-PCR confirmed differential gene expression and immunotherapy response., Conclusions: The model in our work is a novel assessment tool to measure immunotherapy's effectiveness and anticipate BLCA patients' prognosis, offering new avenues for immunological biomarkers and targeted treatments., (© 2023. The Author(s).)

Most patients with pancreatic cancer are already in the late stages of the disease when they are diagnosed, and pancreatic cancer is a deadly disease with a poor prognosis. With the advancement of research, immunotherapy has become a new focus in the treatment of tumors. To the best of our knowledge, there is currently no reliable diagnostic or prognostic marker for pancreatic cancer; therefore, the present study investigated the potential of eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) as a predictive and diagnostic marker for pancreatic cancer. Immunohistochemical staining of clinical samples independently verified that EIF2AK2 expression was significantly higher in clinically operated pancreatic cancer tissues than in adjacent pancreatic tissues., and EIF2AK2 expression and differentially expressed genes (DEGs) were identified using downloadable RNA sequencing data from The Cancer Genome Atlas and Genomic Tumor Expression Atlas. In addition, Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses and immune cell infiltration were used for functional enrichment analysis of EIF2AK2-associated DEGs. The clinical importance of EIF2AK2 was also determined using Kaplan-Meier survival, Cox regression and time-dependent survival receiver operating characteristic curve analyses, and a predictive nomogram model was generated. Finally, the functional role of EIF2AK2 was assessed in PANC-1 cells using a short hairpin RNA-EIF2AK2 knockdown approach, including CCK-8, wound healing assay, cell cycle and apoptosis assays. The findings suggested that EIF2AK2 may have potential as a diagnostic and prognostic biomarker for patients with pancreatic cancer. Furthermore, EIF2AK2 may provide a new therapeutic target for patients with pancreatic cancer., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Du et al.)

Chlorophyll is an important indicator of vegetation health status, accurate estimation of which is important for evaluating forest carbon sink. In this study, we estimated the chlorophyll content of coniferous forests, broad-leaved forests and mixed forest stands at stand and individual tree level by unmanned air vehicle (UAV) hyperspectral data combined with light detection and ranging (LiDAR) point clouds, which improved the non-destructive estimation accuracy of forest chlorophyll. We further comprehensively analyzed the spatial distribution of chlorophyll content at different scales. A total of 36 spectral characteristic variables related to chlorophyll content were screened by correlation analysis based on the fusion of UAV hyperspectral data and LiDAR point clouds combining with the empirical data from ground plots. We constructed multiple models for chlorophyll estimation by using statistical model, including multiple stepwise regression, BP neural network, BP neural network optimized by firefly algorithm, random forest and hybrid data-driven PROSPECT mechanism model. The optimal model was selected to estimate the chlorophyll content. The horizontal and vertical distribution of chlorophyll content at the stand level and individual tree level were analyzed. The results showed that the random forest model was superior to the models constructed by multiple stepwise regression, BP neural network and BP neural network optimized by firefly algorithm for chlorophyll estimation with R 2 and RMSE of 0.59-0.64 and 3.79-5.83 μg·cm -2 , respectively. The accuracy of the mechanism model was the highest, with R 2 and RMSE of 0.97 and 3.40 μg·cm -2 . The chlorophyll contents differed across stand types, with that in broad-leaved forest (25.25-31.60 μg·cm -2 ) being higher than mixed forest (13.52-23.93 μg·cm -2 ) and coniferous forest (6.40-13.71 μg·cm -2 ). There were significant differences in chlorophyll contents the in vertical direction among different stands. For individual tree of different species, the chlorophyll content inside the canopy was lower than that outside the canopy in the horizontal direction. In the vertical direction, there was no difference in chlorophyll content among different layers of Pinus sylvestris var. mongolica canopy. However, significant differences were observed among the upper, middle, and lower layers of Juglans mandshurica canopy. Using the fusion of hyperspectral image and LiDAR point cloud data, the mechanism model driven by hybrid data could effectively improve the accuracy and stability of chlorophyll content estimation at different scales.

Wheat is an essential energy and protein source for humans. Climate change brings daunting challenges to wheat yield through environmental stresses, in which phytohormones play critical roles. Nevertheless, the comprehensive understanding of wheat phytohormone responses remains elusive. Here, we investigated the transcriptome response of wheat seedlings to five phytohormones, cytokinin (6-BA), abscisic acid (ABA), gibberellic acid (GA), jasmonate (JA) and salicylic acid (SA). We further selected two JA marker genes and cloned their promoters to drive the expression of 3XEGFP (tandem trimeric enhanced green fluorescent protein) in transgenic lines. The JA fluorescent reporter displayed a fast and stable response to JA treatment as an ideal tool to follow JA dynamics during fungal and cold stresses at a cellular resolution. Overall, this study provided a transcriptional landscape and facilitated generating fluorescent reporters to monitor the dynamics of phytohormones in food crops., (© 2022 John Wiley & Sons Ltd.)

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a very common urological disorder and has been gradually regarded as an immune-mediated disease. Multiple studies have indicated that the gut microflora plays a pivotal part in immune homeostasis and autoimmune disorder development. However, whether the gut microflora affects the CP/CPPS, and the underlying mechanism behind them remain unclear. Here, we built an experimental autoimmune prostatitis (EAP) mouse model by subcutaneous immunity and identified that its Th17/Treg frequency was imbalanced. Using fecal 16s rRNA sequencing and untargeted/targeted metabolomics, we discovered that the diversity and relative abundance of gut microflora and their metabolites were obviously different between the control and the EAP group. Propionic acid, a kind of short-chain fatty acid (SCFA), was decreased in EAP mice compared to that in controls, and supplementation with propionic acid reduced susceptibility to EAP and corrected the imbalance of Th17/Treg cell differentiation in vivo and in vitro . Furthermore, SCFA receptor G-protein-coupled receptor 43 and intracellular histone deacetylase 6 regulated by propionic acid in Th17 and Treg cells were also evaluated. Lastly, we observed that fecal transplantation from EAP mice induced the decrease of Treg cell frequency in recipient mice. Our data showed that gut dysbiosis contributed to a Th17/Treg differentiation imbalance in EAP via the decrease of metabolite propionic acid and provided valuable immunological groundwork for further intervention in immunologic derangement of CP/CPPS by targeting propionic acid., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Yue, Niu, Liu, Zhang, Chen, Chen, Guan, Hua, Li, Chen, Zhang and Liang.)

Objective To study the influence of recombinant bovine basic fibroblast growth factor as an adjuvant therapy on scar alleviation and inflammatory cytokines in patients with atrophic acne scar. Methods The random number table was employed to randomly assign 120 patients with atrophic acne scar into a test group and a control group.Both groups of patients were treated with CO 2 lattice laser.After the operation,the control group was routinely smeared with erythromycin ointment and the test group was coated with recombinant bovine basic fibroblast growth factor gel.The clinical efficacy,clinical indicators,scar alleviation,and inflammatory cytokine levels before and after treatment were compared,and adverse reactions were counted. Results The test group had higher total effective rate( P =0.040) and lower total incidence of adverse reactions( P =0.028) than the control group.Compared with the control group,the test group showcased short erythema duration after treatment( P =0.025),early scab forming( P =0.002),and early edema regression( P <0.001).After treatment,the proportion of grade 1 scars graded by Goodman and Baron's acne scar grading system in the test group and control group increased( P =0.001, P =0.027),and the proportion of grade 4 scars decreased( P <0.001, P =0.034).Moreover,the proportion of grade 1 scars in the test group was higher than that in the control group( P =0.031) after treatment,and the proportion of grade 4 scars presented an opposite trend( P =0.031).After treatment,the levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in both groups declined(all P <0.001),and the test group had lower TNF-α and IL-1β levels than the control group(all P <0.001). Conclusion The recombinant bovine basic fibroblast growth factor gel as an adjuvant therapy of CO 2 lattice laser can effectively alleviate the atrophic acne scar,relieve local inflammatory reaction,and has good curative effect and less adverse reactions.

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common male genitourinary system disease. As a neuroendocrine hormone, melatonin possesses a variety of biological functions, among which its anti-inflammatory effects have recently drawn substantial attention. The purpose of the current research was to study the effect of melatonin on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP)., Methods: The EAP mouse model was successfully established by subcutaneously injecting a mixture of prostate antigen and complete Freund's adjuvant. On Day 42, hematoxylin-eosin staining was used to evaluate the histological appearance of prostate tissues. Chronic pelvic pain development was assessed by suprapubic allodynia. The levels of inflammation-related cytokines, such as interferon-γ, interleukin (IL)-17, and IL-1β, were detected by enzyme-linked immunosorbent assay. Then, we explored the anti-inflammatory effects of melatonin on CP/CPPS by Western blotting and immunohistochemical staining, by measuring the expression of silent information regulator 1 (Sirt1) and NLRP3 inflammasome-related proteins in EAP mice., Results: The EAP model mice exhibited severe diffuse leukocyte infiltration and significantly increased pelvic pain compared to the control mice. In the melatonin treatment group, the histological appearance of the prostate tissues, pelvic pain development, and the levels of proinflammatory cytokines were significantly alleviated compared to the EAP + dimethyl sulfoxide group. Furthermore, we found that the protective effects of melatonin were achieved through activation of the Sirt1 pathway and downregulation of the NLRP3 inflammasome., Conclusions: The results indicated that melatonin could attenuate prostate inflammation and pelvic pain by inhibiting the NLRP3 inflammasomes signaling pathway through the activation of Sirt1 in mice with EAP, and these efforts should provide a promising therapeutic strategy for CP/CPPS., (© 2021 Wiley Periodicals LLC.)

Emodin has shown pharmacological effects in the treatment of infection with severe acute respiratory syndrome coronavirus-2, which leads to coronavirus disease 2019 (COVID-19). Thus, we speculated that emodin may possess anti-COVID-19 activity. In this study, using bioinformatics databases, we screened and harvested the candidate genes or targets of emodin and COVID-19 prior to the determination of pharmacological targets and molecular mechanisms of emodin against COVID-19. We discovered core targets for the treatment of COVID-19, including mitogen-activated protein kinase 1 (MAPK1), tumor protein (TP53), tumor necrosis factor (TNF), caspase-3 (CASP3), epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), interleukin 1B (IL1B), mitogen-activated protein kinase 14 (MAPK14), prostaglandin-endoperoxide synthase 2 (PTGS2), B-cell lymphoma-2-like protein 1 (BCL2L1), interleukin-8 (CXCL8), myeloid cell leukemia-1 (MCL1), and colony stimulating factor 2 (CSF2). The GO analysis of emodin against COVID-19 mainly included cytokine-mediated signaling pathway, response to lipopolysaccharide, response to molecule of bacterial origin, developmental process involved in reproduction, and reproductive structure development. The KEGG results exhibited that the molecular pathways mainly included IL-17 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, pertussis, proteoglycans in cancer, pathways in cancer, MAPK signaling pathway, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, etc. Also, molecular docking results revealed the docking capability between emodin and COVID-19 and the potential pharmacological activity of emodin against COVID-19. Taken together, these findings uncovered the targets and pharmacological mechanisms of emodin for treating COVID-19 and suggested that the vital targets might be used as biomarkers against COVID-19.

Background: Liver injury is common and also can be fatal, particularly in severe or critical patients with coronavirus disease 2019 (COVID-19)., Aim: To conduct an in-depth investigation into the risk factors for liver injury and into the effective measures to prevent subsequent mortality risk., Methods: A retrospective cohort study was performed on 440 consecutive patients with relatively severe COVID-19 between January 28 and March 9, 2020 at Tongji Hospital, Wuhan, China. Data on clinical features, laboratory parameters, medications, and prognosis were collected., Results: COVID-19-associated liver injury more frequently occurred in patients aged ≥ 65 years, female patients, or those with other comorbidities, decreased lymphocyte count, or elevated D-dimer or serum ferritin ( P < 0.05). The disease severity of COVID-19 was an independent risk factor for liver injury (severe patients: Odds ratio [OR] = 2.86, 95% confidence interval [CI]: 1.78-4.59; critical patients: OR = 13.44, 95%CI: 7.21-25.97). The elevated levels of on-admission aspartate aminotransferase and total bilirubin indicated an increased mortality risk ( P < 0.001). Using intravenous nutrition or antibiotics increased the risk of COVID-19-associated liver injury. Hepatoprotective drugs tended to be of assistance to treat the liver injury and improve the prognosis of patients with COVID-19-associated liver injury., Conclusion: More intensive monitoring of aspartate aminotransferase or total bilirubin is recommended for COVID-19 patients, especially patients aged ≥ 65 years, female patients, or those with other comorbidities. Drug hepatotoxicity of antibiotics and intravenous nutrition should be alert for COVID-19 patients., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Hypertension is a clinical syndrome characterized by elevated systemic arterial blood pressure, which may be accompanied by functional or organic damage of heart, brain, kidney and other organs. The pathogenesis and development of hypertension are affected by genetic, environmental, epigenetic, intestinal microbiota and other factors. They are the result of multiple factors that promote the change of blood pressure level and vascular resistance. G protein coupled receptors(GPCRs) are the largest and most diverse superfamily of transmembrane receptors that transmit signals across cell membranes and mediate a large number of cellular responses required by human physiology. A variety of GPCRs are involved in the control of blood pressure and the maintenance of normal function of cardiovascular system. Hypertension contributes to the damages of heart, brain, kidney, intestine and other organs. Many GPCRs are expressed in various organs to regulate blood pressure. Although many GPCRs have been used as therapeutic targets for hypertension, their efficacy has not been fully studied. The purpose of this paper is to elucidate the role of GPCRs in blood pressure regulation and its distribution in target organs. The relationship between GPCRs related to intestinal microorganisms and blood pressure is emphasized. It is proposed that traditional Chinese medicine may be a new way to treat hypertension by regulating the related GPCRs via intestinal microbial metabolites.

Context: Yinhuapinggan granule (YHPG) is frequently used for treating fever, cough, and viral pneumonia in traditional Chinese medicine., Objective: This study investigated the antiviral effects of YHPG in H1N1 influenza virus (IFV)-infected mice and its possible mechanism., Materials and Methods: ICR mice were intranasally infected with 10 LD 50 viral dose of IFV and then oral administration of YHPG (6, 12, and 18 g/kg) or oseltamivir (positive control) once a day for 2 or 4 consecutive days, six mice in each group. The lung, spleen and thymus indexes of IFV-infected mice, the expression of viral loads and pathological changes in lung tissues were performed to evaluate the antiviral effects of YHPG. Real-time PCR, immunohistochemistry and western blot assays were used to determine the expression of Bax, Bcl-2 and caspase-3., Results: LD 50 in mice was 10 -3.5 /0.02 mL. YHPG (6, 12, and 18 g/kg) dose-dependently decreased the lung index and viral load; the inhibition ratio of lung index was 5.31, 18.22, and 34.06%, respectively. Further detection revealed that YHPG (12 and 18 g/kg) significantly attenuated lung pathological changes, and increased the spleen and thymus indexes. Moreover, YHPG significantly down-regulated the mRNA and protein expression of Bax and caspase-3 in lung tissues of mice infected with IFV, and up-regulated the expression of Bcl-2., Conclusions: YHPG has significant antiviral effects in IFV-infected mice, partially by inhibiting influenza virus replication and regulating the occurrence of apoptosis induced by influenza virus infection, suggesting that YHPG may be a promising antiviral agent with potential clinical application prospects.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is a complicated syndrome characterized by genitourinary pain in the absence of bacterial infection. Th17 cell-driven autoimmunity has been proposed as a cause of CP/CPPS. However, the factors that promote Th17-driven autoimmunity in experimental autoimmune prostatitis (EAP) and the molecular mechanisms are still largely unknown. Here, we showed that Th17 cells were excessively activated, and blockade of IL-17A could effectively ameliorate various symptoms in EAP. Furthermore, we revealed that calcium/calmodulin-dependent kinase Ⅳ (CaMK4), especially Thr 196 p-CaMK4 was increased in the Th17 cells of the EAP group, which were activated by intracellular cytosolic Ca 2+ . Pharmacologic and genetic inhibition of CaMK4 decreased the proportion of Th17 cells, and the protein and mRNA level of IL-17A, IL-22, and RORγt. The phosphorylation of CaMK4 was dependent on the increase in intracellular cytosolic Ca 2+ concentration in Th17 cells. A mechanistic study demonstrated that inhibition of CaMK4 reduced IL-17A production by decreasing the phosphorylation of Akt-mTOR, which was well accepted to positively regulate Th17 differentiation. Collectively, our results demonstrated that Ca 2+ -CaMK4-Akt/mTOR-IL-17A axis inhibition may serve as a promising therapeutic strategy for CP/CPPS., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: To study the relationship between atrophic glossitis and anemia, anemia types and other related factors(oral candida infection, xerostomia) in 124 consecutive cases., Methods: One hundred and twenty-four cases with atrophic glossitis and 53 healthy controls were collected from Qingdao local population. The main indexes including general status, oral examination findings, hemoglobin (Hb), mean red blood cell volume (MCV), vitamin B12, ferritin, folic acid, anemia and anemia type, xerostomia and candida infection were statistically analyzed using SPSS 20.0 software package for Student's t test., Results: Among 124 cases of glossitis group, 48.39% were found with anemia, 41.94% with xerostomia, 79.03% with Candida infection, 29.03% with Vitamin B12 deficiency, 22.58% with ferritin deficiency, 11.29% with folic acid deficiency. The contents of hemoglobin, ferritin and vitamin B12 in glossitis group were significantly lower than those in the control group(P<0.05), and the number of glossitis patients with anemia, xerostomia and candida infection were significantly higher than those in the control group (P<0.05). There was no significant difference in folic acid content between the two groups(P<0.05)., Conclusions: Occurrence of atrophic glossitis is closely related to anemia, vitamin B12 deficiency, ferritin deficiency, xerostomia, oral candida infection. There is no correlation with folic acid deficiency. Patients with atrophic glossitis accompanied by anemia have a higher proportion of macrocytic anemia.

The animal model of hyperlipidemia in rats was established to investigate the lipid-lowering effect and mechanism of Danhong Injection on hyperlipidemic rats. SD rats were selected as the research object. The rats in normal group were fed with basic diet, and the rats in other groups were fed with high-fat diet to establish hyperlipidemia model. The successfully modeled rats were randomly divided into model group, Danhong Injection low, medium, high dose(1.0, 2.0, 4.0 mL·kg~(-1)) groups, and simvastatin(2.0 mg·kg~(-1)) group. Danhong Injection groups received intraperitoneal administration, and simvastatin group received intragastrical administration, once a day for 4 weeks. At the first, second, third, and fourth weekends after administration, blood was collected from the orbital vein to detect the levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C), and then the atherosclerosis index(AI) was calculated. After 4 weeks of administration, the animals were sacrificed, and their heart, liver, spleen, lung, kidney and adipose tissue were extracted and weighed respectively to calculate the organ index of each group. The expressions of acyl-coaoxidase 1(Acox1), adenosine 5'-monophosphate(AMP)-activated protein kinase alpha(AMPK-α), bile salt export pump(BSEP), peroxisome proliferator-activated receptor gamma(PPAR-γ), catalase(CAT) and superoxide dismutase(SOD) mRNA in liver tissues were detected by fluorescence quantitative PCR; the content of cholesteryl ester transfer protein(CETP) and lecithin cholesterol acyltransferase(LCAT) in serum was detected by ELISA. The results showed that as compared with the normal group, the levels of serum TC, TG and LDL-C in the model group were significantly increased, and the level of HDL-C was significantly decreased, indicating that the hyperlipidemia rat model was successfully constructed. As compared with the model group, Danhong Injection could decrease the contents of TC, TG, LDL-C and increase the content of HDL-C in hyperlipidemia rats; reduce the body weight of hyperlipidemia rats, and reduce the liver weight, liver index, fat weight and fat index; it had no significant effect on the main organ indexes such as heart, spleen, lung and kidney; but it could increase the expressions of Acox1, AMPK-α, BSEP, PPAR-γ, CAT and SOD mRNA in liver tissues of rats; it could also reduce the level of CETP and increase the level of LCAT in serum; and the regulatory effect of Danhong Injection groups all showed a dose-dependent effect. It can be concluded that Danhong Injection can regulate the blood lipid contents, reduce the blood lipid levels and alleviate the accumulation of body fat in rats with hyperlipidemia. The mechanism may be related to inhibiting lipid metabolism disorder and oxidative stress induced by high-fat diet feeding, and improving the imbalance of lipid transport system.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel β-coronavirus, causes severe pneumonia and has spread throughout the globe rapidly. The disease associated with SARS-CoV-2 infection is named coronavirus disease 2019 (COVID-19). To date, real-time reverse-transcription polymerase chain reaction (RT-PCR) is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection., Methods: In this study, we developed a peptide-based luminescent immunoassay that detected immunoglobulin (Ig)G and IgM. The assay cutoff value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2., Results: To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively., Conclusions: Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

We report acute antibody responses to SARS-CoV-2 in 285 patients with COVID-19. Within 19 days after symptom onset, 100% of patients tested positive for antiviral immunoglobulin-G (IgG). Seroconversion for IgG and IgM occurred simultaneously or sequentially. Both IgG and IgM titers plateaued within 6 days after seroconversion. Serological testing may be helpful for the diagnosis of suspected patients with negative RT-PCR results and for the identification of asymptomatic infections.

Our previous study found that microRNA-21a-5p (miR-21a-5p) knockdown could improve the recovery of motor function after spinal cord injury in a mouse model, but the precise molecular mechanism remains poorly understood. In this study, a modified Allen's weight drop was used to establish a mouse model of spinal cord injury. A proteomics approach was used to understand the role of differential protein expression with miR-21a-5p knockdown, using a mouse model of spinal cord injury without gene knockout as a negative control group. We found that after introducing miR-21a-5p knockdown, proteins that played an essential role in the regulation of inflammatory processes, cell protection against oxidative stress, cell redox homeostasis, and cell maintenance were upregulated compared with the negative control group. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis identified enriched pathways in both groups, such as the oxidative phosphorylation pathway, which is relevant to Parkinson's disease, Huntington's disease, Alzheimer's disease, and cardiac muscle contraction. We also found that miR-21a-5p could be a potential biomarker for amyotrophic lateral sclerosis, as miR-21a-5p becomes deregulated in this pathway. These results indicate successful detection of some important proteins that play potential roles in spinal cord injury. Elucidating the relationship between these proteins and the recovery of spinal cord injury will provide a reference for future research of spinal cord injury biomarkers. All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Shandong University of China on March 5, 2014., Competing Interests: None

Background: Depressive symptoms are found in approximately 78% of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) patients, but the pathological mechanisms remain unknown. Increasing evidence suggests that abnormal gut microbiota may play an important role in depression. Thus, we aimed to investigate whether gut microbiota contributes to CP/CPPS-associated depression by using a mouse model of experimental autoimmune prostatitis (EAP)., Methods: Male nonobese diabetic mice were immunized twice by subcutaneous injection of prostate antigen and adjuvant. Behavioral tests consisted of an open field test, sucrose preference test, forced swimming tests, and tail suspension test was used to confirm the depression-like symptoms that were induced by EAP. Then, fecal samples were collected, and 16S ribosomal RNA gene sequencing was performed to detect differences in gut microbiota composition between control and EAP group. Additionally, fecal bacteria from the control and EAP mice were transplanted into antibiotics-induced pseudo-germ-free mice to investigate the effects on host behaviors and the composition of gut bacteria., Results: EAP was successfully established and exhibited depressive-like behaviors in mice. The 16S rRNA analysis of fecal samples indicated the abnormal composition of gut microbiota in the EAP mice compared to the control mice. In the fecal microbiota transplant study, antibiotics-treated pseudo-germ-free mice presented depressive states as compared to naïve mice. Fecal bacteria transplant from EAP mice, but not from control mice, into the pseudo-germ-free mice, significantly exaggerated host depression-like behaviors. Moreover, fecal bacteria transplants from control and EAP mice induced distinct alterations in α-diversity and β-diversity indices. In all, 24 bacteria at six phylogenetic levels were remarkably changed by the fecal bacteria transplantation., Conclusions: Abnormal gut microbiota composition after EAP induction may contribute to the development of depression in mice. A therapeutic strategy that targets gut microbiota may provide an alternative treatment for alleviating this condition., (© 2020 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are considered to be involved in the pathogenic initiation and progression of chronic nonbacterial prostatitis (CNP); however, the comprehensive expression profile of dysregulated miRNAs, relevant signaling pathways, and core machineries in CNP have not been fully elucidated. In the current research, CNP rat models were established through the intraprostatic injection of carrageenan into the prostate. Then, next-generation sequencing was performed to explore the miRNA expression profile in CNP. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatical analyses were conducted to reveal the enriched biological processes, molecular functions, and cellular components and signaling pathways. As a result, 1224, 1039, and 1029 known miRNAs were annotated in prostate tissues from the blank control (BC), normal saline injection (NS), and carrageenan injection (CAR) groups (n = 3 for each group), respectively. Among them, 84 miRNAs (CAR vs BC) and 70 miRNAs (CAR vs NS) with significantly different expression levels were identified. Compared with previously reported miRNAs with altered expression in various inflammatory diseases, the majority of deregulated miRNAs in CNP, such as miR-146b-5p, miR-155-5p, miR-150-5p, and miR-139-5p, showed similar expression patterns. Moreover, bioinformatics analyses have enriched mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate (cAMP), endocytosis, mammalian target of rapamycin (mTOR), and forkhead box O (FoxO) signaling pathways. These pathways were all involved in immune response, which indicates the critical regulatory role of the immune system in CNP initiation and progression. Our investigation has presented a global view of the differentially expressed miRNAs and potential regulatory networks containing their target genes, which may be helpful for identifying the novel mechanisms of miRNAs in immune regulation and effective target-specific theragnosis for CNP.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250 μg/mL) significantly up-regulated levels of IFN-β, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250 μg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

This paper aimed to investigate the effect of Yinhua Pinggan granule and San-ao decoction on the immunologic mechanisms of influenza viral pneumonia mice in vivo , in order to study the activity of the combined administration of different formulas on influenza A/H1N1 virus. The model of pneumonia was established in mice through nasal dropping influenza virus, and then divided randomly into five groups: normal control group, influenza virus model group, oseltamivir control group, Yinhua Pinggan granule group, and San-ao decoction group. The animals were put to death at the 5th day after gavage administration with the corresponding drugs. The contents in mice serum of TNF-α, IL-6 and IFN-γ were respectively measured by ELISA. The mRNA expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in lung tissues were respectively detected by RT-PCR. The protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues were determined by immunohistochemical analysis, respectively. According to the results, Yinhua Pinggan granule and San-ao decoction could significantly decrease the levels of TNF-α and IL-6, increase the level of IFN-γ in mice serum of lung tissues, significantly reduce the gene expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in influenza virus-infected mice lung tissues, and significantly reduce the protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues. Furthermore, the regulatory effect of Yinhua Pinggan granule was superior to that of San-ao decoction. In conclusion, Yinhua Pingan granule and San-ao decoction have the therapeutic effect on pneumonia mice infected by H1N1 virus in vivo . The anti-influenza mechanisms of Yinhua Pinggan granule and San-ao decoction may be the results of interactions by regulating the immunologic function of influenza virus-infected mice and TLR3/7 signaling pathway with multiple links of the gene and protein expressions. Moreover, the combined administration of warm-natured and cold-natured Yinhua Pinggan granule with the effects of detoxification and exhalation has a better effect than the single administration of warm-natured San-ao decoction., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders., (Copyright © 2017, American Association for the Advancement of Science.)

Multigene pathway engineering usually needs amounts of part libraries on transcriptional and translational regulation as well as mutant enzymes to achieve the optimal part combinations of the target pathways. We report a new strategy for multigene pathway engineering with regulatory linkers (M-PERL) focusing on tuning the transcriptional start site (TSS) of yeast promoters. The regulatory linkers are composed of two homologous ends of two adjacent gene parts for assembly and a central regulatory region between them. We investigated the effect of the homologous end's length on multigene assembly, analyzed the influences of truncated, replaced, and elongated TSS and the adjacent region on promoters, and introduced 5 to 40 random bases of N (A/T/C/G) in the central regulatory region of the linkers which effectively varied the promoter's strengths. The distinct libraries of five regulatory linkers were used simultaneously to assemble and tune all five genes in the violacein synthesis pathway. The gene expressions affected the product profiles significantly, and the recombinants for enhanced single component synthesis and varied composition synthesis were obtained. This study offers an efficient tool to assemble and regulate multigene pathways.

Campesterol is an important precursor for many sterol drugs, e.g. progesterone and hydrocortisone. In order to produce campesterol in Yarrowia lipolytica, C-22 desaturase encoding gene ERG5 was disrupted and the heterologous 7-dehydrocholesterol reductase (DHCR7) encoding gene was constitutively expressed. The codon-optimized DHCR7 from Rallus norvegicus, Oryza saliva and Xenapus laevis were explored and the strain with the gene DHCR7 from X. laevis achieved the highest titer of campesterol due to D409 in substrate binding sites. In presence of glucose as the carbon source, higher biomass conversion yield and product yield were achieved in shake flask compared to that using glycerol and sunflower seed oil. Nevertheless, better cell growth rate was observed in medium with sunflower seed oil as the sole carbon source. Through high cell density fed-batch fermentation under carbon source restriction strategy, a titer of 453±24.7 mg/L campesterol was achieved with sunflower seed oil as the carbon source, which is the highest reported microbial titer known. Our study has greatly enhanced campesterol accumulation in Y. lipolytica, providing new insight into producing complex and desired molecules in microbes.

Background: The combination of resting-state functional MRI (R-fMRI) technique and graph theoretical approaches has emerged as a promising tool for characterizing the topological organization of brain networks, that is, functional connectomics. In particular, the construction and analysis of high-resolution brain connectomics at a voxel scale are important because they do not require prior regional parcellations and provide finer spatial information about brain connectivity. However, the test-retest reliability of voxel-based functional connectomics remains largely unclear., Aims: This study tended to investigate both short-term (∼20 min apart) and long-term (6 weeks apart) test-retest (TRT) reliability of graph metrics of voxel-based brain networks., Methods: Based on graph theoretical approaches, we analyzed R-fMRI data from 53 young healthy adults who completed two scanning sessions (session 1 included two scans 20 min apart; session 2 included one scan that was performed after an interval of ∼6 weeks)., Results: The high-resolution networks exhibited prominent small-world and modular properties and included functional hubs mainly located at the default-mode, salience, and executive control systems. Further analysis revealed that test-retest reliabilities of network metrics were sensitive to the scanning orders and intervals, with fair to excellent long-term reliability between Scan 1 and Scan 3 and lower reliability involving Scan 2. In the long-term case (Scan 1 and Scan 3), most network metrics were generally test-retest reliable, with the highest reliability in global metrics in the clustering coefficient and in the nodal metrics in nodal degree and efficiency., Conclusion: We showed high test-retest reliability for graph properties in the high-resolution functional connectomics, which provides important guidance for choosing reliable network metrics and analysis strategies in future studies., (© 2015 John Wiley & Sons Ltd.)

Y‑box binding protein‑1 (YB‑1) has been identified as an oncoprotein in various malignancies. The aim of this study was to investigate the biological role of YB‑1 and its association with epithelial‑to‑mesenchymal transition (EMT) in colorectal cancer (CRC). The expression of YB‑1 and three EMT‑related proteins (E‑cadherin, N‑cadherin and vimentin) was analyzed in 80 CRC and matched normal tissue samples, by immunohistochemistry. The results indicated that the expression of YB‑1 was higher in CRC tissue samples than that in matched normal controls and was significantly correlated with tumor differentiation, tumor invasion, lymph node metastasis and distant metastases. Furthermore, analysis showed that YB‑1 expression was negatively correlated with E‑cadherin and positively correlated with N‑cadherin and vimentin expression. In vitro assays showed that knockdown of YB‑1 inhibited the proliferation, apoptosis resistance, invasion and migration of the HT‑29 CRC cell line. Of note, following knockdown of YB‑1, E‑cadherin expression was elevated whereas N‑cadherin and vimentin expression was reduced. Taken together, these results suggest that YB‑1 promotes the malignant progression of CRC in part through the induction of EMT, and YB‑1 may therefore be a potential novel target for CRC treatment.

β-elemene (β-ELE) is a new anticancer drug extracted from Curcuma zedoaria Roscoe and has been widely used to treat malignant tumors. Recent studies have demonstrated that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of action of β-ELE, we investigated its effects on cisplatin-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining and flow cytometry with Annexin V-FITC/PI double staining. Mitochondrial membrane potential was assessed using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorofluorescein-diacetate staining and flow cytometry. Cytosolic glutathione content was determined using GSH kits. The expression of cytochrome c, caspase-3, procaspase-3 and the Bcl-2 family proteins was assessed by western blotting. The results demonstrated that β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and decreased the cytoplasmic glutathione levels in a time- and dose-dependent manner. The combination of β-ELE and cisplatin enhanced the protein expression of cytochrome c, caspase-3 and Bad, and reduced protein levels of Bcl-2 and procaspase-3 in the A549/DDP lung cancer cells. These results define a pathway of procaspase‑3-β-ELE function that involves decreased mitochondrial membrane potential, leading to apoptosis triggered by the release of cytochrome c into the cytoplasm and the modulation of apoptosis-related genes. The reversal of drug resistance of the A549/DDP cell line by β-ELE may be derived from its effect in inducing apoptosis.

Fuzhuan brick-tea is a special dark tea prepared from the leaves of Camellia sinensis var. sinensis. Its production involves a fungal fermentation stage, which forms the unique flavors and functions by a series of biochemical reactions. Our phytochemical research of the material led to the isolation of two new B-ring fission lactones of flavan-3-ols, fuzhuanins A (1) and B (2). In addition, three other flavan-3-ol derivatives (3-5), three flavone C-glycosides (6-8), eight flavonoid O-glycosides (10-17), five simple phenolics (19-23), two norisoprenoid glycosides (24, 25), two sesquiterpenoids (26, 27), and theobromine (28), as well as two flavonoid anions (9 and 18), were also identified. The structures of these compounds were determined by spectroscopic methods. Compounds 4, 19, 20, 22-24, 26, and 27 were reported for the first time in Camellia spp. and tea. Furthermore, HPLC analysis method was performed to compare the chemical constituents of the before/after fungal fermentation Fuzhuan brick-teas. Compound 1 was indicated as one of the major characteristic constituents generated in the fungal fermentation process. The IC50 value of the antiproliferative activity of 2 on HeLa cells was assayed as 4.48 μM. None of the isolated compounds showed any inhibition activity against the enteric pathogenic microbes at 800 μg/mL by the hole plate diffusion method.

Objective: To evaluate the biological effect on the synthesis of the extracellular matrix (ECM) in the cultivation of adult degenerative nucleus pulposus cells using the stiring microcarrier system in vitro., Methods: Thirty-four specimens were collected after intervertebral fusion operations of the patients with intervertebral disc herniation diseases from September 2005 to May 2009. The specimens were then randomly allocated into 2 groups for in vitro cultivation: monolayer culture group and microcarrier culture group. On the exponential phase, SP-ABC immunohistochemical staining and Western blot quantitative analysis were conducted in the two groups to detect the collagen type I and II. Proteoglycan contents of two groups in different growth phases were detected with (35)S-sulfate incorporation assay., Result: The expressions of collagen type I and II in microcarrier culture group were significantly higher than those in monolayer culture group: SP-ABC immunohistochemical staining (collagen type I: 32.5 ± 4.4 vs. 15.2 ± 1.2, t = 2.871, P < 0.01; collagen type II: 43.6 ± 4.1 vs. 23.1 ± 2.2, t = 2.375, P < 0.05); Western blot quantitative analysis (collagen type I: 0.62 ± 0.08 vs. 0.50 ± 0.06, t = 3.327, P < 0.01; collagen type II: 1.46 ± 0.08 vs. 0.86 ± 0.04, t = 2.453, P < 0.05). Nucleus pulposus cells cultivated in stiring microcarrier system showed significantly increased proteoglycan synthesis than monolayer culture group does on both exponential phase and stationary phase (exponential phase: 34 821 ± 312 vs. 21 046 ± 673, t = 2.134, P < 0.05; stationary phase: 45 134 ± 175 vs. 32 193 ± 713, t = 2.801, P < 0.01)., Conclusions: The expression of collagen type I, II and proteoglycan of adult degenerative nucleus pulposus cells are positive regulated by the stiring microcarrier system, which can be used in the mass amplification of the adult degenerative nucleus pulposus cells.

This study investigated the efficacy of a combination gene therapy to repress interleukin-1 (IL-1) and receptor activator of nuclear factor NF-kappa B ligand (RANKL) for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism of the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra (retroviral vector encoding IL-1 receptor antagonist) and AAV-OPG (adeno-associated viral vectors-osteoprotegerin) individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by enzyme-linked immunosorbent assay, and gene expressions of RANK, IL-1β, c-Fos, TRAF6, JNK1 and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given 3 weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8 weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significant diminishment of TRAF6 expression.

Objective: To compare laparoscopic Nissen fundoplication (LNF)and Toupet laparoscopic fundoplication (LTF) with respect to treatment outcomes and postoperative complications., Methods: PubMed, Medline, Embase and the Cochrane Library were searched. Only randomized controlled trials (RCTs) comparing laparoscopic Nissen and Toupet fundoplication were included. Outcomes evaluation included occurrences of heartburn, reflux, difficulty swallowing, chest pain, abdominal distention, failure to hiccup, diarrhea, and early complications and degree of patient satisfaction at early (three to six months) and later (one to three years) post-operative periods., Results: Of 939 patients in seven RCTs, 478 received LNF and 461 received LTF. For both groups, control of reflux was good and occurrence of heartburn was similar (P>0.05). A lower incidence of postoperative dysphagia for both early and later post-operative periods, but a higher overall complication rate in early post-operative period were observed in the LTF group (P<0.05). Patient satisfaction was similar (P>0.05)., Conclusions: LNF and LTF are both safe and effective. The adoption of procedure should be based on the patient status and surgeon experience.

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.

Circulating tumor cells (CTCs) from peripheral blood are emerging as a useful tool for the detection of malignancy, monitoring disease progression, and measuring response to therapy. We describe a unique microfluidic chip that was capable of efficient and selective separation of CTCs from peripheral whole blood samples. The ability of microfluidic chip to capture CTCs from PBS and whole blood samples was tested. Sixty-eight peripheral blood samples from 68 colorectal cancer patients were investigated for the presence of CTCs by microchip technology. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. We also examined samples from 20 healthy individuals as controls. The calculated capture efficiency was 85.7% and decreased significantly at flow rates above 2.0 ml/h. The number of CTCs isolated ranged from 3 to 236/ml for colorectal patients [99 +/- 64 (mean +/- SD) CTCs/ml]. None of the 20 healthy subjects had any identifiable CTCs. We identified CTCs in 46 (67.65%) of the 68 patients: in two of nine (22.22%) Dukes A, in 10 of 24 (41.67%) Dukes B, in 21 of 22 (95.45%) Dukes C, and in all 13 Dukes D patients. The detection rate in Dukes C and D patients was much higher than in Dukes A and B patients (97.73% vs. 36.36%) (p < 0.01). A significant correlation between detection of CTCs and clinical stage (r = 0.792, p < 0.01) was found, which was higher than carcinoembryonic antigen (r = 0.285, p > 0.01), carbohydrate antigen 19-9 (r = 0.258, p > 0.01), alpha-fetoprotein (r = 0.096, p > 0.01), and cancer antigen 125 (r = 0.134, p > 0.01). Microfluidic chip provides a novel method for capturing CTCs. The presence of CTCs correlated with clinical stage. It is important to evaluate CK-positive and DAPI-stained tumor cells together to determine the role of CTCs in tumor behavior and disease progression.

Cobalt (II) polyamidomine dendrimer was prepared by the reaction of cobalt chloride, glyoxal and polyamidomine dendrimer of 5.0 generation. The interaction of cobalt (II) polyamidomine dendrimer complex with herring sperm (hsDNA) was carried out using methylene blue (MB) as the probe molecule by absorption and fluorescence spectroscopy and synchronous fluorescence spectroscopy. The results showed that the intensity of absorption peaks and fluorescence peaks increased when the complex interacted with hsDNA. The effect of sodium chloride showed that sodium ion can significantly constrain the interaction of cobalt(II) polyamidomine dendrimer with hsDNA. The curves indicated the competitive inhibition of MB binding to hsDNA in the presence of cobalt (II) polyamidomine dendrimer complexes, also MB could insert into interior of cobalt (II) polyamidomine dendrimer complexes. The results suggested that the complex mainly interacted with negatively charged phosphate moieties on hsDNA through electrostatic attraction and stacked on the surface of double stranded hsDNA, which may reduce the binding affinity of MB to hsDNA in the surrounding site. It was indicated that sodium ion might neutralize the negatively charged phosphate backbone of hsDNA, and then weaken the electrostatic attraction between complexes and hsDNA.

With the planted forest ecosystems of Cercidiphyllum japonicum, Betula utilis, Pinus yunnansinsis, and Picea asperata in subalpine area of west Sichuan as test objects, their total biomass and the C and N contents in soils and tree organs were determined. The results showed that the allocation of C in tree organs had less correlation with the age of the organs, while that of N and C/N ratio had closer relationship with the age. The N content in young organs was higher than that in aged ones, whereas the C/N ratio was higher in aged organs than in young organs, and higher in the leaf litters of needle-leaved forests than in those of broad-leaved forests. There was an obvious enrichment of C and N in the topsoil of test forests. The accumulated amounts of C and N in the whole planted forest ecosystem, including tree, litter, and 0-40 cm soil layer, were 176.75-228.05 t x hm(-2) and 11.06-16.54 t x hm(-2), respectively, and the nutrients allocation ratio between soil-litter and tree was (1.9-3.3):1 for C and (15.6-41.5):1 for N. Needle-leaved forests functioned as a stronger "C-sink" than broad-leaved forests. The decomposition rate of the leaf litters in needle-leaved forests was larger than that in broad-leaved forests, with the turnover rate being 2.2-3.7 years and 3.9-4.2 years, respectively. During the decomposition of leaf litter, the C in all of the four forests released at super-speed, with the turnover rate being 1.9-3.4 years. As for N, it also released at super-speed in C. japonicum and B. utilis forests, with the turnover rate being 1.9-3.2 years, but released at low speed in P. yunnansinsis and P. asperata forests, with the turnover rate being 6.7-8.5 years.

Through 2 years leaf litter replacement experiments in 4 typical artificial pure forests Larix kaempferi, Pinus tabulaeformis, Catalpa fargesii, and Quercus aliena var. acuteserrata in Qinling Mountains of China, this paper studied the effects of leaf litter replacement on soil biological and chemical characteristics and the interspecific relationships between different tree species. The results showed that the annual decomposition rate of broad-leaved litter was 33.70% higher than that of needle-leaved litter. The annual decomposition rate of needle-leaved litter increased by 8.35%-12.15% when replaced to broad-leaved forests, whereas that of broad-leaved litter decreased by 5.38%-9.49% when replaced to needle-leaved forests. Leaf litter replacement between needle and broad-leaved forests could increase the contents of soil organic-C and available N, P and K, and the increments were obviously higher in needle-leaved forests (8.70%-35.84%) than in broad-leaved forests (3.73%-10.44%). In needle-leaved forests, the increments with the replacement of C. fargesii litter (24.63%-35.84%) were higher than those with the replacement of Q. aliena var. acuteserrata litter (8.70%-28.15%). Furthermore, the replacement of broad-leaved litter could make the soil pH in needle-leaved forests changed from light-acid to neutral, and increase soil enzyme activities, microbial amounts, and microbial biomass C and N contents. The increments with the replacement of C. fargesii litter were higher than those with the replacement of Q. aliena var. acuteserrata litter. The soil enzyme activities, microbial amounts, and microbial biomass C and N contents in broad-leaved forests after the replacement of needle-leaved litter differed with broadleaved tree species. Q. aliena var. acuteserrata forest had the higher soil enzyme activities and microbial biomass C and N contents, while C. fargesii forest was in adverse. It was suggested that in the control of soil degradation under artificial pure forests, much attention should be paid to the direction of interspecific relationship in mixed forestation and leaf litter replacement.