A practical guide to amplicon and metagenomic analysis of microbiome data

Advances in high-throughput sequencing(HTS)have fostered rapid developments in the field of microbiome research,and massive microbiome datasets are now being generated.However,the diversity of software tools and the complexity of analysis pipelines make it difficult to access this field.Here,we systematically summarize the advantages and limitations of micro-biome methods.Then,we recommend specific pipelines for amplicon and metagenomic analyses,and describe commonly-used software and databases,to help researchers select the appropriate tools.Furthermore,we introduce statistical and visualization methods suit-able for microbiome analysis,including alpha-and beta-diversity,taxonomic composition,difference compar-isons,correlation,networks,machine learning,evolu-tion,source tracing,and common visualization styles to help researchers make informed choices.Finally,a step-by-step reproducible analysis guide is introduced.We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the bio-logical significance behind the data.     

Anti-inflammatory mechanisms and research progress of colchicine in atherosclerotic therapy

Inflammatory responses play a vital role in the onset and development of atherosclerosis, and throughout the entire process of the chronic disease. The inflammatory responses in atherosclerosis are mainly mediated by the NLRP3 inflammasome and its downstream inflammatory factors. As a powerful anti-inflammatory medicine, colchicine has a history of more than 200 years in clinical application and is the first-choice treatment for immune diseases such as gout and familial Mediterranean fever. In atherosclerosis, colchicine can inhibit the assembly and activation of NLRP3 inflammasome via various mechanisms to effectively reduce the expression of inflammatory factors, thereby reducing the inflammation. Recent clinical trials show that a low dose of colchicine (0.5 mg per day) has a certain protective effect in stable angina patients or those with acute myocardial infarction after PCI. This article summarizes and discusses the mechanisms of colchicine in the treatment of atherosclerosis and the latest research progress.     

ARL4C might serve as a prognostic factor and a novel therapeutic target for gastric cancer: bioinformatics analyses and biological experiments

The ADP-ribosylation factor-like proteins (ARLs) have been proved to regulate the malignant phenotypes of several cancers. However, the exact role of ARLs in gastric cancer (GC) remains elusive. In this study, we systematically investigate the expression status, interactive relations, potential pathways, genetic variations and clinical values of ARLs in GC. We find that ARLs are significantly dysregulated in GC and involved in various cancer-related pathways. Subsequently, machine learning models identify ARL4C as one of the two most significant clinical indicators among ARLs for GC. Furthermore, ARL4C silencing remarkably inhibits the growth and metastasis of GC cells both in vitro and in vivo. Moreover, enrichment analysis indicates that ARL4C is highly correlated with TGF-β1 signalling. Correspondingly, TGF-β1 treatment dramatically increases ARL4C expression and ARL4C knockdown inhibits the phosphorylation level of Smads, downstream factors of TGF-β1. Meanwhile, the coexpression of ARL4C and TGF-β1 worsens the prognosis of GC patients. Our work comprehensively demonstrates the crucial role of ARLs in the carcinogenesis of GC and the specific mechanisms underlying the GC-promoting effects of TGF-β1. More importantly, we uncover the great promise of ARL4C-targeted therapy in improving the efficacy of TGF-β1 inhibitors for GC patients.     

Floral fungal-bacterial community structure and co-occurrence patterns in four sympatric island plant species

Flowers’ fungal and bacterial communities can exert great impacts on host plant wellness and reproductive success—both directly and indirectly through species interactions. However, information about community structure and co-occurrence patterns in floral microbiome remains scarce. Here, using culture-independent methods, we investigated fungal and bacterial communities associated with stamens and pistils of four plant species (Scaevola taccada, Ipomoea cairica, Ipomoea pes-caprae, and Mussaenda kwangtungensis) growing together under the same environment conditions in an island located in South China. Plant species identity significantly influenced community composition of floral fungi but not bacteria. Stamen and pistil microbiomes did not differ in community composition, but differed in co-occurrence network topological features. Compared with the stamen network, pistil counterpart had fewer links between bacteria and fungi and showed more modular but less concentrated and connected structure. In addition, degree distribution of microbial network in each host species and each microhabitat (stamen or pistil) followed a significant power-law pattern. These results enhance our understanding in the assembly principles and ecological interactions of floral microbial communities.     

Temperature-related effects of treatments with jasmonic and salicylic acids on Arabidopsis infected with cucumber mosaic virus

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H₂O₂), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H₂O₂ and superoxide radical (O ₂ ^·? ) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

Pharmacokinetics Study of Amoxycillin and Clavulanic acid (8:1)-A New Combination in Healthy Chinese Adult Male Volunteers Using the LC–MS/MS Method

New oral granules of amoxicillin and clavulanic acid in 8:1 ratio have recently been developed and approved to conduct clinical trial in China. To date, there has been no report studying the pharmacokinetic characteristics of amoxicillin and clavulanic acid in man. Therefore, it is urgent to investigate the pharmacokinetic properties of amoxicillin and clavulanic acid in man. The aim of the study was to assess the pharmacokinetic properties of amoxicillin and clavulanic acid in 8:1 with different dosage in healthy volunteers and provide support for this drug to obtain marketing authorization in China. A liquid chromatography-tandem mass spectrometry method for determining the concentration of amoxicillin and clavulanic acid in human plasma was developed and applied to this open-label, single- and multiple-dose Pharmacokinetics study. Subjects were randomized to receive a single dose of 1, 2, and 4 pouches of the test granulation of amoxicillin and clavulanic acid in 8:1 ratio (amoxicillin is 250 mg and clavulanic acid is 31.25 mg per pouch). In the single-dose phase, blood samples were collected before dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 5, 8, 12, and 24 h after drug administration. In the multiple-dose phase, samples were obtained before drug administration on days 1, 2, 3, and 4 to determine the C_min of amoxicillin and clavulanic acid. In the 4th day, samples were collected from 0.25 to 24 h after drug administration. Profiles of the concentration–time curves of amoxicillin and clavulanic acid were best fitted to two-compartment model. In this group of healthy Chinese subjects, the pharmacokinetics of amoxicillin fitted the linear dynamic feature at doses of 250,500 and 1,000 mg, and not obviously about clavulanic acid at doses of 31.25, 62.5, and 125 mg. The t _1/2 of single dose and multidoses were (1.45 ± 0.12) and (1.44 ± 0.26) h of amoxicillin and (1.24 ± 0.23) and (1.24 ± 0.17) of clavulanic acid, respectively; The AUC_0–24 of single dose and multidoses were (27937.85 ± 4265.59) and (24569.80 ± 3663.63) ng h mL^?1 of amoxicillin and (891.45 ± 194.30) and (679.61 ± 284.05) ng h mL^?1 of clavulanic acid, respectively; The C_max of single dose and multidoses were (8414.58 ± 1416.78) and (7929.17 ± 1291.54) ng mL^?1 of amoxicillin and (349.00 ± 89.54) and (289.00 ± 67.36) ng h mL^?1 of clavulanic acid, respectively. t _1/2, AUC_0–24, and C_max were similar after multiple-dose administration and after single-dose administration, suggesting that amoxicillin and clavulanic acid do not accumulate with multiple-dose administration of 500 and 62.5 mg, respectively.     

Increased invasiveness of osteosarcoma mesenchymal stem cells induced by bone-morphogenetic protein-2

To evaluate the different traits of mesenchymal stem cell (MSC) isolated from osteosarcoma (OS) and normal bone marrow (BM) induced by bone-morphogenetic protein-2 (BMP-2). MSCs from implanted osteosarcoma or femur bone marrow were isolated and cultured. Differentiation potency was verified and phenotypes were evaluated by flow cytometry. Increased or decreased expressions of BMP-2 were delivered by adenovirus and lentivirus vector, respectively. Expressions of VEGF, EMMPRIN, and MMP-9 were examined. Cell cycle, apoptosis, invasiveness, and proliferation assays were performed between the transfected groups and controls. Increased BMP-2 induced over-expression of VEGF, EMMPRIN, and MMP-9 in OS- and BM-MSCs both intra- and extra-cellularly. Decreased BMP-2 expression induced inhibition of the factors. Increased BMP-2 also induced less population of cells at G1 phase, more apoptotic cells, more cells that invade through Transwell membrane, and faster proliferation in OSMSC compared to those in BMMSC. BMP-2 induced higher expression of tumorigenic factors, which could be responsible for promoting the proliferation and aggressiveness of OSMSC over BMMSC.     

Developmental changes of BKCa channels depend on differentiation status in cultured podocytes

The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Podocytes are of keen interests because of their key roles in kidney development and disease. Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca channels) are important ion channels located in podocytes and play the essential role in regulating calcium homeostasis cell signaling. In this research, we studied the undergoing developmental changes of BK_Ca channels and their contribution to functional maturation of podocytes. Our results showed that the distribution of BK_Ca channels changed with the maturity of differentiation in a conditionally immortalized mouse podocyte cell line. Additionally, the increase of BK_Ca channel protein expression was detected by immunofluorescence staining with confocal microscopy in podocytes, which was consistent with the increase in the current density of BK_Ca channels examined by whole-cell patch-clamp technique. Our results suggested that the developmental changes of BK_Ca channels may help podocytes adapt to changes in pressure gradients occurring in physiological conditions. Those findings may have implications for understanding the physiology and development of kidney and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in podocytes.