Analysis of Bacterial Community Characteristics, Abundance of Antibiotics and Antibiotic Resistance Genes Along a Pollution Gradient of Ba River in Xi’an, China

The microbial communities in freshwater have raised concerns about the ecosystem and human health. Many ecological environmental problems have been found in urban river because of the unreasonable use and long-term wastewater discharge. In this study, we explored the bacterial community composition, abundance of 14 antibiotics and 21 antibiotic resistance genes (ARGs), and water environment features in seven water samples and seven sediment samples from Ba River in Xi’an, China. Results showed Proteobacteria and Bacteroidetes were the dominant phyla in all samples, and sediment samples had a higher bacterial diversity and richness than it in water. Bacterial communities of site 5 and 6 were clustered in discrepant patterns compared to those at remaining sites from other samples. It might be influenced by nutrients, heavy metals and antibiotics. Antibiotics concentrations ranged from 1.26 to 1.61 × 103 ng L-1 in water samples and 1.55 to 4.05 × 102 μg kg-1 in sediment samples. Sulfamerazine (SM1) and erythromycin (ERY) were the chief antibiotics in water samples, while the level of oxytetracycline (OTC) and cefazolin (CFZ) were higher in sediment samples. Canonical correspondence analysis showed that trimethoprim (TMP) was significantly related to Acinetobacter in W6, and that SM1 and OTC had positive correlation with Arcobacter in W5. The tetC, blaTEM,ermF and sul1 had higher pollution abundance ranging from 10-4 to 100 copies/16S rRNA gene copies in all samples. Significant correlations were observed between ARGs and matching antibiotics, suggesting that antibiotics can pose the selective pressure on ARGs in this river. In summary, these finding might provide some new data to the limited information available on the bacterial community characteristics, abundance of antibiotics and ARGs in urban river of China

Sustained NF-κB Activation and Inhibition in β-Cells Have Minimal Effects on Function and Islet Transplant Outcomes

The activation of the transcription factor NF-κB leads to changes in expression of many genes in pancreatic β-cells. However, the role of NF-κB activation in islet transplantation has not been fully elucidated. The aim of the present study was to investigate whether the state of NF-κB activation would influence the outcome of islet transplantation. Transgenic mice expressing a dominant active IKKβ (constitutively active) or a non-degradable form of IκBα (constitutive inhibition) under control of the rat insulin promoter were generated. Islets from these mice were transplanted into streptozotocin diabetic mice in suboptimal numbers. Further, the effects of salicylate (an inhibitor of NF-κB) treatment of normal islets prior to transplantation, and the effects of salicylate administration to mice prior to and after islet implantation were evaluated. Transplantation outcomes were not affected using islets expressing a non-degradable form of IκBα when compared to wild type controls. However, the transplantation outcomes using islets isolated from mice expressing a constitutively active mutant of NF-κB were marginally worse, although no aberrations of islet function in vitro could be detected. Salicylate treatment of normal islets or mice had no effect on transplantation outcome. The current study draws attention to the complexities of NF-κB in pancreatic beta cells by suggesting that they can adapt with normal or near normal function to both chronic activation and inhibition of this important transcription factor

The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls

Complex disease is characterized by the interplay of multiple genetic and environmental factors. Rheumatoid arthritis (RA) is a complex autoimmune disease with a pronounced genetic component, mainly due to HLA-DRB1 gene, but also a multitude of loci outside the HLA region. In this work we strive to contribute to the understanding of the functional involvement of these susceptibility loci in the pathogenesis of RA. This study is based on a large material of whole blood samples and peripheral blood mononuclear cells (PBMCs) from RA patients and matched healthy controls from Sweden. The main methods used in this work included probe-based genotyping and gene-expression assays, cell cultures, RNA-sequencing, gene-gene interaction and pathway analysis, as well as a plethora of common molecular genetics and bioinformatics methods. We investigated the role of expression of known genetic risk factors PTPN22 and PTPN2 in RA, with a special attention to the splicing profile of these genes. Our data indicates significant differences in the expression ratio of splice variants for PTPN22 in whole blood samples from RA patients and healthy controls. For PTPN2 we demonstrate a significant difference in the relative mRNA expression of' transcript TC48 in PBMCs of healthy controls and RA patients. Additionally, we identified new susceptibility SNPs in the PTPN2 locus: rs657555 and rs11080606, by addressing the interaction of PTPN2 variants with HLA-DRB1 shared-epitope (SE) alleles in autoantibody positive RA patients in two independent cohorts. In this work, we also address the functional genetic role of the members of the MAP signaling pathway upstream of p38 and JNK – crucial enzymes in RA – with a regard to splicing profile and their connection to HLA-DRB1. We found a significant statistical interaction for rs10468473 from MAP2K4 locus with SE alleles in autoantibody-positive RA. Importantly, individuals heterozygous for rs10468473 demonstrated higher expression of total MAP2K4 mRNA in blood, compared to A-allele homozygous. We also describe a novel, putatively translated RNA splice form of MAP2K4, that is differentially expressed in peripheral blood mononuclear cells from 88 RA cases and controls, and is modulated in response to TNF in Jurkat cell line. Finally, we performed an expression analysis of multiple validated RA risk loci, and pathway analysis to assess functional relationship between RA susceptibility genes and predict new potential study candidates. New candidate molecules suggested by the pathway analysis, genes ERBB2 and HSPB1, as well as HLA-DRB1, were differentially expressed between RA patients and healthy individuals in RNA-seq data. ERBB2 expression profile was similar in whole blood of both treated and untreated patients compared to healthy individuals. A similar expression profile was replicated for ERBB2 in PBMCs in an independent material. In this work, we approached the task of elucidating the functional aspects of genetic susceptibility of RA, by integrating genetic epidemiology, transcriptomics, proteomics, cellmodels, and bioinformatics. We maintain, that such integrative approach provides the rationale to prioritize genes and genetic events for further functional studies. Our findings also outline the need to consider potential clinical significance of alternative splicing in gene expression studies

Assessing agonistic potential of a candidate therapeutic anti-IL21R antibody

<p>Abstract</p> <p>Background</p> <p>Selective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under <it>in vitro </it>cross-linking and <it>in vivo </it>conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken.</p> <p>Methods</p> <p><it>In vitro </it>antibody cross-linking and cell culture protocols reported for studies with a human agonistic antibody, TGN1412, were followed for Ab-01. rhIL21, the agonist ligand of the targeted receptor, and cross-linked anti-CD28 were used as positive controls for signal transduction. <it>In vivo </it>agonistic potential of Ab-01 was assessed by measuring expression levels of cytokine storm-associated and IL21 pathway genes in blood of cynomolgus monkeys before and after IV administration of Ab-01.</p> <p>Results</p> <p>Using a comprehensive set of assays that detected multiple activation signals in the presence of the positive control agonists, <it>in vitro </it>Ab-01-dependent activation was not detected in either PBMCs or the rhIL21-responsive cell line Daudi. Furthermore, no difference in gene expression levels was detected in blood before and after <it>in vivo </it>Ab-01 dosing of cynomolgus monkeys.</p> <p>Conclusions</p> <p>Despite efforts to intentionally force an agonistic signal from Ab-01, none could be detected.</p

Clinical presentation of hemophagocytic lymphohistiocytosis in adults is less typical than in children

OBJECTIVE: Hemophagocytic lymphohistiocytosis in adults is largely underdiagnosed. To improve the rate and accuracy of diagnosis in adults, the clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis were analyzed in and compared between adults and children in a Chinese cohort. METHOD: Data from 50 hemophagocytic lymphohistiocytosis patients, including 34 adults and 16 children who fulfilled the 2004 hemophagocytic lymphohistiocytosis diagnostic criteria, were collected and analyzed. RESULTS: 1. Etiological factors: The proportion of Epstein-Barr virus infection was lower in adults compared with children, whereas fungal infection and natural killer/T cell lymphoma were more frequent in adults (

Stable knockout of Mcart-1 down-regulates the proliferation and oxidative phosphorylation of RAW264.7 macrophages

Objective To construct a RAW264.7 macrophage cell strain with stable knockout of Mcart-1 by using CRISPR/Cas9 gene editing technique, and to detect its biological function. Methods Cas9 and sgRNA lentivirus were used to infect RAW264.7 macrophages in two steps. Positive cells were screened with puromycin and hygromycin, and single cells were plated by flow cytometry to obtain monoclonal cells; Expression of Cas9 and Mcart-1 was detected by qPCR and Western blot, and the mutation site was confirmed by sequence analysis. Cells number counting and carboxyfluorescein diacetate succinimdyl ester (CFSE) staining were used to detect cell proliferation; ATP detection kit was used to measure total ATP content in cells; Seahorse bioenergy analyzer was used to detect cell oxygen consumption rate(OCR) and glycolysis rate. Results Successfully constructed RAW264.7 cell strain with Mcart-1 stably knocked out, denoted as Mcart-1-/--RAW264.7; A frameshift mutation occurred in Mcart-1 gene in this cell strain; Compared with the wild-type RAW264.7 cell line (RAW264.7), the OCR increased and the extracellular acidification rate(ECAR)of Mcart-1-/--RAW264.7 cells decreased; The total ATP content in Mcart-1 knocked out cells decreased(P＜0.05),mitochondrial ATP production rate decreased (P＜0.01), while glycolytic ATP production rate increased (P＜0.01); The proliferation activity of RAW264.7 cells decreased after Mcart-1 was knocked out(P＜0.001). Conclusions The proliferation activity and oxidative phosphorylation level of RAW264.7 cells were both down-regulated after the Mcart-1 was stably knocked out. This cell strain is an important tool for exploring the function of cells in the tumor microenvironment

Evolution of the Microstructure and Mechanical Performance of As-Sprayed and Annealed Silicon Coating on Melt-Infiltrated Silicon Carbide Composites

In this study, silicon coating was deposited on melt-infiltrated SiC composites using atmospheric plasma spraying and then annealed at 1100 and 1250 °C for 1–10 h to investigate the effect of annealing on the layer. The microstructure and mechanical properties were evaluated using scanning electron microscopy, X-ray diffractometry, transmission electron microscopy, nano-indentation, and bond strength tests. A silicon layer with a homogeneous polycrystalline cubic structure was obtained without phase transition after annealing. After annealing, three features were observed at the interface, namely β-SiC/nano-oxide film/Si, Si-rich SiC/Si, and residual Si/nano-oxide film/Si. The nano-oxide film thickness was ≤100 nm and was well combined with SiC and silicon. Additionally, a good bond was formed between the silicon-rich SiC and silicon layer, resulting in a significant bond strength improvement from 11 to >30 MPa