LncRNA PVT1 is increased in renal cell carcinoma and affects viability and migration in vitro

BackgroundRenal cell carcinoma is difficult to diagnose and unpredictable in disease course and severity. There are no specific biomarkers for diagnosis and prognosis estimation feasible in clinical practice. Long non‐coding RNAs (lncRNAs) have emerged as potent regulators of gene expression in recent years. Aside from their cellular role, their expression patterns could be used as a biomarker of ongoing pathology.MethodsIn this work, we used next‐generation sequencing for global lncRNA expression profiling in tumor and non‐tumor tissue of RCC patients. The four candidate lncRNAs have been further validated on an independent cohort. PVT1, as the most promising lncRNA, has also been studied using functional in vitro tests.ResultsNext‐generation sequencing showed significant dysregulation of 1163 lncRNAs; among them top 20 dysregulated lncRNAs were AC061975.7, {"type":"entrez-nucleotide","attrs":{"text":"AC124017.1","term_id":"21327652","term_text":"AC124017.1"}}AC124017.1, {"type":"entrez-nucleotide","attrs":{"text":"AP000696.1","term_id":"6705905","term_text":"AP000696.1"}}AP000696.1, AC148477.4, LINC02437, GATA3‐AS, LINC01762, LINC01230, LINC01271, LINC01187, LINC00472, {"type":"entrez-nucleotide","attrs":{"text":"AC007849.1","term_id":"5091570","term_text":"AC007849.1"}}AC007849.1, LINC00982, LINC01543, {"type":"entrez-nucleotide","attrs":{"text":"AL031710.1","term_id":"3821014","term_text":"AL031710.1"}}AL031710.1, and {"type":"entrez-nucleotide","attrs":{"text":"AC019197.1","term_id":"6648325","term_text":"AC019197.1"}}AC019197.1 as down‐regulated lncRNAs; and SLC16A1‐AS1, PVT1, LINC0887, and LUCAT1 as up‐regulated lncRNAs. We observed statistically significant dysregulation of PVT1, LUCAT1, and LINC00982. Moreover, we studied the effect of artificial PVT1 decrease in renal cell line 786–0 and observed an effect on cell viability and migration.ConclusionOur results show not only the diagnostic but also the therapeutic potential of PVT1 in renal cell carcinoma.     

Development of six immune‐related lncRNA signature prognostic model for smoking‐positive lung adenocarcinoma

BackgroundSmoking is one of the most hazardous risk factors for the development of lung adenocarcinoma (LUAD). Many survival and prognosis‐related biomarkers were discovered using database mining. However, the precision of immune‐related long noncoding RNAs (lncRNAs) predictions is insufficient. We identified a novel signature to improve the estimate of smoking‐related LUAD prognosis.MethodsThe Cancer Genome Atlas database (TCGA) was used to obtain the LUAD lncRNA expression profiles. The smoking‐related LUAD cohort was randomly split into discovery and validation cohorts. To determine the risk score, use the LASSO Cox regression technique on the prognostic immune‐related lncRNA. The risk signature has been developed.ResultsA total of 643 immune‐related lncRNAs were identified as potential candidates for a risk signature. Finally, six immune‐related lncRNAs ({"type":"entrez-nucleotide","attrs":{"text":"AL359915.2","term_id":"8894448","term_text":"AL359915.2"}}AL359915.2, {"type":"entrez-nucleotide","attrs":{"text":"AP000695.1","term_id":"6705904","term_text":"AP000695.1"}}AP000695.1, HSPC324, TGFB2‐AS1, {"type":"entrez-nucleotide","attrs":{"text":"AC026355.1","term_id":"7276276","term_text":"AC026355.1"}}AC026355.1, and AC002128.2) were identified and used to carry out risk signature, which showed a close association with overall survival in the discovery cohort. We classified patients as high risk or low risk based on a median risk score of 1.0783. In the discovery cohort, overall survival was marginally longer in the low‐risk group than in the high‐risk category (p = 2.28e08). The area under the curves (AUC) for 1‐, 3‐, and 5‐year survival was 0.67, 0.7, and 0.82, respectively. Furthermore, we successfully validated and combined cohorts using this risk profile. We discovered a strong positive connection between HSPC324 and VIPR1 as a possible novel biomarker for smoking‐related LUAD development in our study.ConclusionsOur research has established a six immune‐lncRNA signature that may be used to predict the prognosis of smoking‐related LUAD with great accuracy.     

AC016405.3 functions as an oncogenic long non‐coding RNA by regulating ERBB3 via sponging miR‐22‐3p in breast cancer

BackgroundIncreasing studies reported that long non‐coding RNAs are involved in regulating breast cancer (BRCA) progression. However, the specific roles and mechanisms of lncRNAs in BRCA remain largely unknown. Here, we sought to explore the functions and mechanisms of {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 in BRCA progression.MethodsBioinformatic analysis for {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3, miR‐22‐3p, and ERBB3 were performed on starBase. The expressions of {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3, miR‐22‐3p, and ERBB3 were examined by RT‐qPCR. The functions of {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 on the proliferation, migration, and invasion of cells were evaluated by conducting CCK‐8, colony formation, wound‐healing, and Transwell assays. The subcellular distribution of {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 in BRCA cells was identified by performing fluorescence in situ hybridization (FISH) and subcellular fractionation techniques. Dual‐luciferase assay was applied to validate the interactions of miR‐22‐3p with {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 or ERBB3. The interaction between ERBB3 and miR‐22‐3p was also tested by Anti‐Ago2 RNA immunoprecipitation (RIP) assay.ResultsThe results showed that {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 is highly expressed in BRCA tissues as well as cells and positively correlated with poor prognosis in BRCA patients. Silencing {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 obviously repressed the malignant behaviors of BRCA cells. Mechanistically, {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 functioned as a competing endogenous RNA (ceRNA) for miR‐22‐3p in the cytoplasm and sponged miR‐22‐3p to release its suppression of ERBB3. Rescue experiments revealed that the suppression role induced by {"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 depletion on malignant behaviors of BRCA cells could be obviously counter by inhibiting miR‐22‐3p or overexpressing ERBB3.Conclusion{"type":"entrez-nucleotide","attrs":{"text":"AC016405.3","term_id":"7341689","term_text":"AC016405.3"}}AC016405.3 promotes BRCA progression by the derepression of ERBB3 via sponging miR‐22‐3p, which may represent a potential target for BRCA treatment.     

A novel prognostic model associated with the overall survival in patients with breast cancer based on lipid metabolism‐related long noncoding RNAs

BackgroundLipid metabolism is closely related to the occurrence and development of breast cancer. Our purpose was to establish a novel model based on lipid metabolism‐related long noncoding RNAs (lncRNAs) and evaluate the potential clinical value in predicting prognosis for patients suffering from breast cancer.MethodsRNA data and clinical information for breast cancer were obtained from the cancer genome atlas (TCGA) database. Lipid metabolism‐related lncRNAs were identified via the criteria of correlation coefficient |R ²| > 0.4 and p < 0.001, and prognostic lncRNAs were identified to establish model through Cox regression analysis. The training set and validation set were established to certify the feasibility, and all samples were separated into high‐risk group or low‐risk group. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were conducted to evaluate the potential biological functions, and the immune infiltration levels were explored through Cibersortx database.ResultsA total of 14 lncRNAs were identified as protective genes ({"type":"entrez-nucleotide","attrs":{"text":"AC022150.4","term_id":"7711716","term_text":"AC022150.4"}}AC022150.4, {"type":"entrez-nucleotide","attrs":{"text":"AC061992.1","term_id":"7630731","term_text":"AC061992.1"}}AC061992.1, AC090948.3, {"type":"entrez-nucleotide","attrs":{"text":"AC092794.1","term_id":"15027722","term_text":"AC092794.1"}}AC092794.1, {"type":"entrez-nucleotide","attrs":{"text":"AC107464.3","term_id":"19339290","term_text":"AC107464.3"}}AC107464.3, AL021707.8, {"type":"entrez-nucleotide","attrs":{"text":"AL451085.2","term_id":"13561296","term_text":"AL451085.2"}}AL451085.2, AL606834.2, FLJ42351, LINC00926, LINC01871, TNFRSF14−AS1, {"type":"entrez-nucleotide","attrs":{"text":"U73166.1","term_id":"1613889","term_text":"U73166.1"}}U73166.1 and USP30−AS1) with HRs < 1 while 10 lncRNAs ({"type":"entrez-nucleotide","attrs":{"text":"AC022150.2","term_id":"7021694","term_text":"AC022150.2"}}AC022150.2, {"type":"entrez-nucleotide","attrs":{"text":"AC090948.1","term_id":"13384352","term_text":"AC090948.1"}}AC090948.1, {"type":"entrez-nucleotide","attrs":{"text":"AC243960.1","term_id":"325121040","term_text":"AC243960.1"}}AC243960.1, AL021707.6, ITGB2−AS1, OTUD6B−AS1, SP2−AS1, TOLLIP−AS1, {"type":"entrez-nucleotide","attrs":{"text":"Z68871.1","term_id":"1164911","term_text":"Z68871.1"}}Z68871.1 and ZNF337−AS1) were associated with increased risk with HRs >1. A total of 24 prognostic lncRNAs were selected to construct the model. The patients in low‐risk group were associated with better prognosis in both training set (p < 0.001) and validation set (p < 0.001). The univariate and multivariate Cox regression analyses revealed that risk score was an independent prognostic factors in both training set (p < 0.001) and validation set (p < 0.001). GO and GSEA analyses revealed that these lncRNAs were related to metabolism‐related signal pathway and immune cells signal pathway. Risk score was negatively correlated with B cells (r = −0.097, p = 0.002), NK cells (r = −0.097, p = 0.002), Plasma cells (r = −0.111, p = 3.329e‐04), T‐cells CD4 (r = −0.064, p = 0.039) and T‐cells CD8 (r = −0.322, p = 2.357e‐26) and positively correlated with Dendritic cells (r = 0.077, p = 0.013) and Monocytes (r = 0.228, p = 1.107e‐13).ConclusionThe prognostic model based on lipid metabolism lncRNAs possessed an important value in survival prediction of breast cancer patients.     

Screening the hub genes and analyzing the mechanisms in discharged COVID‐19 patients retesting positive through bioinformatics analysis

BackgroundAfter encountering COVID‐19 patients who test positive again after discharge, our study analyzed the pathogenesis to further assess the risk and possibility of virus reactivation.MethodsA separate microarray was acquired from the Gene Expression Omnibus (GEO), and its samples were divided into two groups: a “convalescent‐RTP” group consisting of convalescent and “retesting positive” (RTP) patients (group CR) and a “healthy‐RTP” group consisting of healthy control and RTP patients (group HR). The enrichment analysis was performed with R software, obtaining the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the protein–protein interaction (PPI) networks of each group were established, and the hub genes were discovered using the cytoHubba plugin.ResultsIn this study, 6622 differentially expressed genes were identified in the group CR, among which RAB11B‐AS1, DISP1, MICAL3, PSMG1, and DOCK4 were up‐regulated genes, and ANAPC1, IGLV1‐40, SORT1, PLPPR2, and ATP1A1‐AS1 were down‐regulated. 7335 genes were screened in the group HR, including the top 5 up‐regulated genes ALKBH6, AMBRA1, MIR1249, TRAV18, and LRRC69, and the top 5 down‐regulated genes FAM241B, {"type":"entrez-nucleotide","attrs":{"text":"AC018529.3","term_id":"6653321","term_text":"AC018529.3"}}AC018529.3, {"type":"entrez-nucleotide","attrs":{"text":"AL031963.3","term_id":"5650585","term_text":"AL031963.3"}}AL031963.3, {"type":"entrez-nucleotide","attrs":{"text":"AC006946.1","term_id":"4314404","term_text":"AC006946.1"}}AC006946.1, and FAM149B1. The GO and KEGG analysis of the two groups revealed a significant enrichment in immune response and apoptosis. In the PPI network constructed, group CR and group HR identified 10 genes, respectively, and TP53BP1, SNRPD1, and SNRPD2 were selected as hub genes.ConclusionsUsing the messenger ribonucleic acid (mRNA) expression data from {"type":"entrez-geo","attrs":{"text":"GSE166253","term_id":"166253"}}GSE166253, we found TP53BP1, SNRPD1, and SNRPD2 as hub genes in RTP patients, which is vital to the management and prognostic prediction of RTP patients.     

Patient-derived bladder cancer xenograft models reveal VEGF and CDK4 enhancing tumor metastasis behavior

New strategies to treat advanced bladder cancer are urgently required. A patient-derived xenograft (PDX) model has become a useful tool to evaluate chemotherapeutics and investigate personalized cancer treatment options. In this study, fresh human bladder cancer specimens ({"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303 and {"type":"entrez-nucleotide","attrs":{"text":"C21391","term_id":"1622501","term_text":"C21391"}}C21391) were transplanted subcutaneously into nude mice to establish PDX models. These models well retained pathological characteristics and molecular markers of the original tumor. Primary cells derived from {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303 were cultured and perfused into the bladders of nude mice to establish orthotopic xenograft models. Evident signals of lung and kidney metastases were detected by whole-body optical imaging. Gemcitabine displayed a significant therapeutic effect on the subcutaneous or orthotopic xenograft tumors of {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303. In vitro, matrigel invasion assays showed that {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303 primary culture cells are remarkably invasive. To study the metastatic properties of {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303, we sequenced the genomes of {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303 and {"type":"entrez-nucleotide","attrs":{"text":"C21391","term_id":"1622501","term_text":"C21391"}}C21391, and found that the expression of VEGF and CDK4 was significantly upregulated, and VEGF-knockdown or CDK4-knockdown {"type":"entrez-nucleotide","attrs":{"text":"C09303","term_id":"1534374","term_text":"C09303"}}C09303 cells showed attenuated invasiveness and migration. This PDX model revealed that VEGF and CDK4 enhanced tumor metastasis behavior, suggesting them as novel molecular therapeutic targets. This framework provides a tool for research on metastasis mechanism and individualized treatment for bladder cancer.

New strategies to treat advanced bladder cancer are urgently required.     

Neurodegenerative disorder and diffuse brain calcifications due to FARSB mutation in two siblings

Pathogenic mutations in the FARSB gene are associated with neurodevelopmental disorder involving the brain, liver, and lungs. We report genetic analysis of a family including two affected members with this disorder, which revealed a homozygous pathogenic missense variant, FARSB: {"type":"entrez-nucleotide","attrs":{"text":"NM_005687.4","term_id":"743405629","term_text":"NM_005687.4"}}NM_005687.4:c.853G > A:p.E285K in both affected patients. The parents were heterozygous for this variant.     

Novel truncating variant in KMT2E associated with cerebellar hypoplasia and velopharyngeal dysfunction

KMT2E‐related neurodevelopmental disorder is a recently described intellectual disability syndrome often with speech difficulties. Here, we describe an individual with a heterozygous frameshift variant in KMT2E ({"type":"entrez-nucleotide","attrs":{"text":"NM_182931.2","term_id":"91199541","term_text":"NM_182931.2"}}NM_182931.2:c.2334_2337delTTAC, p.[Tyr779AlafsTer41]), intellectual disability, cerebellar hypoplasia, and velopharyngeal dysfunction. This case suggests potential mechanisms of speech disturbance in the disorder, requiring further investigation.     

First Report of a Clinical,Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon,Tn1721

In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain {"type":"entrez-nucleotide","attrs":{"text":"HS102707","term_id":"312647496","term_text":"HS102707"}}HS102707 isolate and an Enterobacter aerogenes strain {"type":"entrez-nucleotide","attrs":{"text":"HS112625","term_id":"313253202","term_text":"HS112625"}}HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla_KPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain {"type":"entrez-nucleotide","attrs":{"text":"HS092839","term_id":"312447864","term_text":"HS092839"}}HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla_KPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla_KPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla_KPC-2 colocated in the same Tn1721-Tn3–like composite transposon on a novel IncP group plasmid.     

Activation of p38 Mitogen-Activated Protein Kinase Promotes Peritoneal Fibrosis by Regulating Fibrocytes

♦ Background: Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, and yet the precise pathogenic mechanisms of peritoneal fibrosis remain unknown. Fibrocytes participate in tissue fibrosis and express chemokine receptors that are necessary for migration. The p38 mitogen-activated protein kinase (MAPK) pathway regulates the production of chemokines and has been demonstrated to contribute to the pathogenesis of various fibrotic conditions. Accordingly, we used an experimental mouse model of peritoneal fibrosis to examine the dependency of fibrocytes on p38MAPK signaling.♦ Methods: Peritoneal fibrosis was induced in mice by the injection of 0.1% chlorhexidine gluconate (CG) into the abdominal cavity. Mice were treated with {"type":"entrez-nucleotide","attrs":{"text":"FR167653","term_id":"258093044","term_text":"FR167653"}}FR167653, a specific inhibitor of p38MAPK, and immunohistochemical studies were performed to detect fibrocytes and cells positive for phosphorylated p38MAPK. The involvement of p38MAPK in the activation of fibrocytes also was also investigated in vitro.♦ Results: Fibrocytes infiltrated peritoneum in response to CG, and that response was accompanied by progressive peritoneal fibrosis. The phosphorylation of p38MAPK, as defined by CD45+ spindle-shaped cells, was detected both in peritoneal mesothelial cells and in fibrocytes. The level of peritoneal expression of CCL2, a chemoattractant for fibrocytes, was upregulated by CG injection, and treatment with {"type":"entrez-nucleotide","attrs":{"text":"FR167653","term_id":"258093044","term_text":"FR167653"}}FR167653 reduced the number of cells positive for phosphorylated p38MAPK, the peritoneal expression of CCL2, and the extent of peritoneal fibrosis. Pretreatment with {"type":"entrez-nucleotide","attrs":{"text":"FR167653","term_id":"258093044","term_text":"FR167653"}}FR167653 inhibited the expression of procollagen type I α1 induced by transforming growth factor-β1.♦ Conclusions: Our results suggest that p38MAPK signaling contributes to peritoneal fibrosis by regulating fibrocyte function.     

Crystal Structures of KPC-2 and SHV-1 β-Lactamases in Complex with the Boronic Acid Transition State Analog S02030

Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to β-lactamases emerges in part via the expression of KPC-2 and SHV-1 β-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this β-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism-based inactivators (e.g., clavulanate). In order to develop new β-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 for its inhibition of KPC-2 and SHV-1. {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54- and 1.87-Å resolution crystal structures of {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 bound to SHV-1 and KPC-2 β-lactamases, respectively, as well as a comparative analysis of the {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 binding modes, including a previously determined {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 class C ADC-7 β-lactamase complex. {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 is able to inhibit vastly different serine β-lactamases by interacting with the conserved features of these active sites, which includes (i) forming the bond with catalytic serine via the boron atom, (ii) positioning one of the boronic acid oxygens in the oxyanion hole, and (iii) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, {"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030 is able to overcome more distantly located structural differences between the β-lactamases. This unique feature is achieved by repositioning the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring, allowing it to potentially form a unique C—H…O 2.9-Å hydrogen bond with S130 in KPC-2.     

Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats.

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.     

Novel splicing‐site mutation in DCAF17 gene causing Woodhouse‐Sakati syndrome in a large consanguineous family

BackgroundWoodhouse‐Sakati syndrome is a rare autosomal recessive disease with endocrine and neuroectodermal aberrations with heterogeneous phenotypes and disease course. The most common phenotypes of the disease are progressive sensorineural hearing loss and alopecia, mild‐to‐moderate mental retardation and hypogonadism. The disease results from mutations in the DCAF17 gene.MethodHere, we reported a large consanguineous pedigree with multiple affected individuals with Woodhouse‐Sakati syndrome phenotypes. Laboratory tests confirmed the endocrine perturbance in affected individuals. To find out the underlying genetic change, whole‐exome sequencing was carried out.ResultAnalysis of the exome data identified a splicing‐site deletion {"type":"entrez-nucleotide","attrs":{"text":"NM_025000.3","term_id":"259906387","term_text":"NM_025000.3"}}NM_025000.3:c.1423‐1_1425delGACA in DCAF17 gene. Sanger sequencing confirmed the co‐segregation of the variant with the disease phenotypes in the family.ConclusionThe variant is predicted to cause aberrant splicing, i.e., exon skipping, resulting in the translation of a truncated functionless protein which results in appearance of typical phenotypic features and clinical laboratory findings of Woodhouse‐Sakati syndrome in affected members of the family.     

Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of {"type":"entrez-nucleotide","attrs":{"text":"A40926","term_id":"2296837","term_text":"A40926"}}A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the β-lactam-sensitive d,d-peptidase/d,d-carboxypeptidase encoded by vanY_n, the only van-like gene found in the {"type":"entrez-nucleotide","attrs":{"text":"A40926","term_id":"2296837","term_text":"A40926"}}A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanY_n gene dosage and expressed vanH_atA_atX_at from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanY_n, complementing a vanY_n mutant, or duplicating vanY_n had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanY_n activity. The heterologous expression of vanH_atA_atX_at increased {"type":"entrez-nucleotide","attrs":{"text":"A40926","term_id":"2296837","term_text":"A40926"}}A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanY_n-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.     

Application of Blood Group Genotyping by Next-Generation Sequencing in Various Immunohaematology Cases

BackgroundNext-generation sequencing (NGS) technology has been recently introduced into blood group genotyping; however, there are few studies using NGS-based blood group genotyping in real-world clinical settings. In this study, we applied NGS-based blood group genotyping into various immunohaematology cases encountered in routine clinical practice.MethodsThis study included 4 immunohaematology cases: ABO subgroup, ABO chimerism, antibody to a high-frequency antigen (HFA), and anti-CD47 interference. We designed a hybridization capture-based NGS panel targeting 39 blood group-related genes and applied it to the 4 cases.ResultsNGS analysis revealed a novel intronic variant ({"type":"entrez-nucleotide","attrs":{"text":"NM_020469.3","term_id":"1482586037","term_text":"NM_020469.3"}}NM_020469.3:c.29-10T>G) in a patient with an A_el phenotype and detected a small fraction of ABO*A1.02 (approximately 3–6%) coexisting with the major genotype ABO*B.01/O.01.02 in dizygotic twins. In addition, NGS analysis found a homozygous stop-gain variant ({"type":"entrez-nucleotide","attrs":{"text":"NM_004827.3","term_id":"1519311581","term_text":"NM_004827.3"}}NM_004827.3:c.376C>T, p.Gln126*; ABCG2*01N.01) in a patient with an antibody to an HFA; consequently, this patient''s phenotype was predicted as Jr(a−). Lastly, blood group phenotypes predicted by NGS were concordant with those determined by serology in 2 patients treated with anti-CD47 drugs.ConclusionNGS-based blood group genotyping can be used for identifying ABO subgroup alleles, low levels of blood group chimerism, and antibodies to HFAs. Furthermore, it can be applied to extended blood group antigen matching for patients treated with anti-CD47 drugs.     

HIV-specific humoral responses benefit from stronger prime in phase Ib clinical trial

BACKGROUND. Vector prime-boost immunization strategies induce strong cellular and humoral immune responses. We examined the priming dose and administration order of heterologous vectors in HIV Vaccine Trials Network 078 (HVTN 078), a randomized, double-blind phase Ib clinical trial to evaluate the safety and immunogenicity of heterologous prime-boost regimens, with a New York vaccinia HIV clade B (NYVAC-B) vaccine and a recombinant adenovirus 5–vectored (rAd5-vectored) vaccine.METHODS. NYVAC-B included HIV-1 clade B Gag-Pol-Nef and gp120, while rAd5 included HIV-1 clade B Gag-Pol and clades A, B, and C gp140. Eighty Ad5-seronegative subjects were randomized to receive 2 × NYVAC-B followed by 1 × 10¹⁰ PFU rAd5 (NYVAC/Ad5_hi); 1 × 10⁸ PFU rAd5 followed by 2 × NYVAC-B (Ad5_lo/NYVAC); 1 × 10⁹ PFU rAd5 followed by 2 × NYVAC-B (Ad5_med/NYVAC); 1 × 10¹⁰ PFU rAd5 followed by 2 × NYVAC-B (Ad5_hi/NYVAC); or placebo. Immune responses were assessed 2 weeks after the final vaccination. Intracellular cytokine staining measured T cells producing IFN-γ and/or IL-2; cross-clade and epitope-specific binding antibodies were determined; and neutralizing antibodies (nAbs) were assessed with 6 tier 1 viruses.RESULTS. CD4⁺ T cell response rates ranged from 42.9% to 93.3%. NYVAC/Ad5_hi response rates (P ≤ 0.01) and magnitudes (P ≤ 0.03) were significantly lower than those of other groups. CD8⁺ T cell response rates ranged from 65.5% to 85.7%. NYVAC/Ad5_hi magnitudes were significantly lower than those of other groups (P ≤ 0.04). IgG response rates to the group M consensus gp140 were 89.7% for NYVAC/Ad5_hi and 21.4%, 84.6%, and 100% for Ad5_lo/NYVAC, Ad5_med/NYVAC, and Ad5_hi/NYVAC, respectively, and were similar for other vaccine proteins. Overall nAb responses were low, but aggregate responses appeared stronger for Ad5_med/NYVAC and Ad5_hi/NYVAC than for NYVAC/Ad5_hi.CONCLUSIONS. rAd5 prime followed by NYVAC boost is superior to the reverse regimen for both vaccine-induced cellular and humoral immune responses. Higher Ad5 priming doses significantly increased binding and nAbs. These data provide a basis for optimizing the design of future clinical trials testing vector-based heterologous prime-boost strategies.TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00961883","term_id":"NCT00961883"}}NCT00961883.FUNDING. NIAID, NIH UM1AI068618, {"type":"entrez-nucleotide","attrs":{"text":"AI068635","term_id":"3391610","term_text":"AI068635"}}AI068635, {"type":"entrez-nucleotide","attrs":{"text":"AI068614","term_id":"3391589","term_text":"AI068614"}}AI068614, and {"type":"entrez-nucleotide","attrs":{"text":"AI069443","term_id":"3392418","term_text":"AI069443"}}AI069443.