Duevega6695

Our results indicate that removal of OCT from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer, and to determine the long-term stability of sphingolipids in OCT-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT-cryopreserved normal lung tissues. This validated sphingolipidomic OCT-removal protocol should be a valuable addition to the lipid biologist's toolbox. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Lipid kinases and phosphatases play key roles in cell signaling and regulation, and are implicated in many human diseases, and are hence thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Due Because of to its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including Class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided the newdetailed mechanistic information about the product inhibition and substrate acyl acyl-chain selectivity of PI3K and allowed enabled rapid evaluation of its small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological functionThe assay also quantitatively determined the substrate specificity of PTEN, thereby providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.BACKGROUND Mendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke. Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate therapeutic management and genetic counselling. Next-generation sequencing makes large-scale sequencing and genetic testing possible. METHODS A systematic literature search was conducted to identify causal genes of Mendelian strokes, which were used to construct a hybridization-based gene capture panel. Genetic variants for target genes were detected using Illumina HiSeq X10 and the Novaseq platform. The sensitivity and specificity were evaluated by comparing the results with Sanger sequencing. RESULTS 53 suspected patients of Mendelian strokes were analysed using the panel of 181 causal genes. According to the American College of Medical Genetics and Genomics standard, 16 likely pathogenic/variants of uncertain significance genetic variants were identified. Diagnostic testing was conducted by comparing the consistency between the results of panel and Sanger sequencing. Both the sensitivity and specificity were 100% for the panel. CONCLUSION This panel provides an economical, time-saving and labour-saving method to detect causal mutations of Mendelian strokes. © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.The elements Zinc (Zn) and cadmium (Cd) have similar chemical and physical properties, but contrasting physiological effects in higher organisms. In plants, Zn/Cd transport is mediated by various transporter proteins belonging to different families. In this study, we functionally characterized two Zn transporter genes in rice (Oryza sativa), ZINC TRANSPORTER5 (OsZIP5) and ZINC TRANSPORTER9 (OsZIP9), which are tandem duplicates and act synergistically in Zn/Cd uptake. Both genes encode plasma membrane-localized proteins with influx transporter activity. The expression profiles of OsZIP5 and OsZIP9 overlap in the root epidermis and respond to the local Zn status in the root. However, OsZIP9 is also regulated by systemic signals of Zn status from the shoot. OsZIP5 functions redundantly to OsZIP9, but has a relatively weaker effect. Plants with the knockout mutations oszip5, oszip9, or oszip5oszip9 show impaired Zn/Cd uptake. The decreased Zn/Cd levels and growth retardation in the oszip5 mutant are less severe than in the oszip9 mutant. However, the double mutant oszip5oszip9 showed an enhanced Zn deficiency phenotype compared with the single mutants, and few double knockout plants were able to survive the entire growth cycle without excessive Zn supply. Transgenic plants overexpressing OsZIP9 had markedly enhanced Zn/Cd levels in the aboveground tissues and brown rice. The results of our study fill a gap in current knowledge of Zn uptake and improve our understanding of Zn/Cd accumulation in rice. copyright, serif 2020 American Society of Plant Biologists. All rights reserved.BACKGROUND There are 43 000 new cases of hepatitis B virus infection and 1000 cases of perinatally acquired infection each year in the United States. National recommendations are to administer hepatitis B (HepB) vaccine to all stable newborns >2000 g within 24 hours of birth. Our primary objective was to increase institutional vaccination rates from a baseline of 52% to goal >85% before hospital discharge. METHODS In February 2017, we instituted a multidisciplinary quality improvement project aimed at increasing HepB vaccination birth dose rates. Interventions included (1) standardizing the process of offering HepB vaccine via scripting and timing, (2) engaging and educating parents, and (3) educating physicians and nurses regarding the importance of HepB vaccination and strategies to discuss HepB vaccination with vaccine-hesitant parents. The main outcome measure was the percentage of newborns receiving HepB vaccination by discharge. The secondary outcome was the percentage of newborns receiving HepB vaccination by 12 hours of life per New York State Department of Health recommendation. Data were analyzed by using statistical process control P-charts. RESULTS A total of 21 108 newborns were included between July 2015 and April 2019. In addition to several upward centerline shifts, implementation of interventions resulted in increased and sustained HepB vaccination rates by hospital discharge from a baseline of 52.4% to 72.5%. Rates by 12 hours of life increased from 21.5% to 42.5%. CONCLUSIONS Multidisciplinary collaboration, scripting, and provider and staff education regarding the risks of hepatitis B virus, benefits of HepB vaccine, and strategies to discuss HepB vaccination with parents significantly increased vaccination rates. Further efforts to improve vaccination rates to within 12 hours are preferable. Copyright © 2020 by the American Academy of Pediatrics.Many transferable quinolone-resistance mechanisms have been already identified in Gram-negative bacteria. The plasmid-encoded 65 amino-acid long ciprofloxacin-modifying enzyme, namely CrpP, was recently identified in Pseudomonas aeruginosa We analyzed a collection of 100 clonally-unrelated and multidrug-resistant P. aeruginosa clinical isolates among which 46 (46%) were found positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. Those crpP-like genes were chromosomally located, as part of PAGI-like pathogenicity genomic islands. Copyright © 2020 American Society for Microbiology.One of the reasons for the lengthy tuberculosis (TB) treatment is the difficult to treat non-multiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our pre-clinical drug development pipeline. In most available dormancy models it takes a long time to create a dormant state and it is difficult to identify and quantify this non-multiplying condition.The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes non-multiplying after 10 days of streptomycin starvation, but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetic model to assess the difference in the activity of isoniazid, rifampicin, moxifloxacin and bedaquiline against log-phase growth compared to the non-multiplying M. tuberculosis subpopulation by CFU counting including a novel AUC-based approach as well as time-to-positivity (TTP) measurements.We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampicin and high dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value as it identifies a specific mycobacterial subpopulation that is non-culturable on solid media.In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline. Copyright © 2020 American Society for Microbiology.Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathologic bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal's "quorum-quenching" activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard of care antibiotics. PR-957 supplier The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis, and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo Similarly, locally-delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of sub-therapeutic vancomycin. However, we also found that the resorbable polyurethane foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard of care antibiotic therapy for S. aureus osteomyelitis, but also highlight potential pitfalls encountered with local drug delivery. Copyright © 2020 American Society for Microbiology.