Best Practice for Identification of Classical 21-Hydroxylase Deficiency Should Include 21 Deoxycortisol Analysis with Appropriate Isomeric Steroid Separation.

There are mixed reports on the inclusion and use of 21 deoxycortisol (21DF) as the primary decision marker for classical 21-hydroxylase deficiency. We hypothesize that this may be due to insufficient recognition of the presence and chromatographic separation of isomeric steroids. The aim of this study was to determine the comparative utility of 21DF for screening and diagnosis of CAH due to classical 21-hydroxylase deficiency using a second-tier LC-MS/MS method that included the separation of isomeric steroids to 17OHP and 21DF. For each baby sample, one 3.2 mm dried blood spot was eluted in a methanolic solution containing isotopically matched internal standards. Data were interrogated by univariate and receiver operator characteristic analysis. Steroid profile results were generated for 924 non-CAH baby samples (median gestational age 37 weeks, range 22 to 43 weeks) and 17 babies with 21-hydroxylase deficiency. The ROC curves demonstrated 21DF to have the best sensitivity and specificity for the diagnosis of classical 21-hydroxylase deficiency with an AUC = 1.0. The heatmap showed the very strong correlation (r = 0.83) between 17OHP and 21DF. Our data support 21DF as a robust marker for CAH due to 21-hydroxylase deficiency. We recommend that 21DF be incorporated into routine newborn screening panels as part of the second-tier LC-MS/MS method, follow-up plasma steroid panels, and external quality assurance material.

The GSDMB rs7216389 SNP is associated with chronic rhinosinusitis in a multi-institutional cohort.

BACKGROUND: Chronic rhinosinusitis (CRS) is a multifactorial disease with a high co-occurrence with asthma. In this multicohort study, we tested whether single nucleotide polymorphisms (SNPs) associated with childhood asthma and rhinovirus (RV)-associated disease are related to an increased susceptibility to adult CRS in a multicohort retrospective case-control study. METHODS: Participants at two tertiary academic rhinology centers, University of Arizona (UofA) and University of Pennsylvania (UPenn) were recruited. Cases were defined as those with physician diagnosed CRS (UofA, n = 149; UPenn, n = 250), and healthy controls were those without CRS (UofA, n = 66; UPenn, n = 275). Genomic DNA was screened for the GSDMB rs7216389 SNP and CDHR3 rs6967330 SNP. Gene dosage, or the number of combined risk alleles in a single subject was calculated. Meta-analysis of the association between GSDMB or CDHR3 genotypes and CRS was performed and additive gene dosage effect for each population calculated using p for trend. RESULTS: A meta-analysis revealed a combined increased risk for CRS in subjects with the GSDMB rs7216389 SNP (odds ratio [OR] 1.40; 95% confidence interval [CI], 1.16-1.76; p = 0.004). Both the UofA (OR 1.73; 95% CI, 1.23-2.43; p = 0.002) and UPenn (OR 1.27; 95% CI, 1.02-1.58; p = 0.035) populations showed a significant positive association between the number of combined risk alleles of GSDMB rs7216389 SNP and CDHR3 rs6967330 SNP and risk for CRS. CONCLUSION: Carriers of the GSDMB rs7216389 SNP and CDHR3 rs6967330 SNP are at increased susceptibility for CRS. These data suggest that therapeutic approaches to target aberrant responses to RV infection may play a role in the treatment of unified airway disease.

Human Disease Ontology 2018 update: classification, content and workflow expansion.

The Human Disease Ontology (DO) (http://www.disease-ontology.org), database has undergone significant expansion in the past three years. The DO disease classification includes specific formal semantic rules to express meaningful disease models and has expanded from a single asserted classification to include multiple-inferred mechanistic disease classifications, thus providing novel perspectives on related diseases. Expansion of disease terms, alternative anatomy, cell type and genetic disease classifications and workflow automation highlight the updates for the DO since 2015. The enhanced breadth and depth of the DO's knowledgebase has expanded the DO's utility for exploring the multi-etiology of human disease, thus improving the capture and communication of health-related data across biomedical databases, bioinformatics tools, genomic and cancer resources and demonstrated by a 6.6× growth in DO's user community since 2015. The DO's continual integration of human disease knowledge, evidenced by the more than 200 SVN/GitHub releases/revisions, since previously reported in our DO 2015 NAR paper, includes the addition of 2650 new disease terms, a 30% increase of textual definitions, and an expanding suite of disease classification hierarchies constructed through defined logical axioms.

A phenotypic screening platform to identify small molecule modulators of Chlamydomonas reinhardtii growth, motility and photosynthesis.

Chemical biology, the interfacial discipline of using small molecules as probes to investigate biology, is a powerful approach of developing specific, rapidly acting tools that can be applied across organisms. The single-celled alga Chlamydomonas reinhardtii is an excellent model system because of its photosynthetic ability, cilia-related motility and simple genetics. We report the results of an automated fitness screen of 5,445 small molecules and subsequent assays on motility/phototaxis and photosynthesis. Cheminformatic analysis revealed active core structures and was used to construct a naïve Bayes model that successfully predicts algal bioactive compounds.

Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies.

Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.

Enhancement of immunocomplex detection and application to assays for DNA adduct of benzo[a]pyrene.

The stability of antibody and formation of immunocomplexes are essential to high-sensitivity capillary electrophoresis immunoassays (CEIA). However, little attention has been paid to enhancing or maintaining immunocomplex formation and antibody stability to improve the performance of CEIA. We report here the use of nonspecific proteins, such as bovine serum albumin (BSA) and rabbit immunoglobulin (rIgG), to enhance immunocomplex formation and to stabilize antibodies and immunocomplexes for immunoassays. Complexes between DNA adducts of benzo[a]pyrenediol epoxide (BPDE) and their antibodies were examined using capillary electrophoresis with laser-induced fluorescence detection (CE-HF). A tetramethylrhodamine (TMR)-labeled single-stranded oligonucleotide (16-mer) containing a single BPDE adduct was used as a fluorescent probe to study its immunocomplexes with a monoclonal antibody (8E11). To examine the formation of larger complexes, a TMR-labeled secondary antibody (anti-mouse), a primary antibody (mouse monoclonal antibody 5D11), and BPDE adducts in cellular DNA were used. We demonstrate that the use of nonspecific proteins stabilized the antibody and greatly enhanced the formation and stability of the immunocomplexes, resulting in substantial improvements in the detection limit (10-fold) and the reproducibility of the analysis. Another advantageous consequence of the stabilization was a 150-fold reduction of the concentration of the antibody needed for the immunoassay, resulting in reduced background and cost. We successfully applied this technique to the determination of DNA adducts of BPDE using a competitive immunoassay. The results from both small complexes (between a primary antibody and an oligonucleotide) and larger complexes (among a secondary antibody, a primary antibody, and cellular DNA) indicate that the technique can be extended to other immunoassays. We suggest that nonspecific proteins may assist the formation and stabilization of antibody-antigen complexes by maintaining the correct conformation of the antibody and antigen for optimum binding.

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.