HIV-1 Particle Release Mediated by Vpu Is Distinct from That Mediated by p6

AbstractVpu and the C-terminal peptide of Gag (p6) are both HIV-1-encoded proteins that augment the release of virus particles from cells. We examined the functional relationship between these proteins and their activities during particle release. Our results indicate that efficient HIV-1 particle release from HeLa and Jurkat cells depends on the presence of Vpu. However, Vpu is dispensable for efficient release from Cos cells. In contrast, p6 is required for efficient release from Cos cells but not from Jurkat or HeLa cells. These data suggest that Vpu and p6 have distinct activities in virus exit from different cell lines. Intracellular proteolytic processing of Gag precursor protein is more complete in Cos cells than in HeLa cells. However, this processing has little or no effect on Vpu- or p6-mediated particle release. p6 is required for incorporation of yet another virus protein (Vpr) into cells but our data suggest that Vpr plays no role in p6-dependent particle release. Vpu also facilitates the degradation of CD4 in virus producing cells but, in contrast to particle release, the ability of Vpu to facilitate the degradation of CD4 is not cell line-dependent

Prediction of congenital hypothyroidism based on initial screening thyroid-stimulating-hormone

Background: In thyroid-stimulating-hormone (TSH)-based newborn congenital hypothyroidism (CH) screening programs, the optimal screening-TSH cutoff level is critical to ensuring that true cases of CH are not missed. Screening-TSH results can also be used to predict the likelihood of CH and guide appropriate clinical management. The purpose of this study is to evaluate the predictive value of various screening-TSH levels in predicting a diagnosis of CH in the Ontario Newborn Screening Program (ONSP). Methods: The initial screening and follow-up data of 444,744 full term infants born in Ontario, Canada from April 1, 2006 to March 31, 2010 were analyzed. Confirmed CH cases were based on local endocrinologists\u27 report and initiation of thyroxine treatment. Results: There were a total of 541 positive screening tests (~1/822 live births) of which 296 were true positives (~1:1,500 live births). Subjects were further subdivided based on screening-TSH and positive predictive values (PPV) were calculated. Twenty four percent in the 17-19.9 mIU/L range were true positives. In the 17-30 mIU/L range, 29 % were true positives with a significantly higher PPV for those sampled after (43 %) rather than before (25 %) 28 h of age (p \u3c 0.02). Seventy three percent of neonates with an initial screening-TSH of ≥ 30 mIU/L and 97 % of those with ≥ 40 mIU/L were later confirmed to have CH. Conclusions: Infants with modestly elevated screening positive TSH levels between 17 and 19.9 mIU/L have a significant risk (24 %) of having CH. The very high frequency of true positives in term newborns with initial TSH values ≥ 30mIU/L suggests that this group should be referred directly to a pediatric endocrinologist in an effort to expedite further assessment and treatment. Screen positives with a modestly elevated TSH values (17-19.9 mIU/L) need to be examined in more detail with extended follow-up data to determine if they have transient or permanent CH

Three novel mutations in KIF21A highlight the importance of the third coiled-coil stalk domain in the etiology of CFEOM1

<p>Abstract</p> <p>Background</p> <p>Congenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3) are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in <it>KIF21A</it>. <it>KIF21A </it>encodes a kinesin motor involved in anterograde axonal transport, and the familial and <it>de novo </it>mutations reported to date predictably alter one of only a few KIF21A amino acids – three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of <it>KIF21A </it>mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in <it>KIF21A</it>.</p> <p>Results</p> <p>Sixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported <it>de novo </it>KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in <it>KIF21A</it>; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands.</p> <p>Conclusion</p> <p>Analysis of sixteen CFEOM1 probands revealed three novel <it>KIF21A </it>mutations and confirmed three reported mutations, bringing the total number of reported <it>KIF21A </it>mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1.</p

Enantiomer-specific pharmacokinetics of D,L-3-hydroxybutyrate:Implications for the treatment of multiple acyl-CoA dehydrogenase deficiency

D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased Cmax and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for Cmax: 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (Cmax: 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB

The clinical application of genome-wide sequencing for monogenic diseases in Canada: Position statement of the Canadian College of medical geneticists

Purpose and scope: The aim of this Position Statement is to provide recommendations for Canadian medical geneticists, clinical laboratory geneticists, genetic counsellors and other physicians regarding the use of genome-wide sequencing of germline DNA in the context of clinical genetic diagnosis. This statement has been developed to facilitate the clinical translation and development of best practices for clinical genome-wide sequencing for genetic diagnosis of monogenic diseases in Canada; it does not address the clinical application of this technology in other fields such as molecular investigation of cancer or for population screening of healthy individuals. Methods of statement development: Two multidisciplinary groups consisting of medical geneticists, clinical laboratory geneticists, genetic counsellors, ethicists, lawyers and genetic researchers were assembled to review existing literature and guidelines on genome-wide sequencing for clinical genetic diagnosis in the context of monogenic diseases, and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the Canadian College of Medical Geneticists (CCMG) membership-at-large and, following incorporation of feedback, approved by the CCMG Board of Directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. Results and conclusions: Recommendations include (1) clinical genome-wide sequencing is an appropriate approach in the diagnostic assessment of a patient for whom there is suspicion of a significant monogenic disease that is associated with a high degree of genetic heterogeneity, or where specific genetic tests have failed to provide a diagnosis; (2) until the benefits of reporting incidental findings are established, we do not endorse the intentional clinical analysis of disease-associated genes other than those linked to the primary indication; and (3) clinicians should provide genetic counselling and obtain informed consent prior to undertaking clinical genome-wide sequencing. Counselling should include discussion of the limitations of testing, likelihood and implications of diagnosis and incidental findings, and the potential need for further analysis to facilitate clinical interpretation, including studies performed in a research setting. These recommendations will be routinely reevaluated as knowledge of diagnostic and clinical utility of clinical genome-wide sequencing improves. While the document was developed to direct practice in Canada, the applicability of the statement is broader and will be of interest to clinicians and health jurisdictions internationally

Accelerating Innovation in the Creation of Biovalue : The Cell and Gene Therapy Catapult

The field of regenerative medicine (RM) has considerable therapeutic promise that is proving difficult to realize. As a result, governments have supported the establishment of intermediary agencies to “accelerate” innovation. This paper examines in detail one such agency, the UK's Cell and Gene Therapy Catapult (CGTC). We describe CGTC’s role as an accelerator agency and its value-narrative, which combines both “health and wealth.” Drawing on the notion of socio-technical imaginaries, we unpack the tensions within this narrative and its instantiation as the CGTC cell therapy infrastructure is built and engages with other agencies, some of which have different priorities and roles to play within the RM field

Health Care for Mitochondrial Disorders in Canada: A Survey of Physicians

Background: An improved understanding of diagnostic and treatment practices for patients with rare primary mitochondrial disorders can support benchmarking against guidelines and establish priorities for evaluative research. We aimed to describe physician care for patients with mitochondrial diseases in Canada, including variation in care. Methods: We conducted a cross-sectional survey of Canadian physicians involved in the diagnosis and/or ongoing care of patients with mitochondrial diseases. We used snowball sampling to identify potentially eligible participants, who were contacted by mail up to five times and invited to complete a questionnaire by mail or internet. The questionnaire addressed: personal experience in providing care for mitochondrial disorders; diagnostic and treatment practices; challenges in accessing tests or treatments; and views regarding research priorities. Results: We received 58 survey responses (52% response rate). Most respondents (83%) reported spending 20% or less of their clinical practice time caring for patients with mitochondrial disorders. We identified important variation in diagnostic care, although assessments frequently reported as diagnostically helpful (e.g., brain magnetic resonance imaging, MRI/MR spectroscopy) were also recommended in published guidelines. Approximately half (49%) of participants would recommend mitochondrial cocktails for all or most patients, but we identified variation in responses regarding specific vitamins and cofactors. A majority of physicians recommended studies on the development of effective therapies as the top research priority. Conclusions: While Canadian physicians\u27 views about diagnostic care and disease management are aligned with published recommendations, important variations in care reflect persistent areas of uncertainty and a need for empirical evidence to support and update standard protocols

Identification of a Thymic Epithelial Cell Subset Sharing Expression of the Class Ib HLA-G Molecule with Fetal Trophoblasts

HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the α2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G–expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium