Recipients’ Perspectives Regarding Expanded Carrier Screening of Gamete Donors

Purpose: This study explored the perspectives of intended parents regarding genetic carrier screening of a gamete donor. The main goal of this study was to determine how much genetic carrier screening information a recipient would prefer to receive about potential donors. The study also aimed to identify factors that potentially influence a recipient’s choice of donor based on genetic screening results. Methods: An online questionnaire was developed to assess intended parents’ preferences regarding expanded carrier screening (ECS) of their donors. Participants were recruited from various online support groups and were eligible if they had previously utilized or were currently utilizing donor gametes. A total of 58 usable responses were collected and reflect insight into the perspectives of intended parents regarding which factors associated with genetic carrier screening influence their choice of donor. The questionnaire consisted of demographic questions, general questions about carrier screening, a genetics knowledge quiz, and questions about hypothetical scenarios in which a donor was a carrier for one of four distinct conditions: hemochromatosis, Usher syndrome, Bardet-Biedl syndrome, and GRACILE syndrome. Results: The majority of women (91.4%, 53/58) opted for ECS of their potential gamete donor, preferred over traditional ethnicity- or family history-based screening. Participants were comfortable proceeding with a donor with the knowledge that he/she was a carrier for a mild genetic condition (hemochromatosis, 83.3%). Fewer respondents were comfortable proceeding with a donor who was a carrier for a more severe condition (Usher, 37.0%; BBS, 39.1%; GRACILE, 39.1%). Conclusion: Intended parents prefer ECS for their donors over traditional ethnicity- or family history-based screening. Participants were uncomfortable with a donor who is a carrier for severe, lifelimiting conditions, regardless of statistical risk. Expanded carrier screening is desired and could be beneficial for use in gamete donation; however, given the overall discomfort with identification of positive carrier status, ECS would significantly alter clinical decision making in these settings. Increased genetic education of recipients on the implications of ECS carrier results is indicated, and access to genetic counseling services may be indicated for optimal implementation

Characterization of degeneration in the retina, brain and spinal cord of the Cln1 knockout mouse

Abstract only availableThe neuronal-ceroid lipofuscinoses (NCLs; often referred to as Battens Disease) are a group of hereditary disorders of childhood. Symptoms of NCLs are characterized by neurodegeneration with progressive neural cell death in the retina and central nervous system (CNS). The infantile form of NCL results from a deficiency in the protein, palmitoyl protein thioesterase-1 (PPT-1). PPT-1, encoded by the Cln1 gene, removes long chain fatty acids from modified cysteine residues in proteins. Mutations in the Cln1 gene are associated with an accumulation of autofluorescent lysosomal lipopigments in various tissues such as the retina and CNS. In the current study, we use a transgenic mouse model in which the gene for Cln1 has been mutated, i.e., 'knocked out'. Our goal is to perform histological experiments to assess the functional progression of neurodegenerative changes in the retina, brain and spinal cord as the subject ages. The retina, brain and spinal cord of the mice at different ages were fixed and embedded in plastic resin and/or paraffin. Thick sections (1 mm or 10 mm, respectively) were stained with toluidine blue or propidium iodide to detect neuronal loss and/or apoptosis as a result of the PPT-1 deficiency. Fluorescent images of the stained sections were obtained to document changes in tissue structure and the extent of degeneration. These studies provide information that will aid future studies in which stem cell transplants will be made into the Cln1 knockout mouse model. Ultimately, this approach will determine whether combined gene and stem cell therapies can be applied to patients with Battens Disease.Molecular Biology Progra

Essential Role of Cdc42 in Ras-Induced Transformation Revealed by Gene Targeting

The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies

Association between depression, anxiety and weight change in young adults

Background To investigate whether there are bi-directional associations between anxiety and mood disorders and body mass index (BMI) in a cohort of young adults. Methods We analysed data from the 2004–2006 (baseline) and 2009–2011 (follow-up) waves of the Childhood Determinants of Adult Health study. Lifetime DSM-IV anxiety and mood disorders were retrospectively diagnosed with the Composite International Diagnostic Interview. Potential mediators were individually added to the base models to assess their potential role as a mediator of the associations. Results In males, presence of mood disorder history at baseline was positively associated with BMI gain (β = 0.77, 95% CI: 0.14–1.40), but baseline BMI was not associated with subsequent risk of mood disorder. Further adjustment for covariates, including dietary pattern, physical activity, and smoking reduced the coefficient (β) to 0.70 (95% CI: 0.01–1.39), suggesting that the increase in BMI was partly mediated by these factors. In females, presence of mood disorder history at baseline was not associated with subsequent weight gain, however, BMI at baseline was associated with higher risk of episode of mood disorder (RR per kg/m2: 1.04, 95% CI: 1.01–1.08), which was strengthened (RR per kg/m2 = 1.07, 95% CI: 1.00–1.15) after additional adjustment in the full model. There was no significant association between anxiety and change in BMI and vice-versa. Conclusion The results do not suggest bidirectional associations between anxiety and mood disorders, and change in BMI. Interventions promoting healthy lifestyle could contribute to reducing increase in BMI associated with mood disorder in males, and excess risk of mood disorder associated with BMI in females

Renal nerves contribute to hypertension in Schlager BPH/2J mice

Schlager mice (BPH/2J) are hypertensive due to a greater contribution of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). The kidneys of BPH/2J are hyper-innervated suggesting renal nerves may contribute to the hypertension. We therefore determined the effect of bilateral renal denervation (RD) on hypertension in BPH/2J. Mean arterial pressure (MAP) was measured by radiotelemetry before and for 3 weeks after RD in BPH/2J and BPN/3J. The effects of pentolinium and enalaprilat were examined to determine the contribution of the SNS and RAS, respectively. After 3 weeks, MAP was −10.9 ± 2.1 mmHg lower in RD BPH/2J compared to baseline and −2.1 ± 2.2 mmHg in sham BPH/2J (P < 0.001, n = 8–10). RD had no effect in BPN/3J (P > 0.1). The depressor response to pentolinium was greater in BPH/2J than BPN/3J, but in both cases the response in RD mice was similar to sham. Enalaprilat decreased MAP more in RD BPH/2J compared to sham (−12 vs −3 mmHg, P < 0.001) but had no effect in BPN/3J. RD reduced renal noradrenaline in both strains but more so in BPH/2J. RD reduced renin mRNA and protein, but not plasma renin in BPH/2J to levels comparable with BPN/3J mice. We conclude that renal nerves contribute to hypertension in BPH mice as RD induced a sustained fall in MAP, which was associated with a reduction of intrarenal renin expression. The lack of inhibition of the depressor effects of pentolinium and enalaprilat by RD suggests that vasoconstrictor effects of the SNS or RAS are not involved

Critical Role of Constitutive Type I Interferon Response in Bronchial Epithelial Cell to Influenza Infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead