Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role <it>san-1</it>, the MAD3 homologue, has in <it>Caenorhabditis elegans </it>embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable <it>san-1(ok1580) </it>deletion mutant.</p> <p>Results</p> <p>The <it>san-1(ok1580) </it>animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of <it>san-1(ok1580) </it>embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of <it>san-1(ok1580) </it>embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of <it>san-1(ok1580);hcp-1(RNAi) </it>embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the <it>san-1(ok1580);hcp-1(RNAi) </it>animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of <it>mdf-2(RNAi);hcp-1(RNAi) </it>embryos but not <it>bub-3(RNAi);hcp-1(RNAi) </it>embryos was also compromised. Finally, we found that <it>san-1(ok1580) </it>and <it>bub-3(RNAi)</it>, but not <it>hcp-1(RNAi) </it>embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.</p> <p>Conclusion</p> <p>Together, these data suggest that in the <it>C. elegans </it>embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that <it>san-1(ok1580);hcp-1(RNAi) </it>animals had a severe viability defect whereas in the <it>san-1(ok1580);hcp-2(RNAi) </it>and <it>san-1(ok1580);hcp-2(ok1757) </it>animals the viability defect was not as severe suggesting that <it>hcp-1 </it>and <it>hcp-2 </it>are not completely redundant.</p

Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson’s disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states

Soluble fms-Like Tyrosine Kinase 1 (sFlt1), Endoglin and Placental Growth Factor (PlGF) in Preeclampsia among High Risk Pregnancies

Background: Differences in circulating concentrations of antiangiogenic factors sFlt1 and soluble endoglin (sEng) and the pro-angiogenic growth factor PlGF are reported to precede the onset of preeclampsia weeks to months in low-risk pregnant women. The objective of this study was to investigate whether similar changes can be detected in pregnant women at high-risk to develop the syndrome. Methods: This study is a secondary analysis of the NICHD MFMU trial of aspirin to prevent preeclampsia in high-risk pregnancies. Serum samples were available from 194 women with pre-existing diabetes, 313 with chronic hypertension, 234 with multifetal gestation, and 252 with a history of preeclampsia in a previous pregnancy. Samples collected across pregnancy were analyzed in a blinded fashion for sFlt1, sEng and PlGF. Results: The odds of developing preeclampsia were significantly increased among women with multiple fetuses for each 2- fold elevation in sFlt1, sEng and the ratio of angiogenic factors (e.g. OR 2.18, 95% CI 1.46-3.32), and significantly decreased for each 2-fold elevation in circulating PlGF (OR 0.50, 95% CI 0.30-0.82) between 7 and 26 weeks' gestation. Cross-sectional analysis of the angiogenic factors across gestation showed significant differences during the third trimester in women who develop preeclampsia compared with appropriate controls in all high-risk groups. However, when data were examined in relation to the gestational week when preeclampsia was diagnosed only sFlt1 was significantly higher 2 to 5 weeks before the clinical onset of preeclampsia and only in women with previous preeclampsia. Conclusions: The pattern of elevated concentrations of sFlt1 and sEng, and low PlGF in high-risk pregnant subjects who develop preeclampsia is similar to that reported in low-risk pregnant women. However, differences in these factors among high-risk women who do and do not develop preeclampsia are modest, and do not appear to be clinically useful predictors in these high-risk pregnant women

Growth patterns of infants with in- utero HIV and ARV exposure in Cape Town, South Africa and Lusaka, Zambia

Background Infants born HIV-exposed yet remain uninfected (HEU) are at increased risk of poorer growth and health compared to infants born HIV-unexposed (HU). Whether maternal antiretroviral treatment (ART) in pregnancy ameliorates this risk of poorer growth is not well understood. Furthermore, whether risks are similar across high burden HIV settings has not been extensively explored. Methods We harmonized data from two prospective observational studies conducted in Cape Town, South Africa, and Lusaka, Zambia, to compare weight-for-age (WAZ), length-for-age (LAZ) and weight-for-length (WLZ) Z-scores between infants who were HEU and HU, converting infant anthropometric measures using World Health Organisation Growth Standards adjusted for age and sex. Linear mixed effects models were fit to identify risk factors for differences in anthropometrics at 6–10 weeks and 6 months by infant HIV exposures status and by timing of exposure to maternal ART, either from conception or later in gestation. Results Overall 773 mother-infant pairs were included across two countries: women living with HIV (WLHIV), 51% (n = 395) with 65% on ART at conception and 35% initiating treatment in pregnancy. In linear mixed effects models, WAZ and WLZ at 6–10 weeks were lower among infants who were HEU vs HU [β = − 0.29 (95% CI: − 0.46, − 0.12) and [β = − 0.42 (95% CI: − 0.68, − 0.16)] respectively after adjusting for maternal characteristics and infant feeding with a random intercept for country. At 6 months, LAZ was lower [β = − 0.28 CI: − 0.50, − 0.06)] among infants who were HEU, adjusting for the same variables, with no differences in WAZ and WLZ. Within cohort evaluations identified different results with higher LAZ among infants who were HEU from Zambia at 6–10 weeks, [β = + 0.34 CI: + 0.01, + 0.68)] and lower LAZ among infants who were HEU from South Africa [β = − 0.30 CI: − 0.59, − 0.01)] at 6 months, without other anthropometric differences at either site. Conclusion Infant growth trajectories differed by country, highlighting the importance of studying contextual influences on outcomes of infants who were HEU

Socio-cultural influences on the behaviour of South Asian women with diabetes in pregnancy: qualitative study using a multi-level theoretical approach

BACKGROUND: Diabetes in pregnancy is common in South Asians, especially those from low-income backgrounds, and leads to short-term morbidity and longer-term metabolic programming in mother and offspring. We sought to understand the multiple influences on behaviour (hence risks to metabolic health) of South Asian mothers and their unborn child, theorise how these influences interact and build over time, and inform the design of culturally congruent, multi-level interventions. METHODS: Our sample for this qualitative study was 45 women of Bangladeshi, Indian, Sri Lankan, or Pakistani origin aged 21-45 years with a history of diabetes in pregnancy, recruited from diabetes and antenatal services in two deprived London boroughs. Overall, 17 women shared their experiences of diabetes, pregnancy, and health services in group discussions and 28 women gave individual narrative interviews, facilitated by multilingual researchers, audiotaped, translated, and transcribed. Data were analysed using the constant comparative method, drawing on sociological and narrative theories. RESULTS: Key storylines (over-arching narratives) recurred across all ethnic groups studied. Short-term storylines depicted the experience of diabetic pregnancy as stressful, difficult to control, and associated with negative symptoms, especially tiredness. Taking exercise and restricting diet often worsened these symptoms and conflicted with advice from relatives and peers. Many women believed that exercise in pregnancy would damage the fetus and drain the mother's strength, and that eating would be strength-giving for mother and fetus. These short-term storylines were nested within medium-term storylines about family life, especially the cultural, practical, and material constraints of the traditional South Asian wife and mother role and past experiences of illness and healthcare, and within longer-term storylines about genetic, cultural, and material heritage - including migration, acculturation, and family memories of food insecurity. While peer advice was familiar, meaningful, and morally resonant, health education advice from clinicians was usually unfamiliar and devoid of cultural meaning. CONCLUSIONS: 'Behaviour change' interventions aimed at preventing and managing diabetes in South Asian women before and during pregnancy are likely to be ineffective if delivered in a socio-cultural vacuum. Individual education should be supplemented with community-level interventions to address the socio-material constraints and cultural frames within which behavioural 'choices' are made

Incident Tuberculosis during Antiretroviral Therapy Contributes to Suboptimal Immune Reconstitution in a Large Urban HIV Clinic in Sub-Saharan Africa

Antiretroviral therapy (ART) effectively decreases tuberculosis (TB) incidence long-term, but is associated with high TB incidence rates in the first 6 months. We sought to determine the incidence and the long-term effects of TB during ART on HIV treatment outcome, and the risk factors for incident TB during ART in a large urban HIV clinic in Uganda.Routinely collected longitudinal clinical data from all patients initiated on first-line ART was retrospectively analysed. 5,982 patients were included with a median baseline CD4+ T cell count (CD4 count) of 117 cells/mm(3) (interquartile range [IQR]; 42, 182). In the first 2 years, there were 336 (5.6%) incident TB events in 10,710 person-years (py) of follow-up (3.14 cases/100 pyar [95% CI 2.82-3.49]); incidence rates at 0-3, 3-6, 6-12 and 12-24 months were 11.25 (9.58-13.21), 6.27 (4.99-7.87), 2.47 (1.87-3.36) and 1.02 (0.80-1.31), respectively. Incident TB during ART was independently associated with baseline CD4 count of <50 cells/mm(3) (hazard ratio [HR] 1.84 [1.25-2.70], P = 0.002) and male gender (HR 1.68 [1.34-2.11], P<0.001). After two years on ART, the patients who had developed TB in the first 12 months had a significantly lower median CD4 count increase (184 cells/mm(3) [IQR; 107, 258, n = 118] vs 209 cells/mm(3) [124, 309, n = 2166], P = 0.01), a larger proportion of suboptimal immune reconstitution according to two definitions (increase in CD4 count <200 cells/mm(3): 57.4% vs 46.9%, P = 0.03, and absolute CD4 count <200 cells/mm(3): 30.4 vs 19.9%, P = 0.006), and a higher percentage of immunological failure according to the WHO criteria (13.6% vs 6.5%, P = 0.003). Incident TB during ART was independently associated with poor CD4 count recovery and fulfilling WHO immunological failure definitions.Incident TB during ART occurs most often within 3 months and in patients with CD4 counts less than 50 cells/mm(3). Incident TB during ART is associated with long-term impairment in immune recovery

General Practice and Pandemic Influenza: A Framework for Planning and Comparison of Plans in Five Countries

BACKGROUND: Although primary health care, and in particular, general practice will be at the frontline in the response to pandemic influenza, there are no frameworks to guide systematic planning for this task or to appraise available plans for their relevance to general practice. We aimed to develop a framework that will facilitate planning for general practice, and used it to appraise pandemic plans from Australia, England, USA, New Zealand and Canada. METHODOLOGY/PRINCIPAL FINDINGS: We adapted the Haddon matrix to develop the framework, populating its cells through a multi-method study that incorporated the peer-reviewed and grey literature, interviews with general practitioners, practice nurses and senior decision-makers, and desktop simulation exercises. We used the framework to analyse 89 publicly-available jurisdictional plans at similar managerial levels in the five countries. The framework identifies four functional domains: clinical care for influenza and other needs, public health responsibilities, the internal environment and the macro-environment of general practice. No plan addressed all four domains. Most plans either ignored or were sketchy about non-influenza clinical needs, and about the contribution of general practice to public health beyond surveillance. Collaborations between general practices were addressed in few plans, and inter-relationships with the broader health system, even less frequently. CONCLUSIONS: This is the first study to provide a framework to guide general practice planning for pandemic influenza. The framework helped identify critical shortcomings in available plans. Engaging general practice effectively in planning is challenging, particularly where governance structures for primary health care are weak. We identify implications for practice and for research