Effect of Comprehensive Oncogenetics Training Interventions for General Practitioners,Evaluated at Multiple Performance Levels

General practitioners (GPs) are increasingly called upon to identify patients at risk for hereditary cancers, and their genetic competencies need to be enhanced. This article gives an overview of a research project on how to build effective educational modules on genetics, assessed by randomized controlled trials (RCTs), reflecting the prioritized educational needs of primary care physicians. It also reports on an ongoing study to investigate long-term increase in genetic consultation skills (1-year follow-up) and interest in and satisfaction with a supportive website on genetics among GPs. Three oncogenetics modules were developed: an online Continuing Professional Development (G-eCPD) module, a live genetic CPD module, and a “GP and genetics” website (huisartsengenetica.nl) providing further genetics information applicable in daily practice. Three assessments to evaluate the effectiveness (1-year follow-up) of the oncogenetic modules were designed: 1.An online questionnaire on self-reported genetic competencies and changes in referral behaviour, 2.Referral rates from GPs to clinical genetics centres and 3.Satisfaction questionnaire and visitor count analytics of supportive genetics website. The setting was Primary care in the Netherlands and three groups of study participants were included in the reported studies:. Assessment 1. 168 GPs responded to an email invitation and were randomly assigned to an intervention or control group, evaluating the G-eCPD module (n = 80) or the live module (n = 88). Assessment 2. Referral rates by GPs were requested from the clinical genetics centres, in the northern and southern parts of the Netherlands (Amsterdam and Maastricht), for the two years before (2010 [n = 2510] and 2011 [n = 2940]) and the year after (2012 [n = 2875]) launch of the oncogenetics CPD modules and the website. Assessment 3. Participants of the website evaluation were all recruited online. When they visited the website during the month of February 2013, a pop-up invitation came up. Of the 1350 unique visitors that month, only 38 completed the online questionnaire. Main outcomes measure showed long-term (self-reported) genetic consultation skills (i.e. increased genetics awareness and referrals to clinical genetics centres) among GPs who participated in the oncogenetic training course, and interest in and satisfaction with the supportive website. 42 GPs (52%) who previously participated in the G-eCPD evaluation study and 50 GPs (57%) who participated in the live training programme responded to the online questionnaire on long-term effects of educational outcome. Previous RCTs showed that the genetics CPD modules achieved sustained improvement of oncogenetic knowledge and consultation skills (3-months follow-up). Participants of these RCTs reported being more aware of genetic problems long term; this was reported by 29 GPs (69%) and 46 GPs (92%) participating in the G-eCPD and live module evaluation studies, respectively (Chisquare test, p<0.005). One year later, 68% of the respondents attending the live training reported that they more frequently referred patients to the clinical genetics centres, compared to 29% of those who attended the online oncogenetics training (Chisquare test, p<0.0005). However, the clinical genetics centres reported no significant change in referral numbers one year after the training. Website visitor numbers increased, as did satisfaction, reflected in a 7.7 and 8.1 (out of 10) global rating of the website (by G-eCPD and live module participants, respectively). The page most often consulted was “family tree drawing”. Self-perceived genetic consultation skills increased long-term and GPs were interested in and satisfied with the supportive website. Further studies are necessary to see whether the oncogenetics CPD modules result in more efficient referral. The results presented suggest we have provided a flexible and effective framework to meet the need for effective educational programmes for non-geneticist healthcare providers, enabling improvement of genetic medical care.     

The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO₂ transport facilitator

Cellular exchange of carbon dioxide (CO₂) is of extraordinary importance for life. Despite this significance, its molecular mechanisms are still unclear and a matter of controversy. In contrast to other living organisms, plants are physiologically limited by the availability of CO₂. In most plants, net photosynthesis is directly dependent on CO₂ diffusion from the atmosphere to the chloroplast. Thus, it is important to analyze CO₂ transport with regards to its effect on photosynthesis. A mutation of the Arabidopsis thaliana AtPIP1;2 gene, which was characterized as a non‐water transporting but CO₂ transport‐facilitating aquaporin in heterologous expression systems, correlated with a reduction in photosynthesis under a wide range of atmospheric CO₂ concentrations. Here, we could demonstrate that the effect was caused by reduced CO₂ conductivity in leaf tissue. It is concluded that the AtPIP1;2 gene product limits CO₂ diffusion and photosynthesis in leaves.     

Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization

The suggestion that genetic divergence can arise and/or be maintained in the face of gene flow has been contentious since first proposed. This controversy and a rarity of good examples have limited our understanding of this process. Partially reproductively isolated taxa have been highlighted as offering unique opportunities for identifying the mechanisms underlying divergence with gene flow. The African malaria vector, Anopheles gambiae s.s., is widely regarded as consisting of two sympatric forms, thought by many to represent incipient species, the M and S molecular forms. However, there has been much debate about the extent of reproductive isolation between M and S, with one view positing that divergence may have arisen and is being maintained in the presence of gene flow, and the other proposing a more advanced speciation process with little realized gene flow because of low hybrid fitness. These hypotheses have been difficult to address because hybrids are typically rare (<1%). Here, we assess samples from an area of high hybridization and demonstrate that hybrids are fit and responsible for extensive introgression. Nonetheless, we show that strong divergent selection at a subset of loci combined with highly asymmetric introgression has enabled M and S to remain genetically differentiated despite extensive gene flow. We propose that the extent of reproductive isolation between M and S varies across West Africa resulting in a 'geographic mosaic of reproductive isolation'; a finding which adds further complexity to our understanding of divergence in this taxon and which has considerable implications for transgenic control strategies.     

Live covisualization of competing adeno-associated virus and herpes simplex virus type 1 DNA replication: molecular mechanisms of interaction

We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.     

Apoptosis effects of Xrel3 c-Rel/Nuclear Factor-kappa B homolog in human cervical cancer cells

Cervical cancer is considered a common yet preventable cause of death in women. In this report, we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. The expression of Xrel3/c-Rel slowed cell growth 6-fold, consistent with an upregulated expression of the cell cycle inhibitor p21. The activated PARP apoptosis effector was significantly increased (P<0.01). Based on cell viability assays Xrel3 provided an anti-apoptotic effect in 1 microM cisplatin, and this was associated with significantly lower levels of the apoptotic proteins Bax and MDM-2 (P<0.05). Furthermore, there was a 3-fold drop in the level of the tumor suppressor protein p53. In 5 microM cisplatin, expression of HeLa Xrel3 enhanced apoptosis by significantly increasing the expression of the apoptotic proteins Bax and MDM-2 (P<0.05). However, the tumor suppressor protein p53 showed a significant decrease (P<0.05) relative to the control. Thus, c-Rel/NF-kappaB may potentially be of clinical significance, especially in tumors exhibiting resistance to high-level chemotherapy.     

Reverse and conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 microg/l) and nonanal (0.15 ng/microl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture.     

Targeting residual inflammatory risk in coronary disease: to catch a monkey by its tail

Patients with coronary disease remain at high risk for future cardiovascular events, even with optimal risk factor modification, lipid-lowering drugs and antithrombotic regimens. A myriad of inflammatory pathways contribute to progression of the atherosclerotic burden in these patients. Only in the last few years has the inflammatory biology of atherosclerosis translated into clinical therapeutic options. Low-dose colchicine can provide a clinically relevant reduction in the risk for composite and individual major cardiovascular outcomes in patients with acute and chronic coronary syndromes. Among others, its anti-inflammatory effects in atherosclerosis seem to be related to neutrophil recruitment and adhesion, inflammasome inhibition, and morphological changes in platelets and platelet aggregation. Future research is aimed at further elucidating its particular mechanism of action, as well as identifying patients with the highest expected benefit and evaluating efficacy in other vascular beds. These data will help to formulate the role of colchicine and other anti-inflammatory drugs in patients with coronary disease and atherosclerosis in general in the near future.