A genome-wide association study of educational attainment was conducted in a discovery sample of 101,069 individuals and a replication sample of 25,490. Three independent SNPs are genome-wide significant (rs9320913, rs11584700, rs4851266), and all three replicate. Estimated effects sizes are small (R2 ≈ 0.02%), approximately 1 month of schooling per allele. A linear polygenic score from all measured SNPs accounts for ≈ 2% of the variance in both educational attainment and cognitive function. Genes in the region of the loci have previously been associated with health, cognitive, and central nervous system phenotypes, and bioinformatics analyses suggest the involvement of the anterior caudate nucleus. These findings provide promising candidate SNPs for follow-up work, and our effect size estimates can anchor power analyses in social-science genetics.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
Equity focused health impact assessments (EFHIAs), or health equity impact assessments, are being increasingly promoted internationally as a mechanism for enhancing the consideration of health equity in the development of policies, programs and projects. Despite this there are relatively few examples of examples of completed EFHIAs available. This paper presents a case study of a rapid EFHIA that was conducted in Australia on a health promotion policy implementation plan. It briefly describes the process and findings of the EFHIA and evaluates the impact on decision-making and implementation.
The rapid EFHIA was undertaken in four days, drawing on an expert panel and limited review of the literature. A process evaluation was undertaken by email one month after the EFHIA was completed. An impact evaluation was undertaken two years later based on five semi-structured interviews with members of the EFHIA working group and policy officers and managers responsible for implementing the plan. A cost estimation was conducted by the EFHIA working group.
The EFHIA made both general and specific recommendations about how the health equity impacts of the policy implementation plan could be improved. The impact evaluation identified changes to development and implementation that occurred as a result of the EFHIA, though there was disagreement about the extent to which changes could be attributed solely to the EFHIA. Those responsible considered the recommendations of the EFHIA in the next versions of their ABHI implementation plans. Factors that influenced the impact of the EFHIA included consolidating understandings of equity, enabling discussion of alternatives, and differing understandings of the purpose of the EFHIA. The EFHIA cost US$4,036 to undertake.
This EFHIA was conducted in a short timeframe using relatively few resources. It had some reported impacts on the development of the implementation plan and enhanced overall consideration of health equity. This case highlights some of the factors and preconditions that may maximise the impact of future EFHIAs on decision-making and implementation.
Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.
Diabetes is a chronic condition associated with many long-term complications. People with diabetes need to actively manage their condition, which can be complex. In consultations with healthcare professionals, patients receive advice about their diabetes but do not always discuss things which concern them, perhaps because of the perceived limited time or embarrassment. We want to test a ‘preconsultation’ intervention in which the patient is supported by a healthcare assistant to complete a web-based intervention aimed at producing an agenda to help them identify important areas for discussion in the consultation. Use of this agenda may enable the patient to play a more active role in that consultation and consequently become more confident, and hence more successful, in managing their condition.
Methods and analysis
In this pilot randomised controlled trial, 120 people with diabetes will be randomised with equal allocation to receive the intervention or usual clinical care. The primary outcome is reduction in glycosylated haemoglobin(HbA1c). Secondary outcomes are patient-reported communication, enablement, self-care activity, diabetes-dependent quality of life, empowerment, satisfaction, health-related quality of life and resource use. The aim of the pilot study was to estimate parameters to inform the design of the definitive trial. Follow-up on quantitative outcomes will be at 3 and 6 months. A nested qualitative study will collect data on the patients’ experiences of producing an agenda. Resource use data and medication use will also be collected via a review of medical records for a sample of participants.
Ethics and dissemination
Approval was granted by the NHS Research Ethics Committee North West—Preston (13/NW/0123). Dissemination will include publication of quantitative and qualitative findings, and experience of public involvement in peer-reviewed journals. Results will also be disseminated to trial participants via workshops led by lay coapplicants.
Quality in health care < HEALTH SERVICES ADMINISTRATION & MANAGEMENT; World Wide Web technology < BIOTECHNOLOGY & BIOINFORMATICS; Qualitative Research
Our objective was to compare long-term outcomes of deep brain stimulation (DBS) of the globus pallidus interna (GPi) and subthalamic nucleus (STN) for patients with Parkinson disease (PD) in a multicenter randomized controlled trial.
Patients randomly assigned to GPi (n = 89) or STN DBS (n = 70) were followed for 36 months. The primary outcome was motor function on stimulation/off medication using the Unified Parkinson's Disease Rating Scale motor subscale. Secondary outcomes included quality of life and neurocognitive function.
Motor function improved between baseline and 36 months for GPi (41.1 to 27.1; 95% confidence interval [CI] −16.4 to −10.8; p < 0.001) and STN (42.5 to 29.7; 95% CI −15.8 to −9.4; p < 0.001); improvements were similar between targets and stable over time (p = 0.59). Health-related quality of life improved at 6 months on all subscales (all p values significant), but improvement diminished over time. Mattis Dementia Rating Scale scores declined faster for STN than GPi patients (p = 0.01); other neurocognitive measures showed gradual decline overall.
The beneficial effect of DBS on motor function was stable and comparable by target over 36 months. Slight declines in quality of life following initial gains and gradual decline in neurocognitive function likely reflect underlying disease progression and highlight the importance of nonmotor symptoms in determining quality of life.
Classification of Evidence:
This study provides Class III evidence that improvement of motor symptoms of PD by DBS remains stable over 3 years and does not differ by surgical target. Neurology® 2012;79:55–65
Infections with Bartonella spp. have been recognized as emerging zoonotic diseases in humans. Large knowledge gaps exist, however, relating to reservoirs, vectors, and transmission of these bacteria. We describe identification by culture, PCR, and housekeeping gene sequencing of Bartonella spp. in fed, wingless deer keds (Lipoptena cervi), deer ked pupae, and blood samples collected from moose, Alces alces, sampled within the deer ked distribution range in Norway. Direct sequencing from moose blood sampled in a deer ked-free area also indicated Bartonella infection but at a much lower prevalence. The sequencing data suggested the presence of mixed infections involving two species of Bartonella within the deer ked range, while moose outside the range appeared to be infected with a single species. Bartonella were not detected or cultured from unfed winged deer keds. The results may indicate that long-term bacteremia in the moose represents a reservoir of infection and that L. cervi acts as a vector for the spread of infection of Bartonella spp. Further research is needed to evaluate the role of L. cervi in the transmission of Bartonella to animals and humans and the possible pathogenicity of these bacteria for humans and animals.
Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7+ (Osterix+) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA+ cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKOSp7-Cre-EGFP. Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKOSp7-Cre-EGFP. These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.
Bmp2 gene; cementum; dentinogenesis; periodontium development; root formation
In August 2009, during the swine flu pandemic, a 55-year-old male presented to the Emergency Department (ED) with shortness-of-breath and chest pain. He had experienced diarrhoea and vomiting during the 3 weeks preceding admission, and so had sought the advice of his General Practitioner (GP) who had prescribed a course of oseltamivir (Tamiflu). Despite this, his symptoms had worsened, and on arrival in the ED he was found to be tachypnoeic, tachycardic, feverish, anuric and in type I respiratory failure with a compensated metabolic acidosis. He subsequently became hypotensive, and so was transferred to the Intensive Care Unit (ICU) for supportive treatment of three organ failures.
Investigation revealed that he was suffering from Legionnaires disease. This required a prolonged inpatient stay owing to both disease and treatment related complications.
Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.
DMP1; Cre-loxP; gene targeting; mouse; bone
Unlike healthy adult tissues, cancers produce energy mainly by aerobic glycolysis instead of oxidative phosphorylation1. This adaptation, called the Warburg effect, may be a feature of all dividing cells, both normal and cancerous2, or it may be specific to cancers3. Whether in a normally growing tissue during development, proliferating and postmitotic cells produce energy in fundamentally different ways is not known. Here we show in the embryonic Xenopus retina in vivo, that dividing progenitor cells depend less on oxidative phosphorylation for ATP production than non-dividing differentiated cells, and instead use glycogen to fuel aerobic glycolysis. The transition from glycolysis to oxidative phosphorylation is connected to the cell differentiation process. Glycolysis is indispensable for progenitor proliferation and biosynthesis, even when it is not used for ATP production. These results suggest that the Warburg effect can be a feature of normal proliferation in vivo, and that the regulation of glycolysis and oxidative phosphorylation is critical for normal development.
There are significant gaps in the implementation and uptake of evidence-based guideline recommendations for cardiovascular disease (CVD) and diabetes in Australian general practice. This study protocol describes the methodology for a cluster randomised trial to evaluate the effectiveness of a model that aims to improve the implementation of these guidelines in Australian general practice developed by a collaboration between researchers, non-government organisations, and the profession.
We hypothesise that the intervention will alter the behaviour of clinicians and patients resulting in improvements of recording of lifestyle and physiological risk factors (by 20%) and increased adherence to guideline recommendations for: the management of CVD and diabetes risk factors (by 20%); and lifestyle and physiological risk factors of patients at risk (by 5%). Thirty-two general practices will be randomised in a 1:1 allocation to receive either the intervention or continue with usual care, after stratification by state. The intervention will be delivered through: small group education; audit of patient records to determine preventive care; and practice facilitation visits adapted to the needs of the practices. Outcome data will be extracted from electronic medical records and patient questionnaires, and qualitative evaluation from provider and patient interviews.
We plan to disseminate study findings widely and directly inform implementation strategies by governments, professional bodies, and non-government organisations including the partner organisations.
Primary care; Family medicine; Guidelines; Preventive care; Cardiovascular disease
Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKOod) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKOod do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.
bone morphogenetic protein 2; blood vessels; dentinogenesis; dental pulp stem cells; odontogenesis; pericytes
Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8+ T cells have been implicated in the pathogenesis of vitiligo and expression of interferon-gamma (IFN-γ) is increased in the lesional skin of patients, however it is currently unknown what role IFN-γ plays in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8+ T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8+ T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents CD8+ T cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.
CSF-1, a key regulator of mononuclear phagocyte production, is highly expressed in the skeleton by osteoblasts/osteocytes and in a number of nonskeletal tissues such as uterus, kidney and brain. The spontaneous mutant op/op mouse has been the conventional model of CSF-1 deficiency and exhibits a pleiotropic phenotype characterized by osteopetrosis, and defects in hematopoiesis, fertility and neural function. Studies to further delineate the biologic effect of CSF-1 within various tissues have been hampered by the lack of suitable models. To address this issue, we generated CSF-1 floxed/floxed mice and demonstrate that Cre-mediated recombination using Meox2Cre, a Cre line expressed in epiblast during early embryogenesis, results in mice with ubiquitous CSF-1 deficiency (CSF-1KO). Homozygous CSF-1KO mice lacked CSF-1 in all tissues and displayed, in part, a similar phenotype to op/op mice that included: failure of tooth eruption, osteopetrosis, reduced macrophage densities in reproductive and other organs and altered hematopoiesis with decreased marrow cellularity, circulating monocytes and B cell lymphopoiesis. In contrast to op/op mice, CSF-1KO mice showed elevated circulating and splenic T cells. A striking feature in CSF-1KO mice was defective osteocyte maturation, bone mineralization and osteocyte-lacunar system that was associated with reduced dentin matrix protein 1 (DMP1) expression in osteocytes. CSF-1KO mice also showed a dramatic reduction in osteomacs along the endosteal surface that may have contributed to the hematopoietic and cortical bone defects. Thus, our findings show that ubiquitous CSF-1 gene deletion using a Cre-based system recapitulates the expected osteopetrotic phenotype. Moreover, results point to a novel link between CSF-1 and osteocyte survival/function that is essential for maintaining bone mass and strength during skeletal development.
colony stimulating factor-1 (CSF-1); osteocytes; osteoblasts; dentin matrix protein 1 (DMP1); knockout mouse
Benzene is employed in large quantities in the chemical industry and is a ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH4. The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, and N-termini of peptides with the dGuo-muconaldehyde monoadduct.
benzene; E,E-muconaldehyde; dGuo adduct; cross-link; leukemia
Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation.
Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn’s disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression.
Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.
DMP1; MEPE; Mechanical Loading; Osteocyte
Pulmonary function measures reflect respiratory health and predict mortality, and are used in the diagnosis of chronic obstructive pulmonary disease (COPD). We tested genome-wide association with the forced expiratory volume in 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in 48,201 individuals of European ancestry, with follow-up of top associations in up to an additional 46,411 individuals. We identified new regions showing association (combined P<5×10−8) with pulmonary function, in or near MFAP2, TGFB2, HDAC4, RARB, MECOM (EVI1), SPATA9, ARMC2, NCR3, ZKSCAN3, CDC123, C10orf11, LRP1, CCDC38, MMP15, CFDP1, and KCNE2. Identification of these 16 new loci may provide insight into the molecular mechanisms regulating pulmonary function and into molecular targets for future therapy to alleviate reduced lung function.
The purpose of this study was to use a community-based participatory research (CBPR) approach to translate the original Diabetes Prevention Program (DPP) to be age and culturally specific for American Indian (AI) youth.
Tribally enrolled members on 2 Montana Indian reservations conducted focus groups and interviews to discuss community members’ perspectives of factors that encouraged or were barriers to healthy diet and exercise behaviors in AI youth. In total, 31 community members, aged 10 to 68 years old, participated in 4 focus groups and 14 individual interviews. Participants were self-identified as elder, cultural expert, tribal health worker, educator, parent/guardian, youth, or school food service worker. Researchers analyzed transcripts based on inductive methods of grounded theory.
Data analysis revealed translating the DPP to youth was contingent on the lessons incorporating cultural strategies for healthy behaviors in youth such as berry picking, gardening, horseback riding, and dancing; improving knowledge and access to healthy foods and physical activity for youth and their parents; having interactive, hands-on learning activities for healthy lifestyles in the DPP lessons; using a group format and tribal members to deliver the DPP lessons; and having tribal elders talk to youth about the importance of adopting healthy behaviors when they are young.
A CBPR approach engaged community members to identify strategies inherent in their culture, tradition, and environment that could effectively translate the DPP to Montana Indian youth living in rural reservation communities.
Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are α,β-unsaturated aldehyde (enal) endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive enals covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N2 -positions of deoxyguanosine (dG). The bifunctional nature of these enals enables them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS3 or MS4 scan stage. The lower limit of quantification of the cross-linked adducts is ~1 adduct per 108 DNA bases, when 50 µg of DNA is assayed. The cross-linked adducts occur at levels that are ~1–2% of the levels of the monomeric 1,N2-dG adducts in CT DNA treated with either enal.
During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre, the BMP4 floxed and BMP4 knock-out mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process lead to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10–12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, are also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine role of odontoblasts derived BMP4 postnatally on the proper amelogenesis and formation of the enamel.
Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studing of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility.
Western health care systems are facing today increasing movement of genetic knowledge from research labs into clinical practice. This paper reports the results of a survey that addressed the confidence of primary care physicians in their ability to carry out basic medical genetic tasks. The survey was conducted in five countries (France, Germany, The Netherlands, Sweden and the UK). Stratified random samples were drawn from primary care physicians in the five countries representing a sampling frame of 139,579 physicians. Stepwise binary logistic regression procedures were performed to identify the predictor variables for self-reported confidence. Three thousand six hundred eighty-six physicians participated and filled out a self-administered questionnaire. The margin of error for accurate representation of each group of European general practitioners and specialists in the total sample is 2.9% for GP, 2.8% for obstetricians/gynaecologists (OB/GYN) and for paediatricians (PAED) 2.6% (95% confidence level). Confidence in their ability to carry out basic medical genetic tasks is low among participating primary care physicians: 44.2% are not confident, 36.5% somewhat confident, confident or very confident are 19.3%. In each country, those confident/very confident represent less than 33% of the participating physicians. Primary care physicians who report the lowest levels of confidence prove to be those least exposed to medical genetics information and training. Although there are significant differences in the way in which professional education is organised and practice is regulated across European countries, there is a need for a coordinated European effort to improve primary care physicians’ background in medical genetics.
Genetic education; Genetic services; Primary care