aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation

Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na+ channel of Anopheles culicifacies from Orissa, India

<p>Abstract</p> <p>Background</p> <p>Knockdown resistance in insects resulting from mutation(s) in the voltage gated Na⁺channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common <it>kdr </it>mutation in insects, was reported in <it>Anopheles culicifacies</it>-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an <it>An. culicifacies </it>population from Malkangiri district of Orissa, India.</p> <p>Methods</p> <p><it>Anopheles culicifacies sensu lato (s.l.) </it>samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The <it>An. culicifacies </it>population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.</p> <p>Results</p> <p>DNA sequencing of <it>An. culicifacies </it>individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.</p> <p>Conclusions</p> <p>This is the first report of the presence of L1014S (homologous to the <it>kdr-e </it>in <it>An. gambiae</it>) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of <it>An. culicifacies </it>in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.</p

Development and validation of self-reported line drawings for assessment of knee malalignment and foot rotation: a cross-sectional comparative study

<p>Abstract</p> <p>Background</p> <p>For large scale epidemiological studies clinical assessments and radiographs can be impractical and expensive to apply to more than just a sample of the population examined. The study objectives were to develop and validate two novel instruments for self-reported knee malalignment and foot rotation suitable for use in questionnaire studies of knee pain and osteoarthritis.</p> <p>Methods</p> <p>Two sets of line drawings were developed using similar methodology. Each instrument consisted of an explanatory question followed by a set of drawings showing straight alignment, then two each at 7.5° angulation and 15° angulation in the varus/valgus (knee) and inward/outward (foot) directions. Forty one participants undertaking a community study completed the instruments on two occasions. Participants were assessed once by a blinded expert clinical observer with demonstrated excellent reproducibility. Validity was assessed by sensitivity, specificity and likelihood ratio (LR) using the observer as the reference standard. Reliability was assessed using weighted kappa (κ). Knee malalignment was measured on 400 knee radiographs. General linear model was used to assess for the presence of a linear increase in knee alignment angle (measured medially) from self-reported severe varus to mild varus, straight, mild valgus and severe valgus deformity.</p> <p>Results</p> <p>Observer reproducibility (κ) was 0.89 and 0.81 for the knee malalignment and foot rotation instruments respectively. Self-reported participant reproducibility was also good for the knee (κ 0.73) and foot (κ 0.87) instruments. Validity was excellent for the knee malalignment instrument, with a sensitivity of 0.74 (95%CI 0.54, 0.93) and specificity of 0.97 (95%CI 0.94, 1.00). Similarly the foot rotation instrument was also found to have high sensitivity (0.92, 95%CI 0.83, 1.01) and specificity (0.96, 95%CI 0.93, 1.00). The knee alignment angle increased progressively from self reported severe varus to mild varus, straight, mild valgus and severe valgus knee malalignment (p_trend<0.001).</p> <p>Conclusions</p> <p>The two novel instruments appear to provide a valid and reliable assessment of self-reported knee malalignment and foot rotation, and may have a practical use in epidemiological studies.</p

The North Staffordshire Osteoarthritis Project – NorStOP: Prospective, 3-year study of the epidemiology and management of clinical osteoarthritis in a general population of older adults

BACKGROUND: The clinical syndrome of joint pain and stiffness in older people is the commonest cause of disability and health care consultation in this age group. Yet there have been few prospective studies of its course over time and its impact on personal and social life. We plan a cohort study in the general population aged 50 years and over to determine the course and prognosis of hand, hip, knee and foot pain, and the impact of these syndromes on participation levels and health care use. METHODS: All patients aged 50 years and over registered with 3 local general practices are to be recruited to a population-based cohort study through the use of a two-stage mailing process. Participants will initially complete a "Health Survey" questionnaire. This will collect information on several areas of life including socio-demographics, general health, physical function, participation, and bodily pain. Those who state that they have experienced any hand problem or any pain in their hands, hips, knees, or feet in the previous 12 months, and also give permission to be re-contacted, will be mailed a "Regional Pains Survey" questionnaire which collects detailed information on the four selected body regions (hand, hips, knees, feet). Follow-up data for the three-year period subsequent to cohort recruitment will be collected through two sources: i) general practice medical records and ii) repeat mailed survey

DNA methylation and mRNA expression of SYN III, a candidate gene for schizophrenia

<p>Abstract</p> <p>Background</p> <p>The synapsin III (<it>SYN III</it>) gene on chromosome 22q is a candidate gene for schizophrenia susceptibility due to its chromosome location, neurological function, expression patterns and functional polymorphisms.</p> <p>Methods</p> <p>This research has established the mRNA expression of <it>SYN III </it>in 22 adult human brain regions as well as the methylation specificity in the closest CpG island of this gene. The methylation specificity studied in 31 brain regions (from a single individual) was also assessed in 51 human blood samples (representing 20 people affected with schizophrenia and 31 normal controls) including a pair of monozygotic twin discordant for schizophrenia and 2 non-human primates.</p> <p>Results</p> <p>The results show that the cytosine methylation in this genomic region is 1) restricted to cytosines in CpG dinucleotides 2) similar in brain regions and blood and 3) appears conserved in primate evolution. Two cytosines (cytosine 8 and 20) localized as the CpG dinucleotide are partially methylated in all brain regions studied. The methylation of these sites in schizophrenia and control blood samples was variable. While cytosine 8 was partially methylated in all samples, the distribution of partial to complete methylation at the cytosine 20 was 22:9 in controls as compared to 18:2 in schizophrenia (p = 0.82). Also, there is no difference in methylation between the affected and unaffected member of a monozygotic twin pair.</p> <p>Conclusion</p> <p>The variation in <it>SYN III </it>methylation studied is 1) not related to schizophrenia in the population sample or a monozygotic twin pair discordant for schizophrenia and 2) not related to the mRNA level of <it>SYN IIIa </it>in different human brain regions.</p

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of ∼200 mm3 significantly increased the time for tumours to grow to 1000 mm3

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Population genomics of cardiometabolic traits: design of the University College London-London School of Hygiene and Tropical Medicine-Edinburgh-Bristol (UCLEB) Consortium.

Substantial advances have been made in identifying common genetic variants influencing cardiometabolic traits and disease outcomes through genome wide association studies. Nevertheless, gaps in knowledge remain and new questions have arisen regarding the population relevance, mechanisms, and applications for healthcare. Using a new high-resolution custom single nucleotide polymorphism (SNP) array (Metabochip) incorporating dense coverage of genomic regions linked to cardiometabolic disease, the University College-London School-Edinburgh-Bristol (UCLEB) consortium of highly-phenotyped population-based prospective studies, aims to: (1) fine map functionally relevant SNPs; (2) precisely estimate individual absolute and population attributable risks based on individual SNPs and their combination; (3) investigate mechanisms leading to altered risk factor profiles and CVD events; and (4) use Mendelian randomisation to undertake studies of the causal role in CVD of a range of cardiovascular biomarkers to inform public health policy and help develop new preventative therapies