Shavanthi Rajatileka

Pyrethroid Resistance in Aedes aegypti from Grand Cayman

The Grand Cayman population of Aedes aegypti is highly resistant to DDT and pyrethroid insecticides. Glutathione transferase, cytochrome P450, and esterase levels were increased in the Grand Cayman population relative to a susceptible laboratory strain, but synergist studies did not implicate elevated insecticide detoxification as a major cause of resistance. The role of target site resistance was therefore investigated. Two substitutions in the voltage-gated sodium channel were identified, V1016I in domain II, segment 6 (IIS6) (allele frequency = 0.79) and F1534C in IIIS6 (allele frequency = 0.68). The role of the F1534C mutation in conferring resistance to insecticides has not been previously established and so a tetraplex polymerase chain reaction assay was designed and used to genotype mosquitoes that had been exposed to insecticides. The F1534C mutation was strongly correlated with resistance to DDT and permethrin.

Molecular Mechanism of Myosin Va Recruitment to Dense Core Secretory Granules

The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide‐producing cells. The molecular composition of the protein complex that recruits BR‐MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG‐associated proteins granuphilin‐a/b (Gran‐a/b), BR‐MyoVa and Rab27a, a member of the Rab family of GTPases. Gran‐a/b–BR‐MyoVa interaction is direct, involves regions downstream of the Rab27‐binding domain, and the C‐terminal part of Gran‐a determines exon specificity. MyoVa and Gran‐a/b are partially colocalised on SGs and disruption of Gran‐a/b–BR‐MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient‐stimulated hormone secretion in pancreatic beta‐cells. These results indicate the existence of at least another binding partner of BR‐MyoVa that was identified as rabphilin‐3A (Rph‐3A). BR‐MyoVa–Rph‐3A interaction is also direct and enhanced when secretion is activated. The BR‐MyoVa–Rph‐3A and BR‐MyoVa–Gran‐a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR‐MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway

Myosin- and Rab-interacting protein is not a classic receptor for MyoVa on large, dense-core secretory granules (SGs), but it aids in PKA-dependent phosphorylation of MyoVa-associated proteins on SGs in endocrine and neuroendocrine cells.

Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing

BackgroundSingle-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns.ResultsUnexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance.ConclusionOur data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.

The oncogenic transcription factor ERG represses the transcription of the tumour suppressor gene PTEN in prostate cancer cells

The oncogene ETS-related gene (ERG) encodes a transcription factor with roles in the regulation of haematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion. The ERG oncogene is activated in >50% of prostate cancer cases, generally through a gene fusion with the androgen-responsive promoter of transmembrane protease serine 2. Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is often inactivated in cancer. ERG overexpression combined with PTEN inactivation or loss is often associated with aggressive prostate cancer. The present study aimed to determine whether or not ERG regulates PTEN transcription directly. ERG was demonstrated to bind to the PTEN promoter and repress its transcription. ERG overexpression reduced endogenous PTEN expression, whereas ERG knockdown increased PTEN expression. The ability of ERG to repress PTEN may contribute to a more cancer-permissive environment.

Association between neonatal resuscitation and a single nucleotide polymorphism rs1835740.

The aim of this work was to test whether three single nucleotide polymorphisms (SNPs) implicated in glutamate homoeostasis or signalling and cellular survival are associated with birth condition.

PO-0474 Impact Of Glutamate Transporter Haplotypes And Clinical Course On Functional Brain Networks In Preterm Infants

Reducing adverse neurodevelopmental outcomes associated with preterm birth is a major challenge facing neonatal medicine, as abnormalities engendered during the perinatal period have lifelong implications. The pathological mechanisms leading to abnormal neurodevelopment in preterm infants involve several pathways. Many direct and indirect effects of preterm birth on neural development occur at the micro structural or neurochemical level, meaning that current methods for assessing neurological injury in preterm infants provide only limited mechanistic and prognostic information. A promising alternative approach is the use of resting state functional MRI (rs-fMRI) to infer integration of neural activity across brain regions (functional connectivity (FC)). Adults who were born preterm show persistent differences in FC, and early detection of such changes offers potential insights into the pathophysiology of preterm brain injury. These functional changes may be influenced by both neonatal course and underlying susceptibilities to abnormal development, including genetic factors. We utilised rapid multiband sequence rs-fMRI acquisition at 3 Tesla, to characterise functional brain connectivity in 30 infants born at <32 weeks gestation, scanned at term. DNA was extracted and sequenced for EAAT2 glutamate transporter haplotypes associated with adverse preterm neurodevelopmental outcomes. Using a multivariate model we identified dissociable and interacting influences of demographic, genetic and clinical variables on functional infant brain networks. We are the first to describe the influence of genetic variability in cerebral glutamate homeostasis on neonatal brain connectivity. We discuss the impact on understanding preterm brain injury, and the potential for predicting neurodevelopmental outcome by non-invasive measurement of functional brain connectivity.

5.8 Clinical and Genetic Influences on Functional Brain Networks in Preterm Infants

Preterm birth is associated with adverse neurodevelopmental outcomes. The pathological mechanisms leading to adverse outcomes involve several pathways, which are not fully understood. Current methods of assessing neurological injury associated with preterm birth have limited scope and low prognostic value. Whilst structural MRI may provide detailed anatomical information about the neonatal brain, there is imperfect mapping between structure and function. A supplementary approach is the use of functional MRI (fMRI) to infer functional connectivity (FC), evaluating integration of neural activity within the brain. There is emerging evidence that children who were born preterm show long term changes in FC, and early detection of such changes offers potential to improve understanding of pathophysiology of preterm brain injury. These functional changes may be influenced by both neonatal course and underlying susceptibilities to abnormal development, including genetic risk factors. We used resting state fMRI (rs-fMRI) at 3T to examine functional brain connectivity in 20 infants born at <32 weeks of gestation, scanned at term. Infants also had genetic testing to examine polymorphisms in the EAAT2 glutamate transporter, previously associated with variation in preterm neurodevelopmental outcomes. Using a multivariate model to examine the independent contributions of demographic, genetic and clinical characteristics of the infants to FC, we identified multiple dissociable influences on functional brain networks. This is the first report of genetic variability in cerebral glutamate homeostasis influencing neonatal brain connectivity. We discuss the impact on understanding preterm brain injury, and the potential for predicting neurodevelopmental outcome by non-invasive measurement of functional brain connectivity.