Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells.

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC.

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

Idiopathic early ovarian aging: is there a relation with premenopausal accelerated biological aging in young women with diminished response to ART?

PURPOSE: To evaluate whether young women with idiopathic early ovarian aging, as defined by producing fewer oocytes than expected for a given age over multiple in vitro fertilization (IVF) cycles, have changes in telomere length and epigenetic age indicating accelerated biological aging (i.e., increased risk of morbidity and mortality). METHODS: A prospective cohort study was conducted at two Danish public fertility clinics. A total of 55 young women (≤ 37 years) with at least two IVF cycles with ≤ 5 harvested oocytes despite sufficient stimulation with follicle-stimulating hormone (FSH) were included in the early ovarian aging group. As controls, 52 young women (≤ 37 years) with normal ovarian function, defined by at least eight harvested oocytes, were included. Relative telomere length (rTL) and epigenetic age acceleration (AgeAccel) were measured in white blood cells as markers of premenopausal accelerated biological aging. RESULTS: rTL was comparable with a mean of 0.46 (± SD 0.12) in the early ovarian aging group and 0.47 (0.14) in the normal ovarian aging group. The AgeAccel of the early ovarian aging group was, insignificantly, 0.5 years older, but this difference disappeared when adjusting for chronological age. Sub-analysis using Anti-Müllerian hormone (AMH) as selection criterion for the two groups did not change the results. CONCLUSION: We did not find any indications of accelerated aging in whole blood from young women with idiopathic early ovarian aging. Further investigations in a similar cohort of premenopausal women or other tissues are needed to fully elucidate the potential relationship between premenopausal accelerated biological aging and early ovarian aging.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

DNA methylation alterations in response to prenatal exposure of maternal cigarette smoking: A persistent epigenetic impact on health from maternal lifestyle?

Despite increased awareness, maternal cigarette smoking during pregnancy continues to be a common habit causing risk for numerous documented negative health consequences in the exposed children. It has been proposed that epigenetic mechanisms constitute the link between prenatal exposure to maternal cigarette smoking (PEMCS) and the diverse pathologies arising in later life. We here review the current literature, focusing on DNA methylation. Alterations in the global DNA methylation patterns were observed after exposure to maternal smoking during pregnancy in placenta, cord blood and buccal epithelium tissue. Further, a number of specific genes exemplified by CYP1A1, AhRR, FOXP3, TSLP, IGF2, AXL, PTPRO, C11orf52, FRMD4A and BDNF are shown to have altered DNA methylation patterns in at least one of these tissue types due to PEMCS. Investigations showing persistence and indications of trans-generational inheritance of DNA methylation alterations induced by smoking exposure are also described. Further, smoking-induced epigenetic manifestations can be both tissue-dependent and gender-specific which show the importance of addressing the relevant sex, tissue and cell types in the future studies linking specific epigenetic alterations to disease development. Moreover, the effect of paternal cigarette smoking and second-hand smoke exposure is documented and accordingly not to be neglected in future investigations and data evaluations. We also outline possible directions for the future research to address how DNA methylation alterations induced by maternal lifestyle, exemplified by smoking, have direct consequences for fetal development and later in life health and behavior of the child.

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance.

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer.

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

Trans-Regulation of Alternative PD-L1 mRNA Processing by CDK12 in Non-Small-Cell Lung Cancer Cells.

Immunotherapy using checkpoint inhibitors targeting the interaction between PD-1 on T cells and PD-L1 on cancer cells has shown significant results in non-small-cell lung cancer (NSCLC). Not all patients respond to the therapy, and PD-L1 expression heterogeneity is proposed to be one determinant for this. The alternative processing of PD-L1 RNA, which depends on an alternative poly-A site in intron 4, generates a shorter mRNA variant (PD-L1v4) encoding soluble PD-L1 (sPD-L1), relative to the canonical PD-L1v1 mRNA encoding membrane-associated PD-L1 (mPD-L1). This study aimed to identify factors influencing the ratio between these two PD-L1 mRNAs in NSCLC cells. First, we verified the existence of the alternative PD-L1 RNA processing in NSCLC cells, and from in silico analyses, we identified a candidate list of regulatory factors. Examining selected candidates showed that CRISPR/Cas9-generated loss-of-function mutations in CDK12 increased the PD-L1v4/PD-L1v1 mRNA ratio and, accordingly, the sPD-L1/mPD-L1 balance. The CDK12/13 inhibitor THZ531 could also increase the PD-L1v4/PD-L1v1 mRNA ratio and impact the PD-L1 transcriptional response to IFN-γ stimulation. The fact that CDK12 regulates PD-L1 transcript variant formation in NSCLC cells is consistent with CDK12's role in promoting transcriptional elongation over intron-located poly-A sites. This study lays the groundwork for clinical investigations to delineate the implications of the CDK12-mediated balancing of sPD-L1 relative to mPD-L1 for immunotherapeutic responses in NSCLC.

The NIZP1 KRAB and C2HR domains cross-talk for transcriptional regulation.

The NSD1 histone methyltransferase is involved in the outgrowth disorders Sotos and Weaver syndromes and childhood acute myeloid leukemia. NSD1 is a bona fida transcriptional co-repressor for Nizp1 which is a protein including SCAN, KRAB, C2HR and zinc-finger domains. In this study the Nizp1 KRAB-domain was identified to possess an intrinsic transcriptional activation capacity suppressed in cis by the presence of the C2HR domain. Oppositely, the KRAB-domain supported C2HR domain mediated transcriptional repression. The presence of the KRAB-domain resulted in increased NSD1 co-repressor association with the C2HR domain. This study shows a new function of the KRAB-domain, C2HR-domain, and the associated factors to confer Nizp1 mediated transcriptional regulation.

Analysis of HP1α regulation in human breast cancer cells.

The three mammalian HP1 proteins, HP1α/CBX5, HP1ß/CBX1, and HPγ/CBX3, are involved in chromatin packing and gene regulation. The HP1α protein is down-regulated in invasive compared to non-invasive breast cancer cells and HP1α is a suppressor of cell migration and invasion. In this report, we examined the background for HP1α protein down-regulation in invasive breast cancer cells. We identified a strict correlation between HP1α down-regulation at the protein level and the mRNA level. The HP1α mRNA down-regulation in invasive cancer cells was not caused by mRNA destabilization. Chromatin immunoprecipitation analysis of the HP1α gene showed a decrease in the histone mark for transcriptional activity H3-K36 tri-methylation and RNA polymerase II in invasive breast cancer cells which correlated with a decreased abundance of basal transcription factors at the HP1α promoter. E2F transcription factors regulate HP1α transcription and we identified that E2F5 depletion increased HP1α expression in invasive breast cancer cells. Finally, we have characterized two HP1α mRNA isoforms and both HP1α mRNA isoforms were down-regulated to a similar extend at the transcriptional level in invasive breast cancer cells. Collectively the presented results show that HP1α down-regulation in invasive breast cancer cells is primary a transcriptional effect and demonstrates a novel set of mechanisms involved in HP1α transcriptional regulation. The finding that HP1α is down-regulated primarily at the transcriptional level provides a new insight for the further elucidation of the detailed molecular mechanisms causing the HP1α down-regulation in invasive breast cancer cells.

DNA methylation of the KLK8 gene in depression symptomatology.

BACKGROUND: Depression is a common, complex, and debilitating mental disorder estimated to be under-diagnosed and insufficiently treated in society. Liability to depression is influenced by both genetic and environmental risk factors, which are both capable of impacting DNA methylation (DNAm). Accordingly, numerous studies have researched for DNAm signatures of this disorder. Recently, an epigenome-wide association study of monozygotic twins identified an association between DNAm status in the KLK8 (neuropsin) promoter region and severity of depression symptomatology. METHODS: In this study, we aimed to investigate: (i) if blood DNAm levels, quantified by pyrosequencing, at two CpG sites in the KLK8 promoter are associated with depression symptomatology and depression diagnosis in an independent clinical cohort and (ii) if KLK8 DNAm levels are associated with depression, postpartum depression, and depression symptomatology in four independent methylomic cohorts, with blood and brain DNAm quantified by either MBD-seq or 450 k methylation array. RESULTS: DNAm levels in KLK8 were not significantly different between depression cases and controls, and were not significantly associated with any of the depression symptomatology scores after correction for multiple testing (minimum p value for KLK8 CpG1 = 0.12 for 'Depressed mood,' and for CpG2 = 0.03 for 'Loss of self-confidence with other people'). However, investigation of the link between KLK8 promoter DNAm levels and depression-related phenotypes collected from four methylomic cohorts identified significant association (p value < 0.05) between severity of depression symptomatology and blood DNAm levels at seven CpG sites. CONCLUSIONS: Our findings suggest that variance in blood DNAm levels in KLK8 promoter region is associated with severity of depression symptoms, but not depression diagnosis.

Reelin expression during embryonic development of the pig brain.

BACKGROUND: Reelin is an extracellular glycoprotein of crucial importance in the developmental organisation of neurons in the mammalian cerebral cortex and other laminated brain regions. The pig possesses a gyrencephalic brain that bears resemblance to the human brain. In order to establish an animal model for neuronal migration disorders in the pig, we have studied the expression pattern and structure of Reelin during pig brain development. RESULTS: We determined the sequence of pig Reelin mRNA and protein and identified a high degree of homology to human Reelin. A peak in Reelin mRNA and protein expression is present during the period of major neurogenesis and neuronal migration. This resembles observations for human brain development. Immunohistochemical analysis showed the highest expression of Reelin in the Cajal-Reztius cells of the marginal zone, in resemblance with observations for the developing brain in humans and other mammalian species. CONCLUSIONS: We conclude that the pig might serve as an alternative animal model to study Reelin functions and that manipulation of the pig Reelin could allow the establishment of an animal model for human neuronal migration disorders.

Changes in HIV needs identified by the National AIDS Hotline of Trinidad and Tobago.

OBJECTIVES: To examine utilization of the National AIDS Hotline of Trinidad and Tobago (AIDSLINE), evaluate its validity as a reliable data source for monitoring national HIV-related needs, and identify changes in caller requests between two different time periods. METHODS: A total of 7 046 anonymous hotline calls in 1998-2002 (T1) and 2 338 calls in 2007 (T2) were analyzed for associations between caller characteristics and call content. A subsample of the data was also analyzed qualitatively. T1 findings were compared with HIV-related data collected by national policy-makers during that period, to evaluate the hotline's validity as a data source, and findings from T2, to reveal changes in call content over time. RESULTS: In T1, the hotline was well utilized for information and counseling by both the general population and those living with HIV/AIDS. Call content from T2 indicated an increase versus T1 in 1) general awareness of HIV and other sexually transmitted diseases; 2) HIV testing; and 3) knowledge of HIV symptoms and transmission. HIV-related mental health needs, and the relationship between HIV and both child sexual abuse (CSA) and intimate partner violence (IPV), were identified as emerging issues. CONCLUSIONS: AIDSLINE is a well-utilized tool for providing information and counseling on national HIV-related issues, and a valid, cost-effective, easily accessed information source for planners and policy-makers involved in HIV management. Over the two study periods, there was an increase in HIV awareness and testing and in requests related to mental health, CSA, and IPV, but no change in sexual behaviors.

Voluntary Physical Exercise Induces Expression and Epigenetic Remodeling of VegfA in the Rat Hippocampus.

A healthy lifestyle, including regular physical exercise, is generally believed to improve cognitive function and enhance neurogenesis. Such physical exercise-induced effects are associated with increased brain expression of neurotrophic and growth factors. In the present study, we investigated Bdnf, Igf-1, Fgf-2, Egf, and VegfA messenger RNA (mRNA) expression levels in the male rat hippocampus and frontal cortex after 2 weeks of voluntary physical exercise. Whereas the expression of Fgf-2 was upregulated in the hippocampus and prefrontal cortex by physical exercise, the expression levels of Bdnf transcript 1, Bdnf transcript 4, Igf-1, and VegfA were upregulated only in the hippocampus. We focused our subsequent analyses on the VegfA gene, which encodes vascular endothelial growth factor, a signaling molecule important for angiogenesis, vasculogenesis, and neurogenesis. To study the epigenetic mechanisms involved in the physical exercise-mediated induction of VegfA expression, we used oxidative and non-oxidative bisulfite pyrosequencing to analyze VegfA promoter DNA methylation and DNA hydroxymethylation. We observed discrete DNA hypomethylation at specific CpG sites in rats that engaged in physical exercise relative to sedentary rats. This is exemplified by a CpG site located within a VegfA promoter Sp1/Sp3 transcription factor recognition element. DNA hydroxymethylation was present at the VegfA promoter, but no differences in DNA hydroxymethylation were observed in rats that engaged in physical exercise relative to sedentary rats. Moreover, we observed increased Tet1 and decreased Dnmt3b mRNA expression in the hippocampi of rats that engaged in physical exercise. The presented results substantiate the involvement of epigenetics as a mediator of the beneficial effects of physical exercise and point to the importance of analyzing factors beyond Bdnf to delineate the mechanisms behind the functional impacts of physical exercise in mediating benefits to the brain.

Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples.

We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking.

Prenatal exposure to maternal cigarette smoking increases the risk of intrauterine growth retardation, adverse pregnancy outcomes, and diseases later in life. Exposure can result in postnatal global and gene-specific DNA methylation changes, with the latter well documented for the CYP1A1 and AHRR genes involved in the detoxification of xenobiotic substances. This study assessed the impact of exposure to maternal smoking on first trimester fetal CYP1A1 and AHRR mRNA expression and DNA methylation for CpG-sites displaying maternal smoking during pregnancy-mediated methylation changes at birth. The analyses included first trimester (6-12 weeks) placentas (N=39) and livers (N=43). For AHRR, exposure to maternal smoking was associated with increased DNA methylation in the placentas of female fetuses; mRNA expression, however, was unchanged. While exposure to maternal smoking was not associated with AHRR DNA methylation changes in fetal livers; mRNA expression was increased. For CYP1A1, exposure to maternal smoking was not associated with fetal DNA methylation changes whereas mRNA expression increased in placentas and male fetal livers. These results show that first trimester exposure to maternal smoking is associated with CYP1A1 and AHRR DNA methylation and mRNA expression changes. However, the results also indicate that maternal smoking during pregnancy-mediated postnatal CYP1A1 and AHRR DNA methylation changes are not imprinted during the first trimester.

Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain.

BACKGROUND: The transmembrane presenilin (PSEN) proteins, PSEN1 and PSEN2, have been proposed to be the catalytic components of the gamma-secretase protein complex, which is an intramembranous multimeric protease involved in development, cell regulatory processes, and neurodegeneration in Alzheimer's disease. Here we describe the sequencing, chromosomal mapping, and polymorphism analysis of PSEN1 and PSEN2 in the domestic pig (Sus scrofa domesticus). RESULTS: The porcine presenilin proteins showed a high degree of homology over their entire sequences to the PSENs from mouse, bovine, and human. PSEN1 and PSEN2 transcription was examined during prenatal development of the brain stem, hippocampus, cortex, basal ganglia, and cerebellum at embryonic days 60, 80, 100, and 114, which revealed distinct temporal- and tissue-specific expression profiles. Furthermore, immunohistochemical analysis of PSEN1 and PSEN2 showed similar localization of the proteins predominantly in neuronal cells in all examined brain areas. CONCLUSION: The data provide evidence for structural and functional conservation of PSENs in mammalian lineages, and may suggest that the high sequence similarity and colocalization of PSEN1 and PSEN2 in brain tissue reflect a certain degree of functional redundancy. The data show that pigs may provide a new animal model for detailed analysis of the developmental functions of the PSENs.

The Effects of Voluntary Physical Exercise-Activated Neurotrophic Signaling in Rat Hippocampus on mRNA Levels of Downstream Signaling Molecules.

Physical exercise results in the increased expression of neurotrophic factors and the subsequent induction of signal transduction cascades with a positive impact on neuronal functions. In this study, we used a voluntary physical exercise rat model to determine correlations in hippocampus mRNA expression of the neurotropic factors Bdnf, VegfA, and Igf1; their receptors TrkB, Igf1R, VegfR1, and VegfrR2; and downstream signal transducers Creb, Syn1, and Vgf. In hippocampi of physically exercised rats, the mRNA expression levels of Bdnf transcript 4 (Bdnf-t4), VegfA, and Igf1, as well as VegfR1, TrkB, Creb, Vgf, and Syn1, were increased. Bdnf-t4 mRNA expression positively correlated with mRNA expression of Creb, Vgf, and Syn1 in hippocampi of exercised rats. A correlation between Bdnf-t4 and Syn1 mRNA expression was also observed in hippocampi of sedentary rats. Igf1 and VegfA mRNA expression was positively correlated in hippocampi of both exercised and sedentary rats. But, neither Igf1 nor VegfA mRNA expression was correlated with the expression of Bdnf-t4 or the expression of the signal transducers Creb, Syn1, and Vgf. In hippocampi of exercised rats, Creb mRNA expression was positively correlated with TrkB, Syn1, and Vgf mRNA expression and with the correlation between Creb and Vgf mRNA expression also observed in hippocampi of sedentary rats. To examine for causality of the in vivo observed correlated mRNA expression levels between Bdnf-t4 and the other examined transcripts, we used nuclease-deactivated CRISPR-Cas9 fused with VP64 to induce mRNA expression of endogenous Bdnf-t4 in rat PC12 cells. Following Bdnf-t4 mRNA induction, we observed increased Creb mRNA expression. This in vitro result is in accordance with the in vivo results and supports that under specified conditions, an increase in Creb mRNA expression can be a downstream signal transduction event due to induction of endogenous Bdnf mRNA expression.

A review of the public-funded primary health care facilities for children in the pluralistic health care settings of Barbados, a Caribbean island.

UNLABELLED: Aim The major objectives of this study were to evaluate the existing pediatrics health care service provisions and utilizations of the public polyclinics in Barbados. Furthermore, the aim was to assess if the existing manpower resources were adequate. BACKGROUND: Barbados has a mixed health care system consisting of both a socialized and a private health care system. The Ministry of Health commissioned a needs assessment survey of the pediatrics primary health care at the public polyclinics. METHODS: Primary data were collected through interviews with the public primary health care providers. Secondary data were collected from the Barbados Census Data and Ministry of Health statistics. Data were analyzed to assess the pediatrics primary health care service utilization and adequacy of existing resources at the polyclinics. Findings In 2012, there were 62 934 visits from children <16 years of age to the public polyclinics in Barbados and this accounted for 39.1% of all visits (both adults and children) to the polyclinics. An overall 16.7% of the visits were from children less than five years old to the Well Child Clinic for immunization and for growth and development monitoring; 32% of all physician consultations at the polyclinics were for children <16 years. Utilization of health services by children at the polyclinics was 5245 visits/month. Given an expected monthly demand for 10 822 visits from children, the polyclinics serve 48.5% of the primary health care demand for children in Barbados. CONCLUSIONS: The public polyclinics play a pivotal role in the pluralistic primary health care system in Barbados. They fulfill nearly half of all the primary care demand and more importantly provides for almost the entire immunization demand, and thereby ensuring high coverage. The existing resources, if used optimally, would reduce the long consultation time observed in this setting, and thereby increase the capacity considerably.