Light and circadian regulation of clock components aids flexible responses to environmental signals

The circadian clock measures time across a 24h period, increasing fitness by phasing biological processes to the most appropriate time of day. The interlocking feedback loop mechanism of the clock is conserved across species; however, the number of loops varies. Mathematical and computational analyses have suggested that loop complexity affects the overall flexibility of the oscillator, including its responses to entrainment signals. We used a discriminating experimental assay, at the transition between different photoperiods, in order to test this proposal in a minimal circadian network (in Ostreococcus tauri) and a more complex network (in Arabidopsis thaliana). Transcriptional and translational reporters in O.tauri primarily tracked dawn or dusk, whereas in A.thaliana, a wider range of responses were observed, consistent with its more flexible clock. Model analysis supported the requirement for this diversity of responses among the components of the more complex network. However, these and earlier data showed that the O.tauri network retains surprising flexibility, despite its simple circuit. We found that models constructed from experimental data can show flexibility either from multiple loops and/or from multiple light inputs. Our results suggest that O.tauri has adopted the latter strategy, possibly as a consequence of genomic reduction

Light and circadian regulation of clock components aids flexible responses to environmental signals

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThe circadian clock measures time across a 24 h period, increasing fitness by phasing biological processes to the most appropriate time of day. The interlocking feedback loop mechanism of the clock is conserved across species; however, the number of loops varies. Mathematical and computational analyses have suggested that loop complexity affects the overall flexibility of the oscillator, including its responses to entrainment signals. We used a discriminating experimental assay, at the transition between different photoperiods, in order to test this proposal in a minimal circadian network (in Ostreococcus tauri) and a more complex network (in Arabidopsis thaliana). Transcriptional and translational reporters in O. tauri primarily tracked dawn or dusk, whereas in A. thaliana, a wider range of responses were observed, consistent with its more flexible clock. Model analysis supported the requirement for this diversity of responses among the components of the more complex network. However, these and earlier data showed that the O. tauri network retains surprising flexibility, despite its simple circuit. We found that models constructed from experimental data can show flexibility either from multiple loops and/or from multiple light inputs. Our results suggest that O. tauri has adopted the latter strategy, possibly as a consequence of genomic reduction.This research was supported by EU FP7 collaborative project TiMet (award 245143), BBSRC and EPSRC awards BB/F005237/1, BB/D019621/1 and BB/J009423 (to A.J.M. and others) and EPSRC award EP/I017445/1 (to O.E.A. and others). C.T.'s work was supported by the Human Frontiers Science Program and the Swedish Research Council (award 2010-5219)

Long-term culture of human breast cancer specimens and their analysis using optical projection tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account. We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies. We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.Publisher PDFPeer reviewe

An Integrated Surveillance System to Examine Testing, Services, and Outcomes for Sexually Transmitted Diseases

Despite laws that require reporting of sexually transmitted diseases (STDs) to governmental health agencies, integrated surveillance of STDs remains challenging. Data and information about testing are fragmented from information on treatment and outcomes. To overcome this fragmentation, data from multiple electronic systems spanning clinical and public health environments were integrated to create an STD surveillance registry. Electronic health records, disease case records, and birth registry records were linked and then stored in a de-identified, secure server for use by health officials and researchers. The registry contains nearly 6 million tests for 628,138 individuals over a 12-year period. The registry supports efforts to understand the epidemiology of STDs as well as health services and outcomes for those diagnosed with STDs. Specialized disease registries hold promise for collaboration across clinical and public health domains to improve surveillance efforts, reduce health disparities, and increase prevention efforts at the local level

Early life stress influences acute and sensitised responses of adult mice to cocaine by interacting with GABAA α2 receptor expression

Early life stress (ELS) is known to exert long term effects on brain function, with resulting deleterious consequences for several aspects of mental health, including the development of addiction to drugs of abuse. One potential mechanism in humans is suggested by findings that ELS interacts with polymorphisms of the GABRA2 gene, encoding alpha2 subunits of GABAA receptors, to increase risk for both posttraumatic stress disorder, and vulnerability to cocaine addiction. We used a mouse model, in which the amount of material for nest building was reduced during early postnatal life, to study interactions between ELS and expression of alpha2-containing GABAA receptors in influencing cocaine-related behaviour. Breeding of parents heterozygous for deletion of alpha2 resulted in litters containing homozygous knockout (alpha2-/-), heterozygous knockout (alpha2+/-), and wildtype (alpha2+/+) offspring. Following the ELS procedure, the mice were allowed to develop to adulthood before being tested for the acute effect of cocaine on locomotor stimulation, behavioural sensitisation to repeated cocaine, and to cocaine-conditioned activity. Exposure to ELS resulted in increased acute locomotor stimulant effects of cocaine across all genotypes, with the most marked effects in alpha2-/- mice (which also showed increased activity following vehicle). Repeated cocaine administration to non-stressed mice resulted in sensitisation in alpha2+/+ and alpha2+/- mice, but, in keeping with previous findings, not in alpha2-/- mice. Prior exposure to ELS reduced sensitisation in alpha2+/+ mice, albeit not significantly, and abolished sensitisation in alpha2+/- mice. Conditioned activity was elevated following ELS in all animals, independently of genotype. Thus, while the enhanced acute effects of cocaine following ELS being most marked in alpha2-/- mice suggests a function of alpha2-containing GABAA receptors in protecting against stress, the interaction between ELS and genotype in influencing sensitisation may be more in keeping with ELS reducing expression of alpha2-containing GABAA receptors. The ability of ELS to increase cocaine-conditioned locomotor activity appears to be independent of alpha2-containing GABAA receptors

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent protein kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has an unique consensus substrate phosphorylation motif compared to conventional CDKs. To elucidate the structure and inhibitor binding properties of this atypical CDK we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multi-targeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts

Better reporting of interventions : template for intervention description and replication (TIDieR) checklist and guide

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Spatial Integration of Community Data with Clinical Data in Support of Community Health Research and Practice

poster abstractThis poster will describe the recent integration of one of the nation’s largest health information exchanges, the Indiana Network for Patient Care developed by the Regenstrief Institute, with one of the nation’s most comprehensive community information system, the SAVI CIS developed by The Polis Center at IUPUI. Integrating community data that quantifies the social and physical environment with clinical data has great potential for supporting and advancing community health research and practice. Multi-sector collaboration on the development and evaluation of associated uses cases informed system integration is allowing spatially-aware research and practice to be more quickly realized

Where Do People Go for Gonorrhea and Chlamydia Tests: A Cross-sectional View of the Central Indiana population, 2003-2014

Background Despite major efforts to control their spread, reported sexually transmitted infections (STI) are increasing. Using data from a mid-sized Midwest metropolitan area, we examined the settings in which individuals are tested for gonorrhea and chlamydia in relation to demographics and test result to determine where interventions may best be focused. Methods A de-identified and integrated registry, containing records from all patients tested for an STI from 2003-2014, was created by combining data from a large health information exchange and the reporting district’s STI Program located in Indianapolis, IN. Individual characteristics and visit settings where gonorrhea and chlamydia testing was performed were analyzed. Results We identified 298,946 individuals with 1,062,369 visits where testing occurred at least once between the ages of 13 and 44 years. Females were tested significantly more often than males and received testing more often in outpatient clinics whereas males were most often tested in the STI clinic. Individuals who utilized both STI and non-STI settings were more likely to have a positive test at an STI or ED visit (6.4% - 20.8%) than outpatient or inpatient setting (0.0-11.3%) (p<.0001). Test visits increased over the study period particularly in emergency departments, which showed a substantial increase in the number of positive test visits. Conclusions The most frequent testing sites remain STI clinics for men and outpatient clinics for women. Yet, emergency departments are increasingly a source of testing and morbidity. This makes them a valuable target for public health interventions that could improve care and population health

Deletion of the gabra2 gene results in hypersensitivity to the acute effects of ethanol but does not alter ethanol self administration

Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA(A) α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA(A) α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol's rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchange