Gurpreet Kaur

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of off-targets, and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease. DOI: http://dx.doi.org/10.7554/eLife.06942.001

Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress.

The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.

Predictors of Physical Inactivity Among Elderly Malaysians Recommendations for Policy Planning

Physical inactivity is the fourth leading risk factor for global mortality. Regular moderate-intensity physical activity has significant benefits for health. To determine the socioeconomic predictors of physical inactivity among elderly Malaysian population. A nationwide community-based survey was conducted among 4831 respondents aged ≥60 years with a face-to-face questionnaire. The prevalence of physical inactivity among the elderly was 88.0%, highest in respondents aged older than 80 years (95.4%), females (90.1%), other Bumiputra (92.2%), earning household income less than RM1000 (87.9%), and residing in urban locality (88.4%). In the multivariate model, the predictors of physical inactivity were only sex, ethnicity, locality, and age group (adjusted odds ratio = 1.3-3.6). The predictors of physical inactivity can identify the risk factors to develop policies that will reduce the public health burden of noncommunicable diseases.

Overlapping binding sites for importin β1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impβ1 (importin β1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impβ1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impβ1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impβ1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impβ1 for binding to Gli1, through the fact that the sites for SuFu and Impβ1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impβ1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

Prevalence and Correlates of Depression Among Adolescents in Malaysia

Depression among adolescents has been recognized as a major public health issue. The objective of this study was to determine the prevalence and correlates of depression among school-going adolescents in Malaysia. Data from the Malaysia Global School-based Health Survey (GSHS) 2012 were analyzed with additional data from the validated DASS21 (Depression, Anxiety, and Stress) questionnaire. The study revealed that 17.7% of respondents had depressive symptoms. Multivariate analysis further showed that feeling lonely (adjusted odds ratio [aOR] = 2.99; 95% CI = 2.57-3.47), Indian ethnicity (aOR = 2.00; 95% CI = 1.63-2.44), using drugs (aOR = 1.85; 95% CI = 1.21-2.82), and being bullied (aOR = 1.79; 95% CI = 1.60-1.99) were significantly associated with depressive symptoms. Lack of parental supervision, alcohol use, and tobacco use were also significant risk factors. Addressing depressive symptoms among adolescents may have implications for managing their risks of being bullied and substance use. This study also highlights the need to further investigate depressive symptoms among adolescents of Indian ethnicity.

Correlates of Current Smoking Among Malaysian Secondary School Children

Cigarette smoking in adolescent is a significant public health problem, leading to the risk of addiction, morbidity, and mortality in the long term. This study determined the prevalence and correlates of current smoking among adolescent school children. A nationwide school-based survey among 25 507 students between Forms 1 to 5 (aged 12-17) was conducted using a 2-stage cluster sampling design. The prevalence of current smoking was 11.5%. Multivariable logistic regression analysis revealed that current smoking was significantly associated with males (adjusted odds ratio [aOR] = 3.25; 95% confidence interval [CI] = 1.87, 4.98), current drinking (aOR = 2.34; 95% CI = 1.46, 3.74), drug used (aOR = 2.97; 95% CI = 1.24, 7.11), and being bullied (aOR = 1.41; 95% CI = 1.00, 1.98) at least once in the past 12 months. Smoking is associated with several behaviors that pose risks to adolescents, such as social issues and smoking-related health problems. Thus, early and integrated prevention programs that address multiple risk behaviors simultaneously are required.

Intracellular calcium levels can regulate Importin-dependent nuclear import.

We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

Point mutations in murine Nkx2-5 phenocopy human congenital heart disease and induce pathogenic Wnt signaling

Mutations in the Nkx2-5 gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the Nkx2-5 gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new Nkx2-5 point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of Nkx2-5-driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by Nkx2-5 in the heart and show that Nkx2-5-dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how Nkx2-5 regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by NKX2-5 mutations in patients.

Systematic Review on International Practices in Controlling Waterpipe Tobacco Smoking

BACKGROUNDnWaterpipe tobacco smoking has becoming popular especially among young people worldwide. Smokers are attracted by its sweeter, smoother smoke, social ambience and the misconception of reduced harm. The objective of this study was to systematically review the effects of waterpipe tobacco policies and practices in reducing its prevalence.nnnMATERIALS AND METHODSnA systematic review was conducted electronically using the PubMed, OVID, Science Direct, Proquest and Embase databases. All possible studies from 1980 to 2013 were initially screened based on titles and abstracts. The selected articles were subjected to data extraction and quality rating.nnnRESULTSnThree studies met the inclusion criteria and were eligible for this review. Almost all of the waterpipe tobacco products and its accessories did not comply with the regulations on health warning labelling practices as stipulated under Article 11 of WHO FCTC. In addition, the grisly new warning labels for cigarettes introduced by Food and Drug Administration did not affect hookah tobacco smoking generally. Indoor air quality in smoking lounges was found to be poor and some hookah lounges were operated without smoke shop certification.nnnCONCLUSIONSnOur findings revealed the availability of minimal information on the practices in controlling waterpipe smoking in reducing its prevalence. The lack of comprehensive legislations or practices in controlling waterpipe smoking warrants further research and policy initiatives to curb this burgeoning global epidemic, especially among the vulnerable younger population.