High-sensitivity measurements of multiple kinase activities in live single cells.

Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.

STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

Accelerated discovery via a whole-cell model.

To test the promise of whole-cell modeling to facilitate scientific inquiry, we compared growth rates simulated in a whole-cell model with experimental measurements for all viable single-gene disruption Mycoplasma genitalium strains. Discrepancies between simulations and experiments led to predictions about kinetic parameters of specific enzymes that we subsequently validated. These findings represent, to our knowledge, the first application of whole-cell modeling to accelerate biological discovery.

The role of socio-demographic factors in premature cervical cancer mortality in Colombia.

BACKGROUND: While cervical cancer (CC) is an important cause of premature mortality in Colombia, the impact of socio-demographic factors on CC mortality in young women is not well understood. The primary objective of this study was to identify differences in CC mortality among Colombian women aged 20-49 years associated with education, type of health insurance, urban or rural and region of residence, and to determine whether differences in mortality associated with education or insurance varied by age. METHODS: Cervical cancer deaths for 2005-2013 and risk factors were obtained from the National Administrative Department of Statistics. Populations at risk were calculated from age-stratified population projections and the 2010 National and Demographic Health Survey. Negative binomial regression models, stratified by age, were used to examine associations between socio-demographic factors and mortality rates and whether the effects of education and health insurance varied by age. Multiple imputation was used to examine the importance of missing data. RESULTS: Differences of CC mortality were identified among women with limited to no education compared to highly educated women, with the largest disparity in the youngest age group (IRR 26.8, 95 % CI 6.65-108). Differences in mortality associated with health insurance also varied based on age group. Women with contributory and special health insurance had lower mortality rates than women with subsidised or no health insurance, except in the youngest age group. No differences were observed between women with subsidised and those with no insurance in any age group. Mortality rates were high among women who resided in urban areas and in the Atlantic, Central, Pacific, and Amazon-Orinoquía regions of Colombia. Missing values in the mortality database did not impact the findings from this study. CONCLUSIONS: Limited education was most strongly associated with premature CC mortality in the youngest women. Subsidised insurance did not appear to provide significant protection against CC mortality when compared to not having insurance, suggesting the need to examine diagnostic and treatment services available under the subsidised insurance plan. Our results could be used to target interventions to optimise the impact of resources to prevent premature mortality due to CC in Colombia.

Diacylglycerol, when simplicity becomes complex.

Diacylglycerol (DAG) has unique functions as a basic component of membranes, an intermediate in lipid metabolism and a key element in lipid-mediated signaling. In eukaryotes, for example, impaired DAG generation and/or consumption have severe effects on organ development and cell growth associated with diseases such as cancer, diabetes, immune system disorders and Alzheimer's disease. Although DAG has been studied intensively as a signaling lipid, early models of its function are no longer adequate to explain its numerous roles. The interplay between enzymes that control DAG levels, the identification of families of DAG-regulated proteins, and the overlap among DAG metabolic and signaling processes are providing new interpretations of DAG function. Recent discoveries are also delineating the complex and strategic role of DAG in regulating biochemical networks.

Factors associated with drinking and being satisfied with tap water in Indigenous communities in Saskatchewan, Canada.

Previous studies have described concerns regarding tap water in Indigenous communities, yet there is little information on participants who report drinking their tap water and being satisfied with its quality. This study undertaken with members of 8 Indigenous communities in Saskatchewan, Canada, and identified factors associated with both the decision to drink tap water at home and being satisfied with its quality. We examined the importance of factors such as individual attributes, experiences, attitudes, household and community-based variables. Less than one-quarter of participants (23.4%) drank tap water and were satisfied with its quality. Individuals who did not boil tap water (odds ratio [OR] = 5.76, 95% confidence interval [CI] = 1.68-19.8), those who did not experience tap water odour (OR = 2.38, 95% CI = 1.26-4.50) and participants living in communities away from urban centres (OR = 2.74, 95% CI = 1.63-4.51) were more likely to drink and be satisfied with their tap water. Concerns about the environment had the most impact on community members aged 55+ years. Those not reporting concerns about environmental problems affecting water (OR = 11.4, 95% CI = 3.10-42.2) were much more likely to drink and be satisfied with their tap water. Programmes to improve water quality, reduce the need for boil water advisories and increase community confidence in the environment could improve tap water satisfaction and consumption.

Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes.

Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.

Spatial variations in cervical cancer prevention in Colombia: Geographical differences and associated socio-demographic factors.

We examined spatial variations in the frequencies of women who had not heard of human papillomavirus vaccination (NHrd-Vac) and who had not had Pap testing (NHd-Pap) among Colombian administrative divisions (departments), before and after considering differences in socio-demographic factors. Following global and local tests for clustering, Bayesian Poisson hierarchical models identified department factors associated with NHrd-Vac and NHd-Pap, as well as the extent of the spatially structured and unstructured heterogeneity. Models of spatial variations for both outcomes included the department percentage of women with subsidised health insurance. The relative risks of NHrd-Vac and NHd-Pap were highest in several departments adjacent to the Colombian border. Our finding that the risk of not having adequate access to cervical cancer (CC) prevention programmes in Colombia was location-dependent, could be used to focus resources for CC prevention programmes.

Diacylglycerol kinase ζ limits the polarized recruitment of diacylglycerol-enriched organelles to the immune synapse in T cells.

The antigen-induced formation of an immune synapse (IS) between T cells and antigen-presenting cells results in the rapid generation of the lipid second messenger diacylglycerol (DAG) in T cells. Diacylglycerol kinase ζ (DGKζ) converts DAG into phosphatidic acid (PA). Cytotoxic T lymphocytes (CTLs) from mice deficient in DGKζ have enhanced antiviral and antitumor activities, indicating that the amount of DAG controls the effectiveness of the T cell response. We characterized the second C1 domain of protein kinase Cθ (PKCθ), a DAG-binding protein that is specifically recruited to the IS, as a biological sensor to observe the generation of a DAG gradient during IS formation. In experiments with transgenic mouse CTLs expressing the OT-I T cell receptor (TCR), we showed that both strong and weak interactions between antigen and the TCR led to the rapid generation of DAG, whereas only strong interactions induced the movement of DAG-enriched organelles toward the IS. In DGKζ-deficient CTLs, antigen stimulation led to the enhanced accumulation of DAG-containing organelles at the IS; however, impaired activation of the PA effector PKCζ resulted in lack of reorientation of the microtubule-organizing center toward the IS, a process needed for effective T cell activation. Together, these data suggest that the activation of DGKζ downstream of antigen recognition provides a mechanism that ensures the activation of PA-dependent signaling as a direct result of the strength of TCR-dependent DAG mobilization.

Digital spiral imaging.

A major application of optics is imaging all types of structural, physical, chemical and biological features of matter. Techniques based on most known properties of light have been developed over the years to remotely acquire information about such features. They include the spin angular momentum, encoded in the polarization, but not yet the orbital angular momentum encoded in its spiral spectrum. Here we put forward the potential of such spiral spectra. In particular, we use several canonical examples to show how the orbital angular momentum spectra of a light beam can be used to image a variety of intrinsic and extrinsic properties encoded, e.g., in phase and amplitude gradients, dislocations or delays.

Predictors of having heard about human papillomavirus vaccination: Critical aspects for cervical cancer prevention among Colombian women.

OBJECTIVES: To determine whether the probability of having heard about human papillomavirus (HPV) vaccination differs by socio-demographic characteristics among Colombian women; and whether the effect of predictors of having heard about HPV vaccination varies by educational levels and rural/urban area of residence. METHODS: Data of 53,521 women aged 13-49 years were drawn from the 2010 Colombian National Demographic and Health Survey. Women were asked about aspects of their health and their socio-demographic characteristics. A logistic regression model was used to identify factors associated with having heard about HPV vaccination. Educational level and rural/urban area of residence of the women were tested as modifier effects of predictors. RESULTS: 26.8% of the women had heard about HPV vaccination. The odds of having heard about HPV vaccination were lower among women: in low wealth quintiles, without health insurance, with subsidized health insurance, and those who had children (p<0.001). Although women in older age groups and with better education had higher probabilities of having heard about HPV vaccination, differences in these probabilities by age group were more evident among educated women compared to non-educated ones. Probability gaps between non-educated and highly educated women were wider in the Eastern region. Living in rural areas decreased the probability of having heard about HPV vaccination, although narrower rural/urban gaps were observed in the Atlantic and Amazon-Orinoquía regions. CONCLUSIONS: Almost three quarters of the Colombian women had not heard about HPV vaccination, with variations by socio-demographic characteristics. Women in disadvantaged groups were less likely to have heard about HPV vaccination.

Inequities in cervical cancer screening among Colombian women: a multilevel analysis of a nationwide survey.

OBJECTIVES: To identify factors associated with whether women in Colombia have had a Pap test, evaluate differences in risk factors between rural and urban residence, and evaluate the contextual effect of the lack of education on having ever had a Pap test. METHOD: Data used were from the 2010 Colombian National Demographic and Health Survey; 40,392 women reported whether they have had a Pap test. A multilevel mixed logistic regression model was developed with random intercepts to account for clustering by neighbourhood and municipality. The model evaluated whether having a rural/urban area of residence modified the effect of identified risk factors and if the prevalence of no education at the neighbourhood level acted as a contextual effect. RESULTS: Most women (87.3%) reported having at least one Pap test. Women from lower socioeconomic quintiles (p=0.002), who were unemployed (p<0.001), and whose final health decisions depended on others (p<0.001) were less likely to have had a Pap test. Women with children were more likely to have had the test (p<0.001), and the effects of education (p=0.03), type of health insurance (p=0.01), age (p<0.001), and region (p<0.001) varied with having a rural/urban area of residence. Women living in rural areas (specifically younger ones, with no health insurance, living in the Atlantic and Amazon-Orinoquía regions, and with no education) were less likely to have had a Pap test when compared to those living in urban areas. Furthermore, women living in a neighbourhood with a higher prevalence of no education were less likely to have ever had a Pap test (p=0.005). CONCLUSIONS: In Colombia, the probability of having had a Pap test is associated with personal attributes, area of residence, and prevalence of no education in the neighbourhood. Efforts to improve access to cervical cancer screening should focus on disadvantaged women with limited education, low socioeconomic status, and no health insurance or subsidised insurance, especially those in rural/isolated areas.

Properties of quadratic multi-soliton generation near phase-match in periodically poled potassium titanyl phosphate.

The properties of the multi-quadratic-soliton generation process have been investigated both theoretically and experimentally near and on phase-match in non-critically-phase-matched, periodically poled, potassium titanyl phosphate (PPKTP). It was found that multi-soliton generation occurs primarily due to asymmetry in the input beam and at phase-matching. The number of solitons generated depended on the input intensity in a non-trivial way.

Complex soliton-like pattern generation in Potassium Niobate due to noisy, high intensity, input beams.

We report the generation of complex soliton-like patterns in noncritically-phase-matched potassium niobate which occur in random spatial patterns from shot-to-shot. Up to five spots have been generated at input intensities of 10's GW/cm2, many times the single soliton threshold. The mechanism which leads to the symmetry breaking required for the complex patterns is interpreted to be random noise imprinted on the input light.

Observation of multiple soliton generation mediated by amplification of asymmetries.

We report the experimental observation of the formation of multiple optical quadratic solitons in a process mediated by the amplification of minute asymmetries in the diffraction properties of the input light. Experiments were conducted in phase-matched second-harmonic generation in a bulk crystal of periodically poled potassium titanyl phosphate pumped at 1064 nm. The different mechanisms that influence the process were investigated numerically, and the pulsed nature of the pump light was found to play a key role in the observed light distributions.

A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration.

Ca(2+) signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca(2+)-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca(2+) pulses, local depletion of Ca(2+) in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca(2+) influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca(2+) pump rates in the front relative to the back of the plasma membrane enable effective local Ca(2+) signalling by locally decreasing basal Ca(2+). Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca(2+) control system with polarized Ca(2+) signalling proteins and second messengers to synergistically promote directed cell migration.

Ultrabroadband biphotons generated via chirped quasi-phase-matched optical parametric down-conversion.

We generate ultrabroadband biphotons via the process of spontaneous parametric down-conversion (SPDC) in quasi-phase-matched nonlinear gratings that have a linearly chirped wave vector. By using these ultrabroadband biphotons (300-nm bandwidth), we measure the narrowest Hong-Ou-Mandel dip to date, having a full width at half maximum of 7.1 fs. This enables the generation of a high flux of nonoverlapping biphotons with ultrabroad bandwidth, thereby promoting the use of SPDC light in many nonclassical applications.

Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy.

The signal and idler beams from a picosecond, synchronously pumped optical parametric oscillator (OPO) provide the two colors necessary for coherent anti-Stokes Raman scattering (CARS) microscopy. The OPO provides a continuously tunable frequency difference between the two beams over a broad range of Raman shifts (100-3700 cm(-1)) by varying the temperature of a single nonlinear crystal. The near-infrared output (900-1300 nm) allows for deep penetration into thick samples and reduced nonlinear photodamage. Applications of this light source to in vivo cell and ex vivo tissue imaging are demonstrated.

Broadband light generation by noncollinear parametric downconversion.

Broadband light generation is demonstrated by noncollinear spontaneous parametric downconversion with a cw pump laser. By use of a suitable noncollinear phase-matching geometry and a tightly focused pump beam, downconverted signals that feature a bell-shaped spectral distribution with a bandwidth approaching 200 nm are obtained. As an application of the generated broadband light, submicrometer axial resolution in an optical coherence tomography scheme is demonstrated; a free-space resolution down to 0.8 microm was achieved.