DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America

Species can respond to environmental pressures through genetic and epigenetic changes and through phenotypic plasticity, but few studies have evaluated the relationships between genetic differentiation and phenotypic plasticity of plant species along changing environmental conditions throughout wide latitudinal ranges. We studied inter‐ and intrapopulation genetic diversity (using simple sequence repeats and chloroplast DNA sequencing) and inter‐ and intrapopulation phenotypic variability of 33 plant traits (using field and common‐garden measurements) for five populations of the invasive cordgrass Spartina densiflora Brongn. along the Pacific coast of North America from San Francisco Bay to Vancouver Island. Studied populations showed very low genetic diversity, high levels of phenotypic variability when growing in contrasted environments and high intrapopulation phenotypic variability for many plant traits. This intrapopulation phenotypic variability was especially high, irrespective of environmental conditions, for those traits showing also high phenotypic plasticity. Within‐population variation represented 84% of the total genetic variation coinciding with certain individual plants keeping consistent responses for three plant traits (chlorophyll b and carotenoid contents, and dead shoot biomass) in the field and in common‐garden conditions. These populations have most likely undergone genetic bottleneck since their introduction from South America; multiple introductions are unknown but possible as the population from Vancouver Island was the most recent and one of the most genetically diverse. S. densiflora appears as a species that would not be very affected itself by climate change and sea‐level rise as it can disperse, establish, and acclimate to contrasted environments along wide latitudinal ranges.     

Reliability of Health-Related Physical Fitness Tests among Colombian Children and Adolescents: The FUPRECOL Study

Substantial evidence indicates that youth physical fitness levels are an important marker of lifestyle and cardio-metabolic health profiles and predict future risk of chronic diseases. The reliability physical fitness tests have not been explored in Latino-American youth population. This study’s aim was to examine the reliability of health-related physical fitness tests that were used in the Colombian health promotion “Fuprecol study”. Participants were 229 Colombian youth (boys n = 124 and girls n = 105) aged 9 to 17.9 years old. Five components of health-related physical fitness were measured: 1) morphological component: height, weight, body mass index (BMI), waist circumference, triceps skinfold, subscapular skinfold, and body fat (%) via impedance; 2) musculoskeletal component: handgrip and standing long jump test; 3) motor component: speed/agility test (4x10 m shuttle run); 4) flexibility component (hamstring and lumbar extensibility, sit-and-reach test); 5) cardiorespiratory component: 20-meter shuttle-run test (SRT) to estimate maximal oxygen consumption. The tests were performed two times, 1 week apart on the same day of the week, except for the SRT which was performed only once. Intra-observer technical errors of measurement (TEMs) and inter-rater (reliability) were assessed in the morphological component. Reliability for the Musculoskeletal, motor and cardiorespiratory fitness components was examined using Bland–Altman tests. For the morphological component, TEMs were small and reliability was greater than 95% of all cases. For the musculoskeletal, motor, flexibility and cardiorespiratory components, we found adequate reliability patterns in terms of systematic errors (bias) and random error (95% limits of agreement). When the fitness assessments were performed twice, the systematic error was nearly 0 for all tests, except for the sit and reach (mean difference: -1.03% [95% CI = -4.35% to -2.28%]. The results from this study indicate that the “Fuprecol study” health-related physical fitness battery, administered by physical education teachers, was reliable for measuring health-related components of fitness in children and adolescents aged 9–17.9 years old in a school setting in Colombia.     

Epidemiology of Cholera in the Philippines

Background

Despite being a cholera-endemic country, data on cholera in the Philippines remain sparse. Knowing the areas where cholera is known to occur and the factors that lead to its occurrence will assist in planning preventive measures and disaster mitigation.

Methods

Using sentinel surveillance data, PubMed and ProMED searches covering information from 2008–2013 and event-based surveillance reports from 2010–2013, we assessed the epidemiology of cholera in the Philippines. Using spatial log regression, we assessed the role of water, sanitation and population density on the incidence of cholera.

Results and Discussion

We identified 12 articles from ProMED and none from PubMed that reported on cholera in the Philippines from 2008 to 2013. Data from ProMed and surveillance revealed 42,071 suspected and confirmed cholera cases reported from 2008 to 2013, among which only 5,006 were confirmed. 38 (47%) of 81 provinces and metropolitan regions reported at least one confirmed case of cholera and 32 (40%) reported at least one suspected case. The overall case fatality ratio in sentinel sites was 0.62%, but was 2% in outbreaks. All age groups were affected. Using both confirmed and suspected cholera cases, the average annual incidence in 2010–2013 was 9.1 per 100,000 population. Poor access to improved sanitation was consistently associated with higher cholera incidence. Paradoxically, access to improved water sources was associated with higher cholera incidence using both suspected and confirmed cholera data sources. This finding may have been due to the breakdown in the infrastructure and non-chlorination of water supplies, emphasizing the need to maintain public water systems.

Conclusion

Our findings confirm that cholera affects a large proportion of the provinces in the country. Identifying areas most at risk for cholera will support the development and implementation of policies to minimize the morbidity and mortality due to this disease.     

A genomewide admixture mapping panel for Hispanic/Latino populations

Admixture mapping (AM) is a promising method for the identification of genetic risk factors for complex traits and diseases showing prevalence differences among populations. Efficient application of this method requires the use of a genomewide panel of ancestry-informative markers (AIMs) to infer the population of origin of chromosomal regions in admixed individuals. Genomewide AM panels with markers showing high frequency differences between West African and European populations are already available for disease-gene discovery in African Americans. However, no such a map is yet available for Hispanic/Latino populations, which are the result of two-way admixture between Native American and European populations or of three-way admixture of Native American, European, and West African populations. Here, we report a genomewide AM panel with 2,120 AIMs showing high frequency differences between Native American and European populations. The average intermarker genetic distance is ~1.7 cM. The panel was identified by genotyping, with the Affymetrix GeneChip Human Mapping 500K array, a population sample with European ancestry, a Mesoamerican sample comprising Maya and Nahua from Mexico, and a South American sample comprising Aymara/Quechua from Bolivia and Quechua from Peru. The main criteria for marker selection were both high information content for Native American/European ancestry (measured as the standardized variance of the allele frequencies, also known as "f value") and small frequency differences between the Mesoamerican and South American samples. This genomewide AM panel will make it possible to apply AM approaches in many admixed populations throughout the Americas.     

Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides

BACKGROUND AND AIMS: Atriplex (Halimione) portulacoides is a halophytic, C(3) shrub. It is virtually confined to coastal salt marshes, where it often dominates the vegetation. The aim of this study was to investigate its growth responses to salinity and the extent to which these could be explained by photosynthetic physiology. METHODS: The responses of young plants to salinity in the range 0-700 mol m(-3) NaCl were investigated in a glasshouse experiment. The performance of plants was examined using classical growth analysis, measurements of gas exchange (infrared gas analysis), determination of chlorophyll fluorescence characteristics (modulated fluorimeter) and photosynthetic pigment concentrations; total ash, sodium, potassium and nitrogen concentrations, and relative water content were also determined. KEY RESULTS: Plants accumulated Na(+) approximately in proportion to external salinity. Salt stimulated growth up to an external concentration of 200 mol m(-3) NaCl and some growth was maintained at higher salinities. The main determinant of growth response to salinity was unit leaf rate. This was itself reflected in rates of CO(2) assimilation, which were not affected by 200 mol m(-3) but were reduced at higher salinities. Reductions in net photosynthetic rate could be accounted for largely by lower stomatal conductance and intercellular CO(2) concentration. Apart from possible effects of osmotic shock at the beginning of the experiment, salinity did not have any adverse effect on photosystem II (PSII). Neither the quantum efficiency of PSII (Phi(PSII)) nor the chlorophyll fluorescence ratio (F(v)/F(m)) were reduced by salinity, and lower mid-day values recovered by dawn. Mid-day F(v)/F(m) was in fact depressed more at low external sodium concentration, by the end of the experiment. CONCLUSIONS: The growth responses of the hygro-halophyte A. portulacoides to salinity appear largely to depend on changes in its rate of photosynthetic gas exchange. Photosynthesis appears to be limited mainly through stomatal conductance and hence intercellular CO(2) concentration, rather than by effects on PSII; moderate salinity might stimulate carboxylation capacity. This is in contrast to more extreme halophytes, for which an ability to maintain leaf area can partially offset declining rates of carbon assimilation at high salinity.     

Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.