Are genetic polymorphisms in OGG1, XRCC1 and XRCC3 genes predictive for the DNA strand break repair phenotype and genotoxicity in workers exposed to low dose ionising radiations?

Identification of higher risk individuals carrying genetic polymorphisms responsible for reduced DNA repair capacity has substantial preventive implications as these individuals could be targeted for cancer prevention. We have conducted a study to assess the predictivity of the OGG1, XRCC1 and XRCC3 genotypes and the in vitro single strand break repair phenotype for the induction of genotoxic effects. At the population level, a significant contribution of the OGG1 genotypes to the in vitro DNA strand break repair capacity was found. At an individual level, the OGG1 variants Ser/Cys and Cys/Cys genotypes showed a slower in vitro DNA repair than the Ser/Ser OGG1genotype. A multivariate analysis performed with genotypes, age, cumulative dose, exposure status and smoking as independent variables indicated that in the control population, repair capacity is influenced by age and OGG1 polymorphisms. In the exposed population, DNA damage is greater in older men and in smokers. Repair capacity is slower in individuals with Ser/Cys or Cys/Cys OGG1 genotypes compared to those with the Ser/Ser OGG1 genotype. Micronuclei (MN) frequencies increased with age and the cumulative dose of gamma-rays. Analysis of the total population revealed that genetic polymorphisms in XRCC1 resulted in higher residual DNA (RDNA) values and the Met/Met variant of XRCC3 resulted in an increased frequency of micronuclei. The analysis confirms that MN frequencies are reliable biomarkers for the assessment of genetic effects in workers exposed to ionising radiation (IR). A combined analysis of the three genotypes, OGG1, XRCC1 and XRCC3 polymorphisms is advised in order to assess individual susceptibility to ionising radiation. As an alternative or complement, the in vitro DNA strand break repair phenotype which integrates several repair pathways is recommended. Smokers with OGG1 polymorphisms who are exposed to ionising radiation represent a specific population requiring closer medical surveillance because of their increased mutagenic/carcinogenic risk.     

High-efficiency immunomagnetic isolation of solid tissue-originated integrin-expressing adult stem cells

Isolation of highly pure specific cell types is crucial for successful adult stem cell-based therapy. As the number of such cells in adult tissue is low, an extremely efficient method is needed for their isolation. Here, we describe cell-separation methodologies based on magnetic-affinity cell sorting (MACS) MicroBeads with monoclonal antibodies against specific membrane proteins conjugated to superparamagnetic particles. Cells labeled with MACS MicroBeads are retained in a magnetic field within a MACS column placed in a MACS separator, allowing fast and efficient separation. Both positively labeled and non-labeled fractions can be used directly for downstream applications as the separated cell fractions remain viable with no functional impairment. As immunomagnetic separation depends on the interaction between a cell's membrane and the magnetically labeled antibody, separation of specific cells originating from solid tissues is more complex and demands a cell-dissociating pretreatment. In this paper, we detail the use of immunomagnetic separation for the purpose of regenerating damaged salivary gland (SG) function in animal and human models of irradiated head and neck cancer. Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients lose SG function following irradiation therapy. SGs contain integrin α6β1-expressing epithelial stem cells. We hypothesized that these cells can be isolated, multiplied in culture and auto-implanted into the irradiated SGs to regenerate damaged SG function.     

Generation of some new mutant xylitol producing yeast strains

Xylitol, a sugar alcohol with various utilisations in food, pharmaceutical and cosmetics industry can be produced by yeasts via biotechnologies far more economically efficient and environmentally friendly than chemical separation from natural sources. The present paper reports on a successful attempt to identify high performance xylitol producers among the representatives of the Candida and Rhodotorula genera, followed by the enhancement of their capacities by mutagenesis. The strain designated as C. boidinii ICCF-UV10 was finally selected as the best xylitol producer from the parental and mutant strains.     

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established¹, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca²⁺ homeostasis², bioenergetics and redox signaling ³.mtPTP opening is triggered by matrix Ca²⁺ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids⁴. In vitro, mtPTP opening can be achieved by increasing Ca²⁺ concentration inside the mitochondrial matrix through exogenous additions of Ca²⁺ (calcium retention capacity). When Ca²⁺ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca²⁺ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca²⁺ handling (uptake and release, as measured by Ca²⁺ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca²⁺ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca²⁺, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.     

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors

Background

Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence.

Methodology/Principal Findings

197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy.

Conclusion/Significance

RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.     

Continuity of primary care and emergency department utilization among elderly people

Background

People aged 65 years or more represent a growing group of emergency department users. We investigated whether characteristics of primary care (accessibility and continuity) are associated with emergency department use by elderly people in both urban and rural areas.

Methods

We conducted a cross-sectional study using information for a random sample of 95 173 people aged 65 years or more drawn from provincial administrative databases in Quebec for 2000 and 2001. We obtained data on the patients'' age, sex, comorbidity, rate of emergency department use (number of days on which a visit was made to an amergency department per 1000 days at risk [i.e., alive and not in hospital] during the 2-year study period), use of hospital and ambulatory physician services, residence (urban v. rural), socioeconomic status, access (physician: population ratio, presence of primary physician) and continuity of primary care.

Results

After adjusting for age, sex and comorbidity, we found that an increased rate of emergency department use was associated with lack of a primary physician (adjusted rate ratio [RR] 1.45, 95% confidence interval [CI] 1.41–1.49) and low or medium (v. high) levels of continuity of care with a primary physician (adjusted RR 1.46, 95% CI 1.44–1.48, and 1.27, 95% CI 1.25–1.29, respectively). Other significant predictors of increased use of emergency department services were residence in a rural area, low socioeconomic status and residence in a region with a higher physician:population ratio. Among the patients who had a primary physician, continuity of care had a stronger protective effect in urban than in rural areas.

Interpretation

Having a primary physician and greater continuity of care with this physician are factors associated with decreased emergency department use by elderly people, particularly those living in urban areas.Canada is reforming its health care system, with primary care as a major focus.¹ The population of Canadians aged 65 years or older is expected to double by 2026² and already accounts for the largest share of total health care expenditures.³ Thus, it is important to evaluate primary care services in this population. Because the emergency department often acts as a safety net for patients receiving inadequate primary care,⁴ emergency department use may be an important indicator of the adequacy of primary care services.The main determinants of emergency department use by elderly people are the severity and the nature of the medical needs of the patient (overall and specific comorbidities).⁵ After adjustment for need, increased access to and continuity of primary care may also be associated with lower emergency department use.⁵ However, most studies that investigated the impact of access and continuity of primary care were carried out in the United States, where the health care system is fundamentally different from Canada''s.^5–8 Furthermore, most of these studies used self- reported measures of access and continuity of primary care.^5,7,9We sought to identify determinants of emergency department use in a population-based sample of elderly people in Quebec, with particular focus on measures of access to and continuity of primary care. Access was defined by 2 measures: (a) presence of a primary physician and (b) physician: population ratio. Relational continuity was defined as the proportion of primary care visits with the primary physician.^10,11 Finally, because primary care services in Quebec are organized differently in urban and rural areas,¹² we also compared the association between emergency department use and continuity of care for urban and rural areas.     

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ?50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.