Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

PURPOSE: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, > or =3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. EXPERIMENTAL DESIGN: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). RESULTS: Overall, 13 and 21 patients were found to possess a total of <4 and > or =4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had > or =4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had > or =4 SNPs (P < 0.001). CONCLUSIONS: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol.

Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10(-9). There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.

Face Short-Term Memory-Related Electroencephalographic Patterns can Differentiate Multi- versus Single-Domain Amnestic Mild Cognitive Impairment.

Amnestic mild cognitive impairment (aMCI) is characterized by memory deficits alone (single-domain, sd-aMCI) or associated with other cognitive disabilities (multi-domain, md-aMCI). The present study assessed the patterns of electroencephalographic (EEG) activity during the encoding and retrieval phases of short-term memory in these two aMCI subtypes, to identify potential functional differences according to the neuropsychological profile. Continuous EEG was recorded in 43 aMCI patients, whose 16 sd-aMCI and 27 md-aMCI, and 36 age-matched controls (EC) during delayed match-to-sample tasks for face and letter stimuli. At encoding, attended stimuli elicited parietal alpha (8-12 Hz) power decrease (desynchronization), whereas distracting stimuli were associated with alpha power increase (synchronization) over right central sites. No difference was observed in parietal alpha desynchronization among the three groups. For attended faces, the alpha synchronization underlying suppression of distracting letters was reduced in both aMCI subgroups, but more severely in md-aMCI cases that differed significantly from EC. At retrieval, the early N250r recognition effect was significantly reduced for faces in md-aMCI as compared to both sd-aMCI and EC. The results suggest a differential alteration of working memory cerebral processes for faces in the two aMCI subtypes, face covert recognition processes being specifically altered in md-aMCI.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Sensitivity of single-voxel 1H-MRS in investigating the metabolism of the activated human visual cortex at 7 T.

Proton magnetic resonance spectroscopy (1H-MRS) has been used in a number of studies to noninvasively assess the temporal changes of lactate in the activated human brain. However, the results have not been consistent. The aim of the present study was to test the sensitivity of 1H-MRS during functional experiments at the highest magnetic field currently available for human studies (7 T). Stability and reproducibility of the measurements were evaluated from LCModel analysis of time series of spectra measured during a visual stimulation paradigm and by examination of the difference between spectra obtained at rest and during activation. The sensitivity threshold to detect concentration changes was 0.2 micromol/g for most of the quantified metabolites. The possible variations of metabolite concentrations during visual stimulation were within the same range (+/-0.2 micromol/g). In addition, the influence of a small line-narrowing effect due to the blood oxygenation level-dependent (BOLD) T2* changes on the estimated concentrations was simulated. Quantification of metabolites was, in general, not affected beyond 1% by line-width changes within 0.5 Hz.

The timing of exploratory decision-making revealed by single-trial topographic EEGanalyses.

Decision-making in an uncertain environment is driven by two major needs: exploring the environment to gather information or exploiting acquired knowledge to maximize reward. The neural processes underlying exploratory decision-making have been mainly studied by means of functional magnetic resonance imaging, overlooking any information about the time when decisions are made. Here, we carried out an electroencephalography (EEG) experiment, in order to detect the time when the brain generators responsible for these decisions have been sufficiently activated to lead to the next decision. Our analyses, based on a classification scheme, extract time-unlocked voltage topographies during reward presentation and use them to predict the type of decisions made on the subsequent trial. Classification accuracy, measured as the area under the Receiver Operator's Characteristic curve was on average 0.65 across 7 subjects. Classification accuracy was above chance levels already after 516 ms on average, across subjects. We speculate that decisions were already made before this critical period, as confirmed by a positive correlation with reaction times across subjects. On an individual subject basis, distributed source estimations were performed on the extracted topographies to statistically evaluate the neural correlates of decision-making. For trials leading to exploration, there was significantly higher activity in dorsolateral prefrontal cortex and the right supramarginal gyrus; areas responsible for modulating behavior under risk and deduction. No area was more active during exploitation. We show for the first time the temporal evolution of differential patterns of brain activation in an exploratory decision-making task on a single-trial basis.

Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index.

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.

Bone mineral density (BMD), micro-architecture estimation (TBS) and vertebral fracture assessment (VFA) extracted from a single DXA device in combination with clinical risk factors improve significantly the identification of women at high risk of fracture.

Osteoporosis (OP) is a systemic skeletal disease characterized by a low bone mineral density (BMD) and a micro-architectural (MA) deterioration. Clinical risk factors (CRF) are often used as a MA approximation. MA is yet evaluable in daily practice by the trabecular bone score (TBS) measure. TBS is very simple to obtain, by reanalyzing a lumbar DXA-scan. TBS has proven to have diagnosis and prognosis values, partially independent of CRF and BMD. The aim of the OsteoLaus cohort is to combine in daily practice the CRF and the information given by DXA (BMD, TBS and vertebral fracture assessment (VFA)) to better identify women at high fracture risk. The OsteoLaus cohort (1400 women 50 to 80 years living in Lausanne, Switzerland) started in 2010. This study is derived from the cohort COLAUS who started in Lausanne in 2003. The main goal of COLAUS is to obtain information on the epidemiology and genetic determinants of cardiovascular risk in 6700 men and women. CRF for OP, bone ultrasound of the heel, lumbar spine and hip BMD, VFA by DXA and MA evaluation by TBS are recorded in OsteoLaus. Preliminary results are reported. We included 631 women: mean age 67.4 ± 6.7 years, BMI 26.1 ± 4.6, mean lumbar spine BMD 0.943 ± 0.168 (T-score − 1.4 SD), and TBS 1.271 ± 0.103. As expected, correlation between BMD and site matched TBS is low (r2 = 0.16). Prevalence of VFx grade 2/3, major OP Fx and all OP Fx is 8.4%, 17.0% and 26.0% respectively. Age- and BMI-adjusted ORs (per SD decrease) are 1.8 (1.2-2.5), 1.6 (1.2-2.1), and 1.3 (1.1-1.6) for BMD for the different categories of fractures and 2.0 (1.4-3.0), 1.9 (1.4-2.5), and 1.4 (1.1-1.7) for TBS respectively. Only 32 to 37% of women with OP Fx have a BMD < − 2.5 SD or a TBS < 1.200. If we combine a BMD < − 2.5 SD or a TBS < 1.200, 54 to 60% of women with an osteoporotic Fx are identified. As in the already published studies, these preliminary results confirm the partial independence between BMD and TBS. More importantly, a combination of TBS subsequent to BMD increases significantly the identification of women with prevalent OP Fx which would have been misclassified by BMD alone. For the first time we are able to have complementary information about fracture (VFA), density (BMD), micro- and macro architecture (TBS and HAS) from a simple, low ionizing radiation and cheap device: DXA. Such complementary information is very useful for the patient in the daily practice and moreover will likely have an impact on cost effectiveness analysis.

Information from single-cell bacterial biosensors: what is it good for?

Bacterial reporter cells (i.e. strains engineered to produce easily measurable signals in response to one or more chemical targets) can principally be used to quantify chemical signals and analytes, physicochemical conditions and gradients on a microscale (i.e. micrometer to submillimeter distances), when the reporter signal is determined in individual cells. This makes sense, as bacterial life essentially thrives in microheterogenic environments and single-cell reporter information can help us to understand the microphysiology of bacterial cells and its importance for macroscale processes like pollutant biodegradation, beneficial bacteria-eukaryote interactions, and infection. Recent findings, however, showed that clonal bacterial populations are essentially always physiologically, phenotypically and genotypically heterogeneous, thus emphasizing the need for sound statistical approaches for the interpretation of reporter response in individual bacterial cells. Serious attempts have been made to measure and interpret single-cell reporter gene expression and to understand variability in reporter expression among individuals in a population.

Initial experience with hybrid surgery in congenital heart disease in a single center : P171

Introduction: A hybrid intervention is a joint procedure involving the interventional cardiologist and the cardiac surgeon. At our institution we have opted for this type of approach in congenital heart disease since 2005. We report here our initial experience. Cases: 1. A 3 year old boy with double aortic arch and multiple muscular ventricular septal defects (VSD),was readdressed for pulmonary band (PAB) removal and residual VSD closure after previous palliation. After surgical removal of the PAB, the surgeon provided a minimal transventricular access for placement of a 6mm Amplatzer® muscular VSD occluder by the cardiologist under transoesophageal guidance. The patient was extubated the same day and discharged after 5 days. 2. An 8 year old girl with Williams syndrome was followed for two large VSDs and severe peripheral pulmonary arteries (PA) stenosis. The membranous VSD was closed surgically, the muscular VSD during the same operation by direct placement of a 12 mm Amplatzer® muscular VSD occluder. During rewarming, balloon angioplasty of peripheral PA stenosis was achieved under fluoroscopy. Patient was extubated the following day and discharged after 8 days. 3. A 9 year old boy post tetralogy of Fallot repair had severe distal stenosis of the right ventricular to PA conduit.With patient on partial cardiopulmonary bypass, an incision was made on the conduit and a CP 8 Zig 16 stent placed on the stenosis. The child passed on full bypass and the definitive placement of the stent achieved. The child was extubated at the end of the intervention and discharged after 6 days. 4. A newborn presented at 2 days life with complex aortic arch anatomy: left aortic arch and right descending thoracic aorta perfused directly from a right arterial duct and left PA atresia. The arterial duct was stented with a Genesis XD stent dilated at 7mm. Two days later the cardiac surgeon made banded the right PA. The child was extubated after the operation and discharged a week later. Conclusion: Hybrid approach opens new ways of correction or palliation in congenital heart disease with encouraging results and less morbidity.

Hidden diversity in honey bee gut symbionts detected by single-cell genomics.

Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.

Analysis of genetic parentage in the tawny owl (Strix aluco) reveals extra-pair paternity is low

We have investigated genetic parentage in a Swiss population of tawny owls (Strix aluco). To this end, we performed genetic analysis for six polymorphic loci of 49 avian microsatellite loci tested for cross-species amplification. We found one extra-pair young out of 137 (0.7%) nestlings in 37 families (2.7%). There was no intra-specific brood parasitism. Our results are in accordance with previous findings for other raptors and owls that genetic monogamy is the rule. Female tawny owls cannot raise offspring without a substantial contribution by their mates. Hence one favoured hypothesis is that high paternal investment in reproduction selects for behaviour that prevents cuckoldry.

Endovascular treatment of symptomatic intracranial arterial stenosis: six-year experience in a single-center series of 42 consecutive patients with acute and mid-term results.

BACKGROUND: The limitations of the medical management of symptomatic intracranial arterial stenosis encourage the development of new therapeutic strategies such as intracranial stenting. OBJECTIVE: To report and analyze the results of a series of 42 patients treated with 3 different endovascular techniques: isolated angioplasty, balloon-expandable coronary stents, and the Wingspan self-expandable intracranial stent system. METHODS: Forty-two patients presenting with symptomatic intracranial arterial stenosis were treated with one of these techniques. Computed tomography angiography was performed 6 months after the procedure, and the clinical neurological statuses were categorized using the modified Rankin Scale and the National Institutes of Health Stroke Scale. RESULTS: A total of 42 lesions were treated: 9 with isolated angioplasty, 14 with balloon-expandable coronary stents, and 19 with Wingspan self-expandable intracranial stents. The mean patient age was 62.9 years, and the mean arterial diameter stenosis was 73.9%. Technical success was achieved in 97.6% of the patients. The overall incidence of procedural complications was 21.4%, and the postoperative permanent morbidity/mortality rate was 7.1%. There were 3 cases of in-stent thrombosis (1 fatal) and 5 cases of asymptomatic restenosis (11.9%), 3 in the isolated angioplasty group and 2 in the Wingspan self-expandable intracranial stent group (mean follow-up 20.4 months). The rate of restenosis was higher in the angioplasty group (33%) than in the coronary (0%) and Wingspan stent (10.5%) groups. CONCLUSION: Endovascular treatment of symptomatic intracranial stenosis has significant overall morbidity and mortality rates. Nevertheless, the very critical natural history of severe refractory lesions and the relatively favorable postoperative evolution suggest that it should be considered the first alternative strategy in cases in which medical therapy has failed.

Six new loci associated with body mass index highlight a neuronal influence on body weight regulation.

Common variants at only two loci, FTO and MC4R, have been reproducibly associated with body mass index (BMI) in humans. To identify additional loci, we conducted meta-analysis of 15 genome-wide association studies for BMI (n > 32,000) and followed up top signals in 14 additional cohorts (n > 59,000). We strongly confirm FTO and MC4R and identify six additional loci (P < 5 x 10(-8)): TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2 and NEGR1 (where a 45-kb deletion polymorphism is a candidate causal variant). Several of the likely causal genes are highly expressed or known to act in the central nervous system (CNS), emphasizing, as in rare monogenic forms of obesity, the role of the CNS in predisposition to obesity.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.