50nm-Scale Localization of Single Unmodified,Isotopically Enriched,Proteins in Cells

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when ¹⁴N isotopes are replaced with ¹⁵N. ¹⁵N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few ¹⁵N-enriched proteins - and even a single protein - within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in ¹⁵N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise ¹⁵N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXRα formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.     

Mapping Cortico-Striatal Connectivity onto the Cortical Surface: A New Tractography-Based Approach to Study Huntington Disease

Huntington disease (HD) is associated with early and severe damage to the basal ganglia and particularly the striatum. We investigated cortico-striatal connectivity modifications occurring in HD patients using a novel approach which focuses on the projection of the connectivity profile of the basal ganglia onto the cortex. This approach consists in computing, for each subcortical structure, surface connectivity measures representing its strength of connections to the cortex and comparing these measures across groups. In this study, we focused on Huntington disease as an application of this new approach. First, surface cortico-striatal connectivity measures of a group of healthy subjects were averaged in order to infer the “normal” connectivity profile of the striatum to the cortex. Second, a statistical analysis was performed from the surface connectivity measures of healthy subjects and HD patients in order to detect the cortical gyri presenting altered cortico-striatal connectivity in HD. Lastly, percentage differences of connectivity between healthy subjects and patients were inferred, for each nucleus of the striatum, from the connectivity measures of the cortical gyri presenting a significant connectivity difference between the two groups. These percentage differences characterize the axonal disruptions between the striatum and the cortex occurring in HD. We found selective region-specific degeneration of cortical connections predominating for associative and primary sensorimotor connections and with relative preservation of limbic connections. Our method can be used to infer novel connectivity-based markers of HD pathological process.     

Adaptation of Maize to Temperate Climates: Mid-Density Genome-Wide Association Genetics and Diversity Patterns Reveal Key Genomic Regions,with a Major Contribution of the Vgt2 (ZCN8) Locus

The migration of maize from tropical to temperate climates was accompanied by a dramatic evolution in flowering time. To gain insight into the genetic architecture of this adaptive trait, we conducted a 50K SNP-based genome-wide association and diversity investigation on a panel of tropical and temperate American and European representatives. Eighteen genomic regions were associated with flowering time. The number of early alleles cumulated along these regions was highly correlated with flowering time. Polymorphism in the vicinity of the ZCN8 gene, which is the closest maize homologue to Arabidopsis major flowering time (FT) gene, had the strongest effect. This polymorphism is in the vicinity of the causal factor of Vgt2 QTL. Diversity was lower, whereas differentiation and LD were higher for associated loci compared to the rest of the genome, which is consistent with selection acting on flowering time during maize migration. Selection tests also revealed supplementary loci that were highly differentiated among groups and not associated with flowering time in our panel, whereas they were in other linkage-based studies. This suggests that allele fixation led to a lack of statistical power when structure and relatedness were taken into account in a linear mixed model. Complementary designs and analysis methods are necessary to unravel the architecture of complex traits. Based on linkage disequilibrium (LD) estimates corrected for population structure, we concluded that the number of SNPs genotyped should be at least doubled to capture all QTLs contributing to the genetic architecture of polygenic traits in this panel. These results show that maize flowering time is controlled by numerous QTLs of small additive effect and that strong polygenic selection occurred under cool climatic conditions. They should contribute to more efficient genomic predictions of flowering time and facilitate the dissemination of diverse maize genetic resources under a wide range of environments.     

Apolipoprotein A-I Is a Potential Mediator of Remote Ischemic Preconditioning

Background

Remote ischemic preconditioning (RIPC) has emerged as an attractive strategy in clinical settings. Despite convincing evidence of the critical role played by circulating humoral mediators, their actual identities remain unknown. In this study, we aimed to identify RIPC-induced humoral mediators using a proteomic approach.

Methods

and Results Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5′) or 10-min (RIPC 10′) reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were analyzed using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). Three protein peaks were selected for their significant increase in RIPC 10′. They were identified and confirmed as apolipoprotein A-I (ApoA-I). Additional rats were exposed to myocardial ischemia-reperfusion (I/R) and assigned to one of the following groups RIPC+myocardial infarction (MI) (10-min limb ischemia followed by 10-min reperfusion initiated 20 minutes prior to myocardial I/R), ApoA-I+MI (10 mg/kg ApoA-I injection 10 minutes before myocardial I/R), and MI (no further intervention). In comparison with untreated MI rats, RIPC reduced infarct size (52.2±3.7% in RIPC+MI vs. 64.9±2.6% in MI; p<0.05). Similarly, ApoA-I injection decreased infarct size (50.9±3.8%; p<0.05 vs. MI).

Conclusions

RIPC was associated with a plasmatic increase in ApoA-I. Furthermore, ApoA-I injection before myocardial I/R recapitulated the cardioprotection offered by RIPC in rats. This data suggests that ApoA-I may be a protective blood-borne factor involved in the RIPC mechanism.     

Diversity,Ecology and Biogeochemistry of Cyst-Forming Acantharia (Radiolaria) in the Oceans

Marine planktonic organisms that undertake active vertical migrations over their life cycle are important contributors to downward particle flux in the oceans. Acantharia, globally distributed heterotrophic protists that are unique in building skeletons of celestite (strontium sulfate), can produce reproductive cysts covered by a heavy mineral shell that sink rapidly from surface to deep waters. We combined phylogenetic and biogeochemical analyses to explore the ecological and biogeochemical significance of this reproductive strategy. Phylogenetic analysis of the 18S and 28S rRNA genes of different cyst morphotypes collected in different oceans indicated that cyst-forming Acantharia belong to three early diverging and essentially non symbiotic clades from the orders Chaunacanthida and Holacanthida. Environmental high-throughput V9 tag sequences and clone libraries of the 18S rRNA showed that the three clades are widely distributed in the Indian, Atlantic and Pacific Oceans at different latitudes, but appear prominent in regions of higher primary productivity. Moreover, sequences of cyst-forming Acantharia were distributed evenly in both the photic and mesopelagic zone, a vertical distribution that we attribute to their life cycle where flagellated swarmers are released in deep waters from sinking cysts. Bathypelagic sediment traps in the subantarctic and oligotrophic subtropical Atlantic Ocean showed that downward flux of Acantharia was only large at high-latitudes and during a phytoplankton bloom. Their contribution to the total monthly particulate organic matter flux can represent up to 3%. High organic carbon export in cold waters would be a putative nutritional source for juveniles ascending in the water column. This study improves our understanding of the life cycle and biogeochemical contribution of Acantharia, and brings new insights into a remarkable reproductive strategy in marine protists.     

2009 A(H1N1) Seroconversion Rates and Risk Factors among the General Population in Vientiane Capital,Laos

Objective

To assess 2009 A(H1N1) seroconversion rates and their determinants within an unvaccinated population in Vientiane Capital, Laos.

Methods

CoPanFlu Laos, a general population cohort of 807 households and 4,072 participants was established in March 2010. Sociodemographic data, epidemiological data, and capillary blood samples were collected from all the household members in March, and again in October 2010, in order to assess the level of antibodies to 2009 A(H1N1) with the haemagglutination inhibition assay. 2009 A(H1N1) seroconversion was defined as a fourfold or greater increase in titre between inclusion and follow-up. Determinants for pandemic influenza infection were studied using the generalized estimating equations model, taking household clustering into account.

Results

Between March and November 2010, 3,524 paired sera were tested. Prior to the pandemic, our cohort was almost completely vaccine-naive for seasonal influenza. The overall seroconversion rate among nonvaccinated individuals (n = 2,810) was 14.3% (95%CI [13.0, 15.6]), with the highest rate for participants under 20 yo (19.8%, 95%CI [17.4, 22.4]) and the lowest rate for participants over 60 yo (6.5%, 95%CI [3.7, 10.4]). Participants with lower baseline titres had significantly higher infection rates, with a dose-effect relationship. Odds ratios (ORs) ranged from 76.5 (95%CI [27.1, 215.8]), for those with a titre at inclusion of 1∶10, to 8.1 (95%CI [3.3, 20.4]), for those with a titre of 1∶40. Having another household member with a titre ≥1∶80 was associated with a higher likelihood of immunity (OR = 3.3, 95%CI [2.8, 3.9]).

Conclusion

The determinants and age distribution for seroconversion within a vaccine-naive population were similar to those found in developed countries. This pandemic was characterized by strong epidemiological determinants, regardless of geographical zone and level of development. Moreover, we detected pre-existing cross-reacting antibodies in participants over 60 yo, which could not have originated from former multiple vaccination as has been suggested elsewhere.     

Deficient tryptophan catabolism along the kynurenine pathway reveals that the epididymis is in a unique tolerogenic state

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.