Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes

We report an optimized synthesis of all canonical 2'-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology.     

Integration of omics data: how well does it work for bacteria?

In the current omics era, innovative high-throughput technologies allow measuring temporal and conditional changes at various cellular levels. Although individual analysis of each of these omics data undoubtedly results into interesting findings, it is only by integrating them that gaining a global insight into cellular behaviour can be aimed at. A systems approach thus is predicated on data integration. However, because of the complexity of biological systems and the specificities of the data-generating technologies (noisiness, heterogeneity, etc.), integrating omics data in an attempt to reconstruct signalling networks is not trivial. Developing its methodologies constitutes a major research challenge. Besides for their intrinsic value towards health care, environment and industry, prokaryotes are ideal model systems to further develop these methods because of their lower regulatory complexity compared with eukaryotes, and the ease with which they can be manipulated. Several successful examples outlined in this review already show the potential of the systems approach for both fundamental and industrial applications, which would be time-consuming or impossible to develop solely through traditional reductionist approaches.     

Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1

Marker-assisted breeding is a very useful tool for breeders but still lags behind its potential because information on the effect of quantitative trait loci (QTLs) in different genetic backgrounds and ideal molecular markers are unavailable. Here, we report on some first steps toward the validation and application of the major rice QTL Phosphate uptake 1 (Pup1) that confers tolerance of phosphorus (P) deficiency in rice (Oryza sativa L.). Based on the Pup1 genomic sequence of the tolerant donor variety Kasalath that recently became available, markers were designed that target (1) putative genes that are partially conserved in the Nipponbare reference genome and (2) Kasalath-specific genes that are located in a large insertion-deletion (INDEL) region that is absent in Nipponbare. Testing these markers in 159 diverse rice accessions confirmed their diagnostic value across genotypes and showed that Pup1 is present in more than 50% of rice accessions adapted to stress-prone environments, whereas it was detected in only about 10% of the analyzed irrigated/lowland varieties. Furthermore, the Pup1 locus was detected in more than 80% of the analyzed drought-tolerant rice breeding lines, suggesting that breeders are unknowingly selecting for Pup1. A hydroponics experiment revealed genotypic differences in the response to P deficiency between upland and irrigated varieties but confirmed that root elongation is independent of Pup1. Contrasting Pup1 near-isogenic lines (NILs) were subsequently grown in two different P-deficient soils and environments. Under the applied aerobic growth conditions, NILs with the Pup1 locus maintained significantly higher grain weight plant^?1 under P deprivation in comparison with intolerant sister lines without Pup1. Overall, the data provide evidence that Pup1 has the potential to improve yield in P-deficient and/or drought-prone environments and in diverse genetic backgrounds.     

<Emphasis Type="Italic">APOE</Emphasis> ε4 lowers age at onset and is a high risk factor for Alzheimer's disease; A case control study from central Norway

Background

The objective of this study was to analyze factors influencing the risk and timing of Alzheimer's disease (AD) in central Norway. The APOE ε4 allele is the only consistently identified risk factor for late onset Alzheimer's disease (LOAD). We have described the allele frequencies of the apolipoprotein E gene (APOE) in a large population of patients with AD compared to the frequencies in a cognitively-normal control group, and estimated the effect of the APOE ε4 allele on the risk and the age at onset of AD in this population.

Methods

376 patients diagnosed with AD and 561 cognitively-normal control individuals with no known first degree relatives with dementia were genotyped for the APOE alleles. Allele frequencies and genotypes in patients and control individuals were compared. Odds Ratio for developing AD in different genotypes was calculated.

Results

Odds Ratio (OR) for developing AD was significantly increased in carriers of the APOE ε4 allele compared to individuals with the APOE ε3/ε3 genotype. Individuals carrying APOE ε4/ε4 had OR of 12.9 for developing AD, while carriers of APOE ε2/ε4 and APOE ε3/ε4 had OR of 3.2 and 4.2 respectively. The effect of the APOE ε4 allele was weaker with increasing age. Carrying the APOE ε2 allele showed no significant protective effect against AD and did not influence age at onset of the disease. Onset in LOAD patients was significantly reduced in a dose dependent manner from 78.4 years in patients without the APOE ε4 allele, to 75.3 in carriers of one APOE ε4 allele and 72.9 in carriers of two APOE ε4 alleles. Age at onset in early onset AD (EOAD) was not influenced by APOE ε4 alleles.

Conclusion

APOE ε4 is a very strong risk factor for AD in the population of central Norway, and lowers age at onset of LOAD significantly.

     

Subtle social cues, explicit incentives and cooperation in social dilemmas

Subtle contextual social cues and the explicit incentive structure of social dilemmas are two important, but fundamentally different classes of determinants of cooperative behavior. The former provides subjective social information regarding the likelihood of attaining mutual cooperation by shaping one's expectations of the cooperative behavior of the interacting party. The latter provides objective, ecological information about the strategic opportunities offered by the situation. In prior research, both classes have generally been studied in isolation, hampering insight into how social judgments and ecological opportunities interact in shaping cooperation. To fill in this gap we set up a repeated mixed-motive game in which we manipulate subtle social cues and the incentive structure of the game simultaneously. We develop the hypothesis that social information is less important in shaping mutual cooperation of two team members when the incentive structure is such that it contains natural synergies. In contrast, when the incentive structure offers no synergy, “rational contracting” is hampered and social cues are essential for mutual cooperation to develop. The evidence we present in this paper is consistent with this hypothesis.     

Differential proteomic analysis of bovine conceptus fluid proteins in pregnancies generated by assisted reproductive technologies

Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.     

Proteomic analysis of bovine conceptus fluids during early pregnancy

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.     

Genetic diversity for mycorrhizal symbiosis and phosphate transporters in rice

Phosphorus(P) is a major plant nutrient and developing crops with higher P-use efficiency is an important breeding goal.In this context we have conducted a comparative study of irrigated and rainfed rice varieties to assess genotypic differences in colonization with arbuscular mycorrhizal(AM) fungi and expression of different P transporter genes.Plants were grown in three different soil samples from a rice farm in the Philippines.The data show that AM symbiosis in all varieties was established after 4 weeks of growth under aerobic conditions and that,in soil derived from a rice paddy,natural AM populations recovered within6 weeks.The analysis of AM marker genes(AM1,AM3,AM14) and P transporter genes for the direct Pi uptake(PT2,PT6) and AM-mediated pathway(PT11,PT13) were largely in agreement with the observed root AM colonization providing a useful tool for diversity studies.Interestingly,delayed AM colonization was observed in the aus-type rice varieties which might be due to their different root structure and might confer an advantage for weed competition in the field.The data further showed that P-starvation induced root growth and expression of the high-affinity P transporter PT6 was highest in the irrigated variety IR66 which also maintained grain yield under P-deficient field conditions.     

CD70 signaling is critical for CD28-independent CD8+ T cell-mediated alloimmune responses in vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.