'PACLIMS': A component LIM system for high-throughput functional genomic analysis

Background

Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required.

Results

The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured.

Conclusion

Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.     

Prediction of nitrogen responses of corn by soil nitrogen mineralization indicators

Soil nitrogen mineralization potential (N min) has to be spatially quantified to enable farmers to vary N fertilizer rates, optimize crop yields, and minimize N transfer from soils to the environment. The study objectives were to assess the spatial variability in soil N min potential based on clay and organic matter (OM) contents and the impact of grouping soils using these criteria on corn grain (Zea mays L.) yield, N uptake response curves to N fertilizer, and soil residual N. Four indicators were used: OM content and three equations involving OM and clay content. The study was conducted on a 15-ha field near Montreal, Quebec, Canada. In the spring 2000, soil samples (n = 150) were collected on a 30- x 30-m grid and six rates of N fertilizer (0 to 250 kg N ha(-1)) were applied. Kriged maps of particle size showed areas of clay, clay loam, and fine sandy loam soils. The N min indicators were spatially structured but soil nitrate (NO3-) was not. The N fertilizer rate to reach maximum grain yield (N max), as estimated by a quadratic model, varied among textural classes and Nmin indicators, and ranged from 159 to 250 kg N ha(-1). The proportion of variability (R2) and the standard error of the estimate (SE) varied among textural groups and N min indicators. The R2 ranged from 0.53 to 0.91 and the SE from 0.13 to 1.62. Corn grain N uptake was significantly affected by N fertilizer and the pattern of response differed with soil texture. For the 50 kg N ha(-1) rate, the apparent N min potential (ANM) was significantly larger in the clay loam (122 kg ha(-1)) than in the fine sandy loam (80 kg ha(-1)) or clay (64 kg ha(-1)) soils. The fall soil residual N was not affected by N fertlizer inputs. Textural classes can be used to predict N max. The N min indicators may also assist the variable rate N fertilizer inputs for corn production.     

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

Introduction

Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.

Methods

Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using ³⁵S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.

Results

DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.

Conclusions

Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.     

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay

Introduction

Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.

Methods

The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.

Results

Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).

Conclusion

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.     

Kin Preference and Partner Choice

This paper presents a comparison of social kinship (patrilineage) and biological kinship (genetic relatedness) in predicting cooperative relationships in two different economic contexts in the fishing and whaling village of Lamalera, Indonesia. A previous analysis (Alvard, Human Nature 14:129-163, 2003) of boat crew affiliation data collected in the village in 1999 found that social kinship (patrilineage) was a better predictor of crew affiliation than was genetic kinship. A replication of this analysis using similar data collected in 2006 finds the same pattern: lineage is a better predictor than genetic kinship of crew affiliation, and the two together explain little additional variance over that explained by lineage alone. However, an analogous test on food-sharing relationships finds the opposite pattern: biological kinship is a better predictor of food-sharing relationships than is social kinship. The difference between these two cooperative contexts is interpreted in terms of kin preferences that shape partner choice, and the relative autonomy with which individuals can seek to satisfy those preferences. Drawing on stable matching theory, it is suggested that unilineal descent may serve as a stable compromise among multiple individuals' incongruent partner preferences, with patriliny favored over matriliny in the crew-formation context because it leads to higher mean degrees of relatedness among male cooperators. In the context of food-sharing, kin preferences can be pursued relatively autonomously, without the necessity of coordinating preferences with those of other households through the institution of lineage.     

赖氨酰氧化酶与人类疾病

赖氨酰氧化酶(lysyl oxidases,LOXs)是一种能够催化细胞外基质蛋白(如胶原和弹性蛋白)交叉连接的酶类,这一功能使其在组织的稳定、重塑和伤口愈合中发挥重要作用.随着研究的不断深入,LOXs在细胞增殖、细胞趋化以及肿瘤发生等过程中也彰显出十分关键的作用.研究发现,一些诸如结缔组织病、剥脱综合症、铜代谢障碍性疾病及盆腔器官脱垂和骨疾等疾病的发生与LOXs有很大关系.综述了LOXs的生物合成、结构特点、多功能性以及与人类疾病的关系.     

Enhanced detection of human immunodeficiency virus type 1 (HIV-1) Nef-specific T cells recognizing multiple variants in early HIV-1 infection

A human immunodeficiency virus (HIV)-preventive vaccine will likely need to induce broad immunity that can recognize antigens expressed within circulating strains. To understand the potentially relevant responses that T-cell based vaccines should elicit, we examined the ability of T cells from early infected persons to recognize a broad spectrum of potential T-cell epitopes (PTE) expressed by the products encoded by the HIV type 1 (HIV-1) nef gene, which is commonly included in candidate vaccines. T cells were evaluated for gamma interferon (IFN-gamma) secretion using two peptide panels: subtype B consensus (CON) peptides and a novel peptide panel providing 70% coverage of PTE in subtype B HIV-1 Nef. Eighteen of 23 subjects' T cells recognized HIV-1 Nef. In one subject, Nef-specific T cells were detected with the PTE but not with the CON peptides. The greatest frequency of responses spanned Nef amino acids 65 to 103 and 113 to 147, with multiple epitope variants being recognized. Detection of both the epitope domain number and the response magnitude was enhanced using the PTE peptides. On average, we detected 2.7 epitope domains with the PTE peptides versus 1.7 domains with the CON peptides (P = 0.0034). The average response magnitude was 2,169 spot-forming cells (SFC)/10(6) peripheral blood mononuclear cells (PBMC) with the PTE peptides versus 1,010 SFC/10(6) PBMC with CON peptides (P = 0.0046). During early HIV-1 infection, Nef-specific T cells capable of recognizing multiple variants are commonly induced, and these responses are readily detected with the PTE peptide panel. Our findings suggest that Nef responses induced by a given vaccine strain before HIV-1 exposure may be sufficiently broad to recognize most variants within subtype B HIV-1.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.