cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

<p>Abstract</p> <p>Background</p> <p>The completion of the <it>Plasmodium falciparum </it>genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.</p> <p>Results</p> <p>We aimed to obtain cDNA sequences to examine the accuracy of gene prediction <it>in silico</it>. We constructed cDNA libraries from mixed blood stages of <it>P. falciparum </it>parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons <it>versus </it>5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.</p> <p>Conclusion</p> <p>Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.</p

Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray

<p>Abstract</p> <p>Background</p> <p>Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite <it>Plasmodium falciparum</it>. For efficient mapping of a target gene, it is often necessary to genotype a large number of polymorphic markers. Currently, a community effort is underway to collect single nucleotide polymorphisms (SNP) from the parasite genome. Here we evaluate polymorphism detection accuracy of a high-density 'tiling' microarray with 2.56 million probes by comparing single feature polymorphisms (SFP) calls from the microarray with known SNP among parasite isolates.</p> <p>Results</p> <p>We found that probe GC content, SNP position in a probe, probe coverage, and signal ratio cutoff values were important factors for accurate detection of SFP in the parasite genome. We established a set of SFP calling parameters that could predict mSFP (SFP called by multiple overlapping probes) with high accuracy (≥ 94%) and identified 121,087 mSFP genome-wide from five parasite isolates including 40,354 unique mSFP (excluding those from multi-gene families) and ~18,000 new mSFP, producing a genetic map with an average of one unique mSFP per 570 bp. Genomic copy number variation (CNV) among the parasites was also cataloged and compared.</p> <p>Conclusion</p> <p>A large number of mSFP were discovered from the <it>P. falciparum </it>genome using a high-density microarray, most of which were in clusters of highly polymorphic genes at chromosome ends. Our method for accurate mSFP detection and the mSFP identified will greatly facilitate large-scale studies of genome variation in the <it>P. falciparum </it>parasite and provide useful resources for mapping important parasite traits.</p

Mechanical properties and fracture damage law of coal-rock composition under the action of supercritical CO2

The injection of CO2 into deep unrecoverable coal seams is one of the effective ways to achieve CO2 geological sequestration, but CO2 will be in a supercritical state under the action of high pressure and high temperature. In order to investigate the effect of supercritical CO2 action on coal reservoir structure, based on the self-developed supercritical CO2 immersion experimental system, combined with acoustic emission test system and RFPA3D numerical simulation, we studied the three coal seam thicknesses and three top and bottom lithologies of the mechanical damage characteristics and the fracture extension evolution of “Rock-Coal-Rock” (RCR) composite specimens under the action of supercritical CO2 were investigated. The results show that: ① After the action of supercritical CO2, the degradation of compressive strength and elastic modulus of the RCR composite gradually increases and decreases with the increase of coal thickness, while the degradation of compressive strength and elastic modulus are basically the same when the strength ratios of rock and coal are different and do not show large differences; ② The action of supercritical CO2 will promote the plastic damage of the coal body and intensify the transformation of the RCR composite from tensile splitting damage to shear plastic damage, and the degree of plastic damage of RCR assemblage is positively correlated with both coal thickness and rock-to-coal strength ratio; ③ The supercritical CO2 immersion promoted the RCR assemblage to enter the elastic deformation stage earlier, and the destabilization damage occurred after a more brief elastic deformation, the greater the coal thickness, the greater the influence, while the rock-to-coal strength ratio has less influence; ④ The instability potential of RCR assemblage is proportional to coal thickness and inversely proportional to rock-coal strength ratio, and the power intensity of damage is inversely proportional to coal thickness and proportional to rock-coal strength ratio; ⑤ The total energy, dissipative energy, elastic energy and surplus energy of RCR assemblage gradually decrease with the increase of coal thickness and gradually increase with the increase of rock-coal strength ratio, and the supercritical CO2 effect will cause the elastic energy ratio of RCR assemblage specimens to decrease, the dissipative energy ratio to increase and the surplus energy ratio to decreases. Combining the above research results shows that the thicker the coal seam is, the more likely it is to be destabilized, and the higher the strength of the top and bottom rock layer is, the less likely it is to be destabilized, and the dynamic strength of the seam destabilization is inversely proportional to the coal thickness and positively proportional to the rock-to-coal strength ratio. Therefore, in a certain area of stratum that meets the premise of CO2 injection and storage, the area of stratum with higher top and bottom rock strength and thinner coal seam thickness should be selected to store CO2 with higher safety. The research results can provide some theoretical reference for the safety of geological storage of CO2 injected into deep unmineable coal seams

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins

The enteropathogenic bacterium Campylobacter jejuni was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilization. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains 129,108 and NCTC 11168 increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. C. jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion. This article is protected by copyright. All rights reserved

Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes

The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism. Inherited deficiency of the lysosomal acid α-glucosidase (GAA) causes the lysosomal glycogen storage disorder, Pompe disease. Here, we design a synthetic route for fluorescent and biotin-modified ABPs for in vitro and in situ monitoring of α-glucosidases. We show, through mass spectrometry, gel electrophoresis, and X-ray crystallography, that α-glucopyranose configured cyclophellitol aziridines label distinct retaining α-glucosidases including GAA and ER α-glucosidase II, and that this labeling can be tuned by pH. We illustrate a direct diagnostic application in Pompe disease patient cells, and discuss how the probes may be further exploited for diverse applications

Activity-based probes for functional interrogation of retaining β-glucuronidases

Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality

Transposable elements cause the loss of self-incompatibility in citrus

Self-incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S-RNase-based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm-RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self-compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786-bp miniature inverted-repeat transposable element (MITE) insertion in the promoter region of the FhiS2-RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm-RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S-RNase; moreover, transgenic experiments show that deletion of this 786-bp MITE insertion recovers the expression of FhiS2-RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S-locus affecting the SI phenotype. A family-wide survey of the S-locus revealed that MITE insertions occur frequently adjacent to S-RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S-RNase system

Towards broad spectrum activity-based glycosidase probes: synthesis and evaluation of deoxygenated cyclophellitol aziridines

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of [small beta]-glucosidases and [small beta]-galactosidases

Review of thermal management of catalytic converters to decrease engine emissions during cold start and warm up

Catalytic converters mitigate carbon monoxide, hydrocarbon, nitrogen oxides and particulate matter emissions from internal combustion engines, and allow meeting the increasingly stringent emission regulations. However, catalytic converters experience light-off issues during cold start and warm up. This paper reviews the literature on the thermal management of catalysts, which aims to significantly reduce the light-off time and emission concentrations through appropriate heating methods. In particular, methods based on the control of engine parameters are easily implementable, as they do not require extra heating devices. They present good performance in terms of catalyst light-off time reduction, but bring high fuel penalties, caused by the heat loss and unburnt fuel. Other thermal management methods, such as those based on burners, reformers and electrically heated catalysts, involve the installation of additional devices, but allow flexibility in the location and intensity of the heat injection, which can effectively reduce the heat loss in the tailpipe. Heat storage materials decrease catalyst light-off time, emission concentrations and fuel consumption, but they are not effective if the engine remains switched off for long periods of time. The main recommendation of this survey is that integrated and more advanced thermal management control strategies should be developed to reduce light-off time without significant energy penalty