Characterization of Differentially Expressed Genes Involved in Pathways Associated with Gastric Cancer

To explore the patterns of gene expression in gastric cancer, a total of 26 paired gastric cancer and noncancerous tissues from patients were enrolled for gene expression microarray analyses. Limma methods were applied to analyze the data, and genes were considered to be significantly differentially expressed if the False Discovery Rate (FDR) value was < 0.01, P-value was <0.01 and the fold change (FC) was >2. Subsequently, Gene Ontology (GO) categories were used to analyze the main functions of the differentially expressed genes. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found pathways significantly associated with the differential genes. Gene-Act network and co-expression network were built respectively based on the relationships among the genes, proteins and compounds in the database. 2371 mRNAs and 350 lncRNAs considered as significantly differentially expressed genes were selected for the further analysis. The GO categories, pathway analyses and the Gene-Act network showed a consistent result that up-regulated genes were responsible for tumorigenesis, migration, angiogenesis and microenvironment formation, while down-regulated genes were involved in metabolism. These results of this study provide some novel findings on coding RNAs, lncRNAs, pathways and the co-expression network in gastric cancer which will be useful to guide further investigation and target therapy for this disease.     

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.     

Pathway Evolution by Horizontal Transfer and Positive Selection Is Accommodated by Relaxed Negative Selection upon Upstream Pathway Genes in Purple Bacterial Carotenoid Biosynthesis

Horizontal gene transfer and selection are major forces driving microbial evolution. However, interactions between them are rarely studied. Phylogenetic analyses of purple bacterial carotenoid biosynthesis genes suggest two lineages: one producing spheroidenone and the other producing spirilloxanthin. Of the latter lineage, Rubrivivax gelatinosus S1 and Hoeflea phototrophica DFL-43 also or instead produce spheroidenone. Evolution of the spheroidenone pathway from that producing spirilloxanthin theoretically requires changes in the substrate specificity of upstream pathway enzymes and acquisition of a terminal ketolase (CrtA). In R. gelatinosus and likely also in H. phototrophica, CrtA was acquired from the Bacteroidetes, in which it functions as a hydroxylase. Estimation of nonsynonymous and synonymous mutations using several pairwise methods indicated positive selection upon both genes, consistent with their functional changes from hydroxylases to ketolases. Relaxed negative selection upon all other carotenoid biosynthetic genes in these organisms was also apparent, likely facilitating changes in their substrate specificities. Furthermore, all genes responsible for terminal carotenoid biosynthetic pathway steps were under reduced negative selection compared to those known to govern biosynthetic pathway specificity. Horizontal transfer of crtA into R. gelatinosus and H. phototrophica has therefore likely been promoted by (i) the apparent selective advantage of spheroidenone production relative to spirilloxanthin production, (ii) reduced negative selection upon other carotenoid biosynthetic genes, facilitating changes in their substrate specificities, and (iii) preexisting low enzyme substrate specificities due to relaxed negative selection. These results highlight the importance and complexity of selection acting upon both a horizontally transferred gene and the biochemical network into which it is integrating.Biochemical pathway evolution has been examined extensively, particularly regarding mechanisms by which novel functions can be generated, diversified, and maintained (9, 12). Best studied in this regard is the role of gene duplication followed by divergence, resulting in paralog families that, despite sharing a common evolutionary ancestor, possess different functions (10). In clonally reproducing organisms, such as bacteria and archaea, this type of diversification is further compounded by horizontal gene transfer (18), whereby a divergent ortholog from one organism is introduced into the metabolic network of another, thereby becoming a “xenolog” (29). Horizontal gene transfer between distantly related organisms is especially diversifying due to the likelihood of altering the genome structure or biochemical and regulatory networks of the recipient, in contrast to recombination between close relatives, which may promote genetic cohesion (33).Selection controls phenotypic diversity as a function of evolutionary fitness. Three scenarios can be detected from patterns of nucleotide substitutions (23): (i) positive selection, by which advantageous functionally divergent mutants are further optimized by increased mutational sampling of phenotypic space; (ii) negative (purifying) selection, by which deleterious mutations are purged; and (iii) neutral mutation, in which mutations that do not affect the selected phenotype accumulate, resulting in genetic drift. Considering horizontal transfer, selection will favor fixation of a horizontally transferred gene if its phenotype is advantageous and will disfavor it when either the gene product or the alterations that it causes in the host network are deleterious. Successful horizontal gene transfer resulting in gene fixation is the result of net evolutionary benefit for the host, due both to the horizontally transferred gene itself and to minimal suboptimal alteration of the host metabolic and genetic networks into which it is integrating. Genetic parasites such as plasmids, transposons, and integrated phages are exceptional in directly promoting their own retention.Carotenoids are isoprenoid pigments produced by many bacteria and fungi and all photosynthetic eukaryotes (7, 8). They are typically colored red, orange, and yellow due to their extensively conjugated polyene chains (7). In all photosynthetic organisms, carotenoids facilitate the assembly of the photosynthetic reaction center and interact with it as auxiliary light-harvesting pigments and antioxidant molecules (13-15). Indeed, the niche in which a particular phototroph lives is defined, at least in part, by the absorption spectrum of its pigments, including light-harvesting carotenoids (55). Carotenoids also modulate membrane fluidity and permeability (19); these functions remain poorly understood.In this study, the evolution of carotenoid biosynthesis by horizontal gene transfer and selection is evaluated in the purple bacteria, anoxygenic phototrophic Proteobacteria that can use reduced sulfur compounds as electron sources. Whereas phylogenetically most purple bacteria belong to the Alphaproteobacteria, some belonging to the Betaproteobacteria and Gammaproteobacteria have evolved by horizontal transfer of alphaproteobacterial photosynthetic superoperons, which include carotenoid biosynthetic genes (24, 37). Carotenoid biosynthesis in the purple bacteria (Fig. ​(Fig.1)1) begins with the condensation of two molecules of geranylgeranyl pyrophosphate by the phytoene synthase CrtB, forming phytoene. The phytoene desaturase CrtI then desaturates phytoene either three or four times, producing neurosporene or lycopene, respectively. Both of these intermediates are subsequently hydroxylated at the 1 position by the hydroxylase CrtC, desaturated at the 3 and 4 positions by the CrtI homolog CrtD, methylated at the 1-hydroxyl group by the methyltransferase CrtF, and, in spheroidenone- and 2,2′-diketospirilloxanthin-producing organisms, ketolated at the 2 position by the ketolase CrtA (Fig. ​(Fig.1).1). Considerable subpathway diversity also exists because of the potential for asymmetry between carotenoid ends.Open in a separate windowFIG. 1.Carotenoid biosynthetic pathway in purple bacteria. For simplicity, not all subpathways are shown. Carbon numbers for the ψ end group are shown for phytoene.Carotenoid biosynthesis has been well studied biochemically and genetically in the spheroidene-producing organisms Rhodobacter capsulatus (3, 4, 16, 45) and Rhodobacter sphaeroides (1, 31, 32) and the spirilloxanthin-producing organisms Bradyrhizobium sp. strain ORS278 (17), Thiocapsa roseopersicina (30), and Rubrivivax gelatinosus (16, 21, 22, 40, 41, 43, 52-54). The latter organism produces 2,2′-diketospirilloxanthin (lycopene derived), spheroidenone (neurosporene derived), and their precursors using the same enzymes. Pathway utilization in R. gelatinosus is determined primarily by the substrate specificities of CrtC, CrtD, and CrtI and by the rate of metabolic flux (52-54). These extensive biochemical and genetic studies provide a solid framework for sequence-based evolutionary analyses, making this pathway a valuable model with which to study biochemical pathway evolution.     

Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily

A remarkable diversity of venom peptides is expressed in the genus Conus (known as “conotoxins” or “conopeptides”). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the κA-family, the α4/7-subfamily, and α3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the α4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of α4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.     

Evolution of Rh Blood Group Genes Have Experienced Gene Conversions and Positive Selection

There are two tightly linked loci (D and CE) for the human Rh blood group. Their gene products are membrane proteins having 12 transmembrane domains and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of human and nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We therefore used a modified site-by-site reconstruction method by using two assumed gene trees and detected 9 or 11 converted regions. After eliminating the effect of gene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch of both trees. Whichever gene tree we selected the branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions, an indication of positive selection. Many other branches also showed higher nonsynonymous than synonymous substitutions; this suggests that the Rh genes have experienced some kind of positive selection. Received: 16 March 1999 / Accepted: 17 June 1999     

Positive Selection on Loci Associated with Drug and Alcohol Dependence

Much of the evolution of human behavior remains a mystery, including how certain disadvantageous behaviors are so prevalent. Nicotine addiction is one such phenotype. Several loci have been implicated in nicotine related phenotypes including the nicotinic receptor gene clusters (CHRNs) on chromosomes 8 and 15. Here we use 1000 Genomes sequence data from 3 populations (Africans, Asians and Europeans) to examine whether natural selection has occurred at these loci. We used Tajima’s D and the integrated haplotype score (iHS) to test for evidence of natural selection. Our results provide evidence for strong selection in the nicotinic receptor gene cluster on chromosome 8, previously found to be significantly associated with both nicotine and cocaine dependence, as well as evidence selection acting on the region containing the CHRNA5 nicotinic receptor gene on chromosome 15, that is genome wide significant for risk for nicotine dependence. To examine the possibility that this selection is related to memory and learning, we utilized genetic data from the Collaborative Studies on the Genetics of Alcoholism (COGA) to test variants within these regions with three tests of memory and learning, the Wechsler Adult Intelligence Scale (WAIS) Block Design, WAIS Digit Symbol and WAIS Information tests. Of the 17 SNPs genotyped in COGA in this region, we find one significantly associated with WAIS digit symbol test results. This test captures aspects of reaction time and memory, suggesting that a phenotype relating to memory and learning may have been the driving force behind selection at these loci. This study could begin to explain why these seemingly deleterious SNPs are present at their current frequencies.     

正向选择的生物安全标记基因

标记基因的产生方便了植物的转化,随着转基因植物的迅速发展及商品化,人类更关注抗性标记基因的安全性。目前解决的有效途径是发展正向选择系统,使用非抗性的生物安全标记基因,主要包括糖类代谢酶基因（pmi和xylA）、干扰氨基酸代谢酶基因（ak和dapA）、绿色荧光蛋白基因（gfp）、β-葡萄糖苷酸酶基因（gus）、核糖醇操纵子（rtl）和叶绿素生物合成基因（hemL）等。     

植物进化中的正选择作用

正选择是指将因含有有利突变而提高个体适合度的等位基因固定下来的选择作用, 研究正选择对理解生物进化过程具有重要意义。本文回顾了近年来在植物基因中发现的正选择作用, 分别对陆生植物和藻类中经历正选择作用的基因进行了总结, 其中在陆生植物中发现的正选择位点主要集中在与生殖相关及与抗逆相关的基因上, 这为以后对植物中正选择作用的研究提供了线索。     

植物进化中的正选择作用

正选择是指将因含有有利突变而提高个体适合度的等位基因固定下来的选择作用,研究正选择对理解生物进化过程具有重要意义。本文回顾了近年来在植物基因中发现的正选择作用,分别对陆生植物和藻类中经历正选择作用的基因进行了总结,其中在陆生植物中发现的正选择位点主要集中在与生殖相关及与抗逆相关的基因上,这为以后对植物中正选择作用的研究提供了线索。     

Rapid Intrahost Evolution of Human Cytomegalovirus Is Shaped by Demography and Positive Selection

Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ∼88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13–18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥34 of the 167 open reading frames (∼20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization.     

Synonymous Codon Usage Bias in Plant Mitochondrial Genes Is Associated with Intron Number and Mirrors Species Evolution

Synonymous codon usage bias (SCUB) is a common event that a non-uniform usage of codons often occurs in nearly all organisms. We previously found that SCUB is correlated with both intron number and exon position in the plant nuclear genome but not in the plastid genome; SCUB in both nuclear and plastid genome can mirror the evolutionary specialization. However, how about the rules in the mitochondrial genome has not been addressed. Here, we present an analysis of SCUB in the mitochondrial genome, based on 24 plant species ranging from algae to land plants. The frequencies of NNA and NNT (A- and T-ending codons) are higher than those of NNG and NNC, with the strongest preference in bryophytes and the weakest in land plants, suggesting an association between SCUB and plant evolution. The preference for NNA and NNT is more evident in genes harboring a greater number of introns in land plants, but the bias to NNA and NNT exhibits even among exons. The pattern of SCUB in the mitochondrial genome differs in some respects to that present in both the nuclear and plastid genomes.     

CRISPLD2 Is Expressed at Low Levels during Septic Shock and Is Associated with Procalcitonin

Introduction

Previous studies have shown that cysteine-rich secretory protein containing LCCL domain 2 (CRISPLD2) is a novel lipopolysaccharide (LPS)-binding protein, and the upregulation of CRISPLD2 expression protects mice against LPS-induced lethality. The aim of this study was to examine the expression of CRISPLD2 in patients with sepsis and characterize the association of this protein with procalcitonin.

Methods

The expression of CRISPLD2 was determined in100 healthy volunteers and 119 septic patients. According to the definition of sepsis, patients were divided into three groups sepsis, severe sepsis, and septic shock. The relationship between CRISPLD2 levels and procalcitonin was also examined and statistically analyzed.

Results

The CRISPLD2 levels in healthy individuals were 219.3±69.1 µg/ml. Patients with sepsis exhibited higher CRISPLD2 levels than observed in healthy individuals (p = 0.001), but CRISPLD2 expression was not upregulated in patients with septic shock. No significant differences were observed between the levels of CRISPLD2 in surviving and non-surviving spesis patients. CRISPLD2 levels were negatively correlated with procalcitonin levels(r = −0.334, p<0.001).

Conclusions

The present study is the first to demonstrate the decreased expression of CRISPLD2 in septic shock and its association with PCT in sepsis. Further studies are needed to clarify the potential association between CRISPLD2 expression and clinical outcomes to determine if it could be used as a novel sepsis biomarker.     

Identification of Methylated Genes Associated with Aggressive Bladder Cancer

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.     

Microbial Dysbiosis Is Associated with Human Breast Cancer

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.     

Using Human Microarrays to Identify Differentially Expressed Genes Associated with Increased Steroidogenesis in Boars

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.