Similarity-based gene detection: using COGs to find evolutionarily-conserved ORFs

BACKGROUND: Experimental verification of gene products has not kept pace with the rapid growth of microbial sequence information. However, existing annotations of gene locations contain sufficient information to screen for probable errors. Furthermore, comparisons among genomes become more informative as more genomes are examined. We studied all open reading frames (ORFs) of at least 30 codons from the genomes of 27 sequenced bacterial strains. We grouped the potential peptide sequences encoded from the ORFs by forming Clusters of Orthologous Groups (COGs). We used this grouping in order to find homologous relationships that would not be distinguishable from noise when using simple BLAST searches. Although COG analysis was initially developed to group annotated genes, we applied it to the task of grouping anonymous DNA sequences that may encode proteins. RESULTS: "Mixed COGs" of ORFs (clusters in which some sequences correspond to annotated genes and some do not) are attractive targets when seeking errors of gene predicion. Examination of mixed COGs reveals some situations in which genes appear to have been missed in current annotations and a smaller number of regions that appear to have been annotated as gene loci erroneously. This technique can also be used to detect potential pseudogenes or sequencing errors. Our method uses an adjustable parameter for degree of conservation among the studied genomes (stringency). We detail results for one level of stringency at which we found 83 potential genes which had not previously been identified, 60 potential pseudogenes, and 7 sequences with existing gene annotations that are probably incorrect. CONCLUSION: Systematic study of sequence conservation offers a way to improve existing annotations by identifying potentially homologous regions where the annotation of the presence or absence of a gene is inconsistent among genomes

Large Angle Transient Dynamics (LATDYN) user's manual

A computer code for modeling the large angle transient dynamics (LATDYN) of structures was developed to investigate techniques for analyzing flexible deformation and control/structure interaction problems associated with large angular motions of spacecraft. This type of analysis is beyond the routine capability of conventional analytical tools without simplifying assumptions. In some instances, the motion may be sufficiently slow and the spacecraft (or component) sufficiently rigid to simplify analyses of dynamics and controls by making pseudo-static and/or rigid body assumptions. The LATDYN introduces a new approach to the problem by combining finite element structural analysis, multi-body dynamics, and control system analysis in a single tool. It includes a type of finite element that can deform and rotate through large angles at the same time, and which can be connected to other finite elements either rigidly or through mechanical joints. The LATDYN also provides symbolic capabilities for modeling control systems which are interfaced directly with the finite element structural model. Thus, the nonlinear equations representing the structural model are integrated along with the equations representing sensors, processing, and controls as a coupled system

Tracking the evolution of alternatively spliced exons within the Dscam family

BACKGROUND: The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17), potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. RESULTS: Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee), we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248–283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. CONCLUSION: Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays provide a striking example of how a gene can evolve in a modular fashion rather than as a single unit

Rates of ethanol metabolism decrease in sons of alcoholics following a priming dose of ethanol

Rapid changes in rates of ethanol metabolism in response to acute ethanol administration have been observed in animals and humans. To examine whether this phenomenon might vary by risk for alcoholism, 23 young men with a positive family history of alcoholism (FHP) were compared to 15 young men without a family history of alcoholism (FHN). Rates of ethanol metabolism were measured in all subjects first after an initial ethanol dose (0.85 g/kg) and then, several hours later, a second dose (0.3 g/kg), and the two rates were compared. The two groups of subjects were similar in their histories of ethanol consumption. FHP subjects demonstrated faster initial rates of ethanol metabolism, 148 ± 36 mg/kg/hr, compared to FHN subjects, 124 ± 18 mg/kg/hr, p=.01. However, FHN subjects increased their rate of metabolism by 10 ± 27 percent compared to a decrease of -15 ± 24 percent in FHP subjects, p =.007. Fifty-two percent of the FHP and none of the FHN subjects exhibited a decline in metabolic rate of 20% or more, p=.0008. Since a significant proportion of FHP subjects exhibited a decrease in the second rate of ethanol metabolism, these preliminary data might help to partly explain why FHP individuals differ in their sensitivity to ethanol and are more likely to develop alcohol dependence

Effect of spironolactone and potassium canrenoate on cytosolic and nuclear androgen and estrogen receptors of rat liver

Spironolactone and potassium canrenoate are diuretics that are used widely for management of cirrhotic ascites. The administration of spironolactone frequently leads to feminization, which has been noted less frequently with the use of potassium canrenoate, a salt of the active metabolite of spironolactone. The use of these two drugs has been associated with decreases in serum testosterone levels and spironolactone with a reduction in androgen receptor (AR) activity. This decrease in AR has been cited as the cause of the antiandrogen effect of these drugs. We therefore assessed the effect of both drugs on levels of androgen and estrogen receptors (ER) in the liver, a tissue that is responsive to sex steroids. Three groups of male rats (n = 12 rats each) were studied. Group 1 (control) received vehicle only; group 2 received spironolactone (5 mg/day); group 3 received potassium canrenoate (5 mg/day). After 21 days of treatment, the animals of all groups were killed and liver tissue was assayed for nuclear and cytosolic AR and ER, and for male specific estrogen binder (MEB), an androgen-responsive protein. Both drugs drastically decreased the nuclear AR content, as compared with the control group, but only spironolactone decreased cytosolic AR. When the total hepatic content of AR is considered, a highly significant decrease is observed only in rats treated with spironolactone. This reduction in hepatic AR content suggested loss of androgen responsiveness of liver. We confirmed this by assessing levels of MEB, and found that livers from group 2 animals had no detectable MEB activity, whereas livers from both group 1 and 3 had normal MEB activity. No changes were observed in nuclear ER and cytosolic ER of group 3 as compared with group 1. Nuclear estrogen receptor decreased and cytosolic ER increased in group 2, but with no change in total ER content. These results indicate that (a) only spironolactone appears to act as an antiandrogen in liver, resulting in a decrease in both AR and male specific estrogen binder content, and (b) neither drug results in elevated hepatic ER content, although spironolactone-treated animals show an altered subcellular localization. © 1987

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V

Adolescent THC Usage in Virginia: Post-Legalization Challenges and Strategies for Schools

Recreational marijuana was legalized for anyone over the age of 21 in Virginia in 2021. This research and policy brief explores evidence of the impact of such legalization on Tetrahydrocannabinol (THC) usage in PK-12 aged youth, including smoking, vaping, edibles, and dabbing. It addresses the following questions: 1) What are the recent trends in marijuana usage among PK-12 aged youth? 2) What are the impacts of THC usage in schools, particularly after legalization? 3) How can schools and school systems effectively respond to THC usage? 4) What are relevant federal, state, and school division policies that guide responses to youth THC usage in the MERC region? It concludes with a series of key takeaways and recommendations for curbing THC usage in schools

Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges

We investigated the diagnostic and clinical performance of exome sequencing (ES) in fetuses with sonographic abnormalities with normal karyotype, microarray and, in some cases, normal gene specific sequencing