Trypanosoma evansi ima gen sličan genu za oligosaharil-transferazu klona I protozoona Trypanosoma brucei rhodesiense.

Recent data has shown that there are strong indications that the putative oligosaccharyl transferase genes from Trypanosoma brucei rhodesiense were conserved within the family Trypanosomatidae. Based on these findings, the study endeavored to determine if Trypanosoma evansi also possessed putative oligosaccharyl transferase (OST) clone I previously documented in Trypanosoma brucei rhodesiense. Using the DNA hybridization method (Southern blot analyses), genomic DNAs of Trypanosoma brucei rhodesiense and Trypanosoma evansi were processed using the same set of restriction enzymes and subsequently hybridized by the same set of DNA probes designed from the reported nucleotide sequence of Trypanosoma brucei rhodesiense putative oligosaccharyl transferase clone I. The results exhibited that Trypanosoma evansi also contains a gene similar to the reported Trypanosoma brucei rhodesiense putative OST gene clone I, as shown by the successful hybridization of the DNA probes to their complementary nucleotide sequences in the genome of the Trypanosoma evansi species. In addition, the data also showed that Trypanosoma brucei rhodesiense and Trypanosoma evansi genomes shared some common restriction sites and loci within the genome of each individual parasite species.Nedavna istraživanja pokazala su da su geni za oligosaharil transferazu protozoona Trypanosoma brucei rhodesiense vrlo dobro sačuvani unutar porodice Trypanosomatidae. Cilj istraživanja bio je otkriti je li ista pojava karakteristična za gen za oligosaharil transferazu klona I protozoona Trypanosoma evansi. Genomske DNA vrste Trypanosoma brucei rhodesiense i vrste Trypanosoma evansi bile su pretražene hibridizacijom DNA (Southern Blot analizom) rabeći istu skupinu restrikcijskih enzima kao i iste probe za hibridizaciju DNA pripravljene na temelju objavljenog slijeda nukleotida za oligosaharil-transferazu klona I protozoona Trypanosoma brucei rhodesiense. Rezultati su pokazali da Trypanosoma evansi također sadrži gen koji je vrlo sličan genu za oligosaharil-transferazu protozoona Trypanosoma brucei rhodesiense, što je dokazano uspješnom hibridizacijom DNA proba s komplementarnim nukleotidnim slijedovima u genomu vrste Trypanosoma evansi. Istraživanje je pokazalo da Trypanosoma brucei rhodesiense i Trypanosoma evansi dijele i zajednička restrikcijska mjesta

Genomic Cloning and Sequence Analysis of Trypanosoma brucei rhodesiense gene Encoding Putative N-glycosylation Enzyme

Background:  : : is a haemoflagellate parasite of zoonotic significance. Aside from its public health importance, this parasite subspecies gained notoriety because of their effective system to circumvent the immune response of vertebrate host. The parasite cell surface is covered with millions of VSG dimers, which serve as an almost infinite repertoire of biomolecules needed for evasion of host immune system. Around two decades ago, it was resolved that all trypanosome VSG is associated with one or more N-linked oligosaccharides, with a range of structures including high mannose and complex types. This complex process of protein modification known as N-linked glycosylation is catalyzed by oligosaccharyl transferase (OST). In general, the incorporation of glycan structures can alter protein’s antigenic properties and recently it was established that glycan molecules associated with VSG were found to be important in several aspects of trypanosome-host interaction, especially during parasite evasion of the host defense mechanisms. Therefore, our major interest is to clone and characterize the trypanosome OST. Material, Methods and Results: The template genomic DNA for PCR amplification was extracted as described previously. In an attempt to clone Trypanosoma brucei rhodesiense putative oligosaccharyl transferase, an amplicon of ~2000 bp was obtained having an open reading frame of 2057 bp and deduced primary structure composed of 685 amino acid residues (TbrOST II). Comparison of TbrOST II ORF with annotated putative oligosaccharyl transferase in the genome of other organisms revealed sequence identity to other kinetoplastid. TbrOST II had high nucleotide (Ns) and amino acid (As) sequence similarity with the genomes of T. brucei gambiense (Ns:99%; As:78%) and T. brucei (Ns:95-98%; As:77%-98%). There was also significant nucleotide and amino acid sequence identity in the genomes of T. cruzi (Ns:74%; As:63%), Leishmania infantum (Ns:70-83%; As:46-57%), L. braziliensis (Ns:69-81%; As:46-55%) and L. major (Ns:69-80%; As:46-57%). Sequence similarity (71-77%) from other origins was also exhibited. The nucleotide sequence alignments and analysis were performed using the Oxford University Mac Vector 6.5 sequence analysis software and CLC Workbench 5.6 software. Discussion: The nucleotide BLAST results indicate that sequence identity is higher between species of the same genus rather than of the same family. It is known that T. brucei, T. gambiense and T. rhodesiense are members of the Brucei-complex or Brucei group. Although T. brucei brucei has more similarities with T. brucei rhodesiense than T. brucei gambiense, these parasites are morphologically indistinguishable. This is the probable reason why high sequence identity was displayed by other subspecies of the Brucei group. In addition, the high percent identity possessed by TbrOST II with other trypansomatids agrees with the evolutionarily conserved characteristics of the established OST. The DNA sequence data of TbrOST II showing similar sequences in the genome of other organisms further corroborate the previous reports regarding the ubiquitous nature of OST in other life forms. Based on the size of the amplicon and significant percentage of nucleotide and amino acid sequence identity to homologues within the genome of related species and various organisms, the results strongly indicate that TbrOST II is a trypanosome oligosaccharyl transferase gene candidate that should be fully characterized and subjected to functional genomic studies. The study reports the molecular cloning and sequencing of a potential oligosaccharyl transferase gene in T. brucei rhodesiense (TbrOST II). The sequence data has been deposited in the GenBank with accession number GU475126.Trypanosoma brucei rhodesiens

Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis

Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium

<p>Abstract</p> <p>Background</p> <p>The rickettsial bacterium <it>Ehrlichia ruminantium </it>is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus <it>Amblyomma</it>. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of <it>E. ruminantium</it>.</p> <p>Results</p> <p>Two sets of LAMP primers were designed from the pCS20 and <it>sodB </it>genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for <it>sodB</it>, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of <it>E. ruminantium </it>from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic <it>Ehrlichia </it>species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.</p> <p>Conclusions</p> <p>Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of <it>E. ruminantium</it> in both heartwater-endemic areas and regions that are at risk of contracting the disease.</p

衣服設計のための若年女性の肩部形状の3次元的把握

The torsos of 39 young women were measured using two three-dimensional devices (VIVID 910, Konica Minolta Sensing Co.). The shape of the shoulder was represented with a 3D model which was constructed 83 points on the body surface of the shoulder include neck line, shoulder seam line, armhole line, across back line and across chest line. The average model of shoulder for young Japanese women was obtained. But the individual shape of shoulder varied widely. Then the principal component analysis was performed to clarify the factors that characterize individual shoulder shapes. The principal components obtained were "forward thrust shoulder or not,""sloping shoulder or square shoulder,""thick or thin upper back" and "pigeon breasted or flat breasted." These results will help to design more fitted dress dummies and to improve apparel fit

IOP elevation after STTA

Purpose To evaluate real-world evidence for intraocular pressure (IOP) elevation after subtenon triamcinolone acetonide injection (STTA) in 1252 Japanese patients (1406 eyes) in the Japan Clinical REtina STudy group (J-CREST). Methods This was a multicentre retrospective study of the medical records of 1252 patients (676 men (758 eyes); mean age: 63.8 ± 12.9 years) who received STTA in participating centres between April 2013 and July 2017. Results IOP elevation was observed in 206 eyes (14.7%) and IOP increase ≥ 6 mmHg was found in 328 eyes (23.3%). In total, 106 eyes (7.5%) needed medication and two eyes (0.14%) needed surgical procedures. Younger age, higher baseline IOP, and steroid dose were risk factors associated with IOP elevation. Risk factors associated with IOP increase ≥ 6 mmHg were younger age, lower baseline IOP, steroid dose, and higher incidences of diabetic macular oedema (DME) and uveitis. In contrast, with steroid dose fixed at 20 mg, a lower incidence of DME was a risk factor for increased IOP, suggesting that STTA had dose-dependent effects on IOP increase, especially in patients with DME. Conclusion Our real-world evidence from a large sample of Japanese patients who received STTA showed that the incidence of IOP elevation after STTA was 14.7%, and was associated with younger age, higher baseline IOP, and steroid dose. Thus, IOP should be monitored, especially in patients with younger age, higher baseline IOP, and higher incidences of DME and uveitis

Molecular epidemiology of camel trypanosomiasis based on ITS1 rDNA and RoTat 1.2 VSG gene in the Sudan

<p>Abstract</p> <p>Background</p> <p>Internal transcribed spacer one (ITS1) of the ribosomal DNA is known to be a suitable target for PCR-based detection of trypanosomes. The analysis of this region provides a multi-species-specific diagnosis by a single PCR. Using ITS1 primer-based PCR, a cross sectional study was carried out in the period from September to November 2009 on samples collected from 687 camels from geographically distinct zones in the Sudan to detect all possible African trypanosomes, which can infect camels.</p> <p>Results</p> <p>The results showed that all PCR-positive camels were infected with a single parasite species; <it>Trypanosoma evansi</it>. The highest prevalence, 57.1% (117/205), was observed in the Butana plains of mid-Eastern Sudan and the lowest, 6.0% (4/67), was in the Umshadeeda eastern part of White Nile State. In another experiment, the RoTat 1.2 gene encoding the variable surface glycoprotein (VSG) of <it>T. evansi </it>was analyzed for its presence or absence by a polymerase chain reaction (PCR) using <it>T. evansi </it>species-specific primers. The study showed that the RoTat 1.2 VSG gene was absent in thirteen out of thirty <it>T. evansi</it>-positive samples.</p> <p>Conclusions</p> <p>It is concluded that camel trypanosomiasis in Sudan is apparently caused by a single parasite species <it>T. evansi </it>and there were no other typanosomes species detected. In addition, the disease is highly prevalent in the country, which strengthens the need to change control policies and institute measures that help prevent the spread of the parasite. To our knowledge, this is the first molecular diagnosis report, which gives a picture of camel trypanosomiasis covering large geographical areas in Sudan.</p