Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Main features of DNA-based immunization vectors

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

ELEVATED ALANINE AMINOTRANSFERASE (ALT) IN BLOOD DONORS: AN ASSESSMENT OF THE MAIN ASSOCIATED CONDITIONS AND ITS RELATIONSHIP TO THE DEVELOPMENT OF HEPATITIS C

The determination of aminotranferases levels is very useful in the diagnosis of hepatopathies. In recent years, an elevated serum ALT level in blood donors has been associated with an increased risk of post-transfusion hepatitis (PTH). The purpose of the study was to research the factors associated with elevated ALT levels in a cohort of voluntary blood donors and to evaluate the relationship between increased ALT levels and the development of hepatitis C (HCV) infection. 166 volunteer blood donors with elevated ALT at the time of their first donation were studied. All of the donors were questioned about previous hepatopathies, exposure to hepatitis, exposure to chemicals, use of medication or drugs, sexual behaviour, contact with blood or secretions and their intake of alcohol. Every three months, the serum levels of AST, ALT, alkaline phosphatase, gamma glutamyl transpeptidase, cholesterol, triglyceride and glycemia are assessed over a two year follow-up. The serum thyroid hormone levels as well as the presence of auto-antibodies were also measured. Abdominal ultrasound was performed in all patients with persistently elevated ALT or AST levels. A needle biopsy of liver was performed in 9 donors without definite diagnostic after medical investigation. The presence of anti-HCV antibodies in 116 donors were assayed again the first clinical evaluation. At the end of follow-up period (2 years later) 71 donors were tested again for the presence of anti-HCV antibodies. None of donors resulted positive for hepatitis B or hepatitis C markers during the follow-up. Of the 116 donors, 101 (87%) had persistently elevated ALT serum levels during the follow-up. Obesity and alcoholism were the principal conditions related to elevated ALT serum levels in 91/101 (90.1%) donors. Hypertriglyceridemia, hypercholesterolemia, hypothyroidism and diabetes mellitus also were associated with increased ALT levels. Only 1/101 (0.9%) had mild chronic active non A-G viral hepatitis and 3/101 (2.9%) had liver biopsy with non-specific reactive hepatitis. The determination of ALT levels was not useful to detect donors infected with HCV at donation in Brazil, including the initial seronegative anti-HCV phase.

Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in Northern Brazil

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

IntroductionLeishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.MethodsAll rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.ResultsOf 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.ConclusionsThe results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Laboratory assessment of the hypertensive individual. Value of the main guidelines for high blood pressure

OBJECTIVE: To determine if abnormal laboratory findings are more common in individuals with hypertension and in those with other risk factors, such as obesity, smoking and alcohol ingestion. METHODS: A study was carried out in the general outpatient clinics of a university hospital (145 individuals without previous diagnosis of hypertension) and the following variables were assessed: high blood pressure (as defined by the VI Joint National Committee on Prevention, Detection and Treatment of High Blood Pressure - VI JNC), obesity [calculated using body mass index (BMI)], tobacco use, and alcoholic ingestion. The laboratory examinations consisted of the following tests: hemogram, glycemia, uric acid, potassium, total/HDL-fraction cholesterol, triglycerides, calcium and creatinine. RESULTS: High blood pressure was not associated with a higher number of abnormal laboratory tests. Hypertensive individuals with a BMI > or = 25kg/m² or normotensive obese individuals, however, had a higher frequency of diabetes (12X), hypertriglyceridemia (3X), and hypercholesterolemia (2X), as compared with hypertensive individuals with BMI <25kg/m² and preobese/normal weight normotensive individuals. CONCLUSION: High blood pressure is not associated with a higher frequency of abnormal laboratory tests. The association of high blood pressure and obesity, however, increases the detection of diabetes and dyslipidemias.

Stenting of unprotected left main coronary artery in a patient with cardiogenic shock

A 64-year-old female presented with pulmonary edema and cardiogenic shock after coronary arteriography that showed severe suboclusive lesion in the left main coronary artery (LMCA) in a dominant left coronary system. The patient succesfully underwent urgent angioplasty with stent deployment in the LMCA. After an uneventful period, the patient was discharged at day six.

Aneurysm of the left main coronary artery

Aneurysm of the left main coronary artery is a rare angiographic finding, with few cases described in the international literature. We report the case of a 42-year-old male with a previous history of acute myocardial infarction, whose coronariography indicated triple vessel coronary disease and an aneurysm of the left main coronary artery. A review of the etiology, clinical aspects, and surgical management of coronary arterial aneurysm is presented.

Can patients with left main coronary artery disease wait for myocardial revascularization surgery?

OBJECTIVE: To assess the occurrence of cardiac events in patients diagnosed with left main coronary artery disease on diagnostic cardiac catheterization and waiting for myocardial revascularization surgery. METHODS: All patients diagnosed with left main coronary artery disease (stenosis > or = 50%) consecutively identified on diagnostic cardiac catheterization during an 8-month period were selected for the study. The group comprised 56 patients (40 males and 16 females) with a mean age of 61±10 years. The cardiac events included death, nonfatal acute myocardial infarction, acute left ventricular failure, unstable angina, and emergency surgery. RESULTS: While waiting for surgery, patients experienced the following cardiac events: 7 acute myocardial infarctions and 1 death. All events occurred within the first 60 days after the diagnostic cardiac catheterization. More patients, whose indication for diagnostic cardiac catheterization was unstable angina, experienced events as compared with those with other indications [p=0.03, relative risk (RR) = 5.25, 95% confidence interval = 1.47 - 18.7]. In the multivariate analysis of logistic regression, unstable angina was also the only factor that independently contributed to a greater number of events (p = 0.02, OR = 8.43, 95% CI =1.37 - 51.7). CONCLUSION: Unstable angina in patients with left main coronary artery disease acts as a high risk factor for cardiac events, emergency surgery being recommended in these cases.