Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2.

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

Parainfluenza virus as a cause of acute respiratory infection in hospitalized children.

BACKGROUND: Human parainfluenza viruses account for a significant proportion of lower respiratory tract infections in children. OBJECTIVE: To assess the prevalence of Human parainfluenza viruses as a cause of acute respiratory infection and to compare clinical data for this infection against those of the human respiratory syncytial virus. METHODS: A prospective study in children younger than five years with acute respiratory infection was conducted. Detection of respiratory viruses in nasopharyngeal aspirate samples was performed using the indirect immunofluorescence reaction. Length of hospital stay, age, clinical history and physical exam, clinical diagnoses, and evolution (admission to Intensive Care Unit or general ward, discharge or death) were assessed. Past personal (premature birth and cardiopathy) as well as family (smoking and atopy) medical factors were also assessed. RESULTS: A total of 585 patients were included with a median age of 7.9 months and median hospital stay of six days. No difference between the HRSV+ and HPIV+ groups was found in terms of age, gender or length of hospital stay. The HRSV+ group had more fever and cough. Need for admission to the Intensive Care Unit was similar for both groups but more deaths were recorded in the HPIV+ group. The occurrence of parainfluenza peaked during the autumn in the first two years of the study. CONCLUSION: Parainfluenza was responsible for significant morbidity, proving to be the second-most prevalent viral agent in this population after respiratory syncytial virus. No difference in clinical presentation was found between the two groups, but mortality was higher in the HPIV+ group.

Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies.

A rapid test based on an immunochromatography assay - Determine Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Inability to detect human T cell lymphotropic virus type 2-specific antibodies in a patient coinfected with HIV-1, human T cell lymphotropic virus type 1, human T cell lymphotropic virus type 2, and hepatitis C virus.

HIV-1, human T cell lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) and hepatitis C virus (HCV) are common among intravenous drug users (IDUs) and can cause chronic infections in the host. Usually, the diagnosis of such viruses employs serological assays; however, some difficulties in confirming HTLV-2 infection have been reported in high-risk populations in Brazil. We present data of an unusual case of coinfection with HIV-1, HTLV-1, HTLV-2, and HCV in a male IDU in which HTLV-2 was detected only by molecular assays. Comparative analysis of retroviruses from 2002 and 2012 showed identical HTLV-1 and HTLV-2 sequences (LTR, env, and tax), and a change in HIV-1 tropism from CXCR4 to CCR5. No mutation was detected in the hot points of the env region of the HTLV-2 isolate that justified the lack of rgp46-II-specific antibodies. These data emphasize the need for molecular assays to diagnose HTLV-2 in high-risk populations in Brazil.

Parainfluenza virus as a cause of acute respiratory infection in hospitalized childrens

Background: Human parainfluenza viruses account for a significant proportion of lower respiratory tract infections in children.Objective: To assess the prevalence of Human parainfluenza viruses as a cause of acute respiratory infection and to compare clinical data for this infection against those of the human respiratory syncytial virus.Methods: A prospective study in children younger than five years with acute respiratory infection was conducted. Detection of respiratory viruses in nasopharyngeal aspirate samples was performed using the indirect immunofluorescence reaction. Length of hospital stay, age, clinical history and physical exam, clinical diagnoses, and evolution (admission to Intensive Care Unit or general ward, discharge or death) were assessed. Past personal (premature birth and cardiopathy) as well as family (smoking and atopy) medical factors were also assessed.Results: A total of 585 patients were included with a median age of 7.9 months and median hospital stay of six days. No difference between the HRSV+ and HPIV+ groups was found in terms of age, gender or length of hospital stay. The HRSV+ group had more fever and cough. Need for admission to the Intensive Care Unit was similar for both groups but more deaths were recorded in the HPIV+ group. The occurrence of parainfluenza peaked during the autumn in the first two years of the study.Conclusion: Parainfluenza was responsible for significant morbidity, proving to be the second-most prevalent viral agent in this population after respiratory syncytial virus. No difference in clinical presentation was found between the two groups, but mortality was higher in the HPIV+ group.

Profile of anti-Tp47 antibodies in patients with positive serology for syphilis analized by Western Blot.

In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

Diagnóstico laboratorial da neurocisticercose: revisão e perspectivas / Laboratory diagnosis of neurocysticercosis: review and perspectives

A neurocisticercose é causada por Cysticercus cellulose, a forma larval de Taenia solium, quando este se aloja no sistema nervoso central. O seu diagnóstico é realizado com base em dados clínicos, epidemiológicos, demonstração do agente etiológico pelas técnicas de imagem e testes laboratoriais. No presente estudo, apresentamos uma revisão do diagnóstico laboratorial, com ênfase no desempenho dos testes para pesquisa de anticorpos específicos e detecção de antígenos circulantes, utilização de antígeno homólogo ou heterólogo, nativo e recombinante, bem como a aplicação de métodos moleculares.

Neurocysticercosis is caused by Cysticercus cellulosae, the larval form of Taenia solium, when it lodges in the central nervous system. The diagnosis of neurocysticercosis is based on clinical and epidemiological data, neuroimaging findings of etiological agent and serologic test results. Herein we present a review of clinical diagnosis, emphasizing test performance for specific antibody and antigen detection, the use of homologous or heterologous antigen, native and recombinant antigens as well as the application of molecular methods.

Reatividade do anticorpo IgM anti-Treponema pallidum na soroconversão e na resposta sorológica ao tratamento de sífilis / IgM antibody reactivity to Treponema pallidum in seroconversion and in the serologic response to syphilis treatment

INTRODUÇÃO: A utilidade da detecção de anticorpos da imunoglobulina da classe M (IgM) no diagnóstico da sífilis tem sido discutida há tempos. OBJETIVO: No presente estudo foi analisada a ocorrência de anticorpo IgM anti-T. pallidum (Tp-IgMAc) nas amostras de pacientes com sífilis recente, na fase de soroconversão e no monitoramento da resposta sorológica pós-tratamento. MÉTODOS: Amostras séricas de 11 indivíduos. RESULTADOS: Na soroconversão, o Tp-IgMAc foi detectado nas amostras de 10 indivíduos, e em um paciente a reatividade IgM ocorreu anteriormente ao Venereal Disease Research Laboratory (VDRL). A sororreversão foi evidenciada nas amostras de três pacientes com sífilis secundária tratada, e em um indivíduo com reinfecção. CONCLUSÃO: A detecção de Tp-IgMAc mostrou ser um potencial marcador diagnóstico de sífilis ativa e o desempenho do ensaio imunoenzimático de captura de IgM (ELISA-IgM) para o monitoramento pós-tratamento foi similar ao da VDRL.

INTRODUCTION: The appropriateness of IgM antibody detection in the diagnosis of syphilis has been extensively discussed. OBJECTIVE: This study aimed at assessing the detection of anti-T. pallidum IgM antibody (TP-IgMAb) in serum samples from patients with recent syphilis in seroconversion and in the monitoring of post-treatment serological response. METHODS: Serum samples from 11 individuals. RESULTS: At seroconversion, positive Tp-IgMAb was detected in 10 samples and IgM reactivity previous to Venereal Disease Research Laboratory (VDRL) was detected in one sample. Seroreversion was found in samples from three treated patients with secondary syphilis and in one individual with reinfection. CONCLUSION: Tp-IgMAb detection proved to be a potential diagnostic marker for active syphilis, and IgM capture enzyme linked immunosorbent assay (ELISA-IgM) performance was similar to VDRL in post-treatment monitoring.

Conserved loop sequence of helix 69 in Escherichia coli 23 S rRNA is involved in A-site tRNA binding and translational fidelity.

Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(Psi)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process.

Systematic deletion of rRNAs for investigating ribosome architecture and function.

Ribosomal RNAs (rRNAs) form a fundamental structure of ribosome, and play crucial roles in protein biosynthesis. To investigate functional importance of RNA helices in both 16S and 23S rRNAs, we devised an E. coli genetic system that allowed us to identify and select functional deletions in rRNAs. We here describe many RNA helices in rRNAs to be shortened or completely truncated. These results suggest architectural flexibility of rRNAs in ribosome assembly and function.

The A-site finger in 23 S rRNA acts as a functional attenuator for translocation.

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center.

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.

Comparação de técnicas imunológicas in-house empregadas na rotina diagnóstica de cisticercose humana / Comparative analysis on in-house immunologic assays used for performing the routine laboratorial diagnosis of human cysticercosis

Neste estudo foram analisados os resultados obtidos do diagnóstico de cisticercose no Centro de Imunologia do Instituto Adolfo Lutz (IAL), no período de março/2007 a julho/2010. A detecção de anticorpos específicos em 522 amostras de soro e líquido cefalorraquidiano (LCR)foi realizada pelas técnicas de imunofluorescência indireta (IFI) e hemaglutinação indireta (HAI). A frequência de amostras reagentes foi de 11,0% no LCR e 8,2% no soro. Em 50% das amostras não houve informações sobre suspeita clínica de neurocisticercose dos pacientes, sendo disponíveis nos18,3% e 16,4%, em amostras, respectivamente, de LCR e soro. Nas amostras de paciente com suspeita de neurocisticercose, a positividade foi de 22,6% (LCR) e de 18,4% (soro). Houve associação entre a suspeita clínica e a positividade dos testes (p>0.05). A maioria das amostras testadas foi proveniente do Estado de São Paulo, e 16,9% de amostras de LCR e 35,9% de amostras séricas foram enviados de outros Estados do país. Os ensaios de IFI e HAI apresentaram teste de concordância Kappa de 86%. Pela indisponibilidade de kits de reagentes diagnósticos de cisticercose em amostras de LCR no mercado, os testes in-house produzidos no IAL têm sido de grande relevância para os serviços de saúde pública.

Functional genetic selection of the decoding center in E. coli 16S rRNA.

Ribosomal RNAs (rRNAs) play crucial roles in protein biosynthesis. The decoding center of 16S rRNA in 30S subunit affords a place for interaction between mRNA and tRNA, and contributes to the fidelity of the decoding by monitoring the codon-anticodon base pairing. The helices 18 and 44 in 16S rRNA are known to be major components of the decoding center. To investigate functional role of the conserved sequence in rRNAs, we employed a new genetic method that allows us to identify and select from randomized E. coli rRNA libraries those rRNA sequences absolutely required for the ribosome function. Functional consensus sequences were identified in both helices, providing us with a new insight into the decoding mechanism.

Profile of Anti-Tp47 antibodies in patients with positive serology for syphilis analized by western blot

In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - DetermineÕ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The DetermineÕ test presented the sensitivity of 93.6 percent, specificity of 92.5 percent, and positive predictive value and negative predictive value of 95.2 percent and 93.7 percent, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. DetermineÕ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Analysis of Treponema pallidum recombinant antigens for diagnosis of syphilis by Western blotting technique

Os antigenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusao com glutationa S-transferase (GST) em E. coli, foram analisados quanto ao potencial diagnostico da sifilis pela tecnica de Western blotting. Foram testadas 53 amostras, sendo 25 pacientes em diferentes estagios clinicos da sifilis, com resultados positivos no teste treponemico classico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doenca sexualmente transmissivel nao relacionado a sifilis. Todas as amostras de pacientes com sifilis apresentaram alta reatividade com o antigeno GST-rTp17...

Novo teste de immunoblot (IB-rTp) com antígenos recombinantes de Treponema pallidum para o diagnóstico da sífilis / Novel immunoblot treponemal assay using recombinat antigens for immunodiagnosis of syphilis

A sífilis ainda é um problema de saúde pública, apesar dos esforços empreendidos para a sua prevençäo e a existência de tratamento eficiente. Os testes sorológicos säo fundamentais para o diagnóstico da doença. O presente trabalho descreve um novo teste de immunoblot )IB-rTp), utilizando três antígenos recombinantes de Treponema pallidum (GST-rTp47, GST-rTp17 e GST-rTp15), além da glutathiona S-tranferase (GST) purificada. FOram testadas 114 amostras séricas (53 de pacientes com sífilis, 50 de indivíduos sadios e 11 de pacientes com outras doenças) verificou-se concordância de 100 por cento em o IB-rTp e os testes treponêmicos convencionais, que utilizam antígeno nativo. A reatividade, quando presente, foi especificada contra a fraçäo treponêmica de cada proteína de fusäo. O teste IB-rTp apresentou alta especificidade; é de fácil execuçäo, näo requerendo absorçäo prévia do soro, sendo de utilidade como teste confirmatório de sífilis

Clonagem, expressão e caracterização de antígenos recombinantes do Treponema pallidum com potencial imunodiagnóstico / Cloning, expression and characterization pf Treponema pallidum recombinant antigen with potential for imunodiagnosis

A obtenção do Treponema pallidum para fins de pesquisa e de diagnósticos tem sido através da propagação in vivo, em testículos de coelhos. Considerando o alto custo e baixo rendimento da produção em animais, propomos realizar a clonagem dos principais antígenos do T. pallidum para estabelecer uma fonte constante de antígenos em nosso meio. Três antígenos recombinantes de Treponema pallidum cepa Nichols foram obtidos através da clonagem em PCR. O gene que codifica a proteína madura do Tp47, Tp17 e Tp15, amplificado a partir do DNA cromossômico foi clonado no pGEX4T-2 e transformado em E. coli resultando na expressão de GST-rTp47 e GST-rTp17. A expressão do GST-rTp15 foi obtida com suplementação de `tRNA POT. AGGAGA'. A identidade da proteína recombinante foi confirmada por seqüenciamento de DNA e mapeamento peptídico...

Clonagem, expressão e caracterização de antígenos recombinantes do Treponema pallidum com potencial imunodiagnóstico / Cloning, expression and characterization of Treponema pallidum recombinant antigen with potential for immunodiagnosis

A obtenção do Treponema pallidum para fins de pesquisa e de diagnóstico tem sido através da propagação in vivo, em testículos de coelhos. Considerando o alto custo e baixo rendimento da produção em animais, propomos realizar a clonagem dos principais antígenos do T. pallidum para estabelecer uma fonte constante de antígenos em nosso meio. Três antígenos recombinantes de Treponema pallidum cepa Nichols foram obtidos através da clonagem em PCR. O gene que codifica a proteína madura do Tp47, Tp17 e Tp15, amplificado a partir do DNA cromossômico foi clonado no pGEXeT-2 e transformado em E.coli resultando na expressão de GST-rTp47 e GST-rTp17. A expressão do GST-rTp15 foi obtida com suplementação de tRNA AGGAGA. A identidade da proteína recombinante foi confirmada por sequenciamento de DNA e mapeamento peptídico. Os rendimentos obtidos para o GST-rTp47, GST-rTp17 e GST-rTp15 foram respectivamente de 1,9; 3,5 e 7,0 mg por litro de cultura. A mistura equimolar dos três antígenos de fusão e GST purificados foi empregada para a avaliação da imunoreatividade, por Western blotting. No estudo com 128 soros de pacientes com sífilis, outras patologias e indivíduos clínicamente sadios, nenhuma amostra reagiu com a fração GST purificada e os resultados obtidos foram concordantes com os testes sorológicos convencionais para o diagnóstico da sífilis. Os clones construídos são importante fonte antígeno treponêmico específico para uso em testes com melhor desempenho imunodiagnóstico da sífilis