A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.

Isolation of MLL1 Inhibitory RNA Aptamers

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder

MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3’ untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5’-CAGUCG-3’) in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3’ UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Preoperative Imaging Modalities to Predict the Risk of Regional Nodal Recurrence in Well-Differentiated Thyroid Cancers

Introduction Thyroid cancer incidence has increased in the previous 2 decades. Preoperative identification of lymph node metastasis is a suggested risk factor associated with recurrence following thyroidectomy. Objectives We aimed to evaluate the accuracy of preoperative radiologic investigations of nodal status in determining the postoperative risk of regional nodal recurrence in cases of well-differentiated thyroid cancer. Methods This is a case series. We retrospectively reviewed data, including preoperative ultrasonography and/or computed tomography results, on patients who underwent total thyroidectomy for thyroid cancer at our hospital between 2006 and 2012. Prognostic factors for predicting recurrence, including age, sex, tumor diameter, and nodal diameter, were evaluated. Results Total thyroidectomy was performed on 24 male and 74 female patients (median age, 43 years). The median follow-up time was 21 months. Sixty-eight patients had papillary thyroid cancer, and 30 had follicular cancer. Nodal recurrence was evident in 30% of patients, and 4% of patients died. Identification of lymph node involvement during preoperative radiologic investigations was strongly prognostic for recurrence: 35.3% of patients with positive preoperative ultrasonography findings and 62.5% of those with positive preoperative computed tomography findings had recurrence (p = 0.01). Conclusions Preoperative identification of lymph node metastasis on radiologic studies was correlated with an increased risk of regional nodal recurrence in well-differentiated thyroid cancer. Computed tomography was superior to ultrasonography in detecting metastatic nodal involvement preoperatively and is therefore recommended for preoperative assessment and postoperative follow-up. .

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Effect of glycemic index on obesity control

Objective Evaluate the effect of glycemic index (GI) on biochemical parameters, food intake, energy metabolism, anthropometric measures and body composition in overweight subjects.Materials and methods Simple blind study, in which nineteen subjects were randomly assigned to consume in the laboratory two daily low GI (n = 10) or high GI (n = 9) meals, for forty-five consecutive days. Habitual food intake was assessed at baseline. Food intake, anthropometric measures and body composition were assessed at each 15 days. Energy metabolism and biochemical parameters were evaluated at baseline and the end of the study.Results Low GI meals increased fat oxidation, and reduced waist circumference and HOMA-IR, while high GI meals increased daily dietary fiber and energy intake compared to baseline. There was a higher reduction on waist circumference and body fat, and a higher increase on postprandial fat oxidation in response to the LGI meals than after high GI meals. High GI meals increased fasting respiratory coefficient compared to baseline and low GI meals.Conclusion The results of the present study showed that the consumption of two daily low GI meals for forty-five consecutive days has a positive effect on obesity control, whereas, the consumption of high GI meals result has the opposite effect. Arch Endocrinol Metab. 2015;59(3):245-51.

Assessment of adherence to the guidelines for the management of nausea and vomiting induced by chemotherapy / Avaliação da aderência à diretriz de cuidados para náuseas e vômitos induzidos por quimioterapia

ABSTRACT Objective: To assess adherence of the prescribing physicians in a private cancer care center to the American Society of Clinical Oncology guideline for antiemetic prophylaxis, in the first cycle of antineoplastic chemotherapy. Methods: A total of 139 chemotherapy regimens, of 105 patients, were evaluated retrospectively from 2011 to 2013. Results: We observed 78% of non-adherence to the guideline rate. The main disagreements with the directive were the prescription of higher doses of dexamethasone and excessive use of 5-HT3 antagonist for low risk emetogenic chemotherapy regimens. On univariate analysis, hematological malignancies (p=0.005), the use of two or more chemotherapy (p=0.05) and high emetogenic risk regimes (p=0.012) were factors statistically associated with greater adherence to guidelines. Treatment based on paclitaxel was the only significant risk factor for non-adherence (p=0.02). By multivariate analysis, the chemotherapy of high emetogenic risk most correlated with adherence to guideline (p=0.05). Conclusion: We concluded that the adherence to guidelines is greater if the chemotherapy regime has high emetogenic risk. Educational efforts should focus more intensely on the management of chemotherapy regimens with low and moderate emetogenic potential. Perhaps the development of a computer generated reminder may improve the adherence to guidelines. .

RESUMO Objetivo: Avaliar a adesão dos médicos prescritores, de um centro privado especializado em oncologia, à diretriz de antiêmese profilática da American Society of Clinical Oncology, no primeiro ciclo de quimioterapia antineoplásica. Métodos: Foram avaliados retrospectivamente 139 esquemas de quimioterapia, de 105 pacientes, tratados no período de 2011 a 2013. Resultados: Foram observados 78% de taxa de não adesão à diretriz. As principais discordâncias com a diretriz foram prescrição de doses mais elevadas de dexametasona e uso excessivo de antagonista 5-HT3 para regimes de quimioterapia de risco emetogênico baixo. Pela análise univariada, malignidades hematológicas (p=0,005), uso de dois ou mais quimioterápicos (p=0,05) e regimes de alto risco emetogênico (p=0,012) foram fatores estatisticamente associados a maior adesão à diretriz. O tratamento baseado em paclitaxel foi o único fator estatisticamente significativo para a não adesão (p=0,02). Pela análise multivariada, a quimioterapia de alto risco emetogênico apresentou maior correlação com a adesão à diretriz (p=0,05). Conclusão: Houve maior aderência para a quimioterapia de alto risco emetogênico. Esforços educacionais devem se concentrar mais intensamente na gestão de regimes de quimioterapia com potencial emetogênico baixo e moderado. Talvez o desenvolvimento de lembretes gerados por sistemas informatizados possa melhorar a aderência à diretriz. .

Different Extracts of Zingiber officinale Decrease Enterococcus faecalis Infection in Galleria mellonella

Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

Extratos seco, fresco e glicólico de Zingiber officinale foram obtidos para avaliar suas ações por meio de ensaio de sobrevivência em G. mellonella contra infecção por Enterococcus faecalis. Oitenta larvas foram divididas em: 1) Suspensão de E. faecalis (controle); 2) E. faecalis + extrato fresco de Z. officinale (FEO); 3) E. faecalis + extrato seco de Z. officinale (DEO); 4) E. faecalis + extrato glicólico de Z. officinale (GEO); 5) Solução tampão fosfato salina (PBS). Para o grupo de controle, 5 µL de inóculo de suspensão padronizada (107 células/mL) de E. faecalis (ATCC 29212) foi injetado na última proleg esquerda de cada lagarta. Para os grupos com tratamento, após a injeção de E. faecalis, os extratos foram injetados na última proleg direita. Após as injeções, as lagartas foram armazenadas a 37 °C e o número de animais mortos foi registrado diariamente em 168 h (7 dias) para analisar a curva de sobrevivência. As lagartas foram consideradas mortas quando elas não mostraram qualquer movimento após o toque. A infecção por E. faecalis levou à morte de 85% das lagartas após 168 h. Não obstante, nos grupos de tratamento com associação dos extratos, houve um aumento nas taxas de sobrevivência de 50% (GEO), 61% (FEO) e 66% (DEO) das lagartas. Em todos os grupos com tratamento, as lagartas apresentaram um aumento na sobrevivência, com diferença estatisticamente significativa em relação ao grupo controle (p=0,0029). Não houve diferença estatisticamente significativa entre os tratamentos com os diferentes extratos (p=0,3859). Pode concluir-se que os extratos testados mostraram atividade antimicrobiana contra a infecção por E. faecalis, aumentando a sobrevivência das lagartas de G. mellonella.

Precios de los medicamentos: cómo se establecen y cuáles son sus sistemas de control / Drug prices: how they are established and existing price control systems

El precio es una de las principales barreras de acceso a los medicamentos. Por ello es importante conocer cómo se forman los precios y qué factores determinan su cuantía y también qué formas de intervención y regulación son las más adecuadas teniendo en cuenta sus efectos, tanto sobre el acceso, como sobre la innovación, la producción local y otros posibles objetivos de la política de medicamentos. El análisis económico ha desarrollado un conjunto de modelos de mercado que permiten explicar el comportamiento de los precios, aunque los mercados reales divergen sustancialmente de los modelos teóricos. La regulación de precios está justificada por los llamados "fallos de mercado"; la regulación de precios basada en el costo de producción, la modalidad de control de precios más tradicional, ha caído en desuso a favor de los sistemas de precios de referencia internacionales y por la fijación del precio basada en el valor.

Price is one of the main barriers of access to medicines. It is therefore important to understand how prices are formed and what factors determine the amount, as well as what interventions and regulations are the most appropriate considering their effects on access, innovation, local production and other potential objectives of drug policy. Economic analysis has developed a set of market models that can explain the behavior of prices, although actual markets diverge substantially from the theoretical models. Price regulation is justified by the so-called "market failures." Price regulation based on the cost of production, the most traditional form of price control, has fallen into disuse in favor of systems of international reference pricing and value-based pricing.

A possible role of Drosophila CTCF in mitotic bookmarking and maintaining chromatin domains during the cell cycle

BACKGROUND: The CCCTC-binding factor (CTCF) is a highly conserved insulator protein that plays various roles in many cellular processes. CTCF is one of the main architecture proteins in higher eukaryotes, and in combination with other architecture proteins and regulators, also shapes the three-dimensional organization of a genome. Experiments show CTCF partially remains associated with chromatin during mitosis. However, the role of CTCF in the maintenance and propagation of genome architectures throughout the cell cycle remains elusive. RESULTS: We performed a comprehensive bioinformatics analysis on public datasets of Drosophila CTCF (dCTCF). We characterized dCTCF-binding sites according to their occupancy status during the cell cycle, and identified three classes: interphase-mitosis-common (IM), interphase-only (IO) and mitosis-only (MO) sites. Integrated function analysis showed dCTCF-binding sites of different classes might be involved in different biological processes, and IM sites were more conserved and more intensely bound. dCTCF-binding sites of the same class preferentially localized closer to each other, and were highly enriched at chromatin syntenic and topologically associating domains boundaries. CONCLUSIONS: Our results revealed different functions of dCTCF during the cell cycle and suggested that dCTCF might contribute to the establishment of the three-dimensional architecture of the Drosophila genome by maintaining local chromatin compartments throughout the whole cell cycle.

Sequencing and Phylogenetic Analyses of Structural and Accessory Proteins of Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China, 2015 / 病毒学报

To investigate the genetic character and origin of the first imported infection case of middle East respiratory syndrome coronavirus (named as MERS-CoV_China GD01), RNA was extracted from swabs of this patient followed by RT-PCR amplification. All coding gene of structural (S, E, M, E) and accessory (ORF3, ORF4a, ORF4b, ORF5, ORF8b) proteins were sequenced and analyzed. Phylogenetic analyses of structural protein coding genes of MERS-CoV_ China GD01 indicates that several substitutes exists in S coding gene and its origin belong group 5 of MERS-CoV, which were recent circulated in Saudi Arabia area, while other three structural genes (N, E, M) were very conserved. Phylogenetic analyses of accessory protein coding genes of MERS-CoV China GD01 indicates that several substitutes exists among ORF3, ORF4a, ORF4b and ORF5, while ORF8b was conserved. In conclusion, genome of MERS-CoV_ China GD01 was general conserved although several genetic variations were found among structural and accessory protein coding genes. This is the first report on sequencing and phylogenetic analyses of the first imported MERS case in China, which may pay the way for prevention and control of imported MERS-CoV infection.

Structure, Immunogenicity and Clinical Value of Chlamydiaphage Capsid Protein 3 / 病毒学报

We wished to assess the role of chlamydia micro virus capsid protein Vp3 in recombinant molecules, chart its molecular evolution, screen the wild-type strain, and reveal its value in clinical research. Using a protein BLAST multiple-alignment program, we compared various strains of Chlamydia micro virus capsid protein Vp3 sequences. Using a "distance tree" of those results, we created a phylogenetic tree. We applied the Karplus-Schulz method of flexible-region analyses for highly conserved alignments of amino-acid sequences. Gamier-Robson and Chou-Fasman methods were employed to analyze two-level structures of sequences. The Emini method was used for analyses of the accessibility of surface epitopes. Studies of hydrophilic proteins were undertaken using Kyte-Doolittle and Hopp-Woods methods. Analyses of antigen epitopes helped to reveal the antigen index using the Jameson-Wolf method. All sequences in the six strains of chlamydia micro virus capsid protein Vp3 were highly conserved, with the main differences being between Vp3 protein in Chp1 and the other five strains of the micro virus. The viral strain of Vp3 protein was based mainly on micro-alpha helix structures, and multiple epitopes were noted in highly conserved regions. Vp3 protein was highly conserved structurally, and was an important protein of the chlamydiaphage capsid. Vp3 protein has a complicated molecular structure, highly conserved regions with strong immunogenicity, and has considerable research value.

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.