[Correlation between results of the residency admission test and of pediatric certification test in Buenos Aires, Argentina]. / Relación entre el desempeño en el examen de ingreso a la residencia y en el de especialización en pediatría en la Ciudad de Buenos Aires.

In the city of Buenos Aires (CABA), pediatric residents enter the residency program after taking a unified admission test. After completion of the program and passing a final test, the Universidad de Buenos Aires (UBA) provides a professional certification. The objective of this study is to determine if the results obtained in the residency admission test (RAT) and those of the professional certification test (PCT) correlated. This is a cross-sectional study, that included all subjects who passed the pediatrics RAT in CABA in 2004-2009, and that attended the pediatric PCT of the UBA. The score for each subject in both tests was obtained and the corresponding correlation was calculated. Results were divided in quintiles, and the proportion of subjects who improved their position in the PCT with respect to the RAT was calculated. Data from 303 subjects was obtained. The RAT showed a median of 45.0 (over 60 maximum) (IC-range: 43.0-48.7), and the PCT showed a median of 6 points (over 10 max.)(IC-range: 6-8). A significative correlation between results in RAT and PCT was observed (r = 0.37, p < 0.001). Based on their position in the RAT, 43.8% of subjects improved their position in the PCT, without differences between residents attending pediatric and general hospitals (45.6 vs. 31.5%; p = 0.1). In the case of pediatric residents, results of the residency admission test correlate with those obtained in the professional certification test.

Optogenetic strategies for the control of gene expression in yeasts.

Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Awareness of the right to Argentine Sign Language interpretation services among pediatricians practicing in the Autonomous City of Buenos Aires. / Conocimiento del derecho al servicio de interpretación de Lengua de Señas Argentina por pediatras que ejercen en la Ciudad Autónoma de Buenos Aires.

INTRODUCTION: Barriers in communication between physicians and Deaf or hard of hearing people may be risk factors for health. The services of a professional Argentine Sign Language interpreter (PASLI), by virtue of Act No. 26378, may mitigate this. OBJECTIVE: To estimate the proportion of pediatricians who have knowledge of Act No. 26378 and whether this is associated with the recommendation of a PASLI. POPULATION AND METHODS: Cross-sectional study with a selfadministered electronic questionnaire among pediatricians members of the Sociedad Argentina de Pediatría who practiced in the Autonomous City of Buenos Aires (CABA). RESULTS: A total of 381 surveys were assessed. Of these, 15.1 % knew the Act, and this was associated with the recommendation of a PASLI (OR: 3.7; 95 % CI: 1.1-12.9; p < 0.05). CONCLUSIONS: The proportion of pediatricians working in CABA who knew the Act was 15.1 %. This was significantly associated with the recommendation of a PASLI.

Introducción. Los obstáculos en la comunicación entre médicos y personas sordas/hipoacúsicas pueden comportarse como factores de riesgo para la salud. El servicio de un intérprete profesional de Lengua de Señas Argentina (IPLSA), según la Ley N.º 26378, podría mitigar esto. Objetivo. Estimar la proporción de pediatras que conocen la Ley N.º 26378 y explorar si se asocia a recomendar a un IPLSA. Población y métodos. Estudio transversal con cuestionario electrónico autoadministrado a pediatras registrados en la Sociedad Argentina de Pediatría, que ejercían en la Ciudad Autónoma de Buenos Aires. Resultados. Se evaluaron 381 respuestas. El 15,1 % conocía la Ley, y esto se asoció con recomendar a un IPLSA (OR: 3,7; IC 95 %: 1,1-12,9; p < 0,05). Conclusiones. La proporción de pediatras de la Ciudad Autónoma de Buenos Aires que conocía la ley fue el 15,1 %. Esto se asoció significativamente con recomendar a un IPLSA.

Prognostic impact of retropharyngeal lymphadenopathy in early-stage HPV-associated oropharyngeal cancer: Implications for staging optimization.

OBJECTIVES: We analyzed the prognostic impact of retropharyngeal lymphadenopathy (RPL) in stage I node-positive HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). MATERIALS AND METHODS: We performed a centralized and blinded radiographic review of the pre-treatment images of 234 consecutive patients with AJCC 8th edition stage I cT1-2N1 HPV-associated OPSCC treated with definitive chemoradiation from 2006 to 2016. Five-year disease control and survival outcomes were reported. The prognostic significance of RPL was evaluated through multivariable analysis adjusting for age, smoking history (<10 vs. >10 pack-years), and systemic regimen received. RESULTS: Median follow-up for surviving patients was 49 months (range: 16-121). RPL was associated with increased locoregional recurrence (LRR) (17.0% v. 3.4%, p = 0.01) and distant metastasis (DM) (29.1% v. 5.9%, p = 0.001) and inferior progression-free survival (PFS) (55.6% v. 88.2%, p < 0.001) and overall survival (OS) (60.6% v. 91.2%, p < 0.001). In stage I patients who did not receive high-dose cisplatin (HDC), RPL was associated with worse LRR (p = 0.04), DM (p = 0.03), PFS (p < 0.001), and OS (p < 0.001), whereas in those who did receive HDC, RPL was only associated with increased DM (p = 0.002) and inferior PFS (p = 0.04). CONCLUSION: This study suggests that RPL portends a poor prognosis in stage I node-positive HPV-associated OPSCC. The negative impact on LRR may have been mitigated by receipt of HDC. Outcomes of stage I disease with RPL were comparable to historical reports of patients with more advanced-stage disease. Incorporation of RPL into future disease staging should be considered in order to optimize risk-stratification and exclude unsuitable candidates from treatment de-intensification efforts.

Metastatic meningioma: Case report of a WHO grade I meningioma with liver metastases and review of the literature.

Meningioma represents the most frequently diagnosed primary brain tumor, accounting for over one-third of central nervous system neoplasms. The majority of tumors are categorized as benign. However, albeit rarely, meningiomas may metastasize to distant sites. We describe a 78-year-old man with a history of recurrent World Health Organization grade I meningioma managed who presented for evaluation of weakness and urinary retention. A computed tomography scan obtained in the emergency department revealed multiple scattered low-density liver lesions. Subsequent magnetic resonance imaging showed a 5.5-centimeter heterogeneous enhancing mass with 2 smaller enhancing lesions suspicious for a primary or secondary malignant neoplasm. Microscopic examination of a tissue sample obtained via liver biopsy demonstrated a metastatic spindle cell neoplasm with histologic features compatible with a diagnosis of World Health Organization grade I transitional meningioma. The patient was referred to hematology/oncology for systemic therapy.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Prevalence of deaf people who have a professional Argentine Sign Language interpreter during their children's medical consultations. / Prevalencia de personas sordas que cuentan con un intérprete profesional de Lengua de Señas Argentina en la consulta médica de sus hijos.

INTRODUCTION: In Argentina, hearing disability accounts for 18% of disabilities. Argentine Sign Language (ASL) is the communication system of choice used by deaf people (DP). An incorrect communication with physicians hampers the adequate provision of health care. DP require a professional ASL interpreter (PASLI) in the context of health care provided to their children. National Act no. 26378 stipulates the provision of a PASLI to facilitate access to public facilities. However, most DP neither have an interpreter nor know this right. OBJECTIVES: To describe the prevalence of DP who have a PASLI present during their children's medical consultations and who know their right to access one at no charge. MATERIAL AND METHOD: Approved and registered, descriptive, cross-sectional study with a structured, self-administered survey. Participants: DP who knew ASL and had children aged 1 month to 18 years old. Outcome variables: to have a PASLI and to know the right to have one. Estimated sample size: 220. Convenience sampling selected from the Autonomous City of Buenos Aires and the province of Chaco. The prevalence and 95% confidence interval (95% CI) were estimated. RESULTS: Two hundred and twenty-two surveys were analyzed. Age: 34.9 ± 10.1 years old. Among all participants, 15.3% (95% CI: 11.1-20.6) sometimes had a PASLI during their children's medical consultations. Also, 48.6% (95% CI: 42.155.1) knew their right to have one. CONCLUSION: The prevalence of DP who had a PASLI present during their children's medical consultations was low. Less than a half knew their right to have one.

Introducción. En Argentina, la discapacidad auditiva representa 18% de las discapacidades. La Lengua de Señas Argentina (LSA) es el sistema de comunicación de elección en personas sordas (PS). La incorrecta comunicación con el médico impide una adecuada asistencia. Las PS precisan a un intérprete profesional de LSA (IPLSA) durante la atención médica de sus hijos. La Ley Nacional 26378 establece ofrecer IPLSA para facilitar el acceso a instalaciones públicas. Sin embargo, la mayoría de las PS no cuentan con intérprete ni conocen este derecho. Objetivos. Describir la prevalencia de PS que cuentan con IPLSA en la consulta médica de sus hijos y que conocen su derecho a tener acceso gratuito a un IPLSA. Material y método. Estudio descriptivo, transversal, con encuesta estructurada autoadministrada. Se incluyeron PS que supieran LSA, con hijos entre 1 mes y 18 años. Variables de resultado: contar con IPLSA y conocer el derecho a tenerlo. Tamaño de muestra calculado: 220. Muestreo por conveniencia en la Ciudad de Buenos Aires y la provincia del Chaco. Se calculó prevalencia e intervalo de confianza del 95% (IC95%). Estudio aprobado y registrado. Resultados. Se analizaron 222 encuestas. Edad: 34,9 ± 10,1 años. El 15,3% (IC95%: 11,1-20,6) contaron con un IPLSA en alguna consulta de sus hijos. El 48,6% (IC95%: 42,1-55,1) conocía su derecho a contar con uno. Conclusión. La prevalencia de PS que han contado con un IPLSA en la consulta médica de sus hijos fue baja. Casi la mitad conocía su derecho de contar con un IPLSA.

Measuring adverse events in pediatric inpatients with the Global Trigger Tool. / Detección de eventos adversos en pacientes pediátricos hospitalizados mediante la herramienta de disparadores globales Global Trigger Tool.

INTRODUCTION: The safety of inpatients is a priority in the health care system. The Global Trigger Tool seems to be suitable to estimate the incidence of adverse events (AE) in pediatric inpatients. OBJETIVES: To describe the incidence and categories of AE in pediatric inpatients using the Global Trigger Tool and to identify risk factors associated to their development. POPULATION AND METHODS: Retrospective study. Medical records of 200 patients hospitalized at Hospital Elizalde during 2013 were included. Outcome measures: number of AE/100 admissions and distribution of harm. A chi² test, Student´s t test and Pearson's correlation test were carried out. Significance level = p < 0.05. RESULTS: The study detected 289 triggers (1.4/patient); 52 AEs (26 AEs/100 patients, 95% CI: 20.4-32.5). There was at least one AE every 36 patients; 7 patients had more than one AE; 45 AEs were in the E and F categories (temporary harm). Medical care triggers were associated to AEs (OR 8.1; 95% CI: 3.7-17.3, p < 0.001). A positive correlation was found between the number of triggers and the number of AEs per patient (R= 0.46; p < 0.001). Being hospitalized in a closed unit (OR 2.8; 95% CI: 1.2-6.5; p= 0.03) and a longer hospital stay were associated to AEs (p < 0.001). CONCLUSION: An AE frequency of 26% was identified, and most AEs resulted in temporary harm. The presence of AEs was associated to hospitalization in a closed unit, longer hospital stay, higher number of triggers and general care triggers.

INTRODUCCIÓN: La seguridad del paciente hospitalizado es prioritaria para el sistema de salud. La herramienta de disparadores globales parecería ser adecuada para estimar la incidencia de eventos adversos (EA) en niños hospitalizados. OBJETIVOS: Describir la incidencia y la categorización de EA en niños hospitalizados utilizando la herramienta de disparadores globales e identificar los factores de riesgo asociados a su aparición. POBLACIÓN Y MÉTODOS: Estudio retrospectivo. Se incluyeron 200 historias clínicas de pacientes internados en el Hospital Elizalde en el año 2013. Variables de resultado: número de EA/100 ingresos y distribución del daño. Se utilizó la prueba de chi cuadrado, la prueba de t y la correlación de Pearson. Nivel de significación de p < 0,05. RESULTADOS: Se detectaron 289 disparadores (1,4/paciente); 52 EA (26 EA cada 100 pacientes, IC 95%: 20,4-32,5). Presentaron, al menos, un EA 36 pacientes; 7 pacientes sufrieron más de uno; 45 EA fueron categoría E y F (daño temporario). Los disparadores de cuidados médicos se asociaron con EA (OR 8,1; IC 95%: 3,7-17,3; p < 0,001). Se encontró una correlación positiva entre el número de disparadores y el número de EA por paciente (R= 0,46; p < 0,001). Los pacientes internados en unidad cerrada (OR 2,8; IC 95%: 1,2-6,5; p= 0,03) y el mayor promedio de días de internación se asociaron a EA (p < 0,001). CONCLUSIÓN: Se identificó una frecuencia de 26% de EA; la mayoría fue daño temporario. La internación en una unidad cerrada, la mayor cantidad de días de internación, el mayor número de disparadores y los disparadores de cuidados generales se asociaron con la presencia de EA.

Conocimiento del derecho al servicio de interpretación de Lengua de Señas Argentina por pediatras que ejercen en la Ciudad Autónoma de Buenos Aires / Awareness of the right to Argentine Sign Language interpretation services among pediatricians practicing in the Autonomous City of Buenos Aires

Introducción. Los obstáculos en la comunicación entre médicos y personas sordas/hipoacúsicas pueden comportarse como factores de riesgo para la salud. El servicio de un intérprete profesional de Lengua de Señas Argentina (IPLSA), según la Ley N.º 26378, podría mitigar esto.Objetivo. Estimar la proporción de pediatras que conocen la Ley N.º 26378 y explorar si se asocia a recomendar a un IPLSA.Población y métodos. Estudio transversal con cuestionario electrónico autoadministrado a pediatras registrados en la Sociedad Argentina de Pediatría, que ejercían en la Ciudad Autónoma de Buenos Aires.Resultados. Se evaluaron 381 respuestas. El 15,1 % conocía la Ley, y esto se asoció con recomendar a un IPLSA (OR: 3,7; IC 95 %: 1,1-12,9; p < 0,05).Conclusiones. La proporción de pediatras de la Ciudad Autónoma de Buenos Aires que conocía la ley fue el 15,1 %. Esto se asoció significativamente con recomendar a un IPLSA

Introduction. Barriers in communication between physicians and Deaf or hard of hearing people may be risk factors for health. The services of a professional Argentine Sign Language interpreter (PASLI), by virtue of Act No. 26378, may mitigate this.Objective. To estimate the proportion of pediatricians who have knowledge of Act No. 26378 and whether this is associated with the recommendation of a PASLI.Population and methods. Cross-sectional study with a self-administered electronic questionnaire among pediatricians members of the Sociedad Argentina de Pediatría who practiced in the Autonomous City of Buenos Aires (CABA).Results. A total of 381 surveys were assessed. Of these, 15.1 % knew the Act, and this was associated with the recommendation of a PASLI (OR: 3.7; 95 % CI: 1.1-12.9; p < 0.05).Conclusions. The proportion of pediatricians working in CABA who knew the Act was 15.1 %. This was significantly associated with the recommendation of a PASLL.

A Novel Structurally Stable Multiepitope Protein for Detection of HCV.

Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756.

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

Overview and perspectives the transcriptome of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.

Hydrolytic enzymes in Paracoccidioides brasiliensis--ecological aspects.

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and ß-sheet, similar to α/ß hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus.

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).

Impact assessment of a decision rule for using antibiotics in pneumonia: a randomized trial.

BACKGROUND: Although more than half of the cases of pneumonia in children can be due to virus, most of them receive antibiotic treatment. A previously published clinical prediction rule bacterial pneumonia score (BPS) allows the identification of children with pneumonia who do not require antibiotics, but its impact has not been evaluated. We assessed whether the use of the BPS for the initial management of patients with pneumonia results in decreasing the use of antibiotics than under standard management of this condition without increasing patients' risks. METHODS: This was a randomized, parallel-group, observer-blind, controlled clinical trial comparing the use of antibiotics in children aged 3-60 months treated for pneumonia in an outpatient setting, according to two methods of initial management. Patients were assigned randomly to management according to the BPS (antibiotic indication with a BPS ≥ 4 points) or routine management (antibiotic indication based on the institutional guidelines). We calculated the proportion of the use of antibiotics in each group and evaluated each patient's clinical outcome. RESULTS: We included 120 patients (60 BPS and 60 controls) with a mean age of 24.2 ± 14.1 months. The use of antibiotics was significantly lower in the BPS group (46.6% vs. 86.6; OR 0.13; 95% CI: 0.05-0.35; P < 0.001). We observed an unfavorable outcome in 10 patients (8.3%), 5 in each group (P = 1.0; OR: 1.0 95% CI: 0.2-3.6). CONCLUSION: The use of antibiotics was significantly lower in the group managed according to the BPS compared to the conventionally treated group, without increasing the rate of treatment failure.

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.