Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies.

Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.

Meaning of Life Therapy: A Pilot Study of a Novel Psycho-Existential Intervention for Palliative Care in Cancer.

Intervention in Palliative Care aims to provide physical, psychosocial, and spiritual relief for patients and family members. Brief interventions with a psycho-existential approach have shown positive responses; however, cultural adaptations are needed. This pilot study aimed to develop the Meaning of Life Therapy (MLT), a novel psycho-existential intervention, rooted in the Dignity Therapy, Life Review, and Meaning-Centered Psychotherapy. MLT was culturally adapted to the Portuguese context to include questions about forgiveness, apology, reconciliation, farewell, and a legacy document, i.e., the Life Letter. Nine PC cancer patients answered a 14-question MLT protocol, intended to help patients find purpose and meaning in life. Eight themes emerged: Family, Preservation of Identity, Life Retrospective, Clinical Situation, Achievements, Socio-Professional Valorization, Forgiveness/Apology/Reconciliation, and Saying Goodbye. MLT has proved its ability to respond to the psycho-existential needs of PC patients. Further studies should be conducted to gain extensive knowledge of the effectiveness of culturally responsive interventions.

Peripheral dentinogenic ghost cell tumour.

The dentinogenic ghost cell tumour (DGCT) is a rare benign neoplasm, which histologically presents itself as an aberrant keratinisation of the epithelium, ghost cells and dentinoid material. Depending on its location there are two different types of DGCT, central or peripheral, with different clinical characteristics. By 2019, there were only 57 cases of DGCT published: 39 of the central type and 18 of the peripheral type.In this clinical case, the authors describe the case of a 78-year-old man with a painless and slow growing mandibular lump. The diagnosis of peripheral DGCT was made by incisional biopsy and the treatment consisted of radical excision with upper marginal mandibulectomy.The aim of the article is to report a clinical case of a rare pathology and, consequently, to help diagnose and better understand its biological behaviour.

Common polymorphic OTC variants can act as genetic modifiers of enzymatic activity.

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.

Targeting Germ Cell Tumors with the Newly Synthesized Flavanone-Derived Compound MLo1302 Efficiently Reduces Tumor Cell Viability and Induces Apoptosis and Cell Cycle Arrest.

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC50s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.

Cross-cultural adaptation and validation of the Swallowing Disturbance Questionnaire and the Sialorrhea Clinical Scale in Portuguese patients with Parkinson's disease.

INTRODUCTION: To date, no valid outcome measure has been developed in European Portuguese (EP) to evaluate the Parkinsons' Disease (PD) patients' (PwP) reports regarding their swallowing disturbances. OBJECTIVES: The aim of this study was to translate and cross-culturally adapt the Swallowing Disturbance Questionnaire (SDQ) and the Sialorrhea Clinical Scale for PD (SCS-PD) into EP and to determine its clinimetric properties in PwP. MATERIALS AND METHODS: The original English SDQ and SCS-PD versions were cross-culturally adapted following recommendations established in international guidelines. The validation process involved 75 PwP and 65 healthy sex- and age-matched participants. RESULTS: The EP versions of the SDQ and SCS-PD are equivalent to the original versions (content, depth, and scoring). Statistical analyses for the SDQ tool revealed good feasibility (missing data <5%), acceptability (no floor or ceiling effects), excellent internal consistency (Cronbach´s α = 0.95), good construct validity (78.5% revealed large to moderate loadings), moderate convergent validity (r = 0.60), good divergent validity (r = 0.40), good known-groups validity (p-value < .05) and a fair sensitivity and specificity (AUC = 0.700). Statistical analyses for the SCS-PD tool shows good feasibility, reasonable acceptability (floor effect), good internal consistency (Cronbach´s α = 0.85), good construct validity (85.7% showed between large to moderate loadings), good convergent validity (r = 0.78), good divergent validity (r = 0.39), good known groups validity (p-value < .05) and a fair sensitivity and specificity (AUC = 0.704). CONCLUSIONS: The EP versions of the SDQ and SCS-PD maintained the characteristics of the original versions and therefore consistent tools to be used in PwP.

Epigenetic alterations as therapeutic targets in Testicular Germ Cell Tumours : current and future application of 'epidrugs'.

Testicular germ cell tumours (TGCTs) are heterogeneous neoplasms mostly affecting young-adult men. Despite high survival rates, some patients with disseminated disease acquire cisplatin resistance, entailing the need for less toxic therapies. Epigenetic alterations constitute an important feature of TGCTs, which are also implicated in resistance mechanism(s). These alterations might be used as potential targets to design epigenetic drugs. To date, several compounds have been explored and evaluated regarding therapeutic efficacy, making use of pre-clinical studies with in vitro and in vivo models, and some have already been explored in clinical trials. This review summarizes the several epigenetic mechanisms at play in these neoplasms, the current challenges in the field of TGCTs and critically reviews available data on 'epidrugs' in those tumours.

Health system collapse 45 days after the detection of COVID-19 in Ceará, Northeast Brazil: a preliminary analysis.

INTRODUCTION: COVID-19 emerged in late 2019 and quickly became a serious public health problem worldwide. This study aim to describe the epidemiological course of cases and deaths due to COVID-19 and their impact on hospital bed occupancy rates in the first 45 days of the epidemic in the state of Ceará, Northeastern Brazil. METHODS: The study used an ecological design with data gathered from multiple government and health care sources. Data were analyzed using Epi Info software. RESULTS: The first cases were confirmed on March 15, 2020. After 45 days, 37,268 cases reported in 85.9% of Ceará's municipalities, with 1,019 deaths. Laboratory test positivity reached 84.8% at the end of April, a period in which more than 700 daily tests were processed. The average age of cases was 67 (<1 - 101) years, most occurred in a hospital environment (91.9%), and 58% required hospitalization in an ICU bed. The average time between the onset of symptoms and death was 18 (1 - 56) days. Patients who died in the hospital had spent an average of six (0 - 40) days hospitalized. Across Ceará, the bed occupancy rate reached 71.3% in the wards and 80.5% in the ICU. CONCLUSIONS: The first 45 days of the COVID-19 epidemic in Ceará revealed a large number of cases and deaths, spreading initially among the population with a high socioeconomic status. Despite the efforts by the health services and social isolation measures the health system still collapsed.

Health system collapse 45 days after the detection of COVID-19 in Ceará, Northeast Brazil: a preliminary analysis

Abstract INTRODUCTION: COVID-19 emerged in late 2019 and quickly became a serious public health problem worldwide. This study aim to describe the epidemiological course of cases and deaths due to COVID-19 and their impact on hospital bed occupancy rates in the first 45 days of the epidemic in the state of Ceará, Northeastern Brazil. METHODS: The study used an ecological design with data gathered from multiple government and health care sources. Data were analyzed using Epi Info software. RESULTS: The first cases were confirmed on March 15, 2020. After 45 days, 37,268 cases reported in 85.9% of Ceará's municipalities, with 1,019 deaths. Laboratory test positivity reached 84.8% at the end of April, a period in which more than 700 daily tests were processed. The average age of cases was 67 (<1 - 101) years, most occurred in a hospital environment (91.9%), and 58% required hospitalization in an ICU bed. The average time between the onset of symptoms and death was 18 (1 - 56) days. Patients who died in the hospital had spent an average of six (0 - 40) days hospitalized. Across Ceará, the bed occupancy rate reached 71.3% in the wards and 80.5% in the ICU. CONCLUSIONS: The first 45 days of the COVID-19 epidemic in Ceará revealed a large number of cases and deaths, spreading initially among the population with a high socioeconomic status. Despite the efforts by the health services and social isolation measures the health system still collapsed.

Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

Fetal and infant mortality of congenital syphilis reported to the Health Information System.

BACKGROUND: Congenital syphilis (CS) is a major cause of mortality in several countries, especially in Latin America and the Caribbean. This study aimed to analyze fetal and infant mortality of CS reported to the Health Information System in a State in Northeastern Brazil. METHODS AND RESULTS: This was a cross-sectional study that analyzed the deaths of CS from 2010 to 2014 through the linkage of the Mortality Information System (SIM) and the Notifiable Diseases Information System (Sinan). The Statistical Package for the Social Sciences (SPSS) version 23.0 was used to calculate the rates of Fetal, Perinatal, Neonatal (early and late), and Postneonatal Mortality. Simple linear regression was performed. Fisher's exact test or Pearson's chi-square test were used for comparison of proportions and Student's t-test was used for comparison of means. Of the 414 cases reported to the SIM as deaths possibly caused by CS, 44 (10.6%) presented CS as the underlying cause. From 2010 to 2014 the Infant Mortality Rate of CS was 16.3 per 100,000 live births (y = 0.65x + 14.33, R2 = 0.2338, p = 0.003). There was an 89.4% underreporting of deaths. Perinatal deaths and fetal deaths of CS accounted for 87.7% and 73.9% of total deaths, respectively. CONCLUSIONS: The results of the study revealed a significant Fetal and Infant Mortality rate of CS and demonstrated the importance of using the linkage method in studies that involve the analysis of secondary data obtained from mortality and disease reporting systems. The underreporting of CS as a cause of fetal and infant mortality leads to unawareness of the reality of deaths from this disease, hindering the development of public policies aimed at its prevention.

Optical and Structural Characterization of a Chronic Myeloid Leukemia DNA Biosensor.

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.

Analysis of cases of gestational and congenital syphilis between 2008 and 2010 in Fortaleza, State of Ceará, Brazil. / Análise dos casos de sífilis gestacional e congênita nos anos de 2008 a 2010 em Fortaleza, Ceará, Brasil.

This study analyzes the reported cases of syphilis in pregnant women and the possible outcomes for fetuses and the newborn in Fortaleza, Ceará. It is a cross-sectional study that analyzed 175 reported cases of syphilis in pregnant women matched with the corresponding reports of congenital syphilis during the years 2008-2010. Descriptive statistics with absolute and relative frequencies, central tendency and dispersion measures, and the Pearson's chi-square test were used to analyze the statistical significance using the p-value <0.05. Sociodemographic variables of pregnant/postpartum women, the assistance provided to newborns and the outcome of cases were analyzed. The results showed the occurrence of syphilis in young women with more than 85% of inappropriate treatment, 62.9% of untreated sexual partners or lack of statistics and high percentages of non-realization of the recommended tests for congenital syphilis investigation in children. Among the fetuses, five were stillborn, one miscarried and there were three neonatal deaths. The lack of adequate treatment of pregnant women may be associated with morbidity and mortality of fetuses, maintaining this infection as a burden on the list of public health problems.

O presente estudo analisa os casos notificados de sífilis em gestantes e os possíveis desfechos para o feto e o recém-nascido em Fortaleza, Ceará. Estudo transversal que analisou 175 casos notificados de sífilis em gestantes, pareados com as correspondentes notificações de sífilis congênita durante os anos de 2008 a 2010. Utilizou-se estatística descritiva com frequências absolutas e relativas, medidas de tendência central e dispersão e qui-quadrado de Pearson para analisar a significância estatística, utilizando o valor de p < 0,05. Foram analisadas variáveis sociodemográficas das gestantes/puérperas, da assistência prestada aos recém-nascidos e o desfecho dos casos. Os resultados mostraram a ocorrência da sífilis em mulheres jovens com mais de 85,0% de tratamentos inadequados, 62,9% dos parceiros sexuais não tratados ou com informação ignorada e percentuais elevados da não realização dos exames preconizados para a investigação de sífilis congênita nas crianças. Dentre os conceptos, cinco foram natimortos, um aborto e três óbitos neonatais. A falta de tratamento adequado dos casos de sífilis em gestantes pode estar associada à morbimortalidade dos conceptos, mantendo essa infecção como um fardo no rol dos problemas de saúde pública.

Análise dos casos de sífilis gestacional e congênita nos anos de 2008 a 2010 em Fortaleza, Ceará, Brasil / Analysis of cases of gestational and congenital syphilis between 2008 and 2010 in Fortaleza, State of Ceará, Brazil

Resumo O presente estudo analisa os casos notificados de sífilis em gestantes e os possíveis desfechos para o feto e o recém-nascido em Fortaleza, Ceará. Estudo transversal que analisou 175 casos notificados de sífilis em gestantes, pareados com as correspondentes notificações de sífilis congênita durante os anos de 2008 a 2010. Utilizou-se estatística descritiva com frequências absolutas e relativas, medidas de tendência central e dispersão e qui-quadrado de Pearson para analisar a significância estatística, utilizando o valor de p < 0,05. Foram analisadas variáveis sociodemográficas das gestantes/puérperas, da assistência prestada aos recém-nascidos e o desfecho dos casos. Os resultados mostraram a ocorrência da sífilis em mulheres jovens com mais de 85,0% de tratamentos inadequados, 62,9% dos parceiros sexuais não tratados ou com informação ignorada e percentuais elevados da não realização dos exames preconizados para a investigação de sífilis congênita nas crianças. Dentre os conceptos, cinco foram natimortos, um aborto e três óbitos neonatais. A falta de tratamento adequado dos casos de sífilis em gestantes pode estar associada à morbimortalidade dos conceptos, mantendo essa infecção como um fardo no rol dos problemas de saúde pública.

Abstract This study analyzes the reported cases of syphilis in pregnant women and the possible outcomes for fetuses and the newborn in Fortaleza, Ceará. It is a cross-sectional study that analyzed 175 reported cases of syphilis in pregnant women matched with the corresponding reports of congenital syphilis during the years 2008-2010. Descriptive statistics with absolute and relative frequencies, central tendency and dispersion measures, and the Pearson's chi-square test were used to analyze the statistical significance using the p-value <0.05. Sociodemographic variables of pregnant/postpartum women, the assistance provided to newborns and the outcome of cases were analyzed. The results showed the occurrence of syphilis in young women with more than 85% of inappropriate treatment, 62.9% of untreated sexual partners or lack of statistics and high percentages of non-realization of the recommended tests for congenital syphilis investigation in children. Among the fetuses, five were stillborn, one miscarried and there were three neonatal deaths. The lack of adequate treatment of pregnant women may be associated with morbidity and mortality of fetuses, maintaining this infection as a burden on the list of public health problems.

The occupational stress in International Business Travellers of a multinational of the automotive sector / O stress ocupacional em Viajantes de Negócios Internacionais de uma multinacional do setor automóvel / El estrés ocupacional en Viajeros de Negocios Internacionales en una multinacional del sector automotriz

International business travelers constitute an emerging type of international mobility, and there is a gap in terms of studies that address well-being. Based on the Holistic Model of Stress, we intend to explore the occupational stress associated with business travel through a qualitative case study using document analysis and semi-structured interviews. We verified a predominance of distress sources in the trip stage and the adoption of coping strategies focused on the problem. Personal and professional factors are assumed to be the main moderators of the stress experienced. We conclude that travel brings mostly negative personal consequences that are accentuated during the traveler's career, and confirm that the trips are a source of stress with impact on the personal and professional life of the traveler. The paper includes a discussion on the theoretical and practical implications of the findings for both the company and the travelers.

Os Viajantes de Negócios Internacionais constituem uma modalidade de mobilidade internacional emergente, existindo uma lacuna ao nível de estudos que abordem o seu bem-estar. Com base no Modelo Holístico de Stress pretendemos explorar o stress ocupacional associado às viagens de negócios através de um estudo de caso qualitativo com recurso à análise documental e à entrevista semiestruturada. Foram predominantes as fontes de distress na fase da viagem e da adoção de estratégias de coping focadas no problema. Fatores pessoais e profissionais assumem-se como principais moderadores do stress percecionado. Concluímos que a viagem tem maioritariamente consequências pessoais e negativas que se acentuam ao longo da carreira do viajante e confirmamos que as viagens são uma fonte de stress com impacto na sua vida pessoal e profissional do viajante. O artigo incluí a discussão acerca das implicações teóricas e práticas dos resultados para a organização e para os viajantes.

Los Viajeros de Negocios Internacionales constituyen una modalidad de movilidad internacional emergente, existiendo una laguna en el nivel de estudios que abordan su bienestar. Con base en el Modelo Holístico de Estrés pretendemos explorar el estrés ocupacional asociado a los viajes de negocios a través de un estudio de caso cualitativo, utilizando el análisis documental y la entrevista semiestructurada. Se verificó un predominio de fuentes de distress y la adopción de estrategias de coping enfocadas en el problema. Los factores personales y profesionales se asumen como principales moderadores del estrés percibido. Concluimos que el viaje tiene mayormente consecuencias personales y negativas, que se acentúan al través de la carrera de viajero. Confirmamos así que los viajes son una fuente de estrés con impacto en la vida personal y profesional. El artículo incluye la discusión sobre las implicaciones teóricas y prácticas de los resultados para la organización y para los viajeros.

Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20.

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO42-, WO42- and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

Underreporting of Congenital Syphilis as a Cause of Fetal and Infant Deaths in Northeastern Brazil.

INTRODUCTION: Of all syphilis-related pregnancy outcomes, fetal death is certainly the most common one, being directly related to the availability, accessibility and quality of prenatal care. The present study aimed to analyze the underreporting of fetal and infant deaths and other maternal factors associated with congenital syphilis (CS) death. METHODS: This cross-sectional study integrated data of infants that were diagnosed and/or died of CS from the Sistema de Informação de Agravos de Notificação-Sinan (Notifiable Diseases Information System) and the Sistema de Informação de Mortalidade-SIM (Mortality Information System) in Fortaleza, Northeastern Brasil to identify unreported cases of congenital syphilis. We assessed data during the period from 2007 to 2013. RESULTS: The underreporting of CS as a cause of fetal or infant death increased from 41 to 415 cases (90.1%) during 2007-2013. Exactly 3,209 cases of CS were identified in Sinan and 6,578 deaths in SIM. After database linkage, we identified 382 cases that were reported in the SIM and SINAN databases consisting of 309 fetal deaths and 73 infant deaths related to CS. From the children notified at Sinan that born alive, 3.0% (78/2,542) died; Out of these, 39 (50.0%) were early and 25 (32.1%) were late neonatal deaths. The proportion of death by CS increased from 0.62 to 5.8 from 2007 to 2013. At logistic regression, the variable that maintained statistical significance with fetal and infant death outcomes was the presence of CS signs and/or symptoms at birth (OR = 3.20; IC 95% 1.54-6.62; p = 0.002). CONCLUSIONS: Neonatal and Infant deaths following CS-associated live births are underreported in Northeastern Brazil. Data base linkage identified unreported fetal and neonatal deaths due to CS leading to an increased awareness of fetal/infant mortality due to this infection.

The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes.

The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.

TupA: a tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20.

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, ß = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.

Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.