An accurate test for homogeneity of odds ratios based on Cochran's Q-statistic

Background: A frequently used statistic for testing homogeneity in a meta-analysis of K independent studies is Cochran's Q. For a standard test of homogeneity the Q statistic is referred to a chi-square distribution with K - 1 degrees of freedom. For the situation in which the effects of the studies are logarithms of odds ratios, the chi-square distribution is much too conservative for moderate size studies, although it may be asymptotically correct as the individual studies become large. Methods: Using a mixture of theoretical results and simulations, we provide formulas to estimate the shape and scale parameters of a gamma distribution to t the distribution of Q. Results: Simulation studies show that the gamma distribution is a good approximation to the distribution for Q. Conclusions: : Use of the gamma distribution instead of the chi-square distribution for Q should eliminate inaccurate inferences in assessing homogeneity in a meta-analysis. (A computer program for implementing this test is provided.) This hypothesis test is competitive with the Breslow-Day test both in accuracy of level and in power

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Evaluation of invasive and non-invasive methods for the diagnosis of Helicobacter pylori infection in symptomatic children and adolescents

CONTEXT: Multiple diagnostic methods are available for the detection of Helicobacter pylori infection, but at present no single one can be used as the gold standard. OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy of 3 invasive and 2 non-invasive methods for detection of Helicobacter pylori infection in symptomatic children and adolescents. DESIGN: Prospective cohort study SETTING: Peptic Disease outpatients service, Discipline of Pediatric Gastroenterology, Universidade Federal de São Paulo (UNIFESP) / Escola Paulista de Medicina. PATIENTS: Forty-seven patients who underwent endoscopy because of dyspeptic symptoms. DIAGNOSTIC METHODS: Endoscopy with gastric biopsies for 3 invasive (rapid urease test, histology and culture) and 2 non-invasive methods (a commercial ELISA serology and 13carbon urea breath test - isotope ratio mass spectrometry) for detection of Helicobacter pylori infection. MAIN MEASUREMENTS: Sensitivity, specificity, positive and negative predictive values of each method and agreement and disagreement rates between the methods. RESULTS: Forty-seven patients [mean age, 11y9mo (SD 2y10mo), 27 female and 20 male]; 62% of them were Helicobacter pylori-positive. All methods agreed in 61%, and were negative in 21% and positive in 40%. The greatest concordance between 2 methods occurred between the invasive methods: histology and rapid urease test (89.6%) and histology and culture (87.5%). The greatest sensitivity, considering Helicobacter pylori-positive cases, for any combination of 3 or more tests, was achieved by the rapid urease test (S=100%), followed by histology, serology and 13carbon-urea breath test (S=93.1%) and lastly by culture (S=79.3%). The highest specificity was obtained by histology (100%) and culture (100%), followed by the rapid urease test (84.2%), serology (78.9%) and 13carbon-urea breath test (78.9%). CONCLUSIONS: Our results suggest that among invasive methods, an association between the rapid urease test and histology constituted the best choice for the detection of Helicobacter pylori infection. If results of histology and the rapid urease test are different, serology may be recommended.CONTEXTO: Vários métodos diagnósticos estão disponíveis para a detecção da infecção por Helicobacter pylori (Hp), porém, até o presente momento, não há um teste que possa ser utilizado isoladamente como padrão-ouro. OBJETIVO: Avaliar a acurácia de três métodos invasivos e dois não-invasivos na detecção da infecção por Hp em crianças e adolescentes sintomáticos. TIPO DE ESTUDO: Estudo coorte prospectivo. LOCAL: Ambulatório de Doença Péptica, Disciplina de Gastroenterologia Pediátrica, Universidade Federal de São Paulo (UNIFESP) / Escola Paulista de Medicina. PACIENTES: 47 pacientes sintomáticos que realizaram exame endoscópico devido a sintomas dispépticos. MÉTODOS DIAGNÓSTICOS: Exame endoscópico com biopsias gástricas para três métodos invasivos (teste rápido da urease, histologia e cultura) e dois métodos não-invasivos (teste sorológico ELISA industrializado e teste respiratório com uréia marcada com Carbono13). VARIÁVEIS ESTUDADAS: Sensibilidade, especificidade, valor preditivo positivo e negativo de cada método e taxas de concordância e discordância entre os métodos. RESULTADOS: 47 pacientes [idade média de 11a9m (DP 2a10m), 27 do sexo feminino e 20 do masculino], 62% deles com infecção por Hp. Todos os 5 métodos concordaram em 61%, sendo negativo em 21% e positivo em 40%. As maiores concordâncias entre dois métodos ocorreram entre os métodos invasivos: histologia e teste rápido da urease (89,6%) e entre a histologia e cultura (87,5%). A maior sensibilidade, considerando como Hp positivo, qualquer combinação de 3 ou mais testes, foi encontrada no teste rápido da urease (S=100%), seguido pela histologia, sorologia e o teste respiratório com uréia marcada com Carbono13 (S=93,1%) e por fim a cultura (S=79,3%). A maior especificidade foi obtida pela histologia e cultura (100%), seguidos pelo teste rápido da urease (84,2%), sorologia (78,9%) e teste respiratório com uréia marcada com Carbono13 (78,9%). CONCLUSÕES: Nossos resultados sugerem que, entre os métodos invasivos, a associação do teste rápido da urease e histologia constituem a melhor escolha para a detecção da infecção por Hp. Se os resultados da histologia e do teste rápido da urease forem discordantes é recomendada a sorologia.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Pediatric DepartmentUNIFESP, EPM, Pediatric DepartmentSciEL

TRAIL-receptor preferences in pancreatic cancer cells revisited: Both TRAIL-R1 and TRAIL-R2 have a licence to kill

Background TRAIL is a potent and specific inducer of apoptosis in tumour cells and therefore is a possible new cancer treatment. It triggers apoptosis by binding to its cognate, death-inducing receptors, TRAIL-R1 and TRAIL-R2. In order to increase its activity, receptor-specific ligands and agonistic antibodies have been developed and some cancer types, including pancreatic cancer, have been reported to respond preferentially to TRAIL-R1 triggering. The aim of the present study was to examine an array of TRAIL-receptor specific variants on a number of pancreatic cancer cells and test the generality of the concept of TRAIL-R1 preference in these cells. Methods TRAIL-R1 and TRAIL-R2 specific sTRAIL variants were designed and tested on a number of pancreatic cancer cells for their TRAIL-receptor preference. These sTRAIL variants were produced in HEK293 cells and were secreted into the medium. After having measured and normalised the different sTRAIL variant concentrations, they were applied to pancreatic and control cancer cells. Twenty-four hours later apoptosis was measured by DNA hypodiploidy assays. Furthermore, the specificities of the sTRAIL variants were validated in HCT116 cells that were silenced either for TRAIL-R1 or TRAIL-R2. Results Our results show that some pancreatic cancer cells use TRAIL-R1 to induce cell death, whereas other pancreatic carcinoma cells such as AsPC-1 and BxPC-3 cells trigger apoptosis via TRAIL-R2. This observation extended to cells that were naturally TRAIL-resistant and had to be sensitised by silencing of XIAP (Panc1 cells). The measurement of TRAIL-receptor expression by FACS revealed no correlation between receptor preferences and the relative levels of TRAIL-R1 and TRAIL-R2 on the cellular surface. Conclusions These results demonstrate that TRAIL-receptor preferences in pancreatic cancer cells are variable and that predictions according to cancer type are difficult and that determining factors to inform the optimal TRAIL-based treatments still have to be identified

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition

High morbid-mortability and reduced level of osteoporosis diagnosis among elderly people who had hip fractures in São Paulo City

OBJECTIVE: To know the morbid-mortality following an osteoporotic hip fracture in elderly patients living in São Paulo. PATIENTS AND METHODS: This study evaluated prospectively all patient over 60 years admitted in 2 school-hospitals in the city of São Paulo in a following 6-month period due to a osteoporotic proximal femur fracture. All of them filled up the Health Assessment Questionnaire (HAQ) and had their chart reviewed. After 6 months they were re-interviewed. Linear regression analysis was utilized to determine the factors related to functional ability. RESULTS: 56 patients were included (mean age 80.7 ± 7.9 years old, 80.4% females). After the 6-month follow up the mortality rate was 23.2%. Only 30% of the patients returned to their previous activities, and 11.6% became totally dependent. Factors related to worse functional ability after fracture were HAQ before fracture, institutionalization after fracture and age (r² 0.482). The diagnosis of osteoporosis was informed only by 13.9% of them, and just 11.6% received any treatment for that. CONCLUSION: Our results showed the great impact of these fractures on mortality and in the functional ability of these patients. Nevertheless, many of our physicians do not inform the patients about the diagnosis of osteoporosis and, consequently, the treatment of this condition is jeopardized.As fraturas osteoporóticas de fêmur proximal trazem graves conseqüências quanto à morbimortalidade e à qualidade de vida, mas desconhece-se este impacto no Brasil. OBJETIVO: Conhecer a morbimortalidade decorrente deste tipo de fraturas em idosos na cidade de São Paulo. MÉTODOS: Foram incluídos todos os pacientes com mais de 60 anos internados por fraturas de fêmur proximal durante seis meses, em dois hospitais de São Paulo. Os pacientes preencheram o questionário de capacidade funcional (HAQ), tiveram seu prontuário examinado e foram reavaliados após seis meses. Utilizou-se a análise de regressão linear para determinar os fatores relacionados à capacidade funcional. RESULTADOS: Cinqüenta e seis pacientes foram incluídos no estudo (80,7 ± 7,9 anos; 80,4% mulheres). A mortalidade em seis meses foi de 23,2%. Apenas 30% retornaram plenamente às suas atividades prévias e 11,6% tornaram-se completamente dependentes. Os fatores que mais bem conseguiram prever pior capacidade funcional após a fratura foram HAQ pré-fratura, institucionalização pós-fratura e idade (r² 0,482). Somente 13,9% receberam o diagnóstico de osteoporose e 11,6% iniciaram algum tratamento. CONCLUSÕES: Os resultados do presente estudo demonstram o impacto deste tipo de fraturas sobre a mortalidade e a capacidade funcional. Entretanto, a falha médica no diagnóstico e na orientação de tratamento da osteoporose permanece elevada.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaSanta Casa de Misericórdia de São Paulo Departamento de OrtopediaUNIFESP-EPM EPMUNIFESP, EPM, EPMSciEL

EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization for photothermal therapy

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer -coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.Support was provided by: Fundacao para a Ciencia e Tecnologia (FCT) for the financial support under the project reference PTDC/BBB-BMC/0611/2012 [https://www.fct.pt/apoios/projectos)]. The work at CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) [https://www.fct.pt/apoios/projectos]; European Commission through the project H2020-644242-SAPHELY (https://saphely.eu/project.php) and the project H2020-634013-2-PHOCNOSIS [http://cordis.europa.eu/project/rcn/193268_en.html].The authors would like to thank Fundacao para a Ciencia e Tecnologia (FCT) for the financial support under the project reference PTDC/BBB-BMC/0611/2012. The work at CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). The authors acknowledge the funding from the European Commission through the project H2020-644242-SAPHELY and the project H2020-634013-2-PHOCNOSIS. Finally, the authors would also like to thank the master student Joao Lopes from Universidade Lusofona (Portugal) for the help with in vitro cytotoxic assays. Isabel Correia acknowledges FCT for Investigator FCT contract.info:eu-repo/semantics/publishedVersio

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of 18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.IS

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.This work is financed by Portuguese national funds through FCT - Fundação para a Ciência e a Tecnologia, I.P, within the project PTDC/BIA-MIC/3458/2020 (DOI:10.54499/PTDC/BIA-MIC/3458/2020) and PhD fellowships 2021.07335.BD to GSO and UI/BD/150830/2021 to CMB; FWO - Research Foundation Flanders, grant n° 1S68720N; Innovative Medicine Initiative 2 Call 16 (IMI2-Call 16) proposal RespiriTB under the agreement number 853903. The authors acknowledge the support of the i3S Scientific Platform BioSciences Screening, member of the national infrastructures PT-OPENSCREEN (NORTE-01-0145-FEDER-085468) and PPBI - Portuguese Platform of Bioimaging (PPBIPOCI-01-0145-FEDER-022122)

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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