Immune Checkpoint Inhibitor-Associated Remitting Seronegative Symmetrical Synovitis With Pitting Edema: Description of a New Entity by CanRIO.

OBJECTIVE: Remitting seronegative symmetrical synovitis with pitting edema (RS3PE) is characterized by symmetrical synovitis with pitting edema and negative rheumatoid factor (RF). It has been described in a setting of malignancy, suggesting a paraneoplastic association. With the increasing use of immune checkpoint inhibitors (ICIs) for the treatment of cancers and emergence of immune-related adverse events (irAEs), our objective was to identify and describe cases of ICI-associated RS3PE (ICI-RS3PE) and compare them to non-ICI-RS3PE. METHODS: The Canadian Research Group of Rheumatology in Immuno-Oncology (CanRIO) network is a collaboration of Canadian rheumatologists with experience in the management of patients with rheumatic irAEs (Rh-irAEs). Standardized data on adult patients with Rh-irAE have been collected as part of retrospective and prospective cohorts. In this study, detailed information on all cases of ICI-RS3PE from both cohorts were extracted and analyzed. RESULTS: We identified 11 cases of ICI-RS3PE. The most frequently observed malignancy was nonsmall cell lung cancer (4 of 11), followed by malignant melanoma (2 of 11) and cutaneous squamous cell carcinoma (2 of 11). The median time to onset of ICI-RS3PE was 26 weeks from ICI start and 52 weeks from diagnosis of malignancy. Seven patients had stable cancer prior to onset of ICI-RS3PE, 3 had partial response, and 1 had complete response. All patients received glucocorticoids. Conventional synthetic disease-modifying antirheumatic drugs (csDMARD) were needed in 10 patients. CONCLUSION: ICI-RS3PE may be an independent Rh-irAE, separate from paraneoplastic RS3PE. The symptoms of ICI-RS3PE responded well to glucocorticoids, but concomitant treatment with csDMARDs may be necessary.

Qualification of the Microsatellite Instability Analysis (MSA) for Bladder Cancer Detection: The Technical Challenges of Concordance Analysis.

Bladder cancer (here we refer to transitional carcinoma of bladder) is a major cause of morbidity and mortality in the Western world, and recent understanding of its etiology, the molecular characteristics associated with its progression, renders bladder cancer an ideal candidate for screening. Cystoscopy is invasive and sometimes carries unwanted complications, but it is the gold standard for the detection of bladder cancer. Urine cytology, while the most commonly used test as an initial screening tool, is of limited value due to its low sensitivity, particularly for low-grade tumors. Several new "molecular assays" for the diagnosis of urothelial cancer have been developed over the last two decades. Here, we have established our new bladder cancer test based on an assay established for the Early Detection Research Network (EDRN) study. As a part of the study, a quality control CLIA/College of American Pathology (CAP) accredited laboratory, (QA Lab), University of Maryland Baltimore Biomarker Reference Laboratory (UMB-BRL), performed quality assurance analysis. Quality assurance measures included a concordance study between the testing laboratory (AIBioTech), also CLIA/CAP accredited, and the QA lab to ensure that the assay was performed and the results were analyzed in a consistent manner. Therefore, following the technical transfer and training of the microsatellite analysis assay to the UMB-BRL and prior to the initiation of analysis of the clinical samples by the testing lab, a series of qualification studies were performed. This report details the steps taken to ensure qualification of the assay and illustrates the technical challenges facing biomarker validation of this kind.

Results Obtained from a Pivotal Validation Trial of a Microsatellite Analysis (MSA) Assay for Bladder Cancer Detection through a Statistical Approach Using a Four-Stage Pipeline of Modern Machine Learning Techniques.

Several studies have shown that microsatellite changes can be profiled in urine for the detection of bladder cancer. The use of microsatellite analysis (MSA) for bladder cancer detection requires a comprehensive analysis of as many as 15 to 20 markers, based on the amplification and interpretations of many individual MSA markers, and it can be technically challenging. Here, to develop fast, more efficient, standardized, and less costly MSA for the detection of bladder cancer, we developed three multiplex-polymerase-chain-reaction-(PCR)-based MSA assays, all of which were analyzed via a genetic analyzer. First, we selected 16 MSA markers based on 9 selected publications. Based on samples from Johns Hopkins University (the JHU sample, the first set sample), we developed an MSA based on triplet, three-tube-based multiplex PCR (a Triplet MSA assay). The discovery, validation, and translation of biomarkers for the early detection of cancer are the primary focuses of the Early Detection Research Network (EDRN), an initiative of the National Cancer Institute (NCI). A prospective study sponsored by the EDRN was undertaken to determine the efficacy of a novel set of MSA markers for the early detection of bladder cancer. This work and data analysis were performed through a collaboration between academics and industry partners. In the current study, we undertook a re-analysis of the primary data from the Compass study to enhance the predictive power of the dataset in bladder cancer diagnosis. Using a four-stage pipeline of modern machine learning techniques, including outlier removal with a nonlinear model, correcting for majority/minority class imbalance, feature engineering, and the use of a model-derived variable importance measure to select predictors, we were able to increase the utility of the original dataset to predict the occurrence of bladder cancer. The results of this analysis showed an increase in accuracy (85%), sensitivity (82%), and specificity (83%) compared to the original analysis. The re-analysis of the EDRN study results using machine learning statistical analysis proved to achieve an appropriate level of accuracy, sensitivity, and specificity to support the use of the MSA for bladder cancer detection and monitoring. This assay can be a significant addition to the tools urologists use to both detect primary bladder cancers and monitor recurrent bladder cancer.

Heat shock factor 1 directly regulates transsulfuration pathway to promote prostate cancer proliferation and survival.

There are limited therapeutic options for patients with advanced prostate cancer (PCa). We previously found that heat shock factor 1 (HSF1) expression is increased in PCa and is an actionable target. In this manuscript, we identify that HSF1 regulates the conversion of homocysteine to cystathionine in the transsulfuration pathway by altering levels of cystathionine-ß-synthase (CBS). We find that HSF1 directly binds the CBS gene and upregulates CBS mRNA levels. Targeting CBS decreases PCa growth and induces tumor cell death while benign prostate cells are largely unaffected. Combined inhibition of HSF1 and CBS results in more pronounced inhibition of PCa cell proliferation and reduction of transsulfuration pathway metabolites. Combination of HSF1 and CBS knockout decreases tumor size for a small cell PCa xenograft mouse model. Our study thus provides new insights into the molecular mechanism of HSF1 function and an effective therapeutic strategy against advanced PCa.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

Colorimetric test for the monitoring of microcystins in cyanobacterial culture and environmental samples from southeast - Brazil

Microcistinas (MC) são heptapeptídeos de ação neuro e hepatotóxica produzidas por alguns gêneros de cianobactérias em determinadas condições físico-químicas do ambiente e são responsáveis pela morte e intoxicação de animais e humanos. A detecção de MC em água destinada ao consumo no Brasil ainda não é realizada na maioria dos estados brasileiros. O teste de inibição de proteína fosfatase tipo 1 (PP1) por MC é um método colorimétrico simples, rápido e de boa reprodutibilidade. Para testar a aplicação do teste PP1 foram realizados estudos de crescimento de cianobactérias em bioreator com meio ASM-1 dentro de condições controladas de crescimento (12/12h luz/escuro usando 30 mE.m2.s-1 de intensidade luminosa e temperatura constante de 23C) utilizando Microcystis aeruginosa (estirpe 1., UFRJ- produtor de MC). Variaram-se as concentrações de fósforo (P) em 24, 6 e 4 mM e de ferro (Fe) em 4 e 1mM. Uma curva padrão de inibição de PP1 pela MC-LR foi construída, tendo como limite de detecção 0.01 ng.mL-1. Em meio normal de crescimento (24 mM P e 4 mM Fe) para Microcystis aeruginosa, a produção de MC foi detectada continuamente durante o crescimento da cultura. A maior concentração de MC foi observada na concentração de 6 mM P e não foi detectada na concentração de 1 mM Fe. Amostras de florações ambientais, da região sudeste do Brasil (Belo Horizonte/MG), coletadas em corpos d'água utilizados para abastecimento e consumo humano, foram testadas e quantificadas para a presença de microcistina pelo teste colorimétrico PP1. A concentração de microcistina variou entre quantidades não detectáveis pelo método até 100 ng.mL-1 em amostras de floração da espécie Microcistis flos-aquae.

Efeito da temperatura sobre a produção de enterotoxina estafilocócica em leite / Effect of temperature on production of staphylococcal enterotoxin in milk

Muitos estudos têm investigado as condições nas quais Staphylococcus aureus está apto para produzir enteroxinas. As necessidades nutricionais (presença ou ausência de aminoácidos e glicose), bem como variações de pH e temperatura, podem resultar na produção de enterotoxinas capazes de causar doenças. Neste estudo foram usadas cepas de S. aureus produtoras de enterotoxinas A e C2 (respectivamente FRI 722 e FRI 361) e um isolado no qual foi detectada a presença concomitante dos genes de enterotoxina A e da Síndrome do Choque Tóxico (TSST-1) em estudo anterior. As cepas foram inoculadas em leite UHT e as amostras submetidas a diferentes situações de estresse térmico (tempo e temperatura). Em nosso trabalho, houve produção das enterotoxinas A e C2 e de TSST-1 quando o leite UHT foi exposto a temperaturas elevadas, na faixa de 37 a 42ºC e/ou quando o leite foi exposto a temperaturas oscilantes. Os resultados obtidos neste estudo parecem alertar para os métodos de conservação do leite, uma vez que o mesmo pode ser responsável por intoxicações alimentares causadas pela presença de S. aureus e suas enterotoxinas se não houver correta manipulação do produto, em todas as fases de produção.

Aplicação da técnica de REP – PCR no rastreamento de Staphylococcus aureus em sala de ordenha, para o monitoramento da qualidade do leite / Using the REP-PCR technic in the tracking of Staphylococcus aureus in milking room, for milk quality production

A identificação e classificação bacteriana são de suma importância no ambiente, na indústria, na medicina veterinária, na microbiologia e na ecologia microbiana. Um número de diferentes métodos genotípicos e fenotípicos estão sendo empregados para identificação e classificação microbiana. A técnica de REP-PCR é baseada no uso de primers sintetizados a partir de sequências repetidas de DNA, chamadas depalindrômicas extragênicas repetidas (REP), e têm sido descrita como um método o qual gera impressões digitais (fingerprints) de DNA que podem diferenciar bactérias entre gêneros e espécies. Neste estudo, o método de fingerprint foi usado para Staphylococcus aureus com o objetivo de avaliar a higiene de ordenha em duas fazendas leiteiras.Foram obtidos vários fingerprints de todos os isolados coletados das diferentes fontes estudadas (mãos de ordenhadores, tetos das vacas,leite e ordenhadeira), e foram obtidos comportamentos muito similares das bandas indicando que os isolados podem ser relatados como clones epidemiológicos. Em nosso estudo, a técnica mostrou ser eficiente para a análise da similaridade entre indivíduos da mesma espécie, no caso, o Staphylococcus aureus, mostrando ser uma ferramenta útil para investigação de falhas no manejo e, em um controle mais eficiente para evitar e/ou diminuir a disseminação de microrganismos causadores de sérias enfermidades em humanos e em animais, que podem ser transmitidas através de produtos como o leite e seus derivados.

The identification and classification of bacteria are of crucial importance in environmental, industrial, veterinary, microbiology and microbial ecology. A number of different phenotypic and genotypic methods are presently being employed for microbial identification and classification. Repetitive-element PCR (Rep-PCR) with primers basedon repetitive extragenic palindromic (REP) repeated DNA sequencesis a recently described method which generates DNA fingerprints that descriminate between bacterial species and strains. In this study, this method was used for genomic fingerprinting of Staphylococcus aureus in control of hygiene in milk line production on two farms. Complex fingerprinting patterns were obtained for all isolates from different sources (milking handlers, cows teats, milk and milk machine) were very similar, and the data indicated that the isolated were closely related.In our study, this technic shows to be usefull for investigation in fails on milk line production and for more efficient control for patogenic microrganisms that cause serious illness in humans and animals.