Benzo[a]pyrene promotes an epithelial-to-mesenchymal transition process in MCF10A cells and mammary tumor growth and brain metastasis in female mice.

Breast cancer is the most frequent neoplasia in developed countries and the leading cause of death in women worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process through which epithelial cells decrease or lose their epithelial characteristics and gain mesenchymal properties. EMT mediates tumor progression, because tumor cells acquire the capacity to execute the multiple steps of invasion and metastasis. Benzo[a]pyrene (B[a]P) is an environmental organic pollutant generated during the burning of fossil fuels, wood, and other organic materials. B[a]P exposition increases the incidence of breast cancer, and induces migration and/or invasion in MDA-MB-231 and MCF-7 breast cancer cells. However, the role of B[a]P in the induction of an EMT process and metastasis of mammary carcinoma cells has not been studied in detail. In this study, we demonstrate that B[a]P induces an EMT process in MCF10A mammary non-tumorigenic epithelial cells. In addition, B[a]P promotes the formation of larger tumors in Balb/cJ mice inoculated with 4T1 cells than in untreated mice and treated with dimethyl sulfoxide (DMSO). B[a]P also increases the number of mice with metastasis to brain and the total number of brain metastatic nodules in Balb/cJ mice inoculated with 4T1 cells compared with untreated mice and treated with DMSO. In conclusion, B[a]P induces an EMT process in MCF10A cells and the growth of mammary tumors and metastasis to brain in Balb/cJ mice inoculated with 4T1 cells.

The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Gene mutation profiling and clinical significances in patients with renal cell carcinoma.

OBJECTIVES: The pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. METHODS: Tissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. RESULTS: The most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. CONCLUSIONS: Wild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.

Electrophoretic characterization of cellular and extracellular proteins from Selenastrum capricornutum cultures degrading benzo(a)pyrene and their identification by UPLC-ESI-TOF mass spectrometry.

Selenastrum capricornutum efficiently degrades benzo(a)pyrene (BaP) but few proteins related to BaP degradation have been identified in this microalgae. So far, it has only been suggested that it could degrade BaP via the monooxygenase and/or dioxygenase pathways. To know more about this fact, in this work, cultures of S. capricornutum incubated with BaP were used to obtain the molecular weights (MWs) of proteins existing in its extra- and cellular extracts by electrophoresis and UPLC-ESI(+)-TOF MS analysis. The results of this proteomic approach indicated that BaP markedly induces the MWs: 6-20, 30, 45, and 65 kDa in cells; 6-20, 30.3, 38-45, and 55 kDa in liquid medium. So, these proteins could be related to BaP biodegradation. An identified protein with monooxygenase activity and rubredoxins (Rds) show to be related to BaP degradation: Rds could participate, together with the monooxygenase in the electron transfer during the formation of monohydroxylated-BaP metabolites. Rds may be also associated with a dioxygenase system that degrades BaP to form dihydrodiol-BaP metabolites. A multi-pass membrane protein was identified too, and it can regulate the transport of molecules like enzymes from inside the cell to the outside environment. At the same time, the presence of a dihydrolipoamide acetyltransferase validated the stress caused by the exposure to BaP. It is noteworthy that these findings provide valuable and original information on the characterization of the proteins of S. capricornutum cultures degrading BaP, whose enzymes have so far not been known. It is important to highlight that the functions of the identified proteins can help in understanding the metabolic and environmental behavior of this microalgae, and the extracts containing the degrading enzymes could be utilized in bioremediation applications.

Gene mutation profiling and clinical significances in patients with renal cell carcinoma

Abstract Objectives The pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. Methods Tissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. Results The most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. Conclusions Wild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.

Quick bioassay test from tillers for detecting ALS herbicide resistance of weedy rice and barnyardgrass

Resistance to acetolactate synthase (ALS) inhibitors have increased recently in South Brazil where the major weeds of flooded rice (barnyardgrass and weedy rice) have evolved resistance to imazapyr+imazapic. The aim of this research was to evaluate a growth medium for tissue regeneration of tillers in barnyardgrass, as well as an agar-based bioassays test (also from tillers) to detect susceptible and resistant biotypes of weedy rice and barnyardgrass to imazapyr+imazapic in vitro. Greenhouse experiments were conducted to detect ALS-resistant (R) and susceptible (S) weedy rice and barnyardgrass biotypes, and bioassays were carried out to evaluate an adequate growth medium for barnyardgrass tiller regeneration and determine the concentration of herbicide to distinguish R and S plants. The culture medium that provided a suitable barnyardgrass growth was MS 50% with the addition of benzylamino-purine. The tissue regeneration in vitro with the growth medium containing imazapyr+imazapic allowed to discriminate between R and S barnyardgrass and weedy rice plants. The concentration required for satisfactory control of susceptible barnyardgrass and weedy rice explants grown in vitro was 0.9 µM and 1.3 µM of imazapyr+imazapic herbicide, respectively. The bioassay in vitro using tiller regeneration provides an opportunity to predict effectively imazapyr+imazapic resistance in barnyardgrass and weedy rice.

Alterações gênicas em mesoteliomas maligno e importância da proteína BAP-1 para o processo tumoral / Genetic alterations in malignant mesotheliomas and the importance of BAP-1 protein for the tumoral process

O asbesto apresenta grande utilidade comercial, no entanto, é o mais importante carcinógeno ocupacional. O mesotelioma maligno é o tumor mais frequente causado pela exposição ao asbesto, porém devido ao tempo de latência entre a exposição e o desenvolvimento da doença pode chegar a 30 anos, o desenvolvimento de políticas de prevenção é muito difícil. Além disso, as dificuldades de diagnóstico e ausência de terapias específicas, fazem com que este tumor tenha altas taxas de mortalidade, com sobrevida estimada em 8 meses. Estima-se que o Brasil ainda deverá observar um aumento na incidência deste tipo de tumor, resultado das décadas de exposição de trabalhadores da indústria do asbesto. Portanto, compreender os mecanismos moleculares associados ao mesotelioma maligno é fundamental para o desenvolvimento de novas estratégias terapêuticas e de diagnóstico. O objetivo deste trabalho foi caracterizar o perfil epidemiológico dos pacientes acometidos com mesotelioma maligno atendidos na instituição, caracterizar a via de sinalização PI3K/AKT/mtor, assim como identificar as alterações gênicas em amostras de mesotelioma disponíveis. O perfil epidemiológico encontrado do portador de mesotelioma pleural maligno é: homem, ECOG1, diagnosticado entre 65-74 anos, sem exposição ao asbesto, de estágio IV, tabagista, com tumor epitelioide, tratados com cirurgia e quimioterapia, sem histórico familiar de cancer, e com histórico pessoal de neoplasia, com progressão local e recidiva da doença. Enquanto o perfil do portador de mesotelioma peritonial maligno é: mulher, ECOG 1, diagnosticado entre 21 e 44 anos, sem exposição ao asbesto, estádio IV, não fumante, não etilista, apresentando tumor de histologia epitelioide, tratado com quimioterapia, sem histórico familiar de câncer, sem progressão da doença e sem recidiva, e com histórico de neoplasia anterior. A partir dos dados clínicos encontramos: indicativos de predominância de síndrome hereditária, relação entre expressão de BAP-1 e sobrevida global, expressão de BAP-1 e histologia dos tumores, e ausência de correlação entre a expressão de BAP-1 e a topografia do tumor. Foram coletados fragmentos de espécimes cirúrgicos de tumor de 5 pacientes para o estabelecimento de linhagens celulares. As células tumorais cultivadas apresentaram preservação de marcadores de mesotelioma como citoqueratina AE1/AE3, podoplanina e WT-1; e ativação das vias PI3K/AKT/mTOR. Destes 5 casos de mesotelioma maligno, conseguimos estabelecer com sucesso 3 linhagens celulares humana e 2 linhagens de xenoenxerto murinho. A partir do sequenciamento destas 3 linhagens encontramos 1220 alterações gênicas, dentre elas 81 inéditas e de potencialmente maligno. Encontramos também 11 alterações correspondentes com as 20 mais encontradas em mesotelioma maligno de acordo com o The Cancer Genome Atlas TCGA. Entretanto, apenas uma alteração em BAP1 foi identificada. Os resultados deste trabalho permitirão realizar futuros ensaios funcionais in vivo e in vitro para elucidar os mecanismos envolvidos no processo tumoral do mesotelioma maligno

The asbestos has great commercial utility. However, they are the most important occupational carcinogens. The malignant mesothelioma is the most common tumor caused by asbestos' exposure, however due to the latency time between exposure and the development of the disease can take up to 30 years, the development of prevention policies is very difficult. In addition, the difficulties of diagnosis and the absence of specific therapies, cause this tumor to have high mortality rates, with an estimated survival of 8 months. It is estimated that Brazil will still see an increase in the incidence of this type of tumor, as the result of decades of exposure of workers in the asbestos industry. Therefore, understanding the molecular mechanisms associated with malignant mesothelioma is essential for the development of new therapeutic and diagnostic strategies. The objective of this work was to characterize the epidemiological profile of patients affected with malignant mesothelioma treated at the institution, to characterize the PI3K/AKT/mTOR signaling pathway, as well as to identify the genetic alterations in available mesothelioma samples. The epidemiological profile found malignant pleural mesothelioma patients is: male, ECOG1, diagnosed between 65­74 years old, without exposure to asbestos, stage IV, smoker, with epithelioid tumor, treated with surgery and chemotherapy, without family history of cancer, and with a personal history of neoplasia, with local progression and disease recurrence. While the profile of malignant peritoneal mesothelioma patient is: woman, ECOG 1, diagnosed between 21 and 44 years old, without exposure to asbestos, stage IV, non­smoker, non­alcoholic, presenting epithelioid histology tumor, treated with chemotherapy, without family history of cancer, without progression of the disease and without recurrence, and with a history of previous neoplasia. From the clinical data we found: indicative of predominance of hereditary syndrome, relationship between BAP­1 expression and overall survival, BAP­1 expression and tumor histology, and absence of correlation between BAP­1 expression and the topography of the tumor in our cohort. Fragments of surgical specimens of tumor were collected from 5 patients for the establishment of cell lines. Cultured tumor cells showed preservation of mesothelioma markers such as cytokeratin AE1/AE3, podoplanin and WT­1; and activation of PI3K/AKT/mTOR pathway. Among these 5 cases of malignant mesothelioma, we were able to successfully establish 3 human cell lines and 2 murine xenograft lines. From the sequencing of these 3 lines we found 1220 gene alterations, 81 of them were unpublished and potentially malignant. We also found 11 changes corresponding to the 20 most commonly found in malignant mesothelioma according to the TCGA. However, only one change in BAP1 was identified. The results of this study will allow future tests in vivo and in vitro to elucidate the components involved in the malignant mesothelioma tumor process.

Benign multicystic peritoneal mesothelioma: literature review and update.

Benign multicystic peritoneal mesothelioma (BMPM) is a rare peritoneal tumor diagnosed predominantly in pre-menopausal women. Associated risk factors include endometriosis and pelvic inflammatory disease in women, and prior abdominal surgery in both genders. To date, the pathogenesis of this disease remains controversial with possible etiologies, including a neoplastic versus a reactive process. Given the risk factors, some authors believe that this disease is secondary to a reactive process. However, because some studies describe cases where there is no prior surgical history or inflammatory milieu present, and because of this entity's predilection for recurrence, some authors believe the origin to be neoplastic. Some genetic and familial associations have also been reported. Malignant transformation is extremely rare, with only two cases reported in the literature, despite the recurrence potential. Like the etiology, the name of this entity is also controversial. Some authors prefer the term "peritoneal inclusion cyst (PCM)" instead of "benign cystic mesothelioma" and argue that the term mesothelioma should only be used when there is evidence of atypia. Most cases of BMPM are discovered incidentally. Others reflect sequela of tumor mass effect. It appears intra-operatively as large, multi-focal, cystic lesions in the peritoneal and pelvic cavity. Diagnosis is achieved through surgical sampling with histopathological examination. Immunobiologically, BMPM exhibits multiple small cystic spaces with flattened lining containing calretinin positive cells without atypical features, mitotic figures, or tissue invasion. Treatment includes cytoreductive surgery. Here we present a case of BMPM in a 60-year-old male - a rare disease in an uncommon patient population.

Effect of methyl jasmonate and salicylic acid on the production of metabolites in cell suspensions cultures of Piper cumanense (Piperaceae).

Elicitation of cell suspensions culture is a strategy that could increase the production of secondary metabolites under controlled conditions. This research evaluated the effect of methyl jasmonate-MeJA and salicylic acid-SA as elicitors on the production of metabolites in cell suspensions of P. cumanense. The type of elicitor (MeJA or SA), the concentration of elicitor (10 µM and 100 µM), and time of exposition (3, 12, 24 h) on cell suspension were evaluated. Metabolic profiles of intracellular and extracellular extracts were analyzed by UHPLC-DAD and GC-MS. Differential production of metabolites was dependent on the type of elicitor, its concentration, and the time of exposition. Treatments with 100 µM SA were conducted to high production of 5-hydroxymethylfurfural (6.3 %), phenol (6.5 %), and (Z)-9-octadecenamide (8.8 %). This is the first report of elicitation on cell suspensions in the Piper genus and contributes to understanding the effect of MeJA and SA on metabolite production in plant cell culture.

Inhibition of 19S proteasome deubiquitinating activity in Schistosoma mansoni affects viability, oviposition, and structural changes.

The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.

Experimental data of a catalytic decolorization of Ponceau 4R dye using the cobalt (II)/NaHCO3/H2O2 system in aqueous solution.

The treatment by Advanced Oxidation Processes (AOPs) of wastewater polluted with dyes is of particular interest in the field of environmental engineering, especially for the removal azo-dyes, representing over 50% of the global annual production of dyes. Unfortunately, most azo-dyes are non-biodegradable and can be toxic to aquatic organisms. This is the first data article that applies the methodology of response surface for the optimization of decolorization of an azo-compound using cobalt in a homogeneous medium as the catalyst of a bicarbonate activated hydrogen peroxide (BAP) system which, in turn, is an emerging technology for wastewater treatment. The Response Surface Methodology (RSM) based on a Central Composite Design (CCD) was used to evaluate and optimize the influence of three experimental variables (stoichiometric dosage of H2O2, molar ratio H2O2/NaHCO3 and cobalt concentration) on the decolorization of Ponceau 4R. Reactions were performed at 25 °C, pH 8.3 with a reaction time of 2 h. Analysis of variance (ANOVA) showed values of R2 and adjusted-R2 of 0.9815 and 0.9648, and experimental data were fit to a second-order regression model. The optimal conditions to achieve a maximum decolorization (96.31%) of a Ponceau 4R aqueous solution of 20 mg/l were: 4.73 times stoichiometric dosage of H2O2, molar ratio H2O2/NaHCO3 of 1.70 and cobalt concentration of 11.16 µM. Under the optimal reaction conditions, the influence of temperature (20, 25, 30 and 35 °C) on decolorization was evaluated and data were adjusted to second order kinetics. To verify the efficiency of the BAP system on the decolorization of Ponceau 4R, under the optimal conditions of reaction, UV-Vis spectra, at different reaction times, were measured. Additionally, blank experiments in order to evaluate the effect of individual factors in the Ponceau 4R decolorization, using BAP system, were carried out. Data showed that the Co(II)-NaHCO3-H2O2 system is a suitable technology for the decolorization of azo-dyes aqueous solutions.

Novel function of transcription factor Uga3 as an activator of branched-chain amino acid permease BAP2 gene expression.

Gene regulation in yeast occurs at the transcription level, i.e. the basal level of expression is very low and increased transcription requires gene-specific transcription factors allowing the recruitment of basal transcriptional machinery. Saccharomyces cerevisiae BAP2 gene encodes the permease responsible for most uptake of leucine, valine and isoleucine, amino acids that this yeast can use as nitrogen sources. Moreover, BAP2 expression is known to be induced by the presence of amino acids such as leucine. In this context, the results presented in this paper show that BAP2 is an inducible gene in the presence of nitrogen-non-preferred source proline but exhibits high constitutive non-inducible expression in nitrogen-preferred source ammonium. BAP2 expression is regulated by the SPS sensor system and transcription factors Leu3, Gcn4 and Dal81. This can be achieved or not through a direct binding to the promoter depending on the quality of the nitrogen source. We further demonstrate here that an interaction occurs in vivo between Uga3 ‒ the transcriptional activator responsible for γ-aminobutyric acid (GABA)-dependent induction of the GABA genes ‒ and the regulatory region of the BAP2 gene, which leads to an increase in BAP2 transcription.

Mecanismos envolvidos na produção e liberação de TNF-α induzidas pela BaP1 / Mechanisms involved in the production and release of TNF-α induced by BaP1

Metalloproteinases are abundant enzymes in snake venoms of Viperidae family and are relevant in the pathophysiology of envenomation, by their hemorrhagic, fibrinogenolytic and inflammatory activities. From Bothrops asper snake venom was isolated the metalloproteinase BaP1, with molecular weight of 22.7 KDa, weak hemorrhagic action and containing only the catalytic domain of metalloproteinases. Previous studies of our group have shown that BaP1 induces important inflammatory events in vivo and in vitro, inducing the release of inflammatory mediators, such as TNF-α (tumor necrosis factor alpha) by macrophages, important cells of the immune system. ADAM-17 or TACE (TNF-α converting enzyme) is responsible for the release of active TNF-α from its precursor. However, the mechanisms involved in the processing of TNF-α, under the action of BaP1, are unknown. In the present study was demonstrated that macrophages of the RAW 264-7 lineage, pretreated or not with SN50 compound, the nuclear transcription factor NF-κB inhibitor, and stimulated with BaP1 (12.5 μg / mL) for a period of 6 hours, showed a significant decrease in protein expression and the release of TNF-α, when compared to the negative control (culture medium RPMI). Pretreatment of RAW 264-7 cells with the TACE inhibitor, TAPI-1 (200 nM), and incubation with BaP1 (12.5 μg / mL) or RPMI, for 3 h, significantly decreased TNF-α release, when compared to the control group (RPMI). The present study demonstrated that the activation of NF-κB is an important mechanism involved in the processing of TNF-α, induced by BaP1 in RAW 264-7 cells and the release of TNF-α induced by BaP1 is dependent on the action of ADAM-17.

As metaloproteinases são enzimas abundantes em venenos de serpentes da família Viperidae e relevantes na fisiopatologia do envenenamento, devido as suas atividades hemorrágica, fibrinogenolítica e inflamatória. A partir do veneno da serpente B. asper, foi isolada a metaloproteinase de veneno (MV) BaP1, com peso molecular de 22,7 KDa, de fraca ação hemorrágica e que contém apenas o domínio catalítico. Estudos anteriores realizados pelo nosso grupo, demonstraram que a BaP1 induz importantes eventos inflamatórios in vivo e in vitro, induzindo a liberação de mediadores inflamatórios, como o TNF-α (fator de necrose tumoral) em macrófagos, importantes células do sistema imune. A ADAM-17 ou TACE (enzima conversora de TNF-α) é a responsável pela liberação de TNF-α ativo, do seu percursor. Entretanto, os mecanismos envolvidos no processamento do TNF-α, sob ação da BaP1, são desconhecidos. No presente estudo foi demonstrado que macrófagos da linhagem RAW 264-7, pré-tratados ou não com o composto SN50, inibidor do fator de transcrição nuclear κB (NF-κB) e estimulados com a BaP1 (12,5 μg/mL) por um período de 6 horas, apresentaram uma diminuição significativa na expressão proteica e na liberação de TNF-α, quando comparados com o controle negativo (meio de cultura incompleto). O pré-tratamento das células RAW 264-7, com o inibidor da enzima TACE, TAPI-1 (200 nM), e incubação com a BaP1 (12,5 μg/mL) ou meio de cultura (RPMI) por um período de 3 h, diminuiu significativamente a liberação de TNF- α quando comparado ao grupo controle (RPMI). O presente trabalho demonstrou que a ativação do NF-κB é um dos mecanismos envolvidos no processamento do TNF-, induzido pela BaP1 nas células RAW 264-7 e a liberação de TNF-α induzida pela BaP1 é dependente da ação da ADAM-17.

Benign multicystic peritoneal mesothelioma: literature review and update

Benign multicystic peritoneal mesothelioma (BMPM) is a rare peritoneal tumor diagnosed predominantly in pre-menopausal women. Associated risk factors include endometriosis and pelvic inflammatory disease in women, and prior abdominal surgery in both genders. To date, the pathogenesis of this disease remains controversial with possible etiologies, including a neoplastic versus a reactive process. Given the risk factors, some authors believe that this disease is secondary to a reactive process. However, because some studies describe cases where there is no prior surgical history or inflammatory milieu present, and because of this entity's predilection for recurrence, some authors believe the origin to be neoplastic. Some genetic and familial associations have also been reported. Malignant transformation is extremely rare, with only two cases reported in the literature, despite the recurrence potential. Like the etiology, the name of this entity is also controversial. Some authors prefer the term "peritoneal inclusion cyst (PCM)" instead of "benign cystic mesothelioma" and argue that the term mesothelioma should only be used when there is evidence of atypia. Most cases of BMPM are discovered incidentally. Others reflect sequela of tumor mass effect. It appears intra-operatively as large, multi-focal, cystic lesions in the peritoneal and pelvic cavity. Diagnosis is achieved through surgical sampling with histopathological examination. Immunobiologically, BMPM exhibits multiple small cystic spaces with flattened lining containing calretinin positive cells without atypical features, mitotic figures, or tissue invasion. Treatment includes cytoreductive surgery. Here we present a case of BMPM in a 60-year-old male - a rare disease in an uncommon patient population.

BAP1 tumor predisposition syndrome case report: pathological and clinical aspects of BAP1-inactivated melanocytic tumors (BIMTs), including dermoscopy and confocal microscopy.

BACKGROUND: BRCA1 associated-protein 1 (BAP1) tumor predisposition syndrome is associated with an increased risk for malignant mesotheliomas, uveal and cutaneous melanomas, renal cell carcinomas, and singular cutaneous lesions. The latter are referred to as BAP1-inactivated melanocytic tumors (BIMTs). When multiple BIMTs manifest, they are considered potential markers of germline BAP1 mutations. CASE PRESENTATION: Here, we report a novel pathogenic BAP1 germline variant in a family with a history of BIMTs, cutaneous melanomas, and mesotheliomas. We also describe singular pathological aspects of the patient's BIMT lesions and their correlation with dermoscopic and reflectance confocal microscopy findings. CONCLUSIONS: This knowledge is crucial for the recognition of BIMTs by dermatologists and pathologists, allowing the determination of appropriate management for high-risk patients, such as genetic investigations and screening for potentially aggressive tumors.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.

Immunoexpression of BAP1, ROS1, and ALK in Spitzoid Melanocytic Tumors.

BACKGROUND: Spitzoid tumors are a heterogeneous group of melanocytic neoplasms that frequently imposes diagnostic difficulties. Lately, several advances in molecular biology afforded significant discoveries on the pathogenesis of these tumors. BAP1 (BRCA-1 associated protein-1) inactivation and anomalous expression of kinase translocation-related proteins are among the main criteria launched by new classification proposals. Our aim was to systematically assess the immunoexpression of BAP1, ROS1 (receptor tyrosine kinase c-Ros oncogene 1), and ALK (anaplastic lymphoma receptor tyrosine kinase) proteins in an unpublished series of spitzoid tumors. METHODS: Retrospective study based on 47 formalin-fixed paraffin-embedded tissue samples from 3 different institutions. BAP1, ROS1, and ALK immunostains were performed in all cases. We included 27 Spitz tumors without significant abnormality, 15 atypical spitzoid tumors, and 5 spitzoid melanomas. RESULTS: We observed loss of BAP1 nuclear immunolabeling in 4.3% of evaluable cases (2/46), both of them atypical spitzoid tumors. The proportional frequency of BAP1-inactivated cases among atypical spitzoid tumors was 14.2% (2/14). No immunoexpression of ROS1 or ALK was found. CONCLUSIONS: Our study revealed 2 additional BAP1-inactived cases and described its respective frequency. The absence of anomalous expression of translocation-related proteins ALK and ROS1 in this series, composed predominantly of low-grade/low-risk tumors, indicates that translocated spitzoid lesions may not be as prevalent as initially suggested, at least in some populations. Furthermore, our findings encourage additional investigation on unequal occurrence of such immunomarkers among different diagnostic categories of spitzoid neoplasms.

Ras oncogene and Hypoxia-inducible factor-1 alpha (hif-1α) expression in the Amazon fish Colossoma macropomum (Cuvier, 1818) exposed to benzo[a]pyrene

Abstract Benzo[a]pyrene (B[a]P) is a petroleum derivative capable of inducing cancer in human and animals. In this work, under laboratory conditions, we analyzed the responses of Colossoma macropomum to B[a]P acute exposure through intraperitoneal injection of four different B[a]P concentrations (4, 8, 16 and 32 μmol/kg) or corn oil (control group). We analyzed expression of the ras oncogene and the Hypoxia-inducible factor-1 alpha (hif-1α) gene using quantitative real-time PCR. Additionally, liver histopathological changes and genotoxic effects were evaluated through the comet assay. Ras oncogene was overexpressed in fish exposed to 4, 8 of 16 μmol/kg B[a]P, showing 4.96, 7.10 and 6.78-fold increases, respectively. Overexpression also occurred in hif-1α in fish injected with 4 and 8 μmol/kg B[a]P, showing 8.82 and 4.64-fold increases, respectively. Histopathological damage in fish liver was classified as irreparable in fish exposed to 8, 16 and 32 μmol/kg μM B[a]P. The genotoxic damage increased in fish injected with 8 and 16 μmol/kg in comparison with the control group. Acute exposure of B[a]P was capable to interrupt the expression of ras oncogene and hif-1α, and increase DNA breaks due to tissue damage.