Análise proteômica de proteínas sintetizadas em resposta acisplatina em células de adenocarcinoma de pulmão humano resistentes e sensíveis a droga

O câncer de pulmão está entre os mais frequentes na população mundial e é o responsável pelo maior número de mortes relacionadas ao câncer. Como o câncer de pulmão é identificado em estágios avançados de desenvolvimento, o principal tratamento é baseado em quimioterapia utilizando moléculas derivadas de platina, principalmente a cisplatina (CDDP). A CDDP é capaz de formar ligações cruzadas no DNA resultando em morte celular, porém muitos pacientes apresentam tumores que são resistentes ao tratamento com CDDP. Esta resistência é uma das principais barreiras para o sucesso do tratamento do câncer de pulmão através de quimioterapia. Para entendermos melhor os mecanismos envolvidas na resistência a CDDP em células de câncer de pulmão, foram utilizadas células sensíveis (A549) e resistentes a CDDP (A549/CDDP) para a identificação de proteínas recém-sintetizadas em resposta ao tratamento com a droga através da técnica BONCAT. Através desta técnica foi possível a identificação de 173 e 136 proteínas reguladas por CDDP nas células A549 e A549/CDDP, respectivamente As proteínas identificadas estão relacionadas a diversos mecanismos moleculares distintos que potencialmente estão envolvidos na resposta a CDDP, como por exemplo splicing alternativo, resposta a estresse oxidativo, manutenção do telômero, regulação da apoptose e reorganização do citoesqueleto. Os resultados mostram que as células A549/CDDP são menos suscetíveis ao dano no DNA causado por CDDP do que as células sensíveis, A549. Além disso, as células A549/CDDP são capazes de aumentar a expressão de proteínas capazes de combater as espécies reativas de oxigênio geradas devido a presença de CDDP. Além disto, CDDP é capaz de induzir diferentes vias apoptóticas em células com diferentes sensibilidades à droga. Nas células A549, CDDP induz a ativação da via extrínseca enquanto que nas A549/CDDP ela é capaz de induzir a ativação da via intrínseca de apoptose. Portanto, nosso estudo foi capaz de fornecer evidencias de proteínas e vias que são diferencialmente expressas e ativadas após o tratamento com CDDP.Lung cancer is among the most frequent cancer in the world population and it is the leading cause of cancer-related deaths. Since lung cancer is identified in advanced stages of development, the main treatment is based in chemotherapy using platinum containing compounds, mainly cisplatin (CDDP). CDDP is able to crosslink with DNA leading to cell death, however many patients show tumor that are resistance to CDDP. This resistance is one of the main barriers for the success of lung cancer treatment by chemotherapy. To understand the mechanisms involved in CDDP resistance in lung cancer, we used CDDPsensitive (A549) and –resistant (A549/CDDP) cells to identify the newly synthesized proteins in response to drug exposure by BONCAT technique. It was possible the identification of 173 and 136 proteins regulated by CDDP in A549 and A549/CDDP cells, respectively. The identified proteins were related to several distinct molecular mechanisms potentially involved in CDDP response, including alternative splicing, response to oxidative stress, telomere maintenance, apoptosis regulation and cystoskeleton reorganization. Our results showed that A549/CDDP cells are less susceptible to DNA damage caused by CDDP than A549 cells. A549/CDDP also are able to increase the expression of proteins that combat the reactive oxygen species generated due to CDDP presence. In addition, CDDP induces different apoptotic pathways in drug-sensitive and resistant cell. In the A549 cells, CDDP induces the activation of the extrinsic pathway, while in A549/CDDP cells CDDP induces the intrinsic apoptotic pathway. So, our study was able to provide evidence of proteins and pathways that are differentially express and activated after CDDP treatment

Urinary endogenous peptides as biomarkers for prostate cancer

Prostate cancer (PCa) is one of the most prevalent types of cancer in men worldwide; however, the main diagnostic tests available for PCa have limitations and a biopsy is required for histopathological confirmation of the disease. Prostate specific antigen (PSA) is the main biomarker used for the early detection of PCa, but an elevated serum concentration is not cancer specific. Therefore, there is a need for the discovery of new non invasive biomarkers that can accurately diagnose PCa. The present study used trichloroacetic acid induced protein precipitation and liquid chromatography mass spectrometry to profile endogenous peptides in urine samples from patients with PCa (n=33), benign prostatic hyperplasia (n=25) and healthy individuals (n=28). Receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of urinary peptides. In addition, Proteasix tool was used for in silico prediction of protease cleavage sites. Five urinary peptides derived from uromodulin were revealed to be significantly altered between the study groups, all of which were less abundant in the PCa group. This peptide panel showed a high potential to discriminate between the study groups, resulting in area under the curve (AUC) values between 0.788 and 0.951. In addition, urinary peptides outperformed PSA in discriminating between malignant and benign prostate conditions (AUC=0.847), showing high sensitivity (81.82%) and specificity (88%). From in silico analyses, the proteases HTRA2, KLK3, KLK4, KLK14 and MMP25 were identified as potentially involved in the degradation of uromodulin peptides in the urine of patients with PCa. In conclusion, the present study allowed the identification of urinary peptides with potential for use as non invasive biomarkers in PCa diagnosis

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Caracterização funcional de isoformas de proteínas 14-3-3 de Echinococcus granulosus

O Echinococcus granulosus é um platelminto parasita da classe Cestoda (família Taeniidae), que, na sua fase larval (cisto hidático ou metacestódeo) causa a hidatidose cística. Esta doença é caracterizada pelo crescimento em longo prazo do cisto hidático em órgãos internos (principalmente pulmões e fígado) dos hospedeiros intermediários, que podem ser ungulados domésticos e, acidentalmente, seres humanos. O cisto hidático é uma estrutura unilocular delimitada por uma parede trilaminar (camadas germinativa, laminar e adventícia) que é preenchido pelo líquido hidático, no qual se encontram moléculas tanto do parasito quanto do hospedeiro e onde também se encontram os protoescólices (fase pré-adulta do parasito). As proteínas 14-3-3 fazem parte de uma família altamente conservada, com uma ou mais isoformas presentes em todos os eucariotos estudados até o momento. As 14-3-3 são proteínas acídicas e de massa molecular entre 28 e 33 kDa, que interagem com diversas proteínas-alvo e desempenham funções em diferentes processos celulares. Em parasitos, os papéis destas proteínas no contexto das relações com o hospedeiro ainda são pouco conhecidos. Em E. granulosus já foram identificados seis genes que codificam as potenciais isoformas de proteínas 14-3-3. Este estudo tem como objetivo a identificação do padrão de expressão das quatro isoformas cuja expressão já foi detectada em protoescólices (Eg14-3-3ε1, Eg14-3-3ε2, Eg14-3-3ζ2 e Eg14-3-3ζ3) e determinar o repertório de ligantes proteicos da isoforma Eg14-3-3ε2, buscando evidências sobre possíveis funções desta proteína em interações parasito-hospedeiro. Através de imunoblot foi possível a detecção destas quatro isoforma de Eg14-3-3 em protoescólices e camada germinativa, a única isoforma detectada em líquido hidático foi a Eg14-3-3ζ2. Foram identificadas 20 proteínas de interação com a isoforma Eg14-3-3ε2 através de ensaio de 2DE gel-overlay. As principais classes funcionais das proteínas de interação são: transporte e metabolismo de carboidratos, produção e conversão de energia e modificações pós-traducionais, turnover de proteínas e chaperonas. Entre as proteínas identificadas podemos citar a major vault protein, gliceraldeído-3-fosfato-desidrogenase, enolase e frutose-bifosfato-aldolase, Experimentos complementares com as quatro isoformas de Eg14-3-3 estão sendo realizados para a ampliação do repertório conhecido das potenciais proteínas de interação e para a confirmação dos resultados obtidos.Echinococcus granulosus is a parasitic flatworm of the class Cestoda (family Taeniidae), which in its larval stage (hydatid cyst or metacestode) causes cystic hydatid disease. This disease is characterized by long-term growth hydatid cyst in internal organs (mainly liver and lungs) of intermediate hosts, which may be domestic ungulates and accidentally humans. The hydatid cyst is a unilocular structure bounded by a trilaminar wall (germinal, laminar and adventitia layers) that is filled by the hydatid fluid, which contains molecules of the parasite as both the host and also where the protoscoleces (pre-adult parasite) are located. 14-3-3 proteins are part of a highly conserved family, with one or more isoforms present in all eukaryotes studied to date. 14-3-3 proteins are acidic and have a molecular mass between 28 and 33 kDa, which interact with different target proteins and perform functions in different cellular processes. In parasites, the roles of these proteins in the context of relations with the host are not yet known. In E. granulosus has been identified six genes responsible for potential isoforms of 14-3-3 proteins. This study aims to identify the expression pattern of the four isoforms of E. granulosus whose expression has been detected in protoscoleces (Eg14-3-3ε1, Eg14-3-3ε2, Eg14-3-3ζ2 e Eg14-3-3ζ3) and determine the repertoire of proteic ligands of Eg14-3-3ε2 isoform for evidence the possible functions of this protein in host-parasite interactions. By immunoblot was detected the presence of these four isoforms of Eg14-3-3 in protoscoleces and germinal layer, the only isoform detected in hydatid fluid was Eg14-3-3ζ2. We identified 20 proteins that interact with Eg14-3-3ε2 isoform by gel-overlay. The main functional classes of protein interaction are: carbohydrate transport and metabolism, energy production and conversion and posttranslational modification, protein turnover, chaperones. Among the proteins identified we can cite major vault protein, glyceraldehyde-3-phosphate dehydrogenase, enolase and fructose-bisphosphate aldolase. Additional experiments with the four Eg14-3-3 isoform are being performed for the expansion of known repertoire of potential interaction proteins and to confirm the results obtained

Análise proteômica de proteínas sintetizadas em resposta acisplatina em células de adenocarcinoma de pulmão humano resistentes e sensíveis a droga

O câncer de pulmão está entre os mais frequentes na população mundial e é o responsável pelo maior número de mortes relacionadas ao câncer. Como o câncer de pulmão é identificado em estágios avançados de desenvolvimento, o principal tratamento é baseado em quimioterapia utilizando moléculas derivadas de platina, principalmente a cisplatina (CDDP). A CDDP é capaz de formar ligações cruzadas no DNA resultando em morte celular, porém muitos pacientes apresentam tumores que são resistentes ao tratamento com CDDP. Esta resistência é uma das principais barreiras para o sucesso do tratamento do câncer de pulmão através de quimioterapia. Para entendermos melhor os mecanismos envolvidas na resistência a CDDP em células de câncer de pulmão, foram utilizadas células sensíveis (A549) e resistentes a CDDP (A549/CDDP) para a identificação de proteínas recém-sintetizadas em resposta ao tratamento com a droga através da técnica BONCAT. Através desta técnica foi possível a identificação de 173 e 136 proteínas reguladas por CDDP nas células A549 e A549/CDDP, respectivamente As proteínas identificadas estão relacionadas a diversos mecanismos moleculares distintos que potencialmente estão envolvidos na resposta a CDDP, como por exemplo splicing alternativo, resposta a estresse oxidativo, manutenção do telômero, regulação da apoptose e reorganização do citoesqueleto. Os resultados mostram que as células A549/CDDP são menos suscetíveis ao dano no DNA causado por CDDP do que as células sensíveis, A549. Além disso, as células A549/CDDP são capazes de aumentar a expressão de proteínas capazes de combater as espécies reativas de oxigênio geradas devido a presença de CDDP. Além disto, CDDP é capaz de induzir diferentes vias apoptóticas em células com diferentes sensibilidades à droga. Nas células A549, CDDP induz a ativação da via extrínseca enquanto que nas A549/CDDP ela é capaz de induzir a ativação da via intrínseca de apoptose. Portanto, nosso estudo foi capaz de fornecer evidencias de proteínas e vias que são diferencialmente expressas e ativadas após o tratamento com CDDP.Lung cancer is among the most frequent cancer in the world population and it is the leading cause of cancer-related deaths. Since lung cancer is identified in advanced stages of development, the main treatment is based in chemotherapy using platinum containing compounds, mainly cisplatin (CDDP). CDDP is able to crosslink with DNA leading to cell death, however many patients show tumor that are resistance to CDDP. This resistance is one of the main barriers for the success of lung cancer treatment by chemotherapy. To understand the mechanisms involved in CDDP resistance in lung cancer, we used CDDPsensitive (A549) and –resistant (A549/CDDP) cells to identify the newly synthesized proteins in response to drug exposure by BONCAT technique. It was possible the identification of 173 and 136 proteins regulated by CDDP in A549 and A549/CDDP cells, respectively. The identified proteins were related to several distinct molecular mechanisms potentially involved in CDDP response, including alternative splicing, response to oxidative stress, telomere maintenance, apoptosis regulation and cystoskeleton reorganization. Our results showed that A549/CDDP cells are less susceptible to DNA damage caused by CDDP than A549 cells. A549/CDDP also are able to increase the expression of proteins that combat the reactive oxygen species generated due to CDDP presence. In addition, CDDP induces different apoptotic pathways in drug-sensitive and resistant cell. In the A549 cells, CDDP induces the activation of the extrinsic pathway, while in A549/CDDP cells CDDP induces the intrinsic apoptotic pathway. So, our study was able to provide evidence of proteins and pathways that are differentially express and activated after CDDP treatment