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Se realizó una revisión sistemática para conocer las implicaciones éticas, legales y sociales de la creación de un biobanco de investigación desde la perspectiva de los principios de confidencialidad, dignidad y autonomía, con la finalidad de aplicarla a la creación de un biobanco de investigación en el Ecuador. Se incluyeron todos aquellos artículos conteniendo los términos (DECS/MESH): “biobancos de investigación” y “ética”, “dignidad”, “autonomía”, “confidencialidad” y “cuestiones éticas, legales y sociales”. Entre enero del 2021 y mayo del 2022, se hizo uso de los motores de búsqueda Pubmed, Science Direct, Scielo y Google académico; incluyendo los artículos redactados en inglés, portugués y español de los últimos diez años. Se analizaron 85 artículos con temas relevantes para los objetivos de la investigación. De estos, solo el 1% correspondió a América Latina, siendo Colombia, Brasil y México los países que más contribuyeron. A pesar de que no se ha llegado a un consenso a nivel mundial sobre las directrices éticas, legales y de gobernanza de los biobancos de investigación en seres humanos, el camino para la estandarización de dichos procesos se debe fundamentar en el entrenamiento de los investigadores, la capacitación continua de los pacientes y la creación de marcos comunes de gobernanza local e internacional. Se comprobó que la ética principialista, específicamente los principios de confidencialidad, dignidad y autonomía forman parte del marco ético, legal y social de los biobancos de investigación en seres humanos.

Escherichia coli is a major player in foodborne illnesses, capable of forming biofilms on dairy facilities, leading to milk contamination. Thus, we examined the capacity of E. coli strains from raw milk bulk tanks to form biofilms on surfaces made of polystyrene, stainless steel, and silicone; the potential links between biofilm formation with genes responsible for fimbriae and virulence factors of extra-intestinal E. coli (ExPEC); and the susceptibility of biofilm-forming isolates to iodine and chlorhexidine digluconate. Out of 149 E. coli strains, 42.28% (63/149) formed biofilm on polystyrene, 56.38% (84/149) on silicone, and 21.48% (32/149) on stainless steel. The frequency of genes was: fimH (100%), hlyA (5.4%), irp2 (2.7%), sitA (10.7%), ompT (43.6%), and traT (98%). No biofilm developed when disinfectants were used prior to biofilm formation. However, iodine and chlorhexidine digluconate allowed 25.40% (16/63) of isolates displaying growth after a mature biofilm was formed. The presence of biofilm on different surfaces emphasizes the vital need for thorough equipment cleaning, both in farms and in industrial dairy settings. Rapid disinfection is crucial, given the reduced susceptibility of potentially pathogenic E. coli after biofilm maturity.

Background: Toxoplasmosis has been of public health concern due to direct associations with socioeconomic vulnerability and inadequate living conditions. Methods: Accordingly, the present study aimed to assess antibodies against T. gondii, historical reported toxoplasmosis cases and associated socio-environmental risk factors in Pinhais, a full urban area of Curitiba, currently the eighth biggest metropolitan area of Brazil. Anti-Toxoplasma gondii antibodies were assessed by an indirect immunofluorescence reaction (RIFI). Owner and dog samples were also tested by IFAT to anti-Leishmania spp. and anti-Trypanosoma cruzi antibodies. Results: Overall, 20/135 (14.8%) persons and 13/133 (9.8%) dogs from 25 different households were considered seropositive to T. gondii. All samples were seronegative to Leishmania spp. and Trypanosoma cruzi. Conclusions: Although no significant covariates were found in the regression model, statistically associated risk factors in the bivariate analysis included no public water use (p = 0.016) and drinking raw milk (p = 0.041) for owners, and obesity (p = 0.028) and tick infestation (p = 0.03) for dogs. In addition, a spatial cluster of T. gondii seropositivity for both owners and their dogs overlapped the location of historic reported cases of human acquired, gestational and congenital toxoplasmosis. Finally, the results herein showed tick infestation as an indicator of socio-environmental risk for T. gondii exposure in the household environment, and dogs may be used as sentinels for human toxoplasmosis cases.

O presente trabalho se refere à resenha do podcast intitulado “Nos Armários dos Vestiários”, produzido em nove episódios pelo Feel The Match da Rede Globo de Televisão. O objetivo deste texto foi descrever e analisar criticamente esse documentário à luz dos estudos clássicos e contemporâneos de gênero e sexualidade. Foi possível identificar a potência desse tipo de recurso comunicativo e audiovisual no sentido de problematizar e visibilizar os processos de homofobia especialmente no campo esportivo/futebolístico.

Em 2011, a população paraense decidiu, em plebiscito, sobre a configuração político-territorial de um Estado. A criação de novos estados voltou a ecoar na cobertura jornalística das eleições em 2014, como posição midiática de forma a contribuir para a escolha eleitoral do público. Neste trabalho analisamos a cobertura jornalística do jornal O Liberal durante o período de campanhas ao governo estadual em 2014, no que tange à reverberação de discursos sobre o plebiscito realizado em 2011. Utilizamos como metodologia a análise crítica do discurso (ACD) aos moldes do modelo teórico-metodológico proposto por van Dijk (2005). O debate foi reduzido à disputa de grupos políticos já sacralizados na região contra a ascensão de outros que buscam o seu quinhão de poder, tendo como consequências o apagamento dos anseios históricos de autonomia da população da região do Carajás e do Tapajós. O papel informativo do jornalismo utilizou-se de estratégias discursivas que, na disputa comunicacional, buscaram legitimar estruturas de poder hegemônicas.

ABSTRACT Background: Leptospirosis represents a One Health issue, affecting humans and animals. This study investigated pathogenic leptospires in small wild rodents in São Paulo, Brazil. Methods: Kidney samples from 164 rodents underwent qPCR testing, targeting pathogenic Leptospira spp. Results: Thirty-five animals (21.34%) tested positive, including five rodent species: Akodon montensis (2/21; 9.5%), Necromys lasiurus (1/4; 25%), Oligoryzomys nigripes (24/92; 26.1%), Oligoryzomys flavescens (5/26; 19.2%), and Sooretamys angouya (3/14; 21.4%). Botucatu municipality exhibited the highest prevalence, with 42.5% (20/47) of the animals testing positive. Conclusions: The presence of Leptospira spp. in wild rodents suggests they may be chronic carriers, contaminating the environment.

Rationale and Objective: Latinx individuals are at a higher risk for kidney failure than non-Latinx White individuals; however, they are less likely to receive pre-kidney failure medical care. The objective of this study was to determine the feasibility and acceptability of a community health worker (CHW) intervention that facilitated access to medical care for Latinx individuals. Study Design: Single-arm prospective study. Setting and Participants: Latinx adults were found to have albuminuria or risk factors for kidney disease at community screening events in Austin, Texas. Intervention: A 6-month CHW intervention that facilitated the following: (1) obtaining medical insurance; (2) medical care coordination with primary and nephrology care; (3) kidney disease education; and (4) connection with local resources to address health-related social needs. Outcomes: Recruitment, retention, medical care linkage, and participant and CHW-reported satisfaction with the intervention. Results: Of the 173 individuals who attended the 2 community screening events, 49 agreed to participate in the study, of whom, 51% were men with a mean ± standard deviation (SD) age of 45 ± 14 years, and all self-identified as Mexican or Chicano. The mean ± SD estimated glomerular filtration rate (eGFR) was 110 ± 21 mL/min/1.73 m2 and 41% of the participants reported a urine albumin-creatinine ratio of ≥30 mg/g. Among those enrolled, 28 of the 49 (57%) completed at least 1 CHW visit, and 20 of 49 (41%) completed the intervention. 7 individuals who needed assistance with insurance obtained insurance, and 15 of 20 (75%) scheduled an appointment with a primary care physician within 180 days. Participants reported that the US health care previously seemed inaccessible but gained insurance, the ability to navigate the system, and the ability to help others in their community to access medical care because of the program. Limitations: Small sample size and a single community may limit generalizability. Conclusions: We reported the acceptability of a CHW intervention. We encountered challenges with feasibility and identified strategies to overcome them. Studies are needed to test the effect of CHW interventions on outcomes and kidney health disparities. Funding: National Kidney Foundation young investigator research grant to Dr Novick. Plain Language Summary: Latinx individuals are at a higher risk for kidney failure than non-Latinx White individuals; however, they are less likely to receive pre-kidney failure medical care. We piloted a community health worker intervention that connected people with risk factors or showed evidence of kidney dysfunction at community screening events with medical care. Our findings indicate the acceptability of the intervention. We encountered challenges with feasibility and identified strategies to overcome them.

Evaluation approaches that aim to support large-scale social change need to address neoliberal logic ingrained in the way evaluation has been institutionalized in the US since the early 1900s. Harmful dynamics resulting from evaluation’s institutional history include (1) a focus on accountability and effectiveness, (2) the perpetuation of deficit-based narratives about communities of color, and (3) a top-down approach to program development, in which funders define program goals and assessment criteria and outside academics are hired to provide research services. In consequence, evaluation contributes to the extraction and devaluation of community expertise rather than fostering learning, collaboration, critical reflection, and healing. This article highlights ways of addressing these harmful dynamics through a case study that exemplifies an innovative evaluation approach focused on community strengths and values, healing ethno-racial trauma, and critical consciousness building. We call for funders to rethink their requirements for evaluation and emphasize the need to support evaluation infrastructure, time for critical reflection, and the development of community- and asset-based, culturally responsive evaluation approaches and tools.

Abstract Background Individuals with hoarding disorder (HD) presented a persistent difficulty in detaching from objects and/or animals. Unhealthy conditions, frequently found in cases of animal HD (AHD), may favour environmental contamination and the spread of zoonotic pathogens. Despite that, only one study of zoonotic diseases in individuals with AHD and their companion animals has been conducted to date. Objectives This study aimed to assess the seroprevalence of anti‐Leptospira spp. antibodies in individuals with AHD and their dogs in a major city of Southern Brazil. Methods Blood samples were obtained from 264 dogs (21 households) and 19 individuals with AHD (11 households) and tested by microscopic agglutination test. Results All human samples were seronegative. Seropositivity was found in 16/264 (6.1%; CI 95% 3.3–9.6%) dogs from 11/21 (52.38%) households, with titres ranging from 100 up to 400, and Copenhageni (10/16; 62.5%) was the most frequent serovar. Surprisingly, seropositivity of hoarded dogs found herein was among the lowest reportedly observed in other dog populations of Brazil. Two epidemiological variables were significantly associated with seropositivity in dogs: the presence of cat hoarding (p = 0.004) and the report of flood occurrence in the household (p = 0.031). Conclusions No individuals with AHD were seropositive, and besides the lower seroprevalence of dogs, they probably had contact with Leptospira spp. at some point in their life. Since dogs can be considered potential sentinels in leptospirosis, public health programs must become aware of the risk of leptospirosis cases in households of individuals with AHD and nearby communities.

Although leptospirosis has been described as a worldwide bacterial zoonosis primarily affecting vulnerable populations, to date no study has focused on concomitant serosurvey of homeless persons and their dogs. The aim of the present study was, to use a One Health approach to serologically assess homeless persons and their dogs in 3 major cities of south Brazil (São Paulo, Curitiba, and Foz do Iguaçu). Environmental information was obtained with an epidemiological questionnaire given to all participants. A total of 200 human and 75 dog samples were tested for anti-Leptospira spp. antibodies to thirty different serovars using the microscopic agglutination test. None of the homeless persons were positive while 5 of the 75 (6.7%) dogs were positive. Among homeless population, 89% (177 of 200) were male, 61% (122 of 200) self-declared Non-white, and 67% (134 of 200) were educated up to the 8th school grade. Lower exposure of homeless persons to Leptospira spp. in the present study when compared to other vulnerable populations (slum and low-income residents) may be result of less direct exposure as they are able to rapidly change locations in response to flooding events. In addition, these results may reflect the effectiveness of a specific healthcare service provided to people living in the streets in the 3 cities. While dogs may be used as environmental sentinels for leptospirosis, the low seropositivity results found in this report may indicate low transmission risk to homeless owners in direct daily contact with their dogs.

Leptospirosis has been a neglected, widespread and reemerging zoonotic disease of global importance. The One Health holistic approach combining human, animal and environmental health has been important for control and prevention of zoonotic disease. An urban municipality in Southern Brazil with a high prevalence of leptospirosis was selected by convenience, with asymptomatic human and canine individuals serologically tested using MAT against 30 Leptospira spp. serovars. Epidemiological questionnaires were assessed along with the historical national database, with associated risk factors to dog and human seropositivity analyzed using multiple logistic regression. The spatial distribution of retrospective human leptospirosis cases was analyzed using a Kernel map and overlapped dog seropositivity and historical flooding areas, demonstrating that domestic and asymptomatic dogs may be important environmental sentinels for leptospirosis in such urban areas, even in the absence of human seropositivity and low canine seropositivity. Associated risk factors for leptospirosis in dogs and humans according to multiple regression included healthy dogs (p = 0.02, odds ratio—OR = 0.17, confidence interval of 95%CI 0.03–0.71) with indoor access (p = 0.059, OR = 5.12, 95%CI 1.10–37.7) and human males (p = 0.042, OR = 2.44, 95%CI 1.08–6.11) with presenting calf pain (p = 0.005, OR = 3.14, 95%CI 1.46–7. 14), jaundice (p < 0.001, OR = 4.61, 95%CI 2.11–10.10) and renal failure (p = 0.008, OR = 4.49, 95%CI 1.49–13.76). The greater the rain precipitation (above 3 mm of average annual precipitation), the higher the number of leptospirosis cases in humans. In conclusion, dog active serosurvey and rain precipitation should be systematically reported and plotted altogether as a basis for the early detection and monitoring of human risk areas. Such findings may serve as a basis for public health policies in Brazil and other endemic countries worldwide.

Abstract Background Data collection consumes a large proportion of clinical trial resources. Each data item requires time and effort for collection, processing and quality control procedures. In general, more data equals a heavier burden for trial staff and participants. It is also likely to increase costs. Knowing the types of data being collected, and in what proportion, will be helpful to ensure that limited trial resources and participant goodwill are used wisely. Aim The aim of this study is to categorise the types of data collected across a broad range of trials and assess what proportion of collected data each category represents. Methods We developed a standard operating procedure to categorise data into primary outcome, secondary outcome and 15 other categories. We categorised all variables collected on trial data collection forms from 18, mainly publicly funded, randomised superiority trials, including trials of an investigational medicinal product and complex interventions. Categorisation was done independently in pairs: one person having in-depth knowledge of the trial, the other independent of the trial. Disagreement was resolved through reference to the trial protocol and discussion, with the project team being consulted if necessary. Key results Primary outcome data accounted for 5.0% (median)/11.2% (mean) of all data items collected. Secondary outcomes accounted for 39.9% (median)/42.5% (mean) of all data items. Non-outcome data such as participant identifiers and demographic data represented 32.4% (median)/36.5% (mean) of all data items collected. Conclusion A small proportion of the data collected in our sample of 18 trials was related to the primary outcome. Secondary outcomes accounted for eight times the volume of data as the primary outcome. A substantial amount of data collection is not related to trial outcomes. Trialists should work to make sure that the data they collect are only those essential to support the health and treatment decisions of those whom the trial is designed to inform.

Resumo: Introdução: Da população brasileira, 5% vivem com algum nível de deficiência auditiva. Apesar desse número, poucas pessoas têm acesso à educação superior. A legislação nacional defende a integração e, principalmente, a inclusão dessas pessoas no ambiente acadêmico brasileiro. Poucos são os dados sobre o acesso dos deficientes auditivos ao curso de Medicina no Brasil. Relato de experiência: Este artigo apresenta relatos de experiências de uma estudante com deficiência auditiva que cursou a disciplina de Introdução à Semiologia Médica e de seus professores e da estudante-monitora que acompanhou a disciplina. Para discutir os temas, foram selecionados 12 artigos das bases de dados MEDLINE, SciELO e LILACS, publicados entre julho de 2019 e outubro de 2021. Também se utilizaram informações publicadas pelo IBGE e Portal MEC. Discussão: A estudante relata sua dificuldade em compreender termos semiológicos, bem como suas alternativas para adquirir habilidades referentes ao exame físico. O relato do professor aborda como a inclusão é possível porém desafiadora ao se ensinar semiologia médica a uma pessoa com deficiência auditiva. Conclusão: Estudantes deficientes auditivos se beneficiam de adequação acadêmica visando à integração e inclusão para obter êxito em adquirir e treinar suas habilidades semiotécnicas. Há urgente necessidade de novas pesquisas brasileiras nessa área, principalmente relacionadas à medicina e ao ensino médico.

Leptospirosis has been described as a disease neglected worldwide. Affecting humans and animals, the disease is often related to poor environmental conditions such as lack of sanitation and presence of synanthropic rodents. Despite being considered as a One Health issue, no study has focused on comparing owner–dog seroprevalence between islands and seashore mainland. Accordingly, the present study assessed anti-Leptospira spp. antibodies by applying microscopic agglutination test (MAT) methods to Leptospira and assessing associated risk factors via univariate and multivariate logistic regression analysis of owners and their dogs in islands and seashore mainland of southern Brazil. No anti-Leptospira spp. Seropositivity was found in 330 owner serum samples, while dogs presented an overall seroprevalence of 5.9%. All seropositive dogs reacted to serogroups of Leptospira interrogans, including 66.7% of Pyrogenes, 44.4% Canicola, 22.2% Icterohaemorrhagiae, 16.7% Australis; six reacted to more than one serogroup. No association was found among seropositivity and epidemiological variables, except that neighborhood dogs were less likely to be seropositive. Although no seropositivity was observed in owners, seropositivity in dogs had the potential to indicate such species as being sentinels for environmental exposure and potential human risk of infection.

Despite being an important public health issue, particularly due to rabies, dog bites and associated risk factors have rarely been assessed by health services from a One Health perspective. Accordingly, the present study aimed to assess dog biting and associated demographic and socioeconomic risk factors in Curitiba, the eighth-largest Brazilian city with approximately 1.87 million people, based on the post-exposure prophylaxis (PEP) rabies reports between January/2010 and December/2015. The total of 45,392 PEP reports corresponded to an average annual incidence of 4.17/1000 habitants, mainly affecting white (79.9%, 4.38/1000 population), males (53.1%, 4.81/1000 population), and children aged 0–9 years (20.1%, 6.9/1000 population), with severe accidents associated with older victims (p < 0.001) and mainly caused by dogs known to the victims. An increase of USD 100.00 in the median neighborhood income was associated with a 4.9% (95% CI: 3.8–6.1; p < 0.001) reduction in dog bites. In summary, dog biting occurrence was associated with victims’ low income, gender, race/color, and age; severe accidents were associated with elderly victims. As dog bites have been described as multifactorial events involving human, animal, and environmental factors, the characteristics presented herein should be used as a basis to define mitigation, control, and prevention strategies from a One Health perspective.

Euphorbia hirta L. is a medicinal plant widely used in the Philippines and across tropical Asia against various diseases, including respiratory disorders. In this study, the phytochemical components of E. hirta were investigated in silico for their potential to inhibit the severe acute respiratory syndrome-coronavirus-2 main protease (SARS-CoV-2 Mpro), a coronavirus disease 2019 (COVID-19) drug target that plays a critical role in the infection process of SARS-CoV-2. Phytochemical mining in tandem with virtual screening (PM-VS) was the strategy implemented in this study, which allows efficient preliminary in silico assessment of the COVID-19 therapeutic potential of the reported phytochemicals from the plant. The main rationale for considering E. hirta in the investigation was its reported efficacy against respiratory disorders. It is very promising to investigate the phytochemicals of E. hirta for their potential efficacy against diseases, such as COVID-19, that also target the respiratory system. A total of 298 E. hirta phytochemicals were comprehensively collected from the scientific literature. One hundred seventy of these phytochemicals were computed through molecular docking and were shown to have comparable or better binding properties (promising inhibitors) toward SARS-CoV-2 Mpro than known in vitro inhibitors. In connection to our previous work considering different medicinal plants, antiviral compounds were also rediscovered from the phytochemical composition of E. hirta. This finding provides additional basis for the potential of the plant (or its phytochemicals) as a COVID-19 therapeutic directly targeting drug targets such as SARS-CoV-2 Mpro and/or addressing respiratory-system-related symptoms. The study also highlights the utility of PM-VS, which can be efficiently implemented in the preliminary steps of drug discovery and development.

Abstract Background The Mucopolysaccharidosis type VI (MPS VI), also known as Maroteaux-Lamy syndrome (OMIM 253200) is an autosomal recessive lysosomal disorder, caused by the deficiency of the enzyme N-acetylgalactosamine 4-sulfatase (also known as arylsulfatase B) due to mutations of the ARSB gene. Cardiologic features are well recognized, and are always present in MPS VI patients. Generally, the onset and the progression of the cardiologic symptoms are insidious, and just a few patients have developed a rapidly progressive disease. Cardiac involvement in MPS VI is a common and progressive feature. For MPS patients, cardiac evaluations are recommended every 1 to 2 years, including blood pressure measurement, electrocardiography and echocardiography. However, congestive heart failure and valvular surgical repair are not frequently seen, and if so, they are performed in adults. Here we report on an atypical MPS VI case with ascites fetalis and a rapidly progressive cardiac disease. Case presentation A 6-month-old Brazilian male, only child of a Brazilian healthy non-consanguineous couple. During pregnancy, second trimester ultrasonography observed fetal ascites and bilateral hydrocele. Physical exam at 6 months-old revealed a typical gibbus deformity and MPS was suspected. Biochemical investigation revealed a diagnosis of MPS type VI, confirmed by molecular test. Baseline echocardiogram revealed discrete tricuspid regurgitation and a thickened mitral valve with posterior leaflet prolapse, causing moderate to severe regurgitation. The patient evolved with mitral insufficiency and congestive heart failure, eventually requiring surgical repair by the first year of age. Conclusions We report the first case of MPS VI whose manifestations started in the prenatal period with fetal ascites, with severe cardiac valvular disease that eventually required early surgical repair. Moreover, in MPS with neonatal presentation, including fetal hydrops, besides MPS I, IVA and VII, clinicians should include MPS VI in the differential diagnosis.

Due to the characteristic of narrow band conversion around a central radio frequency, the Sigma Delta Modulator (ΣΔM) based on LC resonators is a suitable option for use in Software-Defined Radio (SDR). However, some aspects of the topologies described in the state-of-the-art, such as noise and nonlinear sources, affect the performance of ΣΔM. This paper presents the design methodology of three high-order LC-Based single-block Sigma Delta Modulators. The method is based on the equivalence between continuous time and discrete time loop gain using a Finite Impulse Response Digital-to-Analog Converter (FIRDAC) through a numerical approach to defining the coefficients. The continuous bandpass LC ΣΔM simulations are performed at a center frequency of 432 MHz and a sampling frequency of 1.72 GHz. To the proposed modulators a maximum Signal-to-Noise Ratio (SNR) of 51.39 dB, 48.48 dB, and 46.50 dB in a 4 MHz bandwidth was achieved to respectively 4th Order Gm-LC ΣΔM, 4th Order Magnetically Coupled ΣΔM and 4th Order Capacitively Coupled ΣΔM.

Although toxoplasmosis has been considered among the most neglected zoonoses worldwide, no study has focused on the frequency and associated risk factors of owners and their dogs living on an island and mainland seashore areas. Accordingly, anti-Toxoplasma gondii IgG antibodies were screened by indirect fluorescent antibody test (IFAT) in owners and dogs from three oceanic islands and two nearby mainland harbor areas, with associated risk factors for toxoplasmosis assessed by univariable and multivariable logistic regression models. Overall, anti-T. gondii seropositivity was observed in 59/328 (18.0%) owners and 66/283 (23.3%) dogs, with no statistical difference between owners (p = 0.360) and dogs (p = 0.655) from islands and mainland areas. Consumption of local water springs (p = 0.016; OR = 2.11) was an associated risk factor for T. gondii seropositivity, and owners with the habit of spring water intake were twice more likely seropositive (p = 0.014; OR = 2.14). Presence of anti-T. gondii antibodies in dogs was associated with seropositive owners (p = 0.008; OR = 2.81), household consumption of beef meat (p = 0.042; OR = 1.7) and chicken (p = 0.026; OR = 2.9). Despite being lower than the worldwide prevalence, toxoplasmosis seropositivity in owners and their dogs in southern Brazil was influenced by the positive owner, water source, and meat consumption, and not by inhabiting islands or seashore mainland areas, presence of dogs, cats, or both. In addition, drinking water quality should always be considered a critical risk factor for toxoplasmosis on islands.

Este artigo analisa os discursos contra a gordofobia veiculados em três vídeos brasileiros publicados no YouTube em 2018 com grande visualização. O objetivo éexplorar como plataformas digitais tais como YouTube podem dar visibilidade a discursos contra-hegemônicos acerca dos corpos gordos, colaborando na disseminação de informações e estratégias de mobilização e combate às formas de discriminação e preconceito. Os vídeos foram submetidos à análise temática e discutidos à luz de uma literatura crítica sobre a produção da identidade e diferença.A análise identificatrês temas principais: “gordura como falha física e moral”, “normatização do corpo e discriminação contra gordos” e “afirmação da identidade gorda, autoestima e autoaceitação”. As situações apresentadas nos vídeos criticam os processos rotineiros de exclusão dos indivíduos gordos. Concluímos que os vídeos analisados, mediante paródia, ironia e uso de câmera oculta, ajudam a circular narrativas alternativas que suspendem certas verdades naturalizadas sobre os gordos e seus corpos.

Epidemiologic studies have shown that the prevalence of stress-related mood disorders is higher in women, which suggests a different response of neuroendocrine circuits involved in the response to stressful events, as well as a genetic background influence. The aim of this study was to investigate the baseline differences in anxiety-like behaviors of females of two commonly used mice strains. Secondly, we have also aimed to study their behavioral and biochemical alterations following stress. Naïve 3-4 months-old Swiss and C57BL/6 female mice were evaluated in the elevated plus maze (EPM) and in the acoustic startle response (ASR) for anxiety-like behaviors. Besides, an independent group of animals from each strain was exposed to cold-restraint stress (30 min/4 °C, daily) for 21 consecutive days and then evaluated in EPM and in the sucrose consumption tests. Twenty-four hours following behavioral experimentation mice were decapitated and their hippocampi (HP) and cortex (CT) dissected for further Western blotting analysis of glucocorticoid receptor (GR) and glial fibrillary acid protein (GFAP). Subsequent to each behavioral protocol, animal blood samples were collected for further plasma corticosterone analysis. C57BL/6 presented a lower anxiety profile than Swiss female mice in both behavioral tests, EPM and ASR. These phenomena could be correlated with the fact that both strains have distinct corticosterone levels and GR expression in the HP at the baseline level. Moreover, C57BL/6 female mice were more vulnerable to the stress protocol, which was able to induce an anhedonic state characterized by lower preference for a sucrose solution. Behavioral anhedonic-like alterations in these animals coincide with reduced plasma corticosterone accompanied with increased GR and GFAP levels, both in the HP. Our data suggest that in C57BL/6 female mice a dysregulation of the hypothalamus-pituitary-adrenal axis (HPA-axis) occurs, in which corticosterone acting on GRs would possibly exert its pro-inflammatory role, ultimately leading to astrocyte activation in response to stress. Keywords: Stress-related disorders, Female mice, Anxiety-like behavior, Depression-like behavior, Neuroinflammation, Anhedonia

Este trabalho avaliou a utilização da casca de abacaxi (Ananas comosus) como adsorvente para remoção do corante catiônico azul de metileno presente em solução aquosa. O adsorvente foi obtido através da secagem e trituração das cascas da fruta. A granulometria do material foi < 0.2 mm. Os ensaios de adsorção foram realizados em batelada, sob rotação de 150 rpm e à temperatura de 30 e 40°C. O efeito da quantidade de adsorvente (0,1g-0,5g) e concentração inicial de adsorbato (60ppm-100ppm) no percentual de remoção (%R) e na capacidade de adsorção unitária (qe) foram avaliados. Os modelos de isoterma de Langmuir e Freundlich foram utilizados na descrição matemática do sistema. A casca de abacaxi apresentou remoção de até 81 % do corante azul de metileno. O aumento da massa de adsorvente proporcionou um aumento na percentagem de remoção do corante e diminuição da capacidade de adsorção unitária (qe). O aumento da concentração inicial do adsorbato em solução aquosa provocou uma redução na percentagem de remoção e um aumento na capacidade de adsorção unitária (qe). O modelo de isoterma de Freundlich foi o que melhor se ajustou aos dados experimentais. O ajuste do modelo de Langmuir aos dados experimentais revelou a capacidade de adsorção unitária máxima para a casca de abacaxi como sendo de 17,12 mgg-1 e 10,06 mgg-1 à 30C e 40°C, respectivamente. O caráter da reação de adsorção foi exotérmico pois a elevação da temperatura desfavoreceu a remoção do corante. Os resultados apontam que esse resíduo agroindustrial tem potencial para ser utilizado como um adsorvente econômico para a remoção de azul de metileno.

On December 11, 2011, a plebiscite was held in Pará proposing to create two separatist states, Carajás and Tapajós, out of the state of Pará. The public voted against both projects. This article analyzes the narrative identities found on the front pages of O Liberal and Diário do Pará newspapers about the plebiscite in Pará. Depth Hermeneutics (DH), as proposed by Thompson (2011), was used as the methodological reference. DH emphasizes the object of analysis as a meaningful symbolic construction requiring interpretation. Narrative analysis, as proposed by Motta (2007), was the main research technique used to highlight the movement of construction of journalistic (discursive) characters. The narrative identities built by the two newspapers showed that both were against the creation of the new states, but for very different reasons. The flag of Pará was the main object used to induce the idea of unity. Em 11 de dezembro de 2011, o plebiscito no Pará propôs a criação dos estados de Carajás e de Tapajós a partir da divisão do estado do Pará. O resultado da consulta pública foi negativo aos dois projetos. O artigo analisa as identidades narrativas constituídas pelas primeiras páginas dos jornais O Liberal e Diário do Pará sobre o plebiscito no Pará. Utilizou-se como referencial metodológico a Hermenêutica em Profundidade (HP), proposta por Thompson (2011). A HP evidencia o fato de que o objeto de análise é uma construção simbólica significativa, que exige uma interpretação. Como principal técnica de pesquisa, a análise narrativa, proposta por Motta (2007), com ênfase no movimento de construção de personagens jornalísticas (discursivas). As identidades narrativas construídas pelos dois jornais indicaram que ambos eram contrários à criação dos novos estados, porém por motivos divergentes. A bandeira do Pará foi o principal elemento utilizado para evocar a ideia de unidade. En 11 de diciembre de 2011, el plebiscito en Pará propuso la creación de los estados Carajás y Tapajós a partir de la división del estado Pará. El resultado de la consulta pública fue negativo para los dos proyectos. El artículo analiza las identidades narrativas constituidas por las primeras páginas de los periódicos O Liberal y Diario de Pará acerca del plebiscito en Pará. Fue utilizado como referencia metodológica la Hermenéutica en Profundidad (HP), propuesto por Thompson (2011). La HP pone de relieve el hecho de que el objeto de análisis es una construcción simbólica significativa, lo que requiere una interpretación. Como principal técnica de la investigación, el análisis narrativo, propuesto por Motta (2007), con énfasis en el movimiento de construcción de personajes periodísticos (discursivos). Las identidades narrativas construidas por los dos periódicos han indicado que ambos se opusieron a la creación de los nuevos estados, pero por diferentes razones. La bandera de Pará fue el elemento principal que se utilizó para evocar la idea de la unidad.

O estudo observou como se engendraram os discursos sobre a extensão territorial nas campanhas televisivas das frentes pró e contra a criação dos estados do Carajás e do Tapajós no plebiscito de 2011, no Pará. O Horário Gratuito de Propaganda Eleitoral ocorreu de 11 de novembro a 7 de dezembro daquele ano. Ao todo, 80 programas foram produzidos, veiculados entre 12h e 12h10 e entre 19h30 e 19h40. Neste estudo, trabalhamos com 20 programas, vistos a partir da análise do discurso na vertente dialógica de Mikhail Bakhtin e da disputa por legitimação e descredenciamento de discursos, de Eliseo Verón. A análise mostrou que as frentes de campanhas atualizaram discursos históricos sobre o reordenamento de territórios na Amazônia, vozes que debatem a unidade territorial e a reestruturação da região para o desenvolvimento.

Despite vulnerability and unsanitary conditions of animal hoarding may predispose environmental contamination and spread of vectors and pathogens, no study to date has focused on their impact on public health and zoonotic diseases. Accordingly, this study aimed to assess the seroprevalence of anti-Toxoplasma gondii antibodies and associated factors in individuals with animal hoarding disorder (AHD) and their dogs in Curitiba, Southern Brazil. Blood samples were obtained from 264 dogs (21 households) and 19 individuals with AHD (11 households). Their blood was tested by indirect fluorescent antibody test (IFAT). Overall, anti-Toxoplasma gondii seropositivity was found in 21/264 dogs (7.95%; 95% CI: 4.69-11.22) with titers ranging from 16 to 4096, and in 7/19 individuals with AHD (36.84%; CI: 15.15-58.53) with titers ranging from 16 to 64. Serological analysis for anti-T. gondii antibodies were considered positive in at least one individual or dog in 9/11 (81.82%; 95% CI: 59.03-100.00) cases that were thoroughly assessed. Surprisingly, the seropositivity of individuals with AHD and their dogs was among the lowest reportedly observed in human and dog populations of Brazil. There was no significant association between positive owners and positive dogs or the presence of cats in the household. Regard epidemiological variables, a significant association was found between dog's seropositivity and the type of dog food. To the authors' knowledge, the present study represents the first investigation of T. gondii seroprevalence in individuals with hoarding disorder and their dogs. In conclusion, despite low sanitary conditions, anti-Toxoplasma gondii antibodies frequency in individuals with AHD and their dogs are lower than the general population likely due to low protozoan load in such isolated households.

O estado do Pará viveu, em 2011, um evento inédito na história do país, quando, no mês de dezembro, a população paraense foi consultada para se manifestar em um plebiscito sobre a criação de dois novos estados no seu território, Carajás e Tapajós. Na presente proposta, refletimos sobre como as campanhas televisivas do plebiscito para criação desses dois novos estados constituíram-se em estratégias enunciativas com a finalidade de defender as propostas das frentes contrárias à criação do Carajás e do Tapajós e das frentes favoráveis ao surgimento deles. Na análise, foram selecionados os programas televisivos do horário gratuito de propaganda eleitoral a partir da perspectiva discursiva/teórica bakhtiniana e do pensamento do Círculo, considerando que vozes diferentes entre si estabelecem relações dialógicas. O discurso é tomado como um fenômeno social de inscrição valorativa do sujeito e de sua posição frente a outros discursos impregnados de intenções ideológicas, de caráter político-partidário. A análise mostrou que as frentes contrárias à criação dos estados trabalharam com estratégias enunciativas que as colocaram como protetoras da integridade regional e dos valores multiculturais contra as investidas de interesses externos. Já as frentes favoráveis aos estados autonomearam-se defensoras do desenvolvimento do Pará, contra a estagnação econômica do estado.

ABSTRACT The objective of the current study was to evaluate the effects of three calcium:available phosphorus (Ca:aP) ratios and different supplementation with phytases from different origins on performance and ash, Ca, and P contents in the tibia of broilers. A total of 900 male broilers (22 to 35 days old) were fed ten dietary treatments in a completely randomized design. A 3 × 3 + 1 factorial scheme was used, corresponding to three Ca:aP ratios (4.5:1.0, 6.0:1.0, and 7.5:1.0) and three different supplementations with phytases from different origins (isolated or combined supplementation with bacterial and fungal phytase), plus a control diet. Regardless of the dietary Ca:aP ratio, the isolated use of bacterial phytase provided better feed conversion than the fungal phytase but did not differ from combined supplementation with bacterial and fungal phytase. However, regardless of the supplemented phytase, the 7.5:1.0 Ca:aP ratio decreased the feed conversion. Best results for bone P deposition were observed using diets containing the 4.5:1.0 Ca:aP and fungal phytase or the 4.5:1 and 6.0:1 Ca:aP ratios using the bacterial phytase. In general, when the parameters of feed conversion, bone ash, and P content in tibia are considered together, diets containing a 4.5:1.0 or 6.0:1.0 Ca:aP ratio and 1,500 FTU kg−1 bacterial phytase, or a 4.5:1.0 Ca:aP ratio using fungal phytase and only 1.0 g kg−1 available phosphorus provide better results.

Alzheimer's and Parkinson's disease are late onset neurodegenerative diseases that will require therapy over decades to mitigate the effects of disease-driving proteins such tau and α-synuclein (α-Syn). Previously we found that TRIM28 regulates the levels and toxicity of α-Syn and tau (Rousseaux et al., 2016). However, it was not clear how TRIM28 regulates α-Syn and it was not known if its chronic inhibition later in life was safe. Here, we show that TRIM28 may regulate α-Syn and tau levels via SUMOylation, and that genetic suppression of Trim28 in adult mice is compatible with life. We were surprised to see that mice lacking Trim28 in adulthood do not exhibit behavioral or pathological phenotypes, and importantly, adult reduction of TRIM28 results in a decrease of α-Syn and tau levels. These results suggest that deleterious effects from TRIM28 depletion are limited to development and that its inhibition adulthood provides a potential path for modulating α-Syn and tau levels.

O projeto Mini-hospital Veterinário teve por objetivo ensinar às crianças, por meio de brincadeiras e bichos de pelúcia, o respeito aos animais, a prevenção de doenças e conceitos de bem-estar animal. As atividades foram realizadas principalmente em escolas, públicas ou privadas. Em grupos, as crianças passaram pelos módulos do Mini-hospital, para ajudar os "pacientes" e criar soluções para os problemas apresentados. No ano de 2015, foram feitas 19 apresentações, com a participação de 840 crianças. As crianças demonstraram grande interesse e empolgação durante as atividades, e o projeto foi elogiado por suas professoras. Palavras-chave: Animais, Extensão, Escola, Infantil, Lúdico. Veterinary Mini-hospital: responsible pet ownership, animal welfare, zoonoses, and exotic wildlife protection Abstract: The Veterinary Mini-hospital project aimed to teach children concepts of respect for animals, disease prevention and animal welfare through games and stuffed animal toys. The project carried out its activities mainly in schools, public and private. In groups, children went through the Mini-hospital modules, helping the "patients" and creating solutions to the related problems. In 2015, 19 presentations were given, with the participation of 840 children. The children showed great interest and excitement during the activities, and the project was praised by their teachers. Keywords: Animals, Extension, School, Infancy, Ludic Mini-hospital Veterinario: tenencia responsable de mascotas, bienestar animal, zoonosis y protección de la fauna exótica El proyecto Mini-hospital Veterinario tuvo como objetivo enseñar a los niños por medio del juego y de animales de peluche, el respeto a los animales, la prevención de enfermedades y conceptos de bienestar animal. El proyecto llevó a cabo sus actividades principalmente en las escuelas, públicas o privadas. En grupos, los niños pasaron por los módulos del Mini-hospital ayudaran a los ‘pacientes’ y crearon soluciones a los problemas presentados. En 2015, fueron realizadas 19 presentaciones, con la participación de 840 niños. Los niños mostraron gran interés y entusiasmo durante las actividades y el proyecto fue elogiado por sus profesoras. Palabras-clave: Animales, Extensión, Escuela, Infancia, Lúdico. DOI: https://doi.org/10.24317/2358-0399.2017v8i1.3123

The experiment was carried out to evaluate the influence of nutritional correction on the partial diet replacement or pure-food methodology to determine the energy values (apparent metabolizable energy - AME and nitrogen-correct apparent metabolizable energy - AMEn) and coefficient of metabolizability of nutrients of maize. The method of total excreta collection was used, with 120 female Cobb 500® broilers weighing 1,339±3 g and at 28 days of age distributed in a completely randomized design with five replicates and four birds per experimental unit. The treatments were pure maize with three levels of nutritional correction (0, 50 and 100%) and a maize-replaced basal diet (400 g/kg) with two levels of correction (0 and 100%). The corrections were made in order to meet the nutritional requirements of calcium, available phosphorus and sodium yonder trace minerals, vitamins, choline, lysine, methionine + cystine, threonine and tryptophan. The AME and AMEn of maize and coefficient of metabolizability of dry matter were higher when correction at 100% was made, regardless of the methodology used (pure feed or replaced basal diet). The correction at 50% of nutrients increased the coefficient of metabolizability of crude protein in pure maize when compared with 0 or 100% of correction. The values determined with pure maize were lower than those determined with the method of partial replacement. The absence of nutritional correction underestimates the energy values and metabolizability of nutrients from maize.

A soybean protein diet was used to induce vitamin E deficiency in rhesus monkeys. The deficient monkeys had reduced triglyceride concentrations in liver and skeletal muscle, but the cholesterol concentration in their skeletal muscle was increased. A constant amount of radioactively labeled 3H-cholesterol-7α-3H was fed daily for 48-114 days to control and vitamin E-deficient monkeys to study the relationship between plasma, liver, and skeletal muscle cholesterol. Plasma cholesterol reached constant, maximum specific activity by the 42nd day both in control and in vitamin E-deficient monkeys. In control and previously deficient vitamin E-treated monkeys the specific activity of cholesterol in liver and skeletal muscle was approximately equal to that of plasma. In vitamin E-deficient monkeys the liver cholesterol specific activity was equal to that of plasma cholesterol, but the ratio of skeletal muscle cholesterol specific activity to plasma cholesterol specific activity was reduced. It is concluded from these studies that there is a specific defect(s) in cholesterol metabolism in the skeletal muscle of vitamin E-deficient monkeys.

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion. Here, we undertook a genome-wide expression study in PC-3 cells to explore the mechanism and function of this compound. There was significant overlap in the genes modulated by CCG-1423 and Latrunculin B (Lat B), which blocks the Rho/MKL/SRF pathway by preventing actin polymerization. In contrast, the general transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-1H-benzimidazole (DRB) showed a markedly different pattern. Effects of CCG-1423 and Lat B on gene expression correlated with literature studies of MKL knock-down. Gene sets involved in DNA synthesis and repair, G1/S transition, and apoptosis were modulated by CCG-1423. It also upregulated genes involved in endoplasmic reticulum stress. Targets of the known Rho target transcription factor family E2F and genes related to melanoma progression and metastasis were strongly suppressed by CCG-1423. These results confirm the ability of our compound to inhibit expression of numerous Rho/MKL-dependent genes and show effects on stress pathways as well. This suggests a novel approach to targeting aggressive cancers and metastasis.

Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, was found to bind to SOS1, competitively suppress SOS1-Ras interaction, and dose-dependently inhibit SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho-specific inhibitor, Rhosin. Rhosin contains two aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells, Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity, Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily-specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 μM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 μM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 μM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.

We recently identified bis(amide) CCG-1423 (1) as a novel inhibitor of RhoA/C-mediated gene transcription that is capable of inhibiting invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. An initial structure-activity relationship study focusing on bioisosteric replacement of the amides and conformational restriction identified two compounds, 4g and 8, with improved selectivity for inhibition of RhoA/C-mediated gene transcription and attenuated cytotoxicity relative to 1. Both compounds were also capable of inhibiting cell invasion with equal efficacy to 1 but with less attendant cytotoxicity., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Leukemia associated Rho guanine nucleotide exchange factor (LARG) activates RhoA in response to signals received by specific classes of cell surface receptors. The catalytic core of LARG is a Dbl homology (DH) domain whose activity is modulated by an adjacent pleckstrin homology (PH) domain. In this study, we used a transcriptional assay and confocal microscopy to examine the roles of several novel structural features of the LARG DH/PH domains, including a conserved and exposed hydrophobic patch on the PH domain that mediates protein-protein interactions in crystal structures of LARG and its close homolog PDZ-RhoGEF. Mutation of the hydrophobic patch has no effect on nucleotide exchange activity in vitro, but abolished the ability of LARG to activate RhoA and to induce stress fiber formation in cultured cells. The activity of these mutants could be rescued by fusion with exogenous membrane-targeting domains. However, because membrane recruitment by activated G alpha(13) subunits was not sufficient to rescue activity of a hydrophobic patch mutant, the LARG PH domain cannot solely contribute to membrane targeting. Instead, it seems likely that the domain is involved in regulatory interactions with other proteins near the membrane surface. We also show that the hydrophobic patch of the PH domain is likely important for the activity of all Lbc subfamily RhoGEFs.

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.