Mutant KRAS modulates colorectal cancer cells invasive response to fibroblast-secreted factors through the HGF/C-MET axis.

Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblast-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and nonautonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent nonautonomous effect, resulting from the cooperation between fibroblast-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promote invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells.

Mercury levels in parturient and newborns from Aveiro region, Portugal.

Since the outbreak of methylmercury (MeHg) poisoning in Japan and Iraq, mercury (Hg) is classified as well-established teratogen. The Portuguese region of Aveiro faced some decades ago an environmental Hg contamination due to activities from a chlor-alkali plant. Until now, no apparent evaluation was conducted regarding prenatal exposure to Hg in this area. The main objectives of this study were to: i) assess maternal and fetal exposure to Hg in the Aveiro region using noninvasive biological matrices; ii) examine the influence of variables that may contribute to Hg exposure during pregnancy; and iii) improve knowledge regarding metal accumulation and distribution over the maternal-fetal-placental unit. This study was performed in 50 mother-newborn pairs from the Aveiro district. Total Hg (THg) was quantified in maternal scalp hair, placenta, amniotic membrane, and umbilical cord. Maternal hair presented THg levels with a mean value of 900 ng/g, which is lower than the USEPA and WHO acceptable threshold. Regarding THg levels in placenta and umbilical cord, mean values were similar (decidua basalis: 32.84 ng/g; chorionic plate: 30.18 ng/g; umbilical cord: 30.67 ng/g). The amniotic membrane presented the highest THg levels with a mean concentration of 42.35 ng/g, reaching a maximum of 134.1 ng/g. Further, a significant positive correlation was noted between THg levels found in hair, and all matrices analyzed reinforcing the use of hair in biomonitoring studies with respect to maternal exposure to Hg. In general, levels of THg found in our study were lower than those in previous studies performed in Europe. Consumption of fish rich in selenium and bottled water was negatively correlated with THg levels. Finally, data demonstrated that Hg is capable of crossing the placental barrier and accumulate in placental tissues. Amniotic membrane seemed to play a role in metal detoxification, but further investigations are necessary to examine whether this catabolic process affects Hg accumulation.

KRAS silencing alters chromatin physical organization and transcriptional activity in colorectal cancer cells.

Clinical data revealed that KRAS mutant tumors, while initially sensitive to treatment, rapidly bypass KRAS dependence to acquire a drug-tolerant phenotype. However, the mechanisms underlying the transition from a drug-sensitive to a drug-tolerant state still elude us. Here, we show that global chromatin reorganization is a recurrent and specific feature of KRAS-dependent cells that tolerated KRAS silencing. We show that KRAS-dependent cells undergo G0/G1 cell cycle arrest after KRAS silencing, presenting a transcriptomic signature of quiescence. Proteomic analysis showed upregulated chromatin-associated proteins and transcription-associated biological processes. Accordingly, these cells shifted euchromatin/heterochromatin states, gained topologically associating domains, and altered the nanoscale physical organization of chromatin, more precisely by downregulating chromatin packing domains, a feature associated with the induction of quiescence. In addition, they also accumulated transcriptional alterations over time leading to a diversification of biological processes, linking chromatin alterations to transcriptional performance. Overall, our observations pinpoint a novel molecular mechanism of tolerance to KRAS oncogenic loss driven not by specific gene alterations but by global reorganization of genomic information, in which cells transition chromatin domain structure towards a more quiescent state and gain transcriptional reprogramming capacity.

Microplastics in freshwater sediments: Effects on benthic invertebrate communities and ecosystem functioning assessed in artificial streams.

The high levels of microplastics (MPs) found in freshwaters, particularly in riverine sediments, may impose a threat to the macroinvertebrate communities with possible consequences at ecosystem-level. The present study aimed to assess the effects of a mixture of different sizes of polyethylene microplastics (PE-MPs) on the composition and structure of macroinvertebrate communities and key-functions, such as primary production and leaf litter decomposition. MPs were mixed in the sediment at three different concentrations (0.1, 1, and 10 g kg-1) already found in freshwater sediments to enhance the relevance of the work. After eight days of exposure to PE-MPs, the observed changes in macroinvertebrate community structure were mostly due to the reduction in the abundance of deposit-feeders and grazers that were reduced by ca 31-50% and 34-39%, in the two highest MPs concentrations respectively, in comparison with the control treatment after 8 days of exposure. MPs internal concentrations were detected only in organisms exposed to plastic particles within artificial streams with chironomids and mayflies presenting higher MPs internal levels (average of 115 particles/individual found in chironomids, 166/individual for Baetis sp. and 415 particles/individual for Ephemerella sp.) suggesting higher ingestion of plastic microparticles. Nevertheless, the alterations in the community structure did not translate into impairments in the functional endpoints analysed, leaf litter decomposition and primary production, that were expected due to possible sub-lethal effects (e.g., feeding inhibition) on detritivores and grazers. This study represents one of the few assessments of MPs effects on freshwater benthic macroinvertebrate community structure and the first that simultaneously considered ecosystem-level functional endpoints. Further research combining different microplastics and longer exposure periods are needed to raise knowledge on potential ecological consequences of MPs to freshwaters.

Effects of pyrene and benzo[a]pyrene on the reproduction and newborn morphology and behavior of the freshwater planarian Girardia tigrina.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of aquatic ecosystems. Because they are persistent, there is great potential for chronic toxicity to aquatic species, and the evaluation of reproductive effects is fundamental. In this context, planarians are interesting experimental animals, since they can be sensitive to environmental pollutants, and a wide range of reproductive-related endpoints can be assessed. In this work we evaluated fecundity (number of cocoons), fertility (number of newborns), newborn anomalies, adult weight, regenerative abilities and PAH-residues in tissues of the freshwater planarian Girardia tigrina, exposed to either pyrene or benzo[a]pyrene (B[a]P). Pyrene reduced planarian fecundity and fertility at 18.75 µg L-1 and 75.00 µg L-1, while B[a]P reduced planarian fecundity at the 37.50 µg L-1 treatment, which was accompanied by a 33.7% reduction in fertility. Cocoons were kept in clean media and newborns were evaluated for behavioral and morphological anomalies. Many of the newborns resulting from the B[a]P experiment revealed behavioral anomalies, such as spasms and uncoordinated movements. These behavioral anomalies were observed in 12.9% and 38.2% of newborns resulting from the exposure of adult planarians to 9.38 µg L-1 and 37.50 µg L-1 of B[a]P, respectively. This study is the first report on the effects of PAHs in freshwater planarians' sexual reproduction and a decreased reproductive output was evidenced. Moreover, the exposure of adults to B[a]P lead to defects in newborns, raising concern on the possible long-term consequences of these compounds for natural planarian populations.

Effects of pH and nitrites on the toxicity of a cypermethrin-based pesticide to zebrafish embryos.

Physical and chemical water parameters have an important role on the toxicity of environmental contaminants by affecting their degradation, transformation and bioavailability. The objective of this study was to evaluate the effects of pH and nitrite variation on the toxicity of the cypermethrin based pesticide Barrage®. Zebrafish embryos (Danio rerio) were exposed to a combination of pH or nitrite and cypermethrin through the Barrage® formulation for 96 h and lethal and developmental endpoints assessed. Concerning mortality, pH seemed to increase Barrage® toxicity, while nitrite concentrations tested did not seem to change Barrage® lethality. At sublethal level, either for combinations involving pH or nitrites, results suggested some interactive effects of environmental and chemical factors, highlighting the need of further research. This work suggests that sublethal endpoints should be included in the assessment of combined effects of compounds given that they can provide important information not obtained through lethality.

Toxicity of different polycyclic aromatic hydrocarbons (PAHs) to the freshwater planarian Girardia tigrina.

Freshwater planarians have been gaining relevance as experimental animals for numerous research areas given their interesting features, such as high regeneration potential, shared features with the vertebrates' nervous system or the range of endpoints that can be easily evaluated in response to contaminants. Ecotoxicological research using these animals has been steadily increasing in the past decades, as planarians' potentialities for this research area are being recognized. In this work, we used polycyclic aromatic hydrocarbons (PAHs) as model contaminants and evaluated effects of exposure to phenanthrene, pyrene and benzo[a]pyrene (B[a]P) in planarians. The freshwater planarian Girardia tigrina was chosen and mortality, cephalic regeneration (during and post-exposure), behavioral endpoints and presence of PAHs in tissues, were evaluated. Mortality was only observed in planarians exposed to phenanthrene, with an estimated LC50 of 830 µg L-1. Results indicate that planarian behavioral endpoints were very sensitive in response to sub-lethal concentrations of PAHs, showing a greater sensitivity towards B[a]P and pyrene. Briefly, post-exposure locomotion and post-exposure feeding were significantly impaired by sub-lethal concentrations of all compounds, whereas regeneration of photoreceptors was only significantly delayed in planarians exposed to pyrene. Moreover, levels of PAH-type compounds in planarian tissues followed a concentration-dependent increase, showing uptake of compounds from experimental solutions. The present results highlight the importance of studying alternative and complementary endpoints, such as behavior, not only because these may be able to detect effects at lower levels of contamination, but also due to their ecological relevance. The simplicity of evaluating a wide range of responses to contaminants further demonstrates the utility of freshwater planarians for ecotoxicological research.

Targeting the Tumor Microenvironment: An Unexplored Strategy for Mutant KRAS Tumors.

Current evidence strongly suggests that cancer cells depend on the microenvironment in order to thrive. In fact, signals from the surrounding tumor microenvironment are crucial for cancer cells´ aggressiveness, altering their expression profile and favoring their metastatic potential. As such, targeting the tumor microenvironment to impair cancer progression became an attractive therapeutic option. Interestingly, it has been shown that oncogenic KRAS signaling promotes a pro-tumorigenic microenvironment, and the associated crosstalk alters the expression profile of cancer cells. These findings award KRAS a key role in controlling the interactions between cancer cells and the microenvironment, granting cancer a poor prognosis. Given the lack of effective approaches to target KRAS itself or its downstream effectors in the clinic, exploring such interactions may open new perspectives on possible therapeutic strategies to hinder mutant KRAS tumors. This review highlights those communications and their implications for the development of effective therapies or to provide insights regarding response to existing regimens.

Behavioral effects in adult zebrafish after developmental exposure to carbaryl.

Chemical exposure during the early life stages of development may have long lasting effects on organisms that are rarely studied. The present work intended to evaluate the effect of embryonic exposure to the pesticide carbaryl on adult fish behavior. Zebrafish (Danio rerio) embryos were exposed, for 4 days, to sublethal concentrations of carbaryl (0.01, 0.1 and 1.0 mg/L) plus a control and then kept in standard cultivation conditions until adulthood. A battery of behavioral tests was then performed to assess anxiety-like behavior (locomotor activity, thigmotaxis and novel tank diving test), social behavior, and feeding. Developmental exposure of zebrafish to sublethal concentrations of carbaryl produced important behavioral alterations in the adulthood. Main effects included decreased locomotion/hypoactivity (increase in slow movements and decrease of medium and rapid movements), especially in the light periods. Moreover, spatial pattern also changed: while during dark periods control fish increased activity in the outer zone of the tank, this was not observed in exposed fish. Overall, this demonstrated the importance of life stage exposure, clearly demonstrating long lasting effects of a (chemical) stress event at embryonic stages. This data supports the need of considering this scenario in environmental risk evaluations. Further work should focus on the mechanistic effects of developmental disruption responsible for the effects observed.

Long-term effects of oxytetracycline exposure in zebrafish: A multi-level perspective.

Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 µg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms.

Dental mapractice litigance in the city of São Paulo (SP), Brazil

The relationship between patients and dentists today is more worn and less based on trust, which can lead to high rates of lawsuits related to civil liability and dental malpractice. Aim: verify if there has been an increase in the number of lawsuits related to questioned dental treatments, and against dentists registered in the city of São Paulo (SP), Brazil, from 2012 to 2017. Methods: We outlined an overview based on the list from the São Paulo Council of Dentistry containing 30,238 registered dentists in the city of São Paulo, and searched for lawsuits on the public base of the São Paulo State Court's. Results: The search, after the inclusion and exclusion criteria, found 247 lawsuits, with dental implants as the most involved specialty. The total indemnities requested ranged from R$ 227.42 to R$ 937,000.00, but no indemnity granted exceeded the amount of R$ 100,000.00. Conclusion: According to the analysis of cases, there is a progressive increase in the number of civil liability lawsuits against dentists involving dental malpractice litigance

Carbendazim exposure induces developmental, biochemical and behavioural disturbance in zebrafish embryos.

Carbendazim is a widely used broad spectrum benzimidazole fungicide; however, its effects to non-target aquatic organisms are poorly studied. The aim of this study was to investigate the toxic effects of carbendazim to zebrafish early life stages at several levels of biological organization, including developmental, biochemical and behavioural levels. The embryo assay was done following the OECD guideline 236 and using a concentration range between 1.1 and 1.8mg/L. Lethal and developmental endpoints such as hatching, edemas, malformations, heart beat rate, body growth and delays were assessed in a 96h exposure. A sub-teratogenic range (from 0.16 to 500µg/L) was then used to assess effects at biochemical and behavioural levels. Biochemical markers included cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) and were assessed at 96h. The locomotor behaviour was assessed using an automated video tracking system at 120h. Carbendazim (96h-LC50 of 1.75mg/L) elicited several developmental anomalies in zebrafish embryos with EC50 values ranging from 0.85 to 1.6mg/L. ChE, GST and LDH activities were increased at concentrations equal or above 4µg/L. The locomotor assay showed to be extremely sensitive, detecting effects in time that larvae spent swimming at concentrations of 0.16µg/L and thus, being several orders of magnitude more sensitive that developmental parameters or lethality. These are ecological relevant concentrations and highlight the potential of behavioural endpoints as early warning signs for environmental stress. Further studies should focus on understanding how the behavioural disturbances measured in these types of studies translate into fitness impairment at the adult stage.

Uso de acervos de imagens em pesquisas na Odontologia Legal: reflexões éticas, bioéticas e legais / Use of image collections in Forensic Dentistry research: ethical, bioethical and legal aspects

RESUMO: Objetivo: Realizar um levantamento dos artigos científicos que utilizaram acervos de imagem como fonte de pesquisa em Odontologia Legal e discutir essa utilização sob perspectivas éticas, bioéticas e legais. Material e Métodos: Foi realizada uma busca de artigos científicos nas bases de dados Lilacs, PubMed e Scielo entre os anos de 2015 e 2019 utilizando descritores nos idiomas português, inglês e espanhol. Foram incluídos apenas artigos da área de Odontologia Legal que utilizaram acervos de imagem como fonte de informação. Foram excluídos es-tudos laboratoriais ou clínicos, arqueológicos ou antropológicos, relatos de caso ou artigos de revisão, estudos prospectivos ou que utilizaram exames de imagem não pertencentes a acervos. Resultados: Foram selecionados 171 artigos científicos. Desses, 22,80% (n=39) foram realizados por autores vinculados a instituições brasileiras, e 77,19% (n=132) mencionaram aprovação por comitê de ética em pesquisa ou instituição correspondente com (ou sem) necessidade de Termo de Consentimento Livre e Esclarecido (TCLE). Conclusão: Os acervos de imagem compreendem uma importante fonte de informação para pesquisas em Odontologia Legal dada a quantidade de pu-blicações realizadas no período avaliado. A maioria dos artigos mencionou aprovação ética e respeito à privacidade dos participantes, conforme os requisitos estabelecidos para dispensa do TCLE. (AU)

ABSTRACT: Objective: To conduct a quantitative survey of scientific articles that used image collections as source of research in Forensic Dentistry and discuss its use from ethical, bioethical and legal perspectives. Material and Methods:A search for scientific articles published and available on Lilacs, PubMed and Scielo databases was carried out from 2015 to 2019 using keywords in Portuguese, English and Spanish. Articles in the area of Forensic Dentistry that used image collections as a source of information were included. Laboratory or clinical, archaeological or anthro-pological studies, case reports or review articles, prospective studies or that used imaging exams not belonging to collections were excluded. Results: 171 scientific articles were included. 22.80% (n = 39) were carried out by authors from Brazilian institutions, and 77.19% (n = 132) mentioned approval by a research ethics committee or corresponding institution with (or without) use of signed Informed Consent Form (ICF). Conclusion: Image col-lections are an important source of information for research in Forensic Dentistry, considering the number of publi-cations during the study period. Most of the articles mentioned ethical approval and respect for participant privacy according to the requirements established for waiver of the ICF. (AU)

Mapeamento das revistas científicas nacionais e internacionais e suas produções na área da odontologia legal / Mapping of nationals and internationals scientific journals and productions in the area of Forensic Dentistry

Publicações periódicas de artigos científico-acadêmicos buscam a transferência de informação e construção do conhecimento de alta qualidade. A Odontologia Legal, apesar do amplo campo de atuação profissional, envolve vários temas referentes à legislação, bem como especificidades locais, questões essas que, muitas vezes, dificultam a publicação em revistas científicas com alto fator de impacto. Visto isso, o presente estudo objetivou mapear as revistas científicas nacionais e internacionais que apresentem publicações na área da Odontologia Legal, bem como expor as dificuldades existentes na publicação de trabalhos relacionados à área em revistas de maio relevância. Foi acessada a Plataforma Sucupira e selecionada a Odontologia como área de avaliação e, assim, obtido um arquivo de classificações. Todas as revistas que apresentaram temas relacionados à Odontologia Legal foram registradas em duas tabelas com a quantidade de artigos publicados relacionados à área em cada uma delas. Com bases nos critérios de busca, foram encontradas 191 revistas, sendo 75 nacionais e 116 internacionais. As publicações nacionais mostraram escassa produção em revistas com fator de alto impacto, enquanto que as internacionais apresentaram uma maior quantidade, apesar de ser minoria dentro da produção total. Com esse trabalho, foi possível comparar a classificação Qualis entre revistas nacionais e internacionais, destacando a distância entre tais classificações e expondo a dificuldade de publicação em revistas de alta competitividade e internacionalização.

Scientific papers look for the information transference and construction of high quality knowledge. Forensic Dentistry, although the wide field of professional activity, involves several themes related to Law that often makes it difficult to publish in international scientific journals. This study aimed to map the national and international scientific journals with publications in Forensic Dentistry, as well as to expose the difficulties to publish papers in high impact factor journals. It was accessed the Sucupira Platform (Brazil) and selected Dentistry as an evaluation area to get the classification file. All journals that presented issues related to Forensic Dentistry were recorded in two tables with the number of papers published related to the area in each of them. 191 journals were found, 75 national and 116 international. The national publications showed few productions in high impact factor journals, while the international ones presented a larger quantity, but still a minority within the total production. With this study, it was possible to compare the Qualis classification between national and international journals, highlighting the distance between such classifications and exposing the difficulty of publication in journals that present high competitiveness and internationality.