Ocean acidification impact on the uptake of trace elements by mussels and their biochemical effects.

This study delves into the intricate interplay between ocean acidification (OA), metal bioaccumulation, and cellular responses using mussels (Mytilus galloprovincialis) as bioindicators. For this purpose, environmentally realistic concentrations of isotopically labelled metals (Cd, Cu, Ag, Ce) were added to investigate whether the OA increase would modify metal bioaccumulation and induce adverse effects at the cellular level. The study reveals that while certain elements like Cd and Ag might remain unaffected by OA, the bioavailability of Cu and Ce could potentially escalate, leading to amplified accumulation in marine organisms. The present findings highlight a significant rise in Ce concentrations within different mussel organs under elevated pCO2 conditions, accompanied by an increased isotopic fractionation of Ce (140/142Ce), suggesting a heightened potential for metal accumulation under OA. The results suggested that OA influenced metal accumulation in the gills of mussels. Conversely, metal accumulation in the digestive gland was unaffected by OA. The exposure to both trace metals and OA affects the biochemical responses of M. galloprovincialis, leading to increased metabolic capacity, changes in energy reserves, and alterations in oxidative stress markers, but the specific effects on other biomarkers (e.g., lipid peroxidation, some enzymatic responses or acetylcholinesterase activity) were not uniform, suggesting complex interactions between the stressors and the biochemical pathways in the mussels.

Genetic diversity based on multivariate techniques in elephant grass genotypes for bioenergy.

The use of biomass for energy production constitutes a promising strategy that warrants the search for new sources of biomass. Elephant grass has been gaining notoriety due to its high dry matter yield and rapid growth. The present study was carried out to quantify the genetic divergence of nine elephant grass half-sib families in order to identify genotypes with greater genetic divergence and productive potential for hybridization, using the hierarchical clustering methodology based on principal components. Half-sib families were generated using genotypes from the Active Germplasm Bank of Elephant Grass. The experiment was laid out in a randomized-block design with nine half-sib families, three replicates, and eight plants per plot. A total of 216 genotypes of elephant grass were evaluated. Principal component (PC), biplot, and hierarchical clustering analyses for diversity estimation were conducted using R software. The first two PCs of biplot analysis accounted for 64% of the cumulative variation. Dry matter yield was the most important trait for genotype discrimination (0.89), followed by plant height (0.67) and stem diameter (0.61) in PC1. In this analysis, the distances between accessions were considered and there were no family links, which indicates the existence of wide variability within the evaluated families, since genotypes belonging to the same family were not grouped together, but rather distributed into different groups. Crosses between genotypes of group three and genotypes of groups one and two are recommended for the development of high-yielding genotypes when aiming at energy production.

Cardiopulmonary exercise testing data processing and storage tools

ABSTRACT Conducting a clinical study is a complex and challenging task, so using technology to facilitate the process is necessary for the medical and scientific community. Data digitization is needed to store and process it. Digitization speeds up data visualization techniques so scientists can analyze it most effectively depending on their goals. In this project, we developed two digital tools related to the cardiopulmonary exercise test (CPET) data, one for digitizing PDF reports and the other for generating databases. The latter was a web-based database visualization dashboard. Users can select parameters of interest and check for differences among subgroups. Basic statistical tests are performed for each variable under analysis, and its results are presented in numerical and graphical formats. The initial statistical tests and the derived recommendation will guide the research team in deeper statistical analysis and robust analysis supporting more decisive conclusions. We ended up with the first version for both tools and validated it using patients' CPET data from the Dante Pazzanese Institute of Cardiology. We conducted a pilot study to verify if the tools served their purpose and observed that both programs worked as planned. The tools can be further tailored to be clinically or research-oriented. On the analysis of CPET's results, the conclusions for our example study were in line with what is presented in the bibliography for cardiorespiratory physiology.

Biological role of the bidirectional interaction between epithelial-mesenchymal transition and PD-L1 expression in head and neck squamous cell carcinomas: A systematic review.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common head and neck malignant neoplasm. Despite progress in antineoplastic treatment for SCC, there are still high morbidity and mortality rates. Over the years, several tumor biomarkers have been suggested to predict the prognosis of patients with oral SCC. Studies point to a bidirectional association between the epithelial-mesenchymal transition (EMT) and the expression of PD-L1 with the aggressive biological behavior of the neoplastic cell. Thus, this systematic review aimed to explore the biological roles and mechanisms underlying the interaction between EMT and PD-L1 expression in head and neck SCC-derived cell lines. MATERIAL AND METHODS: An electronic search was performed in the PubMed/Medline, Web of Science, Science Direct, Scopus, Embase, and Cochrane Collaboration Library databases. Articles evaluating the in vitro relationship between EMT/PD-L1 interaction and the biological behavior of head and neck SCC cell lines were selected for this systematic review. The quality of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RESULTS: After applying the previously established inclusion/exclusion criteria, 9 articles were included in the qualitative synthesis. The present systematic review suggests the existence of a bidirectional interaction between EMT and PD-L1 expression, which is related to alterations in the cell cycle, proliferation, apoptosis, and cell survival, affecting the migration and invasion ability of tumor cells. CONCLUSIONS: Combined targeting of the two pathways may be potentially effective for immunotherapy in head and neck SCC.

Supervivencia observada en tres familias de tilapia del Nilo (Oreochromis niloticus) infectadas con Streptococcus agalactiae / Survival observed in three families of Nile tilapia (Oreochromis niloticus) infected with Streptococcus agalactiae

RESUMEN La estreptococosis es una de las principales enfermedades en los peces de agua dulce que causa altas tasas de mortalidad. El objetivo de este estudio fue evaluar la respuesta en la supervivencia a la infección por Streptococcus agalactiae en tres familias de tilapia. El experimento se llevó a cabo en el Laboratorio de Enfermedades de los Peces de la Universidad Federal de Lavras. Se utilizaron peces con un peso de 93,7 ± 5,4 g de tres familias diferentes (FA, FB y FC). Se utilizaron 36 peces en cada unidad experimental, inoculados intraperitonealmente con 107 UFC/mL de Streptococcus agalactiae por peces y un grupo control por familia con 9 peces con 1 mL de caldo BHI (Infusión Cerebro Corazón) evaluados durante 15 días. No hubo mortalidad del grupo control. Se observó la presencia de exoftalmia, coloración oscura en todo el cuerpo, letargo y dilatación abdominal antes de la muerte en las tres familias evaluadas expuestas al patógeno. El estimador no paramétrico de Kaplan-Meier se utilizó para observar las curvas de supervivencia. Durante los 15 días del desafío, el tiempo promedio de supervivencia de un individuo en las familias FA, FB y FC fue de 9,4; 6,90 y 8,14 días, respectivamente. Pruebas de Log-rank y Peto & Peto para evaluar la diferencia entre las curvas de supervivencia arrojaron que no hubo diferencias significativas entre las familias evaluadas (P=0,08 y P= 0,09), respectivamente.

ABSTRACT Streptococcosis is one of the main diseases in freshwater fish that causes high mortality rates. The objective of this study was to evaluate the survival response to Streptococcus agalactiae infection in three families of tilapia. The experiment was carried out at the Laboratory of Fish Diseases of the Federal University of Lavras. Fish weighing 93.7 ± 5.4 g from three different families (FA, FB, and FC) were used. 36 fish were used in each experimental unit, intraperitoneally inoculated with 107 CFU/mL of Streptococcus agalactiae per fish and a control group per family with 9 fish with 1 mL of BHI broth (Brain Heart Infusion) evaluated for 15 days. There was no mortality in the control group. The three evaluated families exposed to the pathogen observed the presence of exophthalmia, dark coloration throughout the body, lethargy, and abdominal dilation before death. The Kaplan-Meier nonparametric estimator was used to observe the survival curves. During the 15 days of the challenge, the average survival time of an individual in the FA, FB, and FC families was 9.4, 6.90, and 8.14 days, respectively. Log-rank and Peto & Peto test to evaluate the difference between the survival curves showed no significant differences between the assessed families (P=0.08 and P= 0.09), respectively.

Gestational weight gain and visceral adiposity in adult offspring: Is there a link with the fecal abundance of Acidaminococcus genus?

Intrauterine environment can influence the offspring's body adiposity whose distribution affect the cardiometabolic risk. Underlying mechanisms may involve the gut microbiome. We investigated associations of gestational weight gain with the adult offspring's gut microbiota, body adiposity and related parameters in participants of the Nutritionists' Health Study. METHODS: This cross-sectional analysis included 114 women who had early life and clinical data, body composition, and biological samples collected. The structure of fecal microbiota was analyzed targeting the V4 region of the 16 S rRNA gene. Beta diversity was calculated by PCoA and PERMANOVA used to test the impact of categorical variables into the diversity. Bacterial clusters were identified based on the Jensen-Shannon divergence matrix and Calinski-Harabasz index. Correlations were tested by Spearman coefficient. RESULTS: Median age was 28 (IQR 24-31) years and BMI 24.5 (IQR 21.4-28.0) kg/m2. Fifty-eight participants were assigned to a profile driven by Prevotella and 56 to another driven by Blautia. Visceral adipose tissue was correlated to abundance of Acidaminococcus genus considering the entire sample (r = 0.37; p < 0.001) and the profiles (Blautia: r = 0.35, p = 0.009, and Prevotella: r = 0.38, p = 0.006). In Blautia-driven profile, the same genus was also correlated to maternal gestational weight gain (r = 0.38, p = 0.006). CONCLUSIONS: Association of Acidaminococcus with gestational weight gain could reinforce the relevance with mothers' nutritional status for gut colonization at the beginning of life. Whether Acidaminococcus abundance could be a marker for central distribution of adiposity in young women requires further investigation.

Prognostic and clinicopathological significance of tumor-stroma ratio in head and neck squamous cell carcinoma: A systematic review.

BACKGROUND: Analysis of the tumor microenvironment has been proposed as a strategy for the treatment and prognosis of different neoplastic processes. A grading system based on the tumor-stroma ratio (TSR), which evaluates the proportion of stroma in relation to neoplastic parenchyma at the invasion front, has shown a strong prognostic value in different neoplastic processes. The aim of the present systematic review was to understand the role of the TSR in head and neck squamous cell carcinoma (HNSCC), evaluating its correlation with clinical and prognostic parameters. MATERIAL AND METHODS: An electronic search was performed in PubMed/Medline, Web of Science, Science Direct, Scopus, Embase, and the Cochrane Collaboration Library. Publications assessing the relationship between TSR and prognosis in cases of HNSCC were eligible. The quality of the studies was assessed independently by four evaluators using the Newcastle-Ottawa scale. RESULTS: After application of the previously es+lished inclusion/exclusion criteria, nine articles were included in the qualitative synthesis. With regards to quality on the Newcastle-Ottawa scale, an overall value of 4.55 was obtained. This systematic review demonstrated a strong association between TSR and prognosis in esophageal and oral squamous cell carcinomas. CONCLUSIONS: Histopathological analysis of the TSR can optimize the analysis of the prognosis of cases diagnosed with HNSSC. In addition, the TSR is a reliable and simple parameter that can be evaluated in hematoxylin/eosin-stained slides during routine laboratory examinations, showing high inter- and intraobserver agreement.

Evaluation of miRNAs regulation of BDNF and IGF1 genes in T2DM insulin resistance in experimental models: bioinformatics based approach.

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.

Ferramentas para otimização de digitalização, armazenamento e visualização de dados do teste cardiopulmonar de exercício / Tools for optimizing the digitization, storage and visualization of cardiopulmonary exercise test data

INTRODUÇÃO: O teste cardiopulmonar de exercício (TCPE) é o padrão ouro para função cardiorrespiratória de pacientes. Por conta disso, os dados obtidos por meio deste são extremamente relevantes em pesquisas clínicas. No entanto, há situações em que, por conta de limitações dos equipamentos, a obtenção e armazenamento dos resultados destes exames se torna um processo complicado e dispendioso. OBJETIVO: Desenvolver ferramentas capazes de: 1) Digitalizar os dados nos formatos disponibilizados pelos equipamentos e montar um banco de dados; 2) Permitam uma análise, visualização e comparação otimizadas desses dados. MÉTODOS: Inicialmente, os laudos obtidos diretamente do equipamento são lidos por um programa, os valores dos parâmetros são armazenados durante os tempos mais relevantes do teste (limiares aeróbico e anaeróbico, VO2 máximo etc.) em um arquivo que servirá como banco de dados relacional. Esta base de dados é então utilizada para montar um Dashboard onde se pode selecionar a variável e o intervalo de tempo que se deseja comparar entre os grupos. Nele são mostrados histogramas e gráficos violinos para se comparar visualmente os grupos (figuras 1 e 2) e estatísticas dos dados coletados junto a: 1) Resultado de um teste de normalidade para o parâmetro escolhido; 2) Resultado (p-valor) de um teste de hipótese (paramétrico caso a distribuição seja considerada normal, ou não paramétrico caso contrário); 3) Os tamanhos mínimos que as amostras devem possuir para que os resultados dos testes possam ser considerados estatisticamente significativos. Por fim, é possível analisar, em uma segunda página desta ferramenta os valores individuais de cada paciente, sendo possível filtrar e visualizar apenas os valores dentro de um intervalo desejado. (Figura 4) RESULTADOS: As ferramentas desenvolvidas foram utilizadas na base de dados dos pacientes do Ambulatório de Medicina Cardiovascular Personalizada com os resultados de 102 exames. O Dashboard foi utilizado para realizar um estudo comparativo entre os pacientes homens e mulheres do ambulatório com resultados coerentes com a literatura. CONCLUSÕES: Com estas ferramentas foi possível, rapidamente, digitalizar e armazenar os dados montando um arquivo com as informações de todos pacientes. Ademais, também permitiu realizar um estudo exemplo comparando todos pacientes gerando imagens que auxiliam a visualizar os resultados e os respectivos p-valores para cada comparação.

Ferramenta para processamento e armazenamento de curvas de strain para estudo da mecânica miocárdica / Tool for processing and storing strain curves for studying myocardial mechanics

INTRODUÇÃO: As curvas de deformação cardíacas derivadas de Speckle Tracking, permitem avaliação detalhada de diversos aspectos da mecânica miocárdica, entretanto o estudo de novos parâmetros é limitado por restrições impostas pelos softwares proprietários dos diferentes fabricantes. OBJETIVO: Desenvolver um programa com interface gráfica que permita visualizar e processar as curvas de Strain e Strain Rate facilitando o desenvolvimento de pesquisas. MÉTODOS: Inicia-se com a exportação das curvas de Strain do programa do fabricante. Elas são a entrada do software que as converte em variáveis do Matlab. Pode-se então, selecionar o exame de interesse, ou seja, paciente e janela de aquisição. As curvas de Strain e Strain Rate são exibidas e é possível selecionar os segmentos de interesse e, caso exista, outros exames do mesmo paciente com a mesma janela de aquisição. As curvas de deformação são exibidas sincronizadas com o Eletrocardiograma possibilitando ter em uma única tela o sinal elétrico e o sinal de deformação. Além disso, também é possível anotar o tempo de abertura e fechamento das válvulas com relação ao pico do QRS, que é encontrado automaticamente e pode ser editado caso necessário. As demais abas do programa apresentam as seguintes funcionalidades: em "Curvas Separadas" é possível visualizar as porções das curvas correspondentes à fase do ciclo cardíaco desejada, na aba "Plot Simul" é possível visualizar, simultaneamente, uma janela do ventrículo esquerdo e uma de átrio esquerdo sincronizados pelos respectivos ECGs, e na aba "Doppler" é possível verificar a imagem de um doppler e utilizá-lo para demarcar os tempos de válvula. RESULTADOS: O software funcional foi implementado em um ambulatório, sendo responsável por converter e processar curvas de mais de 2000 arquivos exportados, segmentando-os nas fases específicas do ciclo cardíaco (relaxamento e contração isovolumétricos, períodos de ejeção, enchimento rápido e enchimento lento) de exames utilizados para desenvolvimento de uma linha de pesquisa. CONCLUSÕES: O software livre desenvolvido no IDPC permite explorar as curvas de deformação cardíaca de maneira personalizada sendo adequado ao estudo do ecocardiograma em diversas situações.

B-esterases characterisation in the digestive tract of the common octopus and the European cuttlefish and their in vitro responses to contaminants of environmental concern.

Cephalopods are a group of marine invertebrates that have received little attention as sentinel species in comparison to other molluscs, such as bivalves. Consequently, their physiological and biochemical xenobiotic metabolism responses are poorly understood. Here we undertake a comparative analysis of the enzymatic activities involved in detoxification reactions and neural transmission in the digestive tract of two commercial cephalopods: the Common octopus, Octopus vulgaris, and the European cuttlefish, Sepia officinalis. For methodological purposes, several common B-esterases (five carboxylesterase (CE) substrates and three cholinesterase (ChE) determinations) were assayed as a proxy of metabolic and neuronal activities, respectively. Four components of the digestive tract in each species were considered: salivary glands, the stomach, the digestive gland and the caecum. The in vitro responses of digestive gland homogenates to model chemicals and contaminants of environmental concern were contrasted between both cephalopod species. The baseline biochemical activities in the four digestive tract components were also determined. Moreover, in order to validate the protocol, purified proteins, recombinant human CE (CE1 and CE2) and purified eel acetylcholinesterase (AChE) were included in the analysis. Overall, carboxylesterase activities were higher in octopus than in cuttlefish, with the activity quantified in the digestive tract components in the following order: digestive gland ≈ caecum > stomach ≈ salivary glands, with higher hydrolysis rates reached with naphthyl-derived substrates. In contrast, cuttlefish hydrolysis rates with ChE substrates were higher than in octopus. This trend was also reflected in a higher sensitivity to CE inhibitors in octopus and to AChE inhibitors in cuttlefish. Given the detoxification character of CEs and its protective role preventing AChE inhibition, octopus could be regarded as more efficiently protected than cuttlefish from neurotoxic exposures. A full characterisation of B-esterases in the digestive tract of the two common cephalopods is also provided.

Will extreme weather events influence the toxic impacts of caffeine in coastal systems? Comparison between two widely used bioindicator species.

In the recent years, marine heatwaves (MHWs) have caused devastating impacts on marine life. The understanding of the combined effects of these extreme events and anthropogenic pollution is a vital challenge. In particular, the combined effect of MHWs on the toxicity of pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine (CAF) toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed the absence of CAF accumulation in both species. However, the used contaminant generated in both bivalve species alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. The obtained results seemed magnified under a simulated MHWs, suggesting to a climate-induced toxicant sensitivities' response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife.

Van der Waals materials as dielectric layers for tailoring the near-field photonic response of surfaces.

Epsilon near-zero photonics and surface polariton nanophotonics have become major fields within optics, leading to unusual and enhanced light-matter interaction. Specific dielectric responses are required in both cases, which can be achieved, e.g., via operation near a material's electronic or phononic resonance. However, this condition restricts operation to a specific, narrow frequency range. It has been shown that using a thin dielectric layer can adjust the dielectric response of a surface and, therefore, the operating frequency for achieving specific photonic excitations. Here, we show that a surface's optical properties can be tuned via the deposition/transference of ultra-thin layered van der Waals (vdW) crystals, the thicknesses of which can easily be adjusted to provide the desired response. In particular, we experimentally and theoretically show that the surface phonon resonance of a silica surface can be tuned by ∼50 cm-1 through the simple deposition of nanometer-thick exfoliated flakes of black phosphorus. The surface properties were probed by infrared nanospectroscopy, and results show a close agreement with the theory. The black phosphorus-silica layered structure effectively acts as a surface with a tunable effective dielectric constant that presents an infrared response dependent on the black phosphorus thickness. In contrast, with a lower dielectric constant, hexagonal boron nitride does not significantly tune the silica surface phonon polariton. Our approach also applies to epsilon near-zero surfaces, as theoretically shown, and to polaritonic surfaces operating at other optical ranges.

Caffeine as a contaminant of concern: A review on concentrations and impacts in marine coastal systems.

Caffeine has been identified as emerging contaminant of concern due to its widespread occurrence in the aquatic environment and potential to be biologically active. Recently, these concerns have been translated in an increasing research on its occurrence and effects on biota. However, there is still a limited knowledge on seawater matrices and the implications of caffeine presence in coastal and marine ecosystems are not fully known. The present review aims to fill these knowledge gaps, analysing the existing literature regarding the occurrence, effects and potential risks of caffeine residues to coastal ecosystems, contributing to the risk assessment of this psychoactive drug in the aquatic environment. The analysed literature reported caffeine concentrations in the coastal ecosystems, raising high concerns about the potential adverse impacts on the ecological safety and human health. Caffeine has been found in tissues from coastal and marine biota including microalgae, coral reefs, bivalves and fish due to bioaccumulation after chronic, long-term exposures in a contaminated environment. Additionally, caffeine residues had been demonstrated to have adverse impacts on aquatic organisms, at environmentally realistic concentrations, inducing oxidative stress and lipid peroxidation, neurotoxicity, changing energy reserves and metabolic activity, affecting reproduction and development and, in some cases, causing mortality. Considering the increasing adverse impacts of caffeine pollution in the coastal environment, this review highlights the urgent need to minimize the increasing load of caffeine to the aquatic ecosystems; being imperative the implementation of scientific programs and projects to classify effectively the caffeine as a high-priority environmentally hazardous emerging pollutant.

Design, simulation, and fabrication of an ingestible capsule with gastric balloon for obesity treatment.

The treatment of obesity based only on lifestyle changes has been shown ineffectiveness in a long-term period. The development of more definitive and non-invasive therapies has been the subject of study. In this paper, a magnetically driven ingestible capsule with the capacity to inflate a gastric balloon is devised, simulated, and fabricated. The balloon is inflated to a volume of 150 ml using an acid-base reaction between citric acid and potassium bicarbonate. Finite element method simulations were performed to study the interaction between the permanent external magnet and the ingestible capsule and confirm the magnetic activation mechanism. A fabrication process was proposed to manufacture a polydimethylsiloxane (PDMS) balloon in a simple, functional, and reproducible way. The two layers and 1:8 ratio balloons are the most cost-effective without compromising their mechanical properties. The capsule body parts manufactured by a three-dimensional (3D) printing process - Digital Light Processing (DLP) showed high accuracy and excellent resolution. This study demonstrated that the proposed ingestible capsule would successfully inflate the gastric balloon to treat obesity.

Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9

BACKGROUND AND AIMS: Introduction: The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process. Aim: To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients. METHODS: Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants. RESULTS: HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection. CONCLUSIONS: We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.

Cannabidiol in the prelimbic cortex modulates the comorbid condition between the chronic neuropathic pain and depression-like behaviour in rats: The role of medial prefrontal cortex 5-HT1A and CB1 receptors.

The prelimbic division (PrL) of the medial prefrontal cortex (mPFC) is a cerebral division that is putatively implicated in the chronic pain and depression. We investigated the activity of PrL cortex neurons in Wistar rats that underwent chronic constriction injury (CCI) of sciatic nerve and were further subjected to the forced swimming (FS) test and mechanical allodynia (by von Frey test). The effect of blockade of synapses with cobalt chloride (CoCl2), and the treatment of the PrL cortex with cannabidiol (CBD), the CB1 receptor antagonist AM251 and the 5-HT1A receptor antagonist WAY-100635 were also investigated. Our results showed that CoCl2 decreased the time spent immobile during the FS test but did not alter mechanical allodynia. CBD (at 15, 30 and 60 nmol) in the PrL cortex also decreased the frequency and duration of immobility; however, only the dose of 30 nmol of CBD attenuated mechanical allodynia in rats with chronic NP. AM251 and WAY-100635 in the PrL cortex attenuated the antidepressive and analgesic effect caused by CBD but did not alter the immobility and the mechanical allodynia when administered alone. These data show that the PrL cortex is part of the neural substrate underlying the comorbidity between NP and depression. Also, the previous blockade of CB1 cannabinoid receptors and 5-HT1A serotonergic receptors in the PrL cortex attenuated the antidepressive and analgesics effect of the CBD. They also suggest that CBD could be a potential medicine for the treatment of depressive and pain symptoms in patients with chronic NP/depression comorbidity.

Adiponectin, HOMA-Adiponectin, HOMA-IR in Children and Adolescents: Ouro Preto Study.

OBJECTIVES: To examine the association and predictive capacity of adiponectin levels, HOMA-AD and HOMA-IR indexes with metabolic risk markers in children and adolescents. METHODS: A cross-sectional study was conducted with 691 children and adolescents (7-14 y), of both sexes. Demographic (sex, age), anthropometric (weight, height, body mass index, waist circumference, body fat), biochemical [total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, fasting glycemia, insulin and adiponectin] and clinical parameters (arterial blood pressure) were analyzed. RESULTS: In multiple linear regression models, metabolic risk were analyzed in relation to adiponectin levels, HOMA-AD and HOMA-IR. ROC curve analysis was used to define the cut-off for metabolic syndrome for each method studied. Adiponectin level was inversely correlated with weight (r = -0.12; p = 0.01), waist circumference (WC) (r = -0.12; p = 0.01), and triglycerides (r = -0.11; p = 0.02); it was directly correlated with HDL (r = 0.10; p = 0.03) only in the adolescents. In the final linear regression model, after adjustment, only triglycerides (p = 0.03) and HDL (p = 0.04) remained significant. However, HOMA-AD and HOMA-IR were associated with metabolic risk and were the most suitable methods for metabolic syndrome screening in both age groups. For children, independent variables explained 16.0% and 14.5% of HOMA-AD and HOMA-IR, respectively. For adolescents, R2 was higher in HOMA-AD and HOMA-IR models (R2adjusted = 31.9% and R2adjusted = 29.6%, respectively). CONCLUSIONS: HOMA-AD and HOMA-IR are better explained by metabolic markers than adiponectin levels.