YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del-CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.

"Geographical distribution of risk genotypes in pediatric patients with celiac disease in Spain".

Celiac disease is strongly associated with HLA DQ, specifically with haplotypes. DRB1*03-DQA1*05:01/DQB1*02:01 (DQ2.5),DRB1*07-DQA1*02:01/DQB1*02:02 (DQ2.2), DRB1*11-DQA1*05:05/DQB1*03:01 (DQ7.5), and DRB1*04-DQA1*03:01/DQB1*03:02 (DQ8). The distribution of these risk haplotypes in patients with celiac disease is different in the geographical areas investigated. A high frequency of DRB1*07- DQA1*02:01/DQB1*02:02 (DQ2.2) and DRB1*11-DQA1*05:05/DQB1*03:01 (DQ7.5), has been described in Southern Europe. We analyzed 2102 confirmed CD cases with information on both DQB1* alelles and their distribution by geographical area in Spain. According to the presence of this haplotype in one or two chromosomes, the genotype is classified in: DQ2 homozygous, DQ2 heterozygous (cis or trans), DQ8 homozygous, DQ8/DQ2.5, DQ 2.2 homozygous and genotype known as "half DQ2". Two different patterns of risks related to CD were identified. In the Basque Country and Navarre, the Mediterranean Area (Aragon, Catalonia, Valencia, Balearic Islands, and Murcia), the South of Spain (Andalucía and Extremadura), and the Canary Islands, higher frequency of DQ2.5 trans, and more than 80% of DQ2.5/DQ2.2 homozygosis were described. The Cantabrian Coast (Cantabria, Asturias, and Galicia) and Central Areas (Castilla-León and Castilla-La Mancha) showed a higher percentage of DQ2.5/DQ2.5 homozygosis and a lower DQ2.5 in trans frequency, as in Northern Europe. Madrid has an intermediate model between the two described above. 17 cases (0.8%) did not carry any CD risk haplotypes.

Orchid Micropropagation Using Conventional Semi-Solid and Temporary Immersion Systems: A Review.

Orchids, with their astonishingly stunning flowers, dominate the international floricultural market. They are considered prized assets for commercial applications in pharmaceutical and floricultural industries as they possess high therapeutic properties and superior ornamental values. The alarming depletion of orchid resources due to excessive unregulated commercial collection and mass habitat destruction makes orchid conservation measures an extreme priority. Conventional propagation methods cannot produce adequate number of orchids, which would meet the requirement of these ornamental plants for commercial and conservational purposes. In vitro orchid propagation using semi-solid media offers an outstanding prospect of rapidly producing quality plants on a large scale. However, the semi-solid (SS) system has shortcomings with low multiplication rates and high production costs. Orchid micropropagation using a temporary immersion system (TIS) overcomes the limitations of the SS system by reducing production costs and making scaleup and full automation possible for mass plant production. The current review highlights different aspects of in vitro orchid propagation using SS and TIS and their benefits and drawbacks on rapid plant generation.

Coeliac Disease Case-Control Study: Has the Time Come to Explore beyond Patients at Risk?

The worldwide prevalence of asymptomatic coeliac disease (CD) is increasing, which is in part due to the routine screening of children with risk factors. Both symptomatic and asymptomatic patients with CD are at risk of long-term complications. The objective of this study was to compare the clinical characteristics of asymptomatic and symptomatic children at the time of CD diagnosis. A case-control study was conducted using data from a cohort of 4838 CD patients recruited from 73 centers across Spain between 2011 and 2017. A total of 468 asymptomatic patients (cases) were selected and matched by age and sex with 468 symptomatic patients (controls). Clinical data, including any reported symptoms, as well as serologic, genetic, and histopathologic data were collected. No significant differences were found between the two groups in most clinical variables, nor in the degree of intestinal lesion. However, the asymptomatic patients were taller (height z-score -0.12 (1.06) vs. -0.45 (1.19), p < 0.001) and were less likely to have anti transglutaminase IgA antibodies ≥ 10 times the upper normal limit (66.2% vs. 758.4%, p = 0.002). Among the 37.1% of asymptomatic patients who were not screened for CD due to the absence of risk factors, only 34% were truly asymptomatic, while the remaining 66% reported non-specific CD-related symptoms. Therefore, expanding CD screening to any child who undergoes a blood test could reduce the burden of care for some children, as many of those considered asymptomatic reported non-specific CD-related symptoms.

Immunomodulatory effects of different strains of Lactococcus lactis in DSS-induced colitis.

Inflammatory bowel diseases (IBD) are gastrointestinal disorders characterized by a breakdown in intestinal homeostasis by inflammatory immune responses to luminal antigens. Novel strategies for ameliorating IBD have been proposed in many studies using animal models. Our group has demonstrated that administration of Lactococcus lactis NCDO 2118 can improve clinical parameters of colitis induced by oral administration of dextran sulphate sodium (DSS). However, it is not clear whether other strains of L. lactis can yield the same effect. The objective of present study was to analyze the effects of three different L. lactis strains (NCDO2118, IL1403 and MG1363) in the development of DSS-induced colitis in C57BL/6 mice. Acute colitis was induced in C57/BL6 mice by the administration of 2% DSS during 7 consecutive days. Body weight loss and shortening of colon length were observed in DSS-treated mice, and none of L. lactis strains had an impact in these clinical signs of colitis. On the other hand, all strains improved the global macroscopical disease index and prevented goblet cells depletion as well as the increase of intestinal permeability. TNF-α production was reduced in gut mucosa of L. lactis DSS-treated mice indicating a modulation of a critical pro-inflammatory response by all strains tested. However, only L. lactis NCDO2118 and MG1363 induced a higher frequency of CD11c+CD11b-CD103+ tolerogenic dendritic cells in lymphoid organs of mice at steady state. We conclude that all tested strains of L. lactis improved the clinical scores and parameters of colitis, which confirm their anti-inflammatory properties in this model of colitis.

Analysis of biofilm formation by Candida albicans in different types of orthodontic fixed appliances and devices / Análise da formação de biofilme por Candida albicans em diferentes tipos de aparelhos e dispositivos ortodônticos fixos

Objective: in this study, biofilm formation by Candida albicans in fixed orthodontic appliances was evaluated. Material and Methods: a total of 300 conventional metal brackets (MC), ceramic (CB), self-ligation (SLB), nickel-titanium (NiTi), and nickel-chromium (NiCr) wires, and ligatures types were organized into thirty groups (n=10). To induce biofilm formation, brackets, wires, and ligatures were joined, sterilized, placed in 24-well plates, contaminated with standardized suspensions of C. albicans (107 cells/mL), and incubated at 37 °C for 48 h with shaking. The biofilms formed were detached using an ultrasonic homogenizer, and suspensions were serially diluted and plated on Sabouraud dextrose agar to determine colony-forming units per mL. Scanning electron microscopy was performed before and after the biofilm formation. Results: lower amount of biofilm formation was observed in the MC group than in the CB and SLB groups (p<0.0001). SLB and CB showed similar biofilm formation rates (p=0.855). In general, the cross-sectional wires .018"x.025" showed higher biofilm formation when associated with the three types of brackets. When brackets, wires, and ligatures were associated, the sets with NiCr wires and SSL ligatures with MC brackets (p=0.0008) and CB (p=0.0003) showed higher biofilm formation. Conclusion: thus, brackets of MC with NiTi and NiCr wires showed lower biofilm formation, regardless of the ligature and cross-sectional or gauge of the wire and, MC and CB brackets with NiCr wires and SSL ligatures were more likely to accumulate biofilms (AU)

Objetivo: neste estudo, a formação de biofilme por Candida albicans em aparelhos ortodônticos fixos foi avaliada. Material e Métodos: um total de 300 bráquetes metálicos convencionais (MC), cerâmicos (CB), autoligados (SLB), com fios de níquel-titânio (NiTi) e níquel-cromo (NiCr) e tipos de ligaduras foram organizados em trinta grupos (n=10). Bráquetes, fios e ligaduras foram unidos, esterilizados, colocados em placas de 24 poços, contaminados com suspensões padronizadas de C. albicans (107 células/mL) e incubados a 37°C por 48 h para a formação de biofilmes. Os biofilmes formados foram rompidos por meio de um homogeneizador ultrassônico e suspensões foram diluídas e semeadas em ágar Sabouraud-dextrose para determinar as unidades formadoras de colônias por mL. A microscopia eletrônica de varredura foi realizada antes e após a formação do biofilme. Resultados: foi observada menor formação de biofilme no grupo MC em comparação aos grupos CB e SLB (p<0,0001). A formação de biofilme foi semelhante nos grupos SLB e CB (p=0,855). Em geral, os fios de seção transversal .018"x.025" apresentaram maior formação de biofilme quando associados aos três tipos de bráquetes. Os conjuntos com fios de NiCr e ligaduras SSL com bráquetes MC (p=0,0008) e CB (p=0,0003) apresentaram maior formação de biofilme. Conclusão: bráquetes MC com fios de NiTi e NiCr apresentaram menor formação de biofilme, independente da ligadura e secção transversal ou bitola do fio e, braquetes MC e CB com fios de NiCr e ligaduras SSL foram mais propensos a acumular biofilmes.(AU)

Repurposing HIV Protease Inhibitors Atazanavir and Darunavir as Antifungal Treatments against Candida albicans Infections: An In Vitro and In Vivo Study.

Candida albicans is the chief etiological agent of candidiasis, a mycosis prevalent in individuals with acquired immunodeficiency syndrome (AIDS). In recent years, the introduction of human immunodeficiency virus (HIV) protease inhibitors (HIV-PI) has reduced the prevalence of candidiasis in these patients. Seeking new therapeutic strategies based on the perspective of drug repositioning, we evaluated the effects of two second-generation HIV-PIs, atazanavir (ATV) and darunavir (DRV), on virulence factors of C. albicans and experimental candidiasis. For this, clinical strains of C. albicans were subjected to in vitro and in vivo treatments with ATV or DRV. As a result, ATV and DRV exhibited antifungal activity against fungal cells at 512 µg/mL, reduced the viability and biomass of biofilms, and inhibited filamentation of C. albicans. In addition, these HIV-PIs downregulated the expression of SAP2 and BRC1 genes of C. albicans. In an in vivo study, prophylactic use of ATV and DRV prolonged the survival rate of Galleria mellonella larvae infected with C. albicans. Therefore, ATV and DRV showed activity against C. albicans by reducing cell growth, biofilm formation, filamentation, and expression of virulence genes. Furthermore, ATV and DRV decreased experimental candidiasis, suggesting the repurposing of HIV-PIs as antifungal treatments for C. albicans infections.

Effects of α and ß-adrenergic signaling on innate immunity and Porphyromonas gingivalis virulence in an invertebrate model.

To investigate the role of adrenergic signalling (AS) in the host immune response and Porphyromonas gingivalis virulence, we compared norepinephrine (NE) and isoproterenol (ISO) responses in Galleria mellonella. P. gingivalis infection was evaluated by survival; humoral immune responses (i.e. melanization and cecropin and gloverin mRNA expression); cellular immune responses (i.e. haemocyte count, nodulation by histology); and P. gingivalis recovery (CFU/mL). P. gingivalis was cultivated in the presence of ISO (PgISO) or NE and injected into the larvae for survival evaluation. Finally, we co-injected ISO and PgISO to evaluate the concomitant effects on the immune response and bacterial virulence. None of the ligands were toxic to the larvae; ISO increased haemocyte number, even after P. gingivalis infection, by mobilizing sessile haemocytes in a ß-adrenergic-specific manner, while NE showed the opposite effect. ISO treatment reduced larval mortality and the number of recovered bacteria, while NE increased mortality and showed no effect on bacterial recovery. ISO and NE had similar effects on melanization and decreased the expression of cecropin. Although co-cultivation with NE and ISO increased the gene expression of bacterial virulence factors in vitro, only the injection of PgISO increased larval death, which was partially reversed by circulating ISO. Therefore, α- and ß-adrenergic signalling had opposite effects after P. gingivalis infection. Ultimately, the catecholamine influence on the immune response overcame the effect of more virulent strains. The effect of AS directly on the pathogen found in vitro did not translate to the in vivo setting.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

The Fagerström and AUDIT Tests as Probable Screening Tools in Oral Cancer and Their Correlation with CYP1A1, GSTM1, GSTP1, and GSTT1 Gene Expression.

BACKGROUND: Cancer is currently a major public health problem worldwide, with a marked increase of about 70% in the number of expected diagnosed cases over the next two decades. The amount of tobacco and alcohol consumed is calculated based on the subjective information provided by the user. Tobacco exposure can be assessed using the Fagerström Test for Cigarette Dependence (FTCD) and alcohol consumption by the Alcohol Use Disorder Identification Test (AUDIT). MATERIALS AND METHODS: Forty-eight subjects answered the Fagerström, and AUDIT tests and we studied them as likely screening tools for oral cancer and their correlation with the expression of CYP1A1, GSTM1, GSTP1, and GSTT1 genes by the RT-qPCR method. RESULTS: There were significant differences in the AUDIT score and CYP1A1 expression between cancer and control groups. Participants in advanced stages, whether due to tumor size or regional metastasis, showed significant differences in the duration of tobacco use, FTCD, AUDIT score, and CYP1A1 expression when compared to patients in early stages. Among subjects without cancer, we found a significant correlation between participant age and GSTP1 expression. Furthermore, the expression of GSTP1 was significantly correlated with the number of cigarettes smoked per day, duration of tobacco use, and FTCD. CONCLUSIONS: Questionnaires designed to evaluate the degree of tobacco and alcohol exposure and dependence combined with gene expression tests can be useful to assess the risk of developing oral cancer. Furthermore, raising the awareness of individuals regarding their degree of dependence and encouraging them to participate in cessation programs are important educational measures for the prevention of tobacco-related malignancies.

Essentials of oral manifestations of leukemia for the dental practitioner.

Oral lesions may be the initial or only manifestation of leukemia and can be the key to early diagnosis. The varied nature of presenting signs and dentists' general lack of familiarity with oral presentations makes diagnosis challenging. This retrospective review reports a series of cases of leukemia to familiarize dentists with the oral manifestations and facilitate earlier diagnosis or recognition of relapse of this life-threatening disease. Following institutional review board approval, the University of Florida Oral Pathology Biopsy Service archive from 1994 to 2018 was queried for all oral biopsies resulting in a diagnosis of leukemia. Cases with insufficient diagnostic information or extraoral manifestations were excluded. Demographic, clinical, and histologic findings were tabulated. Ten cases with 12 biopsy sites were identified. Men (n = 6) were affected more commonly. The mean age of the patients was 58.4 years (range of 17 to 88 years). The gingiva was the most frequently biopsied site (n = 6; 50%). Importantly, 40% of the patients (n = 4) had no prior diagnosis of leukemia. A wide spectrum of clinical impressions was rendered, pyogenic granuloma being the most common, and the reported duration of lesions ranged from several weeks to 6 months. The rarity of patients presenting with leukemia may lead to low levels of clinical suspicion, misdiagnosis, and delays in treatment. However, oral lesions may be the first and only manifestation of leukemia, and clinicians should be aware of the clinical characteristics of these oral presentations to ensure early diagnosis and treatment, thereby helping to reduce disease-related morbidity and mortality.

Natural latex serum: characterization and biocompatibility assessment using Galleria mellonella as an alternative in vivo model.

Natural latex serum (NLS) is one of the natural rubber latex fractions from Hevea brasiliensis tree, which is formed by centrifuged serum and is composed of proteins, acids, nucleotides, salts and carbohydrates. The proteins present in NLS have demonstrated several interesting biological properties, including angiogenic, healing, osteogenic, anti-inflammatory, antimicrobial, in addition to inducing neovascularization, bone formation and osseointegration. Thus, we proposed to characterize NLS by physicochemical techniques and to investigate the biocompatibility by toxicological assays and safety test in Galleria mellonella. Infrared spectrum showed vibrational bands characteristic of amide I, II and III that are linked to the protein content, which was confirmed by the High Performance Liquid Chromatography profile and by the Electrophoresis analysis. This material did not exhibit hemolytic (rate <0.5%) and cytotoxic effects (viability >70%) and was able to enhance the proliferation of fibroblasts (>600%) after 3 days. The pronounced proliferative effect observed in fibroblast cells can be explained by the presence of the fibroblast growth factor (FGF) like protein revealed by the Western blot test. Moreover, NLS did not provoke toxic effects (survival ∼ 80%) on the G. mellonella model, indicating that it is a biocompatible and safe material.

Contenção ambiental de idosos nas instituições de longa permanência em tempos de Covid-19: reflexão teórica / Contención ambiental de ancianos en las instituciones de larga permanencia en tiempos de Covid-19: reflexión teórica / Environmental restraint of the elderly in long-term care facilities in times of Covid-19: theoretical reflection

Estudo teórico de cunho reflexivo que aborda o fenômeno da contenção ambiental em instituições de longa permanência para idosos, apresentando o tema à luz dos direitos humanos e da legislação em vigor, que pregam boas práticas de cuidado aos idosos institucionalizados, inclusive frente ao cenário de enfrentamento à Covid-19. Considerando os impactos negativos da contenção ambiental na saúde física e mental dos idosos, sugere-se maior capacitação da equipe técnica das instituições de longa permanência para idosos e prospecção do fenômeno para garantia de condições mais dignas e que respeitem a liberdade dos idosos. (AU)

Estudio teórico de cuño reflexivo que aborda el fenómeno de la contención ambiental en instituciones de larga permanencia para ancianos, presentando el tema a la luz de los derechos humanos y de la legislación en vigor que proponen buenas prácticas de cuidado para los ancianos institucionalizados, incluso ante el escenario del enfrentamiento a la Covid-19. Considerando los impactos negativos de la contención ambiental en la salud física y mental de los ancianos, se sugiere mayor capacitación del equipo técnico de las instituciones de larga permanencia para ancianos y prospección del fenómeno para garantía de condiciones más dignas y que respeten la libertad de los ancianos. (AU)

A theoretical and reflexive study addressing the phenomenon of environmental restraint in long-term care facilities for the elderly, presenting the topic under the light of human rights and the legislation in force, which postulate good care practices to institutionalized elderly even in the face of the Covid-19 situation. By considering the negative impacts of environmental restraint on the physical and mental health of the elderly, it is suggested that the technical team of long-term care institutions for the elderly should be better trained, and that the phenomenon needs to be explored to guarantee more dignified conditions that respect the freedom of the elderly. (AU)

Distribution of physiotherapy professionals and services in public health in Goiás: coverage according to socioeconomic variables / Distribuição dos profissionais e serviços de fisioterapia na saúde pública em Goiás: cobertura assistencial de acordo com variáveis socioeconômicas

Abstract Introduction Public health in Brazil goes beyond social production, with an expanded view of health. Its complex structure includes physical therapy. Objective To characterize physical therapy in Goiás based on the National Registry of Health Facilities (CNES in Portuguese). Methods Data on specialty, municipality, place of employment, working hours, employment relationship, type of contract and establishment, number of physical therapists, administrative setting, macroregion and health region were tabulated. The following were correlated: municipal population; people dependent on the National Health System (SUS in Portuguese); municipal per capita gross domestic product (GDP); municipal Human Development Index (HDI); percentage of inhabitants dependent on the municipal SUS; number of people covered by municipal physiotherapy (NPCAF). Results In the CNES, (reference July/2019) in Goiás, we found 2,187 physiotherapists in public/private facilities, in 3,353 workplaces. In establishments that serve the SUS, we identified 1,673 physiotherapists in 2,436 workplaces, 32.9% of which are linked to primary and 67.1% to specialized care. The weekly workload was 38 hours and 26 hours per workplace. Only 30.1% of the municipalities had adequate NPCAF. Health regions and macroregions had inadequate coverage. The NPCAF showed an inverse correlation with GDP (r = -0.20; p < 0.001) and HDI (r = -0.14; p = 0.02) and a direct correlation with the population (r = 0.46; p < 0.001). Conclusion Goiás has inadequate coverage in 69.9% of the municipalities, in the macroregions and health regions. Expanding the number of workplaces available to physiotherapists is essential to achieve universal and comprehensive health care, reducing inequity and favoring equitable distribution.

Resumo Introdução A saúde pública no Brasil encontra na saúde coletiva uma transcendência com a produção social, com olhar ampliado à saúde. Na sua complexa estrutura, oferta assistência fisioterapêutica. Objetivo Caracterizar a fisioterapia em Goiás a partir do Cadastro Nacional de Estabelecimentos de Saúde (CNES). Métodos Tabularam-se dados de especialidade, município, estabelecimento laboral, carga horária, vinculação, tipo de contrato e estabelecimento, número de fisioterapeutas, esfera administrativa, macrorregião e região de saúde. Correlacionou-se população geral municipal; pessoas dependente/Sistema Único de Saúde (SUS); Produto Interno Bruto (PIB) per capita municipal; Índice de Desenvolvimento Humano (IDH) municipal; percentual populacional de habitantes dependentes do SUS municipal; número de pessoas cobertas por assistência fisioterapêutica (NPCAF) municipal. Resultados No CNES, referência julho/2019, foram encontrados 2187 fisioterapeutas em estabelecimentos públicos/privados de Goiás, em 3353 postos de trabalho. Nos estabelecimentos que atendem ao SUS foram detectados 1673 fisioterapeutas em 2436 postos de trabalho, sendo 32,9% vinculados à atenção básica e 67,1% à atenção especializada. A carga horária semanal foi de 38 horas e por postos de trabalho, 26 horas. Apenas 30,1% dos municípios tinham NPCAF adequados. As regiões e macrorregiões de saúde apresentaram cobertura inadequada. O NPCAF apresentou correlação inversa com o PIB (r = -0,20; p < 0,001) e IDH (r = -0,14; p = 0,02) e direta com a população (r = 0,46; p < 0,001). Conclusão Goiás apresenta cobertura deficitária em 69,9% dos municípios, nas macrorregiões e regiões de saúde. Expandir postos de fisioterapia é fundamental para a efetivação da universalidade e integralidade da atenção à saúde, reduzindo iniquidade e favorecendo distribuição equânime.

Influence of nicotine dependence, current smoking status and gender on CYP1A1, CYP1B1 and CYP2A6 expression in oral squamous cell carcinoma / Influência da dependência nicotínica, parâmetros de consumo de tabaco e gênero na expressão de CYP1A1, CYP1B1 e CYP2A6 em carcinoma de células escamosas oral

Objective: Tobacco smoke is composed of cancer-causing chemicals referred to as carcinogens. These carcinogens are metabolized by the enzymes of the cytochrome P450 (CYP) family. Our objective was to evaluate the correlation of tobacco consumption parameters with CYP1A1, CYP1B1 and CYP2A6 expression using qRT-PCR in samples of oral squamous cell carcinoma (OSCC). Material and Methods: The sample was divided into 2 groups: Cancer (36 subjects) and non-Cancer (12 subjects). The smokers' participants (36) were evaluated regarding their Nicotine dependence (ND) was assessed by the Fagerström test for cigarette dependence (FTCD). Questions regarding tobacco consumption like the number of cigarettes/day (CPD), duration of use, and pack-years were also evaluated. The Mann-Whitney and Spearman correlation tests were used at a significance level of 5%. Results: 48 participants were included, 32 men (66.7%), 36 smokers (75%) and 27 smokers with OSCC (56.3%). Samples of OSCC expressed more CYP1A1, CYP1B1, and CYP2A6. Especially, the CYP1B1 gene was significantly expressed in OSCC samples, regardless gender or tobacco use. No women expressed CYP2A6, as well as, non-smokers did not express the CYP1A1 and CYP2A6 genes. CYP1A1 gene was higher among men (P = 0.021). Conclusion: Lack of exposure to tobacco may justify the absence of CYP1A1 and CYP2A6 expression in non-smokers. The CYP1B1 gene was significantly expressed in the cancer presence despite gender or tobacco use. The assessment of ND and quantification of tobacco consumption are important instruments in monitoring smokers with benign oral lesions and, especially, in the presence of cancer.(AU)

Objetivo: A fumaça do tabaco é composta de substâncias químicas cancerígenas conhecidas como carcinógenos. Esses carcinógenos são metabolizados pelas enzimas da família do citocromo P450 (CYP). Nosso objetivo foi avaliar a correlação dos parâmetros do consumo de tabaco com a expressão de CYP1A1, CYP1B1 e CYP2A6 por qRT-PCR em amostras de carcinoma de células escamosas bucal (CCEB). Material e Métodos: A amostra foi dividida em 2 grupos: Câncer (36 indivíduos) e sem Câncer (12 indivíduos). Os participantes fumantes (36) foram avaliados quanto à dependência nicotínica (DN) pelo teste de Fagerström para dependência de cigarro (TFDC). Questões relacionadas ao consumo de tabaco como número de cigarros / dia (CPD), tempo de uso e anos-maço também foram avaliadas. Os testes de correlação de Mann-Whitney e Spearman foram utilizados com nível de significância de 5%. Resultados: foram incluídos 48 participantes, 32 homens (66,7%), 36 fumantes (75%) e 27 fumantes com CCEB (56,3%). Amostras de CCEB expressaram mais CYP1A1, CYP1B1 e CYP2A6. Especialmente, o gene CYP1B1 foi significativamente expresso em amostras de CCEB, apesar do sexo ou uso de tabaco. Nenhuma mulher expressou CYP2A6, assim como, não fumantes não expressaram os genes CYP1A1 e CYP2A6. O gene CYP1A1 foi maior entre os homens (P = 0,021). Conclusão: A falta de exposição pode justificar a ausência da expressão dos genes CYP1A1 e CYP2A6 entre não fumantes. O gene CYP1B1 foi significativamente expresso na presença de câncer, independentemente do sexo ou do uso de tabaco. A avaliação da DN e a quantificação do consumo de tabaco são importantes instrumentos no acompanhamento de fumantes com lesões bucais benignas e, principalmente, na presença de câncer (AU)

Treinamento cardiorrespiratório e força dos músculos ventilatórios de idosas: ensaio controlado randomizado duplo cego / Cardiorespiratory training and ventilatory muscle strength in elderly women: double blind randomized controlled trial

A dinapenia da musculatura ventilatória relaciona-se com as complicações respiratórias pós-operatórias e aos óbitos de idosos submetidos a procedimentos cirúrgicos de etiologia toracoabdominal. Objetivo: Verificar o efeito crônico do treinamento cardiorrespiratório sobre a força dos músculos ventilatórios de idosas. Métodos: Amostra (n = 24) randomizada nos grupos: controle (n = 8), treinamento ventilatório (n = 7) e treinamento cardiorrespiratório (n = 9). O desfecho primário (PImáx e PEmáx) foi medido por um manovacuômetro digital MDV®300 (MDI/Brasil). Rodou-se a estatística descritiva (média e desvio padrão), seguida de uma ANOVA 3x3 e o testes post-hoc de Bonferroni, todos com significância de (P ≤ 0,05). Resultados: O teste Post Hoc atestou diferença estatisticamente significativa do grupo treinamento cardiorrespiratório no reteste 1 e 2 quando comparados ao teste inicial (PImáx; P = 0,000001 e P = 0,0000001 respectivamente) e (PEmáx; P = 0,000000 em ambas as comparações). Conclusão: O treinamento cardiorrespiratório proposto foi capaz de aumentar significativamente a PImáx, PEmáx, resistência aeróbica e a força de membros inferiores das participantes deste ECR. (AU)

The Anti-Biofilm Efficacy of Caffeic Acid Phenethyl Ester (CAPE) In Vitro and a Murine Model of Oral Candidiasis.

Candida albicans is the main fungal species associated with the development of oral candidiasis. Currently, therapeutic options for these infections are limited by the adverse effects of antifungal drugs and by the emergence of drug resistant strains. Thus, the development of new antifungal agents is needed for the prevention and treatment of oral Candida infections. Caffeic acid phenethyl ester (CAPE) is a natural compound from propolis polyphenolic groups that exhibits many pharmacological properties. In this study, we investigated whether CAPE can have antifungal and immunomodulatory effects on oral candidiasis. Preliminary tests to assess the antifungal activity of CAPE were performed using the Minimum Inhibitory Concentration (MIC) assay that demonstrated inhibition in a range from 16 to 32 µg/mL, confirming its antifungal activity on several C. albicans strains isolated from the oral cavity. Subsequently, we analyzed Candida spp biofilms formed in vitro, in which CAPE treatment at 5 x MIC caused a reduction of 68.5% in the total biomass and ~2.60 Log in the viable cell count (CFU/mL) in relation to the untreated biofilm (p<0.0001). Next, RNA was extracted from untreated and CAPE-treated biofilms and analyzed by real-time qPCR. A series of genes analyzed (ALS1, ECE1, EPA1, HWP1, YWP1, BCR1, BGR1, CPH1, EFG1, NDT80, ROB1, TEC1, UME6, SAP2, SAP5, PBL2, and LIP9) were downregulated by CAPE compared to the untreated control group (p<0.0001). In in vivo studies using Galleria mellonella, the treatment with CAPE prolonged survival of larvae infected by C. albicans by 44.5% (p < 0.05) and accompanied by a 2.07-fold increase in the number of hemocytes. Flow cytometry revealed the most prominent increases were in types P2 and P3 hemocytes, granular cells, which phagocytize pathogens. In addition, CAPE treatment decreased the fungal load in the hemolymph and stimulated the expression of antifungal peptide genes such as galiomicin and gallerimycin. The antifungal and immunomodulatory activities observed in G. mellonella were extended to a murine model of oral candidiasis, in which CAPE decreased the levels of C. albicans colonization (~2 log CFU/mL) in relation to the untreated control group. In addition, CAPE treatment significantly reduced pseudomembranous lesions, invasion of hyphae on epithelium surfaces, tissue damage and inflammatory infiltrate (p < 0.05). CAPE was also able to increase the expression of ß-defensin 3 compared to the infected and untreated group by 3.91-fold (p < 0.0001). Taken together, these results show that CAPE has both antifungal and immunomodulatory effects, making it a promising natural antifungal agent for the treatment and prevention of candidiasis and shows impact to oral candidiasis.

LDLR variants functional characterization: Contribution to variant classification.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. LDLR mutations are the cause of disease in 90% of the cases but functional studies have only been performed for about 15% of all LDLR variants. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142 unique LDLR alterations were identified and 44 (30%) lack functional characterization. The aim of the present work is to increase evidence for variant classification by performing functional characterization of 13 LDLR missense alterations found in Portugal and in 20 other countries. METHODS: Different LDLR mutants were generated by site-directed mutagenesis and expressed in CHO-ldlA7 cells lacking endogenous expression of LDLR. To determine the effects of alterations on LDLR function, cell surface expression, binding and uptake of FITC-LDL were assessed by flow cytometry and Western blot. RESULTS: Of the 13 variants studied 7 were shown to affect LDLR function - expression, binding or uptake, with rates lower than 60%: p.(Cys184Tyr), p.(Gly207_Ser213del); p.(His211Asp); p.(Asp221Tyr); p.(Glu288Lys); p.(Gly592Glu) and p.(Asp601Val)). The remaining 6 variants do not alter the LDLR function. CONCLUSIONS: These studies contributed to an update of these variants classification: from the 9 variants classified as variants of unknown significance, 7 have reached now a final classification and 3 variants have improved classification from likely pathogenic to pathogenic. In Portugal, an additional 55 patients received an FH definite diagnosis thanks to these studies. Since only likely pathogenic and pathogenic variants are clinically actionable, this work shows the importance of functional studies for variant classification.

Antivirulence activity and in vivo efficacy of a thiazole derivative against candidiasis.

Candida albicans is a pathogen equipped with a variety of commensal and virulence traits that help it colonize the microbiota and invade host tissue during infection. In this study, we investigated the potential anticandidal activity of 3-[2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazino)]butan-1-ol (MT), a thiazolylhydrazone compound synthesized by our group, and identified it as a promising antifungal agent. The activity of MT was evaluated in vitro and in vivo against C. albicans as well as its ability to inhibit virulence factors. For this, the ability of MT to inhibit the adhesion of C. albicans to human buccal epithelial cells and biofilm formation and filamentation was tested. In addition, the potential in vivo activity of MT was evaluated in murine models of oral candidiasis. Our results confirmed the antifungal activity of MT, with a minimal inhibitory concentration range of 0.5-2 µg/mL. Indeed, MT treatment in vitro decreased the expression of C. albicans genes involved in biofilm formation and morphogenesis and encoding hydrolytic enzymes, which was also confirmed through phenotypic observations. In addition, MT promoted a decrease in the colony forming units recovered from the tongues of mice with oral candidiasis. In this work, we present a potent antivirulence compound that shows potential for candidiasis therapy, especially for topical use.