Unraveling Drug Delivery from Cyclodextrin Polymer-Coated Breast Implants: Integrating a Unidirectional Diffusion Mathematical Model with COMSOL Simulations.

Breast cancer ranks among the most commonly diagnosed cancers worldwide and bears the highest mortality rate. As an integral component of cancer treatment, mastectomy entails the complete removal of the affected breast. Typically, breast reconstruction, involving the use of silicone implants (augmentation mammaplasty), is employed to address the aftermath of mastectomy. To mitigate postoperative risks associated with mammaplasty, such as capsular contracture or bacterial infections, the functionalization of breast implants with coatings of cyclodextrin polymers as drug delivery systems represents an excellent alternative. In this context, our work focuses on the application of a mathematical model for simulating drug release from breast implants coated with cyclodextrin polymers. The proposed model considers a unidirectional diffusion process following Fick's second law, which was solved using the orthogonal collocation method, a numerical technique employed to approximate solutions for ordinary and partial differential equations. We conducted simulations to obtain release profiles for three therapeutic molecules: pirfenidone, used for preventing capsular contracture; rose Bengal, an anticancer agent; and the antimicrobial peptide KR-12. Furthermore, we calculated the diffusion profiles of these drugs through the cyclodextrin polymers, determining parameters related to diffusivity, solute solid-liquid partition coefficients, and the Sherwood number. Finally, integrating these parameters in COMSOL multiphysics simulations, the unidirectional diffusion mathematical model was validated.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Snake and arthropod venoms: search for inflammatory activity in human cells involved in joint diseases

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1β, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and β-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Evaluation of lymphocyte count, T-cell subsets and neutrophil-to-lymphocyte ratio as early predictors for severity and outcome of COVID-19 disease-a report from a highly complex hospital in Brazil

ABSTRACT Introduction: Lymphopenia is a laboratory marker of poor prognosis and severity of disease in the SARS-CoV-2 infection. This study aims to describe the immune profile of a Brazilian population. Methods: A total of 121 consecutive patients with severe acute respiratory syndrome (SARS) were analyzed between April and June 2020. Routine peripheral blood counts and multiparametric flow cytometry were performed on admission to assess lymphocytes and subsets (CD3, CD4, CD8). Demographic, clinical and laboratory data were collected from hospital sources. Results: The total of 116 patients included 63 (54.3%) males; 76 (62.8%) COVID-19 patients were divided, based on clinical characteristics and mechanical ventilation (MV) use, into moderate (n = 41; no MV) and severe (n = 35; MV) groups. The control group (n = 40) was comprised of patients with SARS of different etiologies. All patients had lymphopenia, with overall lymphocyte counts and their subsets considerably lower in severe patients, when compared to the moderate and controls. Patients with a high neutrophil-to-lymphocyte ratio (> 15.2) and T-cell lymphopenia (CD3 < 593 cells/μL, CD4 < 326 cells/μL, CD8 < 121 cells/μL) had a higher risk of being intubated and progressing to death. A total of 39 patients (95.1%) in the moderate group and 54.3% (n = 19) in the severe group were discharged; 28 patients died. Conclusion: Laboratory assessment of the neutrophil/lymphocyte ratio and T-cell subsets may be predictive of mortality and may be useful for stratifying COVID-19 patients.

Prognostic impact of molecular profiles and molecular signatures in clear cell ovarian cancer.

OBJECTIVE: Ovarian Clear cell carcinomas (OCCC) are characterized by low response to chemotherapy and a poor prognosis in advanced stages. Several studies have demonstrated that OCCC are heterogenous entities. We have earlier identified four molecular profiles based on the mutational status of ARID1A and PIK3CA. In this study we aimed to examine the association between molecular profiles, Tumor Mutational Burden (TMB), and molecular signatures with the clinical outcome in OCCC METHODS: We identified 55 OCCC cases with corresponding data and biological tissue samples in the Danish Gynecological Cancer Database during 2005-2016. Mutational profiling and TMB were performed using the Oncomine Tumor Mutational Load Assay. Chi-square and Cox regression analyses were used. P-values < 0.05 were considered statistically significant. RESULTS: Mutations in the PIK3CA gene (p=0.04) and low TMB (p=0.05) were associated with disease progression. In multivariate analyses adjusted for stage, patients with tumor mutations in the ARID1A and/or PIK3CA genes had a significantly impaired Progression Free Survival (PFS) and Overall Survival (OS) compared to patients who were wildtype ARID1A and PIK3CA (undetermined subgroup) (HR= 5.42 and HR= 2.77, respectively). High TMB status was associated with an improved PFS (HR= 0.36) and OS (HR= 0.46). A trend towards an improved PFS in patients with APOBEC enrichment was observed (HR 0.45). CONCLUSION: TMB-High was associated with decreased risk of progression and with an improved PFS and OS. Furthermore, OCCC with mutations in either ARID1A and/or PIK3CA genes had a significantly impaired prognosis compared to the undetermined subgroup in stage adjusted analyses.

Strategies for data normalization and missing data imputation and consequences for potential diagnostic microRNA biomarkers in epithelial ovarian cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression with diagnostic potential in different diseases, including epithelial ovarian carcinomas (EOC). As only a few studies have been published on the identification of stable endogenous miRNA in EOC, there is no consensus which miRNAs should be used aiming standardization. Currently, U6-snRNA is widely adopted as a normalization control in RT-qPCR when investigating miRNAs in EOC; despite its variable expression across cancers being reported. Therefore, our goal was to compare different missing data and normalization approaches to investigate their impact on the choice of stable endogenous controls and subsequent survival analysis while performing expression analysis of miRNAs by RT-qPCR in most frequent subtype of EOC: high-grade serous carcinoma (HGSC). 40 miRNAs were included based on their potential as stable endogenous controls or as biomarkers in EOC. Following RNA extraction from formalin-fixed paraffin embedded tissues from 63 HGSC patients, RT-qPCR was performed with a custom panel covering 40 target miRNAs and 8 controls. The raw data was analyzed by applying various strategies regarding choosing stable endogenous controls (geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder), missing data (single/multiple imputation), and normalization (endogenous miRNA controls, U6-snRNA or global mean). Based on our study, we propose hsa-miR-23a-3p and hsa-miR-193a-5p, but not U6-snRNA as endogenous controls in HGSC patients. Our findings are validated in two external cohorts retrieved from the NCBI Gene Expression Omnibus database. We present that the outcome of stability analysis depends on the histological composition of the cohort, and it might suggest unique pattern of miRNA stability profiles for each subtype of EOC. Moreover, our data demonstrates the challenge of miRNA data analysis by presenting various outcomes from normalization and missing data imputation strategies on survival analysis.

Somatic Variants in DNA Damage Response Genes in Ovarian Cancer Patients Using Whole-exome Sequencing.

BACKGROUND/AIM: Several clinical trials have investigated homologous recombination deficiency and BRCA1/2 status to select ovarian cancer patients for treatment with poly(ADP-ribose) polymerase-inhibitors (PARPi), but less attention has been given to other DNA-damage response (DDR) pathways. Therefore, we investigated somatic single/multiple nucleotide variants and small insertions/deletions in exonic and splice-site regions of 356 DDR genes to examine whether genes other than BRCA1/2 are altered. MATERIALS AND METHODS: Whole-exome sequencing data from eight high-grade serous adenocarcinoma (HGSC) and four clear cell carcinoma (oCCC) patients were analyzed. RESULTS: Forty-two variants (pathogenic, likely pathogenic or variants of uncertain significance) in 28 genes from DDR pathways were identified. Seven out of nine TP53 variants were previously described in The Cancer Genome Atlas Ovarian Cancer; other variants were found in 23 out of 28 unique genes, whereas no variants were reported in FAAP24, GTF2H4, POLE4, RPA3, and XRCC4. CONCLUSION: As the identified variants were not only limited to well-known TP53, BRCA1/2, and HR-associated genes, our study might contribute to the better understanding of which DDR pathways potentially influence disease progression. Moreover, they may display a potential role as biomarkers to predict platinum-based chemotherapy or PARPi treatment response or disease progression, as differences in disrupted DDR pathways were observed between patients with long and short overall survival in HGSC and oCCC groups.

ICU Admission, Invasive Mechanical Ventilation, and Mortality among Children and Adolescents Hospitalized for COVID-19 in a Private Healthcare System.

Aim: The COVID-19 pandemic devastated healthcare around the world. Data about the COVID-19 outcomes among young people are still scarce. We aim to identify factors associated with the composite outcome among children and adolescents hospitalized due to COVID-19. Methods: We performed a search in the database of a large Brazilian private healthcare system. Insured people aged 21 years or younger who were hospitalized due to COVID-19 from Feb/28th/2020 to Nov/1st/2021 were included. The primary endpoint was the composite outcome consisting of ICU admission, need for invasive mechanical ventilation, or death. Results: We evaluated 199 patients who had an index hospitalization due to COVID-19. The median monthly rate of index hospitalization was 2.7 (interquartile range [IQR], 1.6-3.9) per 100,000 clients aged 21 years or less. The median age of the patients was 4.5 years (IQR, 1.4-14.1). At the index hospitalization, the composite outcome rate was 26.6%. The composite outcome was associated with all the previous coexisting morbidities evaluated. The median follow-up was 249.0 days (IQR, 152.0-438.5). There were 27 readmissions (16 patients) within 30 days after the discharge. Conclusions: In conclusion, hospitalized children and adolescents had a composite outcome rate of 26.6% at the index hospitalization. Having previous chronic morbidity was associated with the composite.

Endodontia guiada: tecnologia aplicada na resolução de tratamentos de canais calcificados / Guided endodontics: technology applied in the resolution of treatments of calcified canals / Endodoncia guiada: tecnología aplicada en la resolución de tratamientos de conductos calcificados

As calcificações pulpares provocam alterações morfológicas no interior dos canais radiculares que dificultam o tratamento endodôntico. Para solucionar essa dificuldade, a ferramenta Endoguide ou Endodontia Guiada foi desenvolvida para a resolução de casos complexos. Esta pesquisa objetivou descrever as aplicações da ferramenta Endoguide no tratamento de canais calcificados na endodontia. Todas as buscas foram realizadas por um único pesquisador na base de dados PubMed/MEDLINE e busca manual utilizando os descritores "Endodontics", "Digital", "Cone beam", "Guided". Foram considerados elegíveis os artigos publicados entre 2010 e 2022 e excluídos os estudos cuja publicação não foi obtida na íntegra e ainda aqueles em que os resultados não apresentaram embasamento teórico e prático suficientes para que pudessem ser incluídos no trabalho. Após as buscas, foram encontrados 47 artigos, selecionados inicialmente pelo título e resumos, excluindo as duplicatas. Ao final, a leitura completa e coleta de dados foi realizada em 6 artigos. As informações relevantes dos artigos selecionados foram transcritas em forma de quadro para sumarizar os achados. A endodontia guiada pode ser uma excelente alternativa para tratamentos de casos complexos, necessitando um investimento financeiro mínimo no consultório, pois os equipamentos de captura de imagem, planejamento virtual e de guias podem ser realizados em laboratórios capacitados. Assim, concluímos que o emprego da Endoguide como ferramenta no tratamento de canais calcificados se mostrou bem-sucedido e sua utilização não requer um conhecimento amplo, podendo ser usada por profissionais menos experientes.

Pulp calcifications cause morphological changes inside the root canals that make endodontic treatment difficult. To solve this difficulty, the tool Endoguide or Guided Endodontics was developed to solve complex cases. This research aimed to describe the applications of the Endoguide tool in the treatment of calcified root canals in endodontics. All searches were performed by a single researcher in the PubMed/MEDLINE database and manual search using the descriptors "Endodontics", "Digital", "Cone beam", "Guided". Articles published between 2010 and 2022 were considered eligible, and studies whose publication was not obtained in full text were excluded, as well as those in which the results did not have sufficient theoretical and practical basis for them to be included in the study. After the searches, 47 articles were found, initially selected by title and abstract, excluding duplicates. In the end, the complete reading and data collection was performed with 6 articles. Relevant information from the selected articles was transcribed for a table to summarize the findings. Guided endodontics can be an excellent alternative for treating complex cases, requiring minimal financial investment in the office, as image capture equipment, virtual planning and guides can be performed in trained laboratories. Thus, we conclude that the use of Endoguide as a tool in the treatment of calcified canals proved to be successful and its use does not require extensive knowledge and can be used by less experienced professionals.

Las calcificaciones pulpares provocan cambios morfológicos en el interior de los conductos radiculares que dificultan el tratamiento endodóntico. Para solventar esta dificultad se desarrolló la herramienta Endoguide o Endodoncia Guiada para resolver casos complejos. El objetivo de esta investigación fue describir las aplicaciones de la herramienta Endoguide en el tratamiento de conductos radiculares calcificados en endodoncia. Todas las búsquedas fueron realizadas por un único investigador en la base de datos PubMed/MEDLINE y búsqueda manual utilizando los descriptores "Endodontics", "Digital", "Cone beam", "Guided". Se consideraron elegibles los artículos publicados entre 2010 y 2022, y se excluyeron los estudios cuya publicación no se obtuvo a texto completo, así como aquellos en los que los resultados no tenían suficiente base teórica y práctica para ser incluidos en el estudio. Tras las búsquedas, se encontraron 47 artículos, seleccionados inicialmente por título y resumen, excluyendo los duplicados. Al final, se realizó la lectura completa y la recogida de datos con 6 artículos. La información relevante de los artículos seleccionados se transcribió para elaborar una tabla que resumiera los hallazgos. La endodoncia guiada puede ser una excelente alternativa para el tratamiento de casos complejos, requiriendo una mínima inversión financiera en el consultorio, ya que los equipos de captura de imágenes, la planificación virtual y las guías pueden realizarse en laboratorios capacitados. Así, concluimos que el uso de la Endoguía como herramienta en el tratamiento de conductos calcificados demostró ser exitoso y su uso no requiere de grandes conocimientos y puede ser utilizado por profesionales menos experimentados.

Evaluation of lymphocyte count, T-cell subsets and neutrophil-to-lymphocyte ratio as early predictors for severity and outcome of COVID-19 disease-a report from a highly complex hospital in Brazil.

INTRODUCTION: Lymphopenia is a laboratory marker of poor prognosis and severity of disease in the SARS-CoV-2 infection. This study aims to describe the immune profile of a Brazilian population. METHODS: A total of 121 consecutive patients with severe acute respiratory syndrome (SARS) were analyzed between April and June 2020. Routine peripheral blood counts and multiparametric flow cytometry were performed on admission to assess lymphocytes and subsets (CD3, CD4, CD8). Demographic, clinical and laboratory data were collected from hospital sources. RESULTS: The total of 116 patients included 63 (54.3%) males; 76 (62.8%) COVID-19 patients were divided, based on clinical characteristics and mechanical ventilation (MV) use, into moderate (n = 41; no MV) and severe (n = 35; MV) groups. The control group (n = 40) was comprised of patients with SARS of different etiologies. All patients had lymphopenia, with overall lymphocyte counts and their subsets considerably lower in severe patients, when compared to the moderate and controls. Patients with a high neutrophil-to-lymphocyte ratio (> 15.2) and T-cell lymphopenia (CD3 < 593 cells/µL, CD4 < 326 cells/µL, CD8 < 121 cells/µL) had a higher risk of being intubated and progressing to death. A total of 39 patients (95.1%) in the moderate group and 54.3% (n = 19) in the severe group were discharged; 28 patients died. CONCLUSION: Laboratory assessment of the neutrophil/lymphocyte ratio and T-cell subsets may be predictive of mortality and may be useful for stratifying COVID-19 patients.

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes.

Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips.

Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-ß-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0−6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.

Potential Targeted Therapies in Ovarian Cancer.

BACKGROUND: We aimed to identify somatic pathogenic and likely pathogenic mutations using next-generation sequencing (NGS). The mutational findings were held against clinically well-described data to identify potential targeted therapies in Danish patients diagnosed with high-grade serous ovarian cancer (HGSC). METHODS: We characterized the mutational profile of 128 HGSC patients. Clinical data were obtained from the Danish Gynecological Database and tissue samples were collected through the Danish CancerBiobank. DNA was analyzed using NGS. RESULTS: 47 (37%) patients were platinum-sensitive, 32 (25%) partially platinum-sensitive, 35 (27%) platinum-resistant, and three (2%) platinum-refractory, while 11 (9%) patients did not receive chemotherapy. Overall, 27 (21%) had known druggable targets. Twelve (26%) platinum-sensitive patients had druggable targets for PARP inhibitors: one for tyrosine kinase inhibitors and one for immunotherapy treatment. Eight (25%) partially platinum-sensitive patients had druggable targets: seven were eligible for PARP inhibitors and one was potentially eligible for alpesilib and hormone therapy. Seven (20%) platinum-resistant patients had druggable targets: six (86%) were potentially eligible for PARP inhibitors, one for immunotherapy, and one for erdafitinib. CONCLUSIONS: PARP inhibitors are the most frequent potential targeted therapy in HGSC. However, other targeted therapies remain relevant for investigation according to our mutational findings.

Increased platelet activation and platelet-inflammasome engagement during chikungunya infection.

Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. We prospectively included 132 patients attended at the Quinta D'Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP3, caspase 4, and cleaved IL-1ß, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.

A Kunitz-type inhibitor from tick salivary glands: A promising novel antitumor drug candidate.

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Proteomic Analysis Identifies Molecular Players and Biological Processes Specific to SARS-CoV-2 Exposure in Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

Platelet proteome reveals features of cell death, antiviral response and viral replication in covid-19.

Coronavirus disease 2019 (COVID-19) has affected over 400 million people worldwide, leading to 6 million deaths. Among the complex symptomatology of COVID-19, hypercoagulation and thrombosis have been described to directly contribute to lethality, pointing out platelets as an important SARS-CoV-2 target. In this work, we explored the platelet proteome of COVID-19 patients through a label-free shotgun proteomics approach to identify platelet responses to infection, as well as validation experiments in a larger patient cohort. Exclusively detected proteins (EPs) and differentially expressed proteins (DEPs) were identified in the proteomic dataset and thus classified into biological processes to map pathways correlated with pathogenesis. Significant changes in the expression of proteins related to platelet activation, cell death, and antiviral response through interferon type-I were found in all patients. Since the outcome of COVID-19 varies highly among individuals, we also performed a cross-comparison of proteins found in survivors and nonsurvivors. Proteins belonging to the translation pathway were strongly highlighted in the nonsurvivor group. Moreover, the SARS-CoV-2 genome was fully sequenced in platelets from five patients, indicating viral internalization and preprocessing, with CD147 as a potential entry route. In summary, platelets play a significant role in COVID-19 pathogenesis via platelet activation, antiviral response, and disease severity.

miRNA Expression in Ovarian Cancer in Fresh Frozen, Formalin-fixed Paraffin-embedded and Plasma Samples.

BACKGROUND/AIM: MicroRNAs (miRNAs) are small noncoding RNAs involved in gene expression regulation and have been investigated as potential biomarkers for various diseases, including ovarian cancer (OC). However, lack of standardized protocols regarding e.g., RNA isolation, cDNA synthesis, spike-in controls for experimental steps, and data normalization, impacts cross validation of results across research groups and hinders implementation of miRNAs as clinical biomarkers. MATERIALS AND METHODS: RNA was isolated from matching fresh-frozen tissue (FF), formalin-fixed paraffin embedded (FFPE) tissue, and plasma samples from twenty women diagnosed with OC using three commercial kits: miRNeasy Tissue/Cells, miRNeasy FFPE, and miRNeasy Serum/Plasma (Qiagen, Copenhagen, Denmark). RNA isolation, cDNA synthesis, and PCR performance were tested using miRCURY LNA miRNA Quality Control PCR (QC) Panels (Qiagen). Finally, miRNA stability was assessed using five algorithms: BestKeeper, Normfinder, GeNorm, comparative delta-Ct and comprehensive ranking provided by a web-based RefFinder tool. RESULTS: RNA from FF, FFPE and plasma was extracted using commercially available kits and the differences in yield and purity were examined. We developed a simple method for identifying and potentially excluding samples based on their crossing point values from RT-qPCR data, which could improve existing manufacture guidelines. Moreover, we discussed how assessment of miRNA stability differs between algorithms, possibly leading to inconsistent results. CONCLUSION: We present guidelines for RNA isolation, cDNA synthesis, and data normalization for successful miRNA expression profiling using RT-qPCR in corresponding biological OC specimens. We recommend QC panels in combination with spike-in controls and interplate controls to monitor process efficiencies.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): Toxicity and elucidation of their anti-inflammatory mechanism(s) of action.

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1ß, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.