Long-COVID olfactory dysfunction: allele E4 of apolipoprotein E as a possible protective factor.

BACKGROUND: Olfactory dysfunction (OD) represents a frequent manifestation of the coronavirus disease 2019 (COVID-19). Apolipoprotein E (APOE) is a protein that interacts with the angiotensin-converting enzyme receptor, essential for viral entry into the cell. Previous publications have suggested a possible role of APOE in COVID-19 severity. As far as we know, no publications found significant associations between this disease's severity, OD, and APOE polymorphisms (E2, E3, and E4). OBJECTIVE: To analyze the epidemiology of OD and its relationship with APOE polymorphisms in a cohort of Long-COVID patients. METHODS: We conducted a prospective cohort study with patients followed in a post-COVID neurological outpatient clinic, with OD being defined as a subjective reduction of olfactory function after infection, and persistent OD being defined when the complaint lasted more than 3 months after the COVID-19 infection resolution. This cross-sectional study is part of a large research with previously reported data focusing on the cognitive performance of our sample. RESULTS: The final sample comprised 221 patients, among whom 186 collected blood samples for APOE genotyping. The persistent OD group was younger and had a lower hospitalization rate during the acute phase of the disease (p < 0.001). Furthermore, the APOE variant E4 allele frequency was lower in this group (p = 0.035). This study evaluated OD in an outpatient population with COVID-19. In the current literature on this disease, anosmia is associated with better clinical outcomes and the E4 allele is associated with worse outcomes. CONCLUSION: Our study provides new information to these correlations, suggesting APOE E4 as a protective factor for OD.

ANTECEDENTES: A disfunção olfatória (DO) é uma manifestação frequente da doença do coronavírus 2019 (COVID-19). A apolipoproteína E (APOE) é uma proteína que interage com o receptor da enzima conversora de angiotensina, essencial para a entrada viral na célula. Publicações anteriores sugeriram um possível papel da APOE na gravidade da COVID-19. Até onde sabemos, nenhuma publicação encontrou associações significativas entre a gravidade dessa doença, DO e polimorfismos da APOE (E2, E3 e E4). OBJETIVO: Analisar a epidemiologia da DO e sua relação com os polimorfismos do gene APOE em uma coorte de pacientes com COVID longa. MéTODOS: Um estudo de coorte prospectiva com pacientes acompanhados em ambulatório neurológico pós-COVID, com DO sendo definida como uma redução subjetiva da função olfativa após a infecção e a DO persistente sendo definida quando a queixa durou mais de 3 meses após a resolução da infecção por COVID-19. Este estudo transversal é parte de uma pesquisa maior com dados anteriormente relatados, focando na performance cognitiva dos pacientes. RESULTADOS: Foram selecionados 221 pacientes para esse estudo, dos quais 186 haviam coletado amostras de sangue para genotipagem APOE. O grupo DO persistente foi mais jovem e apresentou menor taxa de internação na fase aguda da doença (p < 0,001). Além disso, a frequência do alelo E4 da APOE foi menor nesse grupo (p = 0,035). Este estudo avaliou a DO em uma população com COVID longa. Na literatura atual sobre essa doença, a anosmia está associada a melhores desfechos clínicos e o alelo E4 está associado a piores desfechos. CONCLUSãO: Nosso estudo acrescenta novas informações a essas correlações, sugerindo a APOE E4 como um fator de proteção para DO.

Three dimensional reconstruction of skin with melanoma: A model for study of invasion in vitro.

Melanoma is a type of tumor skin with high metastatic potential. Reconstructed human skin, development for pre-clinic assay, are make using primary human cells, but with same limitations. The aim this study was to characterize a cell culture model, with structure similar to human skin containing melanoma cells entirely from cell lines. Reconstructed skin with melanoma were development using human fibroblasts (MRC5), human epidermal keratinocytes (HaCat), and human melanoma (SK-MEL-28) embedded in collagen type I. The structure was characterized by hematoxylin-eosin stained, as well as points of melanoma cell invasion, which was associated with activity of MMPs (MMP-2 and MMP-9) by zymographic method. Then, the gene expression of the target molecular mechanisms involved in melanoma progression were evaluated. Here, the model development showed a region epidermis organized and separated from the dermis, with fibroblast cells confined and melanoma cells form delimited area invasion. MMP-2 and MMP-9 were identified during of cell culture and gene expression of BRAF, NRAS, and Vimentin was confirmed. The proposed model provides one more opportunity to study in vitro tumor biology of melanoma and also to allows the study of new drugs with more reliable results then whats we would find in vivo.

A Review on Anaplastic Lymphoma Kinase (ALK) Rearrangements and Mutations: Implications for Gastric Carcinogenesis and Target Therapy.

Gastric adenocarcinoma is a complex disease with diverse genetic modifications, including Anaplastic Lymphoma Kinase (ALK) gene changes. The ALK gene is located on chromosome 2p23 and encodes a receptor tyrosine kinase that plays a crucial role in embryonic development and cellular differentiation. ALK alterations can result from gene fusion, mutation, amplification, or overexpression in gastric adenocarcinoma. Fusion occurs when the ALK gene fuses with another gene, resulting in a chimeric protein with constitutive kinase activity and promoting oncogenesis. ALK mutations are less common but can also result in the activation of ALK signaling pathways. Targeted therapies for ALK variations in gastric adenocarcinoma have been developed, including ALK inhibitors that have shown promising results in pre-clinical studies. Future studies are needed to elucidate the ALK role in gastric cancer and to identify predictive biomarkers to improve patient selection for targeted therapy. Overall, ALK alterations are a relevant biomarker for gastric adenocarcinoma treatment and targeted therapies for ALK may improve patients' overall survival.

Long-COVID olfactory dysfunction: allele E4 of apolipoprotein E as a possible protective factor / Disfunção olfativa na COVID longa: o alelo E4 da apolipoproteína E como um possível fator protetor

Abstract Background Olfactory dysfunction (OD) represents a frequent manifestation of the coronavirus disease 2019 (COVID-19). Apolipoprotein E (APOE) is a protein that interacts with the angiotensin-converting enzyme receptor, essential for viral entry into the cell. Previous publications have suggested a possible role of APOE in COVID-19 severity. As far as we know, no publications found significant associations between this disease's severity, OD, and APOE polymorphisms (E2, E3, and E4). Objective To analyze the epidemiology of OD and its relationship with APOE polymorphisms in a cohort of Long-COVID patients. Methods We conducted a prospective cohort study with patients followed in a post-COVID neurological outpatient clinic, with OD being defined as a subjective reduction of olfactory function after infection, and persistent OD being defined when the complaint lasted more than 3 months after the COVID-19 infection resolution. This cross-sectional study is part of a large research with previously reported data focusing on the cognitive performance of our sample. Results The final sample comprised 221 patients, among whom 186 collected blood samples for APOE genotyping. The persistent OD group was younger and had a lower hospitalization rate during the acute phase of the disease (p < 0.001). Furthermore, the APOE variant E4 allele frequency was lower in this group (p = 0.035). This study evaluated OD in an outpatient population with COVID-19. In the current literature on this disease, anosmia is associated with better clinical outcomes and the E4 allele is associated with worse outcomes. Conclusion Our study provides new information to these correlations, suggesting APOE E4 as a protective factor for OD.

Resumo Antecedentes A disfunção olfatória (DO) é uma manifestação frequente da doença do coronavírus 2019 (COVID-19). A apolipoproteína E (APOE) é uma proteína que interage com o receptor da enzima conversora de angiotensina, essencial para a entrada viral na célula. Publicações anteriores sugeriram um possível papel da APOE na gravidade da COVID-19. Até onde sabemos, nenhuma publicação encontrou associações significativas entre a gravidade dessa doença, DO e polimorfismos da APOE (E2, E3 e E4). Objetivo Analisar a epidemiologia da DO e sua relação com os polimorfismos do gene APOE em uma coorte de pacientes com COVID longa. Métodos Um estudo de coorte prospectiva com pacientes acompanhados em ambulatório neurológico pós-COVID, com DO sendo definida como uma redução subjetiva da função olfativa após a infecção e a DO persistente sendo definida quando a queixa durou mais de 3 meses após a resolução da infecção por COVID-19. Este estudo transversal é parte de uma pesquisa maior com dados anteriormente relatados, focando na performance cognitiva dos pacientes. Resultados Foram selecionados 221 pacientes para esse estudo, dos quais 186 haviam coletado amostras de sangue para genotipagem APOE. O grupo DO persistente foi mais jovem e apresentou menor taxa de internação na fase aguda da doença (p < 0,001). Além disso, a frequência do alelo E4 da APOE foi menor nesse grupo (p = 0,035). Este estudo avaliou a DO em uma população com COVID longa. Na literatura atual sobre essa doença, a anosmia está associada a melhores desfechos clínicos e o alelo E4 está associado a piores desfechos. Conclusão Nosso estudo acrescenta novas informações a essas correlações, sugerindo a APOE E4 como um fator de proteção para DO.

PARP1 Characterization as a Potential Biomarker for BCR::ABL1 p190+ Acute Lymphoblastic Leukemia.

Detection of t(9;22), and consequent BCR::ABL1 fusion, is still a marker of worse prognosis for acute lymphoblastic leukemia (ALL), with resistance to tyrosine-kinase inhibitor therapy being a major obstacle in the clinical practice for this subset of patients. In this study, we investigated the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL, the most common isoform to afflict ALL patients, and demonstrated the use of experimental PARP inhibitor (PARPi), AZD2461, as a therapeutic option with cytotoxic capabilities similar to that of imatinib, the current gold standard in medical care. We characterized cytostatic profiles, induced cell death, and biomarker expression modulation utilizing cell models, also providing a comprehensive genome-wide analysis through an aCGH of the model used, and further validated PARP1 differential expression in samples of ALL p190+ patients from local healthcare institutions, as well as in larger cohorts of online and readily available datasets. Overall, we demonstrate the effectiveness of PARPi in the treatment of BCR::ABL1 p190+ ALL cell models and that PARP1 is differentially expressed in patient samples. We hope our findings help expand the characterization of molecular profiles in ALL settings and guide future investigations into novel biomarker detection and pharmacological choices in clinical practice.

Post-COVID-19 Cognitive Decline and Apoe Polymorphism: Towards a Possible Link?

APOE ε4 polymorphism has been recently described as a possible association with cognitive deficits in COVID-19 patients. This research aimed to establish the correlation between COVID-19 and cognitive impairment, and the APOE gene polymorphism among outpatients. We performed a cross-sectional study with confirmed COVID-19 patients and neurological symptoms that persisted for more than three months from onset. APOE genotypes were determined. The final number of patients included in this study was 219, of which 186 blood samples were collected for APOE genotyping, evaluated 4.5 months after COVID-19. Among the participants, 143 patients (65.3%) reported memory impairment symptoms as their primary concern. However, this complaint was objectively verified through screening tests (Addenbrooke Cognitive Examination-Revised and Mini-Mental State Examination) in only 36 patients (16.4%). The group experiencing cognitive decline exhibited a higher prevalence of the APOE ε4 allele than the normal group (30.8% vs. 16.4%, respectively, p = 0.038). Furthermore, the APOE ε4 allele and anxiety symptoms remained significant after multivariate analysis. This study assessed an outpatient population where cognitive changes were the primary complaint, even in mild cases. Moreover, the ε4 allele, sleep disorders, and anxiety symptoms were more frequent in the cognitive decline group.

Mebendazole targets essential proteins in glucose metabolism leading gastric cancer cells to death.

Gastric cancer (GC) is among the most-diagnosed and deadly malignancies worldwide. Deregulation in cellular bioenergetics is a hallmark of cancer. Based on the importance of metabolic reprogramming for the development and cancer progression, inhibitors of cell metabolism have been studied as potential candidates for chemotherapy in oncology. Mebendazole (MBZ), an antihelminthic approved by FDA, has shown antitumoral activity against cancer cell lines. However, its potential in the modulation of tumoral metabolism remains unclear. Results evidenced that the antitumoral and cytotoxic mechanism of MBZ in GC cells is related to the modulation of the mRNA expression of glycolic targets SLC2A1, HK1, GAPDH, and LDHA. Moreover, in silico analysis has shown that these genes are overexpressed in GC samples, and this increase in expression is related to decreased overall survival rates. Molecular docking revealed that MBZ modifies the protein structure of these targets, which may lead to changes in their protein function. In vitro studies also showed that MBZ induces alterations in glucose uptake, LDH's enzymatic activity, and ATP production. Furthermore, MBZ induced morphologic and intracellular alterations typical of the apoptotic cell death pathway. Thus, this data indicated that the cytotoxic mechanism of MBZ is related to an initial modulation of the tumoral metabolism in the GC cell line. Altogether, our results provide more evidence about the antitumoral mechanism of action of MBZ towards GC cells and reveal metabolic reprogramming as a potential area in the discovery of new pharmacological targets for GC chemotherapy.

In silico analysis of ACE2 from different animal species provides new insights into SARS-CoV-2 species spillover.

Aim: This study aimed to analyze the phylogenetic relationships between the ACE2 of humans and other animals and investigate the potential interaction between SARS-CoV-2 RBD and ACE2 of different species. Materials & methods: The phylogenetic construction and molecular interactions were assessed using computational models. Results & conclusion: Despite the evolutionary distance, 11 species had a perfect fit for the interaction between their ACE2 and SARS-CoV-2 RBD (Chinchilla lanigera, Neovison vison, Rhinolophus sinicus, Emballonura alecto, Saccopteryx bilineata, Numida meleagris). Among them, the avian N. meleagris was reported for the first time in this study as a probable SARS-CoV-2 host due to the strong molecular interactions. Therefore, predicting potential hosts for SARS-CoV-2 for understanding the epidemiological cycle and proposal of surveillance strategies.

Here, computational analysis was employed to predict the interaction between the Spike protein from SARS-COV-2 with the ACE2 receptor with animals that could serve as a reservoir for SARS-CoV-2 spillover. Our results reported for the first time that N. meleagris could act as a possible host for SARS-CoV-2.

Cytotoxic and mutagenic effects of the food additive tartrazine on eukaryotic cells.

BACKGROUND: Among the food additives used in the food industry, food dyes are considered the most toxic. For instance, tartrazine (TRZ) is a food colorant commercially available with conflicting data regarding its cytotoxic, genotoxic, and mutagenic effects. Therefore, this study aimed to evaluate the cytotoxic and mutagenic potential of TRZ using different eukaryotic cells (in vitro). METHODS: This study employed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), brine shrimp lethality, Allium cepa and Saccharomyces cerevisiae tests. Different concentrations of TRZ and different exposure times were used in this study. RESULTS: The results demonstrate that TRZ induced a concentration-dependent toxic effect on the test systems. It also exerted cytotoxicity in fibroblasts and human gastric cells. In addition, TRZ showed mutagenic effects on the A. cepa test system. However, its toxicogenic effects may not relate to the oxidizing activity, which was confirmed by the S. cerevisiae test model. CONCLUSION: Taken together, TRZ exerted toxicogenic effects on the test systems. Therefore, it may be harmful to health, especially its prolonged use may trigger carcinogenesis.

Evaluation of the anti-diabetic drug sitagliptin as a novel attenuate to SARS-CoV-2 evidence-based in silico: molecular docking and molecular dynamics.

The current outbreak of COVID-19 cases worldwide has been responsible for a significant number of deaths, especially in hospitalized patients suffering from comorbidities, such as obesity, diabetes, hypertension. The disease not only has prompted an interest in the pathophysiology, but also it has propelled a massive race to find new anti-SARS-CoV-2 drugs. In this scenario, known drugs commonly used to treat other diseases have been suggested as alternative or complementary therapeutics. Herein we propose the use of sitagliptin, an inhibitor of dipeptidyl peptidase-4 (DPP4) used to treat type-II diabetes, as an agent to block and inhibit the activity of two proteases, 3CLpro and PLpro, related to the processing of SARS-CoV-2 structural proteins. Inhibition of these proteases may possibly reduce the viral load and infection on the host by hampering the synthesis of new viruses, thus promoting a better outcome. In silico assays consisting in the modeling of the ligand sitagliptin and evaluation of its capacity to interact with 3CLpro and PLpro through the prediction of the ligand bioactivity, molecular docking, overlapping of crystal structures, and molecular dynamic simulations were conducted. The experiments indicate that sitagliptin can interact and bind to both targets. However, this interaction seems to be stronger and more stable to 3CLpro (ΔG = -7.8 kcal mol-1), when compared to PLpro (ΔG = -7.5 kcal mol-1). This study suggests that sitagliptin may be suitable to treat COVID-19 patients, beyond its common use as an anti-diabetic medication. In vivo studies may further support this hypothesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03406-w.

Antitumoral effect of novel synthetic 8-hydroxy-2-((4-nitrophenyl)thio)naphthalene-1,4-dione (CNN16) via ROS-mediated DNA damage, apoptosis and anti-migratory effect in colon cancer cell line.

Colorectal cancer (CRC) is estimated as the third most incident cancer and second in mortality worldwide. Moreover, CRC metastasis reduces patients' survival rates. Thus, the study and identification of new compounds with anticancer activity selectively to tumor cells are encouraged in the CRC treatment. Naphtoquinones are compounds with several pharmacologic activities, including antitumoral properties. Therefore, this study aimed to investigate the anticancer mechanism of synthetic 8-Hydroxy-2-(P-Nitrothiophenol)-1,4-Naphthoquinone (CNN16) in colon cancer cell line HCT-116. CNN16 showed an IC50 of 5.32 µM in HCT-116, and 9.36, 10.77, and 24.57 µM in the non-cancerous cells MRC-5, MNP-01, and PMBC, respectively, evaluated by the MTT assay. CNN16 showed an anticlonogenic effect in HCT-116 and induced cell fragmentation identified by flow cytometry analysis. Furthermore, we observed that CNN16 presented genotoxicity and induces reactive oxygen species (ROS) after 3 h of treatment visualized by alkaline comet assay and DCFH-DA dye fluorescence, respectively. Furthermore, CNN16 caused cellular membrane disruption, reduction in the mitochondrial membrane polarization, and the presence of apoptotic bodies and chromatin condensation was visualized by differential stained (HO/FD/PI) in fluorescent microscopy along with PARP1, TP53, BCL-2, and BAX analyzed by RT-qPCR. Results also evidenced inhibition in the migratory process analyzed by wound healing assay. Therefore, CNN16 can be considered as a potential new leader molecule for CRC treatment, although further studies are still necessary to comprehend the effects of CNN16 in in vivo models to evaluate the anti-migratory effect, and toxicology and assure compound safety and selectively.

Kinase Inhibitor Screening Displayed ALK as a Possible Therapeutic Biomarker for Gastric Cancer.

Despite advances in cancer chemotherapy, gastric cancer (GC) continues to have high recurrence rates and poor prognosis with limited treatment options. Understanding the etiology of GC and developing more effective, less harmful therapeutic approaches are vital and urgent. Therefore, this work describes a novel kinase target in malignant gastric cells as a potential therapeutic strategy. Our results demonstrate that among 147 kinase inhibitors (KI), only three molecules were significantly cytotoxic for the AGP-01 cell line. Hence, these three molecules were further characterized in their cellular mode of action. There was significant cell cycle impairment due to the expression modulation of genes such as TP53, CDKN1A, CDC25A, MYC, and CDK2 with subsequent induction of apoptosis. In fact, the Gene Ontology analysis revealed a significant enrichment of pathways related to cell cycle regulation (GO:1902749 and GO:1903047). Moreover, the three selected KIs significantly reduced cell migration and Vimentin mRNA expression after treatment. Surprisingly, the three KIs share the same target, ALK and INSR, but only the ALK gene was found to have a high expression level in the gastric cancer cell line. Additionally, lower survival rates were observed for patients with high ALK expression in TCGA-STAD analysis. In summary, we hypothesize that ALK gene overexpression can be a promising biomarker for prognosis and therapeutic management of gastric adenocarcinoma.

Long-covid cognitive impairment: Cognitive assessment and apolipoprotein E (APOE) genotyping correlation in a Brazilian cohort.

Introduction: Few studies have objectively evaluated cognitive deficits after the acute phase of COVID-19 disease. Moreover, the role of apolipoprotein E (APOE) genotypes in cognitive decline in patients with COVID-19 has not been evaluated yet. Methods: This cross-sectional study was conducted in confirmed cases of COVID-19 patients with neurological symptoms that persisted for more than 3 months from the onset. We determined APOE genotypes. Results: The final sample consisted of 141 patients. The most frequent APOE genotype was E3/E3 (N = 95; 67.3%). In total, 93 patients (65.9%) had memory impairment symptoms as the main complaint, objectively confirmed through screening tests in 25 patients (17.7%). Patients with cognitive impairment had a lower frequency of anosmia than the normal and subjective cognitive decline (SCD) groups (p = 0.005). In addition, depression was recurrent in the cognitive impairment group and the SCD group (p = 0.046). Cognitive impairment was significantly more frequent in hospitalized patients and those with a lower education level. Cognitive status was not associated with APOE genotypes. Discussion: Hospitalized patients had more severe infection with a greater possibility of systemic complications, greater inflammatory response, and prolonged hospitalization, which could impact cognitive performance. Cognitive impairment in patients with COVID-19 does not necessarily involve specific APOE polymorphisms. However, psychiatric disorders may also be responsible for cognitive complaints. Cognitive complaints are frequent in patients with COVID-19, even after the acute phase of the disease and in mild cases. Hospitalized participants and depressed patients may have a higher risk of cognitive impairment. APOE genotypes or haplotypes may not significantly play a role in COVID-19 cognitive impairment.

Genomic surveillance: Circulating lineages and genomic variation of SARS-CoV-2 in early pandemic in Ceará state, Northeast Brazil.

In the Northeast of Brazil, Ceará was the second state most impacted by COVID-19 in number of cases and death rate. Despite that, the early dynamics of the pandemic in Ceará was not yet well understood due the low genomic surveillance of SARS-CoV-2 in 2020. In this study, we analyze the circulating lineages and the genomic variation of the virus in Ceará state. Thirty-four genomes were sequenced and combined with sequences available in GISAID database from March 2020 to June 2021 to compose the study dataset. The most prevalent lineages detected were B.1.1.33, in 2020, and P.1, in 2021. Other lineages were found, such as P.2, sublineages of P.1, B.1, B.1.1, B.1.1.28 and B.1.212. Analyzing the mutations, a total of 202 single-nucleotide polymorphisms (SNPs) were identified among the 34 genomes sequenced, of which 127 were missense, 74 synonymous, and one was a nonsense mutation. Among the missense mutations, C14408T, A23403G, T27299C, G28881A G28883C, and T29148C were the most prevalent within the dataset. Although SARS-CoV-2 sequencing data was limited in 2020, our results could provide insights to better understand the genetic diversity of the circulating lineages in Ceará.

Anticancer potential of mebendazole against chronic myeloid leukemia: in silico and in vitro studies revealed new insights about the mechanism of action.

Chronic myeloid leukemia (CML) is caused by constitutively active fusion protein BCR-ABL1, and targeting ABL1 is a promising therapy option. Imatinib, dasatinib, and nilotinib have all been shown to work effectively in clinical trials. ABL1 mutations, particularly the T315I gate-keeper mutation, cause resistance in patients. As a result, broad-spectrum ABL1 medicines are desperately needed. In order to screen potential drugs targeting CML, mebendazole (MBZ) was subjected to the in vitro test against CML cell lines (K562 and FEPS) and computational assays. The antiproliferative effect of MBZ and the combination with tyrosine kinase inhibitors (TKIs) was tested using end-point viability assays, cell cycle distribution analysis, cell membrane, and mitochondrial dyes. By interrupting the cell cycle and causing cell death, MBZ and its combination with imatinib and dasatinib have a significant antiproliferative effect. We identified MBZ as a promising "new use" drug targeting wild-type and mutant ABL1 using molecular docking. Meanwhile, we determined which residues in the allosteric site are important in ABL1 drug development. These findings may not only serve as a model for repositioning current authorized medications but may also provide ABL1-targeted anti-CML treatments a fresh lease of life.

1,4-Naphthoquinone (CNN1) Induces Apoptosis through DNA Damage and Promotes Upregulation of H2AFX in Leukemia Multidrug Resistant Cell Line.

The multidrug resistance (MDR) phenotype is one of the major obstacles in the treatment of chronic myeloid leukemia (CML) in advantage stages such as blast crisis. In this scenario, more patients develop resistance mechanisms during the course of the disease, making tyrosine kinase inhibitors (TKIs) target therapies ineffective. Therefore, the aim of the study was to examine the pharmacological role of CNN1, a para-naphthoquinone, in a leukemia multidrug resistant cell line. First, the in vitro cytotoxic activity of Imatinib Mesylate (IM) in K-562 and FEPS cell lines was evaluated. Subsequently, membrane integrity and mitochondrial membrane potential assays were performed to assess the cytotoxic effects of CNN1 in K-562 and FEPS cell lines, followed by cell cycle, alkaline comet assay and annexin V-Alexa Fluor® 488/propidium iodide assays (Annexin/PI) using flow cytometry. RT-qPCR was used to evaluate the H2AFX gene expression. The results demonstrate that CNN1 was able to induce apoptosis, cell membrane rupture and mitochondrial membrane depolarization in leukemia cell lines. In addition, CNN1 also induced genotoxic effects and caused DNA fragmentation, cell cycle arrest at the G2/M phase in leukemia cells. No genotoxicity was observed on peripheral blood mononuclear cells (PBMC). Additionally, CNN1 increased mRNA levels of H2AFX. Therefore, CNN1 presented anticancer properties against leukemia multidrug resistant cell line being a potential anticancer agent for the treatment of resistant CML.

Differences in glucose concentration shows new perspectives in gastric cancer metabolism.

Gastric cancer (GC) is among the deadliest cancers worldwide despite available therapies, highlighting the need for novel therapies and pharmacological agents. Metabolic deregulation is a potential study area for new anticancer targets, but the in vitro metabolic studies are controversial, as different ranges of glucose used in the culture media can influence results. In this study, we evaluated cellular viability, glucose uptake, and LDH activity in gastric cancer cell lines when exposed to different glucose concentrations: high (HG, 25mM), low (LG, 5.5mM), and free (FG, 0mM) glucose media. Moreover, we evaluated how glucose variations may influence cellular phenotype and the expression of genes related to epithelial-mesenchymal transition (EMT), metabolism, and cancer development in metastatic GC cells (AGP-01). Results showed that metastatic cells exposed to FG medium evidenced higher alterations when compared to other cell lines. Most phenotypic assays did not show difference when exposed to either HG or LG media. However, gene expression profile of cells exposed to LG revealed differences in mRNA levels of metabolism-related genes when compared to HG medium. According to our results, we recommend using LG medium for metabolic studies since the glucose concentration is closer to physiological levels. These findings point out new relevant targets in metabolic reprogramming that can be alternatives to current chemotherapies in patients with metastatic GC.

Solid Tumors and Kinase Inhibition: Management and Therapy Efficacy Evolution.

The increasing numbers of cancer cases worldwide and the exceedingly high mortality rates of some tumor subtypes raise the question about if the current protocols for cancer management are effective and what has been done to improve upon oncologic patients' prognoses. The traditional chemo-immunotherapy options for cancer treatment focus on the use of cytotoxic agents that are able to overcome neoplastic clones' survival mechanisms and induce apoptosis, as well as on the ability to capacitate the host's immune system to hinder the continuous growth of malignant cells. The need to avert the highly toxic profiles of conventional chemo-immunotherapy and to overcome the emerging cases of tumor multidrug resistance has fueled a growing interest in the field of precision medicine and targeted molecular therapies in the last couple of decades, although relatively new alternatives in oncologic practices, the increased specificity, and the positive clinical outcomes achieved through targeted molecular therapies have already consolidated them as promising prospects for the future of cancer management. In recent years, the development and application of targeted drugs as tyrosine kinase inhibitors have enabled cancer treatment to enter the era of specificity. In addition, the combined use of targeted therapy, immunotherapy, and traditional chemotherapy has innovated the standard treatment for many malignancies, bringing new light to patients with recurrent tumors. This article comprises a series of clinical trials that, in the past 5 years, utilized kinase inhibitors (KIs) as a monotherapy or in combination with other cytotoxic agents to treat patients afflicted with solid tumors. The results, with varying degrees of efficacy, are reported.

True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

INTRODUCTION: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease has had a catastrophic impact on the world resulting in several deaths. Since World Health Organization declared the pandemic status of the disease, several molecular diagnostic kits have been developed to help the tracking of viruses spread. AREAS COVERED: This review aims to describe and evaluate the currently reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnosis kit. Several processes used in COVID-19 diagnostic procedures are detailed in further depth to demonstrate optimal practices. Therefore, we debate the main factors that influence the viral detection of SARS-COV-2 and how they can affect the diagnosis of patients. EXPERT OPINION: Here is highlighted and discussed several factors that can interfere in the RT-PCR analysis, such as the viral load of the sample, collection site, collection methodology, sample storage, transport, primer, and probe mismatch/dimerization in different brand kits. This is a pioneer study to discuss the factor that could lead to the wrong interpretation of RT-qPCR diagnosis of SARS-CoV-2. This study aimed to help the readers to understand what very likely is behind a bad result of SARS-CoV-2 detection by RT-PCR and what could be done to reach a reliable diagnosis.

Streptococcus mutans in atherosclerotic plaque: Molecular and immunohistochemical evaluations.

OBJECTIVES: To verify the presence of Streptococcus mutans (S. mutans) in atherosclerotic plaque (AP) using techniques with different sensitivities, correlating with histological changes in plaque and immunoexpression of inflammatory markers. MATERIALS AND METHODS: Thirteen AP samples were subjected to real-time polymerase chain reaction (qRT-PCR), histopathological analyses, histochemical analysis by Giemsa staining (GS), and immunohistochemical analysis for S. mutans, IL-1ß, and TNF-α (streptavidin-biotin-peroxidase method). Ten necropsy samples of healthy vessels were used as controls. RESULTS: All AP samples showed histopathological characteristics of severe atherosclerosis and were positive for S. mutans (100.0%) in qRT-PCR and immunohistochemical analyses. GS showed that Streptococcus sp. colonized the lipid-rich core regions and fibrous tissue, while the control group was negative for Streptococcus sp. IL-1ß and TNF-α were expressed in 100% and 92.3% of the AP tested, respectively. The control samples were positive for S. mutans in qRT-PCR analysis, but negative for S. mutans, IL-1ß, and TNF-α in immunohistochemical analyses. CONCLUSION: The detection of S. mutans in AP and the visualization of Streptococcus sp. suggested a possible association between S. mutans and atherosclerosis. The results obtained from the control samples suggested the presence of DNA fragments or innocuous bacteria that were not associated with tissue alteration. However, future studies are necessary to provide more information.