Brain damage serum biomarkers induced by COVID-19 in patients from northeast Brazil.

Neurological symptoms have been often reported in COVID-19 disease. In the present study, we evaluated brain damage associated with the increase of serum levels of neurological biomarkers S100B and neuron-specific enolase (NSE) induced by SARS-CoV-2 infection, in a population from Northeastern Brazil. Thirty-six healthy control (G1) individuals and 141 patients with confirmed COVID-19 were enrolled in this study. Positive-COVID-19 patients were divided into two groups according to the severity of illness by the National Institute of Health (NIH) criteria, 76 patients with mild symptoms for COVID-19 and (G2) and 65 with acute respiratory conditions requiring supplemental oxygenation via intensive care unit (ICU) admission (G3). A follow-up study was conducted with 23 patients from G2 14 (D14) and 21 (D21) days after the onset of symptoms. Serum levels of NSE and S100B were measured using the enzyme-linked immunoassay method (ELISA). Results revealed a significant positive association between G3 patients and S100B serum expression (p = 0.0403). The serum levels of NSE were also significantly enhanced in the G3 group compared to the control (p < 0.0001) and G2 group (p < 0.0001). In addition, clinical features such as symptoms and oxygenation status were not correlated with NSE or S100B serum expression. The follow-up study demonstrated a decrease over time (21 days) in NSE serum expression (p < 0.0001). These results suggest that brain damage is followed by acute virus exposure, with no long-term effects. Future work examining COVID-19 recovery will shed light on chronic neurological damage of SARS-CoV-2 infection.

Synthesis of novel thiazolidinic-phthalimide derivatives evaluated as new multi-target antiepileptic agents.

Epilepsy is a disease that affects millions of people around the globe and has a multifactorial cause. Inflammation is a process that can be involved in the development of seizures. Thus, the present study proposed the design and synthesis of new candidates for antiepileptic drugs that would also control the inflammatory process. Nine new derivatives of the substituted thiazophthalimide hybrid core were obtained with satisfactory purity ≥99% and yields between 27% and 87%. All compounds showed cell viability values greater than 90% in the culture of PBMC cells from healthy volunteers and, therefore, were not considered cytotoxic. These compounds modulated proinflammatory cytokines IFN-y and IL-17A and can mitigate inflammation. Acute toxicity studies of compound 7i in an animal model indicated that the compound has low toxicity and an LD50 greater than 2 g/kg in healthy adult rats. The same compound did not show positive results for anticonvulsant activity through the PTZ test. However, 7i demonstrates the interaction with the target GABA-A receptor in silico, indicating a possible activity as an agonist of that receptor. Thus, further studies are needed to investigate the anticonvulsant activity, in particular, using models in which the inflammatory process triggers epileptic seizures.

Purified anacardic acids exert multiple neuroprotective effects in pesticide model of Parkinson's disease: in vivo and in silico analysis.

Parkinson's disease (PD) induced by environmental toxins involves a multifactorial cascade of harmful factors, thus motivating the search for therapeutic agents able to act on the greatest number of molecular targets. This study evaluated the efficacy of 50 mg/kg purified anacardic acids (AAs), isolated from cashew nut shell liquid, on multiple steps of oxidative stress and inflammation induced by rotenone in the substantia nigra (SN) and striatum. Adult mice were divided into four groups: Control, rotenone, AAs + rotenone, and AAs alone. Lipoperoxidation, nitric oxide (NO) levels, and reduced glutathione (GSH)/oxidized gluthatione (GSSG) ratio were evaluated. NF-kB-p65, pro-IL-1ß, cleaved IL-1ß, metalloproteinase-9, Tissue Inhibitory Factor-1 (TIMP-1), tyrosine hydroxylase (TH), and glial fibrillary acidic protein (GFAP) levels were assessed by Western blot. In silico studies were also made using the SwissADME web tool. Rotenone increased lipoperoxidation and NO production and reduced TH levels and GSH/GSSG ratio in both SN and striatum. It also enhanced NF-kB-p65, pro, and cleaved IL-1ß, MMP-9, GFAP levels compared to control and AAs groups. The AAs alone reduced pro-IL-1ß in the striatum while they augmented TIMP1 and reduced MMP-9 amounts in both regions. AAs reversed rotenone-induced effects on lipoperoxidation, NO production, and GSH/GSSG ratio, as well as increased TH and attenuated pro-IL-1ß and MMP-9 levels in both regions, NF-kB-p65 in the SN and GFAP in the striatum. Altogether, the in vivo and in silico analysis reinforced multiple and defined molecular targets of AAs, identifying that they are promising neuroprotective drug candidates for PD, acting against oxidative and inflammatory conditions induced by rotenone.

Glycobiology Modifications in Intratumoral Hypoxia: The Breathless Side of Glycans Interaction.

Post-translational and co-translational enzymatic addition of glycans (glycosylation) to proteins, lipids, and other carbohydrates, is a powerful regulator of the molecular machinery involved in cell cycle, adhesion, invasion, and signal transduction, and is usually seen in both in vivo and in vitro cancer models. Glycosyltransferases can alter the glycosylation pattern of normal cells, subsequently leading to the establishment and progression of several diseases, including cancer. Furthermore, a growing amount of research has shown that different oxygen tensions, mainly hypoxia, leads to a markedly altered glycosylation, resulting in altered glycan-receptor interactions. Alteration of intracellular glucose metabolism, from aerobic cellular respiration to anaerobic glycolysis, inhibition of integrin 3α1ß translocation to the plasma membrane, decreased 1,2-fucosylation of cell-surface glycans, and galectin overexpression are some consequences of the hypoxic tumor microenvironment. Additionally, increased expression of gangliosides carrying N-glycolyl sialic acid can also be significantly affected by hypoxia. For all these reasons, it is possible to realize that hypoxia strongly alters glycobiologic events within tumors, leading to changes in their behavior. This review aims to analyze the complexity and importance of glycoconjugates and their molecular interaction network in the hypoxic context of many solid tumors.

In Vitro, Oral Acute, and Repeated 28-Day Oral Dose Toxicity of a Mixed-Valence Polyoxovanadate Cluster.

Polyoxovanadates (POV) are a subgroup of polyoxometalates (POM), which are nanosized clusters with reported biological activities. This manuscript describes the first toxicity evaluation of a mixed-valence polyoxovanadate, pentadecavanadate, (Me4N)6[V15O36Cl], abbreviated as V15. Cytotoxicity experiments using peripheral blood mononuclear cells (PBMC), larvae of Artemia salina Leach, and in vivo oral acute and repeated 28-day doses in mice was carried out. The LC50 values in PBMC cells and A. salina were 17.5 ± 5.8 µmol L-1, and 17.9 µg L-1, respectively, which indicates high cytotoxic activity. The toxicity in mice was not observed upon acute exposure in a single dose, however, the V15 repeated 28-day oral administration demonstrated high toxicity using 25 mg/kg, 50 mg/kg and, 300 mg/kg doses. The biochemical and hematological analyses during the 28-day administration of V15 showed significant alteration of the metabolic parameters related to the kidney and liver, suggesting moderate toxicity. The V15 toxicity was attributed to the oxidative stress and lipid peroxidation, once thiobarbituric acid (TBAR) levels significantly increased in both males and females treated with high doses of the POV and also in males treated with a lower dose of the POV. This is the first study reporting a treatment-related mortality in animals acutely administrated with a mixed-valence POV, contrasting with the well-known, less toxic decavanadate. These results document the toxicity of this mixed-valence POV, which may not be suitable for biomedical applications.

alpha2,3 sialic acid processing enzymes expression in gastric cancer tissues reveals that ST3Gal3 but not Neu3 are associated with Lauren's classification, angiolymphatic invasion and histological grade.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Despite progress in the last decades, there are still no reliable biomarkers for the diagnosis of and prognosis for GC. Aberrant sialylation is a widespread critical event in the development of GC. Neuraminidases (Neu) and sialyltransferases (STs) regulate the ablation and addition of sialic acid during glycoconjugates biosynthesis, and they are a considerable source of biomarkers in various cancers. This study retrospectively characterized Neu3 and ST3Gal3 expression by immunohistochemistry in 71 paraffin-embedded GC tissue specimens and analyzed the relationship between their expression and the clinicopathological parameters. Neu3 expression was markedly increased in GC tissues compared with non-tumoral tissues (p<0.0001). Intratumoral ST3Gal3 staining was significantly associated with intestinal subtype (p=0.0042) and was negatively associated with angiolymphatic invasion (p=0.0002) and higher histological grade G3 (p=0.0066). Multivariate analysis revealed that ST3Gal3 positivity is able to predict Lauren's classification. No associations were found between Neu3 staining and clinical parameters. The in silico analysis of mRNA expression in GC validation cohorts corroborates the significant ST3Gal3 association with higher histological grade observed in our study. These findings suggest that ST3Gal3 expression may be an indicator for aggressiveness of primary GC.

Nanostructured sensor platform based on organic polymer conjugated to metallic nanoparticle for the impedimetric detection of SARS-CoV-2 at various stages of viral infection.

The projection of new biosensing technologies for genetic identification of SARS-COV-2 is essential in the face of a pandemic scenario. For this reason, the current research aims to develop a label-free flexible biodevice applicable to COVID-19. A nanostructured platform made of polypyrrole (PPy) and gold nanoparticles (GNP) was designed for interfacing the electrochemical signal in miniaturized electrodes of tin-doped indium oxide (ITO). Oligonucleotide primer was chemically immobilized on the flexible transducers for the biorecognition of the nucleocapsid protein (N) gene. Methodological protocols based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the nanotechnological apparatus. The biosensor's electrochemical performance was evaluated using the SARS-CoV-2 genome and biological samples of cDNA from patients infected with retrovirus at various disease stages. It is inferred that the analytical tool was able to distinguish the expression of SARS-CoV-2 in patients diagnosed with COVID-19 in the early, intermediate and late stages. The biosensor exhibited high selectivity by not recognizing the biological target in samples from patients not infected with SARS-CoV-2. The proposed sensor obtained a linear response range estimated from 800 to 4000 copies µL-1 with a regression coefficient of 0.99, and a detection limit of 258.01 copies µL-1. Therefore, the electrochemical biosensor based on flexible electrode technology represents a promising trend for sensitive molecular analysis of etiologic agent with fast and simple operationalization. In addition to early genetic diagnosis, the biomolecular assay may help to monitor the progression of COVID-19 infection in a novel manner.

Synthesis, Antitumor Activity and Molecular Docking Studies on Seven Novel Thiazacridine Derivatives.

AIM AND OBJECTIVE: In the last decades, cancer has become a major problem in public health all around the globe. Chimeric chemical structures have been established as an important trend on medicinal chemistry in the last years. Thiazacridines are hybrid molecules composed of a thiazolidine and acridine nucleus, both pharmacophores that act on important biological targets for cancer. By the fact it is a serious disease, seven new 3-acridin-9-ylmethyl-thiazolidine-2,4-dione derivatives were synthesized, characterized, analyzed by computer simulation and tested in tumor cells. In order to find out if the compounds have therapeutic potential. MATERIALS AND METHODS: Seven new 3-acridin-9-ylmethyl-thiazolidine-2,4-dione derivatives were synthesized through Michael addition and Knoevenagel condensation strategies. Characterization was performed by NMR and Infrared spectroscopy techniques. Regarding biological activity, thiazacridines were tested against solid and hematopoietic tumoral cell lines, namely Jurkat (acute T-cell leukemia); HL-60 (acute promyelocytic leukemia); DU 145 (prostate cancer); MOLT-4 (acute lymphoblastic leukemia); RAJI (Burkitt's lymphoma); K562 (chronic myelogenous leukemia) and normal cells PBMC (healthy volunteers). Molecular docking analysis was also performed in order to assess major targets of these new compounds. Cell cycle and clonogenic assay were also performed. RESULTS: Compound LPSF/AA-62 (9f) exhibited the most potent anticancer activity against HL-60 (IC50 3,7±1,7 µM), MOLT-4 (IC50 5,7±1,1 µM), Jurkat (IC50 18,6 µM), Du-145 (IC50 20±5 µM) and Raji (IC50 52,3±9,2 µM). While the compound LPSF/AA-57 (9b) exhibited anticancer activity against the K562 cell line (IC50 51,8±7,8 µM). Derivative LPSF/AA-62 (9f) did not interfere in the cell cycle phases of the Molt-4 lineage. However, the LPSF/AA-62 (9f) derivative significantly reduced the formation of prostate cancer cell clones. The compound LPSF/AA-62 (9f) has shown strong anchorage stability with enzymes topoisomerases 1 and 2, in particular due the presence of chlorine favored hydrogen bonds with topoisomerase 1. CONCLUSION: The 3-(acridin-9-ylmethyl)-5-((10-chloroanthracen-9-yl)methylene)thiazolidine-2,4-dione (LPSF/AA-62) presented the most promising results, showing anti-tumor activity in 5 of the 6 cell types tested, especially inhibiting the formation of colonies of prostate tumor cells (DU-145).

Losartan, but not Enalapril and Valsartan, Inhibits the Expression of IFN-γ, IL-6, IL-17F and IL-22 in PBMCs from Rheumatoid Arthritis Patients.

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic and inflammatory disease that affects about 1% of the world's population. Almost 70% of RA patients have a cardiovascular disease such as Systemic Arterial Hypertension (SAH). Inflammatory cytokines are clearly involved in the pathogenesis of RA and correlated with SAH. OBJECTIVE: It is necessary to understand whether the antihypertensive drugs have a dual effect as immunomodulators and which one is the best choice for RA SAH patients. METHODS: Peripheral Blood Mononuclear Cells (PBMCs) from 16 RA patients were purified and stimulated or not stimulated with anti-CD3 and anti-CD28 mAB and were treated with Enalapril, Losartan and Valsartan at 100µM. Patients were evaluated for clinical and laboratory variables including measures of disease activity by Clinical Disease Activity Index (CDAI) and Disease Activity Score (DAS28). Cytokines were quantified by ELISA sandwich. RESULTS: Losartan was able to reduce levels of IFN-γ (p = 0.0181), IL-6 (p = 0.0056), IL-17F (0.0046) and IL-22 (p = 0.0234) in RA patients. In addition, patients in remission and mild score (DAS28<3.2 and CDAI<10) had a better response to treatment. On the other hand, patients in moderate and severe activity had poor response to Losartan in cytokine inhibition. CONCLUSION: PBMCs from RA patients are responsive in inhibiting proinflammatory cytokines using Losartan better than Enalapril and Valsartan and it could be a better antihypertensive choice for patients with RA and systemic arterial hypertension treatment.

IL-17 and related cytokines involved in systemic sclerosis: Perspectives.

Systemic sclerosis (SSc) is a multisystemic, complex, and rare disease of connective tissue, with high morbidity and mortality, and without specific treatment. The disease is characterized by three main principles: vascular disease, autoantibody production and inflammation, and fibrosis. Since it is well defined that SSc is characterized by elevated production of TGF-ß, IL-6, and IL-1, all of them cytokines related to Th17 differentiation, the hypothesis is that this disease may be strongly related to a polarization of the immune response towards the Th17 pathway. Considering the importance of a better understanding of the pathophysiology of Th17 pathway in SSc, this article aims to propose an update for a better understanding of current knowledge on main cytokines secreted by the Th17 cells (IL-17 A, IL-21, and IL-22) and the future prospects in the current disease.

CD4+CD45RA-FOXP3low Regulatory T Cells as Potential Biomarkers of Disease Activity in Systemic Lupus Erythematosus Brazilian Patients.

Heren, we analyzed Treg cells as potential biomarkers of disease activity in systemic lupus erythematosus (SLE) patients. Peripheral blood mononuclear cells from 30 SLE patients (15 active: SLEDAI > 6/15 SLE remission: SLEDAI< 6) and 15 healthy volunteers were purified. Treg immunophenotyping was performed using CD4, CD25, CD45, CD127, and FOXP3 markers. CD4+FOXP3+ Treg activation state was investigated based on CD45RA and FOXP3 expression. To increase the accuracy of our findings, a multivariate linear regression was performed. We showed a significant increase in the frequency of CD4+FOXP3+ Treg cells in SLE patients. However, unlike all other Treg cells phenotypes analyzed, only eTreg (CD4+FOXP3highCD45RA-) (p=0.01) subtype was inversely correlated with disease activity while Foxp3+nontreg (CD4+FOXP3lowCD45RA-) (p=0.003) exerted a direct influence in the outcome of the disease. Foxp3+nontreg cells were the most consistent SLE active indicator, confirmed by multiple linear regression analyses. In summary, our results demonstrate Foxp3+nontreg cells as new biomarkers in the search of an effective therapeutic strategy in SLE.

Synthesis and Anticancer Evaluation of Thiazacridine Derivatives Reveals New Selective Molecules to Hematopoietic Neoplastic Cells.

AIM AND OBJECTIVE: Cancer has become one of the leading causes of morbidity and mortality worldwide. Limitations associated with existing agents increase the need to develop more effective anticancer drugs to improve the therapeutic arsenal available. The aim of this study was to synthesize and evaluate the antiproliferative effects of three new thiazacridine derivatives. MATERIAL AND METHODS: Using a three steps synthesis reaction, three novel thiazacridine derivatives were obtained and characterized: (Z)-5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-4-thioxo-thiazolidin- 2-one (LPSF/AC-99), (Z)-5-acridin-9-ylmethylene-3-(4-chloro-benzyl)-4-thioxo-thiazolidin-2- one (LPSF/AC-119) and (Z)-5-acridin-9-ylmethylene-3-(3-chloro-benzyl)-4-thioxo-thiazolidin-2- one (LPSF/AC-129). Toxicity and selectivity assays were performed by colorimetric assay. Then, changes in cell cycle and cell death induction mechanisms were assessed by flow cytometry. RESULTS: All compounds exhibited cytotoxicity to Raji (Burkitt's lymphoma) and Jurkat (acute T cell leukemia) cells, where LPSF/AC-119 showed best IC50 values (0.6 and 1.53 µ M, respectively). LPSF/AC-129 was the only cytotoxic compound in glioblastoma cell line NG97 (IC50 = 55.77 µ M). None of the compounds were toxic to normal human cells and induced neoplastic cell death primarily by apoptosis. CONCLUSION: All derivatives were more cytotoxic to hematopoietic neoplastic cells when compared to solid tumor derived cells. All three compounds are promising for in vivo and combination therapy studies against cancer.

Prostate apoptosis response-4 is involved in the apoptosis response to docetaxel in MCF-7 breast cancer cells.

Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as PAWR) is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. Par-4 was first identified in prostate cancer cells undergoing apoptosis. Both intracellular and extracellular Par-4 have been implicated in apoptosis. Relatively little is known about the role of Par-4 in breast cancer cell apoptosis. In this study, we sought to investigate the effects of Par-4 expression on cell proliferation, apoptosis and drug sensitivity in breast cancer cells. MCF-7 cells were stably transfected with expression vectors for Par-4, or transiently transfected with siRNA for Par-4 knockdown. Cell proliferation assays were performed using MTT and apoptosis was evaluated using acridine orange staining, fluorescence microscopy and flow cytometry. Par-4 overexpression reduced MCF-7 proliferation rates. Conversely, Par-4 knockdown led to increased MCF-7 proliferation. Par-4 downregulation also led to increased BCL-2 and reduced BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel.

Insulin-like growth factor-1 and 17ß-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells.

The PKC apoptosis WT1 regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17ß-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 µM ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 µM LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 µM SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.