Environmental enrichment affects behavioral and pharmacological response to antidepressants in CF1 mice.

It has been described that environmental enrichment (EE) exerts beneficial effects on cognitive and emotional performances, dendritic branching, synaptic density, neurogenesis and modulation of neurotrophic systems and neurotransmitters in rodents. However, the influence of EE on pharmacological and behavioral responses in animal models of psychiatric disorders has not been fully established. In this context, the aim of this study was to evaluate the influence of exposure to EE on mice behavior in the open field test (OFT) and forced swimming tests (FST), as well as the response to antidepressant drugs (fluoxetine 30 mg/kg and bupropion 30 mg/kg, p.o.). CF1 mice were exposed to an enriched housing condition at different developmental stages: from mating to postnatal day (PND) 55 (lifelong enrichment), from mating to PND21 (perinatal enrichment) and from PND21 to PND55 (post-weaning enrichment). At PND58 the male offspring were evaluated in the OFT and FST. BDNF gene expression in the hippocampus was determined through qPCR. Mice exposed to perinatal enrichment remained longer in the peripheral zone of the OFT and performed fewer grooming than mice housed under standard condition, and these effects were independent of drug treatment. Post-weaning and lifelong enrichment increased grooming behavior. Bupropion reduced grooming in all groups except in perinatal enriched. In turn, fluoxetine decreased grooming only in post-weaning enriched group. None of the enriched housing conditions altered the immobility time in the FST, which indicates that EE had no antidepressant-like effect. However, all enriched housing conditions abolished the anti-immobility effect of bupropion. None of the EE protocols affected BDNF hippocampal expression. The main conclusion is that mice behavior in the OFT is sensitive to alterations in the housing environment and depends on the developmental stage of exposure. Bupropion and fluoxetine yielded divergent responses depending on the housing condition, which suggests that EE modulates monoaminergic neurotransmission pathways.

Betulinic Acid-Brosimine B Hybrid Compound Has a Synergistic Effect with Imatinib in Chronic Myeloid Leukemia Cell Line, Modulating Apoptosis and Autophagy.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the formation of the BCR-ABL (breakpoint cluster region-Abelson) oncoprotein. As many patients display therapeutic resistance, the development of new drugs based on semisynthetic products represents a new potential therapeutic approach for treating the disease. In this study, we investigated the cytotoxic activity, possible mechanism of action of a hybrid compound of betulinic acid (BA) and brosimine B in CML cell lines that are sensitive (K-562) and resistant (K-562R) to imatinib, in addition to evaluating lower doses of imatinib in combination with the hybrid compound. The effects of the compound, and its combination with imatinib, on apoptosis, cell cycle, autophagy and oxidative stress were determined. The compound was cytotoxic in K-562 (23.57 ± 2.87 µM) and K-562R (25.80 ± 3.21 µM) cells, and a synergistic effect was observed when it was associated with imatinib. Apoptosis was mediated by the caspase 3 and 9 intrinsic pathway, and cell cycle evaluation showed arrest at G0/G1. In addition, the hybrid compound increased the production of reactive oxygen species and induced autophagy by increasing LC3II and Beclin-1 mRNA levels. Results suggest that this hybrid compound causes the death of both imatinib-sensitive and -resistant cell lines and may hold potential as a new anticancer treatment against CML.

Oxidative damage by 1,10-phenanthroline-5,6-dione and its silver and copper complexes lead to apoptotic-like death in Trichomonas vaginalis.

Trichomoniasis is a neglected, parasitic, sexually transmitted infection. Resistance to the only approved drugs is increasing worldwide, leaving millions of people without alternative medications. Thus, the search for new therapeutic options against this infection is necessary. Previously, our group reported that 1,10-phenanthroline-5,6-dione (phendione) and its silver(I) and copper (II) complexes (abbreviated as Ag-phendione and Cu-phendione, respectively) presented activity against the amitochondriate parasite T. vaginalis, with Cu-phendione being the most effective (IC50 = 0.84 µM). Methods: qRT-PCR, SEM, flow cytometry. The current study on the effects of Cu-phendione on the antioxidant metabolism of T. vaginalis by qRT-PCR revealed that the complex causes a decrease in the relative expression of mRNA of NADH oxidase, flavin reductase, superoxide dismutase, peroxiredoxin, iron-sulfur flavoprotein, rubrerythrin and osmotically inducible proteins. In contrast, the mRNA expression of flavodiiron protein was increased. Detoxification-related enzymes were downregulated, impairing oxygen metabolism in trophozoites and triggering a subsequent accumulation of the superoxide anion. Although no DNA fragmentation was observed, the treatment of parasites with Cu-phendione led to a significant reduction in cell size and a concomitant increase in granularity. The complex promoted phosphatidylserine exposure at the plasma membrane (as judged by Annexin V binding) and propidium iodide was unable to passively permeate the parasites. All of these outcomes are classical hallmarks of cell death by apoptosis. In essence, the trichomonacidal effect of Cu-phendione operates through redox homeostasis imbalance, which is a mode of action that is quite distinct from that caused by metronidazole.

Evaluation of Clinical Course of Gamma (P.1) Variant of Concern versus Lineages in Hospitalized Patients with COVID-19 in a Reference Center in Brazil.

The SARS-CoV-2 variant of concern (VOC) gamma (P.1) has increased transmissibility and resulted in elevated hospitalization and mortality rates in Brazil. We investigated the clinical course of COVID-19 caused by gamma and non-VOCs at a reference hospital in Brazil in a retrospective cohort study with nonelderly hospitalized patients from two periods, before and after the emergence of gamma. Cohort 1 included patients from both periods whose samples would be eligible for whole-genome sequencing (WGS). Cohort 2 was composed of randomly selected patients from Cohort 1 whose samples were submitted to WGS. A total of 433 patients composed Cohort 1: 259 from the first and 174 from the second period. Baseline characteristics were similar, except for a higher incidence of severe distress respiratory syndrome at admission in patients from the second period. Patients from the second period had significantly higher incidence rates of advanced respiratory support (adjusted hazard ratio [aHR]: 2.04; 95% confidence interval [CI], 1.60-2.59), invasive ventilatory support (aHR: 2.72; 95% CI: 2.05-3.62), and 28-day mortality from the onset of symptoms (aHR: 2.62; 95% CI: 1.46-4.72). A total of 86 (43 gamma and 43 non-gamma) patients composed Cohort 2. Patients with confirmed gamma VOC infections had higher advanced ventilatory support and mortality rates than non-gamma-infected patients. Our study suggests that non-elderly patients hospitalized for COVID-19 in the second period (used as a proxy of gamma infection) had a more severe clinical course. This might have contributed to higher hospitalization and death rates observed in the second wave in Brazil.

Genomic Surveillance of SARS-CoV-2 Lineages Indicates Early Circulation of P.1 (Gamma) Variant of Concern in Southern Brazil.

The SARS-CoV-2 P.1 lineage emerged in Amazonas (AM), North Brazil and its evolution has been dynamically reported associated with increased transmissibility and/or immune evasion. Here, we evaluated the lineages circulating in 29 cities in Rio Grande do Sul (RS), Southern Brazil between March 2020 and May 2021 and investigated the genetic events associated with the emergence of the P.1. A total of 202 oro/nasopharyngeal SARS-CoV-2 specimens from patients during routine hospital care were submitted to whole-genome sequencing. Phylogenetic and Bayesian Evolutionary Analyses of the P.1 lineage were carried out to determine the relationship between sequences from RS and AM and dated their common ancestor and origin. One hundred six (53%) sequences were assigned as P.1 and most carried the 22 lineage-defining mutations. All the P.1 sequences included other important mutations, such as P314L and R203K/G204R, and revealed a high genetic diversity in the phylogenetic tree. The time-scaled inference suggests that the oldest P.1 sequences from different Brazilian states share a ancestor with those from AM, but the origin of some sequences from RS is unknown. Further, the common ancestor of sequences from RS is dated to mid-June/July 2020, earlier than those previously reported from AM. Our results demonstrate that there is a high degree of genetic diversity among P.1 sequences, which suggests a continuous evolution and community spread of the virus. Although the first P.1 outbreak was reported in AM, the lineage was associated with multiple introductory events and had already been circulating in Southern Brazil prior to November 2020. IMPORTANCE The SARS-CoV-2 P.1 lineage is associated with increased transmissibility and/or immune evasion and presents a dynamic evolution in Brazil. The significance of our research relies in the fact that we evaluated the SARS-CoV-2 lineages circulating in Southern Brazil between March 2020 and May 2021. This evaluation allowed us to detect the genetic events associated with the emergence of the P.1 and its sublineages. This study is important because we were able to establish that the common ancestor of P.1 sequences from Rio Grande do Sul, Southern Brazil, is dated of mid-June/July 2020, earlier than the P.1 sequences previously reported from Amazonas (AM) state. Noteworthy, the high degree of genetic diversity among P.1 sequences found in this study suggests a continuous evolution and community spread of the virus. Moreover, the oldest P.1 sequences from different Brazilian states share a ancestor with those from AM.

RT-qPCR half-reaction optimization for the detection of SARS-CoV-2.

INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

Taste-masked nanoparticles containing Saquinavir for pediatric oral administration.

This research has aimed to improve the stability and taste-masking properties by developing nanostructured dosage forms containing Saquinavir. Liquid formulations were developed using Eudragit RS100® and Pullulan as polymers. The physicochemical characteristics, stability, in vitro drug release, morphology, mucoadhesion and taste masking capacity were evaluated. The Saquinavir-nanoparticles had average diameters between 136 and 158 nm, with a Span below 1.4. These formulations presented a drug content above 80%, a high encapsulation efficiency (>97%), slightly acidic pH levels, low dynamic viscosity and controlled drug release. Electron microscopy revealed irregular spherical nanoparticles. The formulations prepared with higher amounts of Eudragit RS100® had greater mucoadhesion. Both polymers were able to improve drug stabilization, taste-masking properties and protection against drug cytotoxicity. The Saquinavir-nanoparticles exhibited stability and control releasing properties, thus making it a promising liquid dosage form with taste-masking properties intended for application in pediatric treatment.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

DPPIV/CD26: a tumor suppressor or a marker of malignancy?

Dipeptidyl peptidase IV (DPPIV/CD26) is a multifunctional protein with intrinsic peptidase activity that inactivates or degrades some bioactive peptides. It is the main cellular binding protein for ecto-adenosine deaminase and interacts with extracellular matrix proteins, besides participating in different signaling pathways. Due to these multiple functions, DPPIV/CD26 has been shown to be closely related to the tumor process. It has been reported that the progression of certain types of cancer is accompanied by a decrease in DPPIV/CD26 expression, and studies have shown that the malignant phenotype can be reverted when DPPIV/CD26 expression is induced in these cancer cells, characterizing this protein as a tumor suppressor. On the other hand, DPPIV/CD26 was described as a protein associated with invasion and metastatic spread, characterizing it as a marker of malignancy. Thus, this review explores the roles of DPPIV/CD26 expression in tumor progression in different types of cancer and demonstrates the importance of this protein as a promising therapeutic target and tumor biomarker.

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.