Diphenyl Diselenide and SARS-CoV-2: in silico Exploration of the Mechanisms of Inhibition of Main Protease (Mpro) and Papain-like Protease (PLpro).

The SARS-CoV-2 pandemic has prompted global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro) and the papain-like protease (PLpro) are essential for viral replication and are key targets for therapeutic development. In this work, we investigate the mechanisms of SARS-CoV-2 inhibition by diphenyl diselenide (PhSe)2 which is an archetypal model of diselenides and a renowned potential therapeutic agent. The in vitro inhibitory concentration of (PhSe)2 against SARS-CoV-2 in Vero E6 cells falls in the low micromolar range. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations [level of theory: SMD-B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ] are used to inspect non-covalent inhibition modes of both proteases via π-stacking and the mechanism of covalent (PhSe)2 + Mpro product formation involving the catalytic residue C145, respectively. The in vitro CC50 (24.61 µM) and EC50 (2.39 µM) data indicate that (PhSe)2 is a good inhibitor of the SARS-CoV-2 virus replication in a cell culture model. The in silico findings indicate potential mechanisms of proteases' inhibition by (PhSe)2; in particular, the results of the covalent inhibition here discussed for Mpro, whose thermodynamics is approximatively isoergonic, prompt further investigation in the design of antiviral organodiselenides.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Aurones: A Promising Scaffold to Inhibit SARS-CoV-2 Replication.

Aurones are a small subgroup of flavonoids in which the basic C6-C3-C6 skeleton is arranged as (Z)-2-benzylidenebenzofuran-3(2H)-one. These compounds are structural isomers of flavones and flavonols, natural products reported as potent inhibitors of SARS-CoV-2 replication. Herein, we report the design, synthesis, and anti-SARS-CoV-2 activity of a series of 25 aurones bearing different oxygenated groups (OH, OCH3, OCH2OCH3, OCH2O, OCF2H, and OCH2C6H4R) at the A- and/or B-rings using cell-based screening assays. We observed that 12 of the 25 compounds exhibit EC50 < 3 µM (8e, 8h, 8j, 8k, 8l, 8m, 8p, 8q, 8r, 8w, 8x, and 8y), of which five presented EC50 < 1 µM (8h, 8m, 8p, 8q, and 8w) without evident cytotoxic effect in Calu-3 cells. The substitution of the A- and/or B-ring with OCH3, OCH2OCH3, and OCF2H groups seems beneficial for the antiviral activity, while the corresponding phenolic derivatives showed a significant decrease in the anti-SARS-CoV-2 activity. The most potent compound of the series, aurone 8q (EC50 = 0.4 µM, SI = 2441.3), is 2 to 3 times more effective than the polyphenolic flavonoids myricetin (2) and baicalein (1), respectively. Investigation of the five more active compounds as inhibitors of SARS-CoV-2 3CLpro based on molecular dynamic calculations suggested that these aurones should detach from the active site of 3CLpro, and, probably, they could bind to another SARS-CoV-2 protein target (either receptor or enzyme).

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case.

BACKGROUND: According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE: To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS: We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS: We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS: Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Antiviral Effect of 5'-Arylchalcogeno-3-aminothymidine Derivatives in SARS-CoV-2 Infection.

The understanding that zidovudine (ZDV or azidothymidine, AZT) inhibits the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and that chalcogen atoms can increase the bioactivity and reduce the toxicity of AZT has directed our search for the discovery of novel potential anti-coronavirus compounds. Here, the antiviral activity of selenium and tellurium containing AZT derivatives in human type II pneumocytes cell model (Calu-3) and monkey kidney cells (Vero E6) infected with SARS-CoV-2, and their toxic effects on these cells, was evaluated. Cell viability analysis revealed that organoselenium (R3a-R3e) showed lower cytotoxicity than organotellurium (R3f, R3n-R3q), with CC50 ≥ 100 µM. The R3b and R3e were particularly noteworthy for inhibiting viral replication in both cell models and showed better selectivity index. In Vero E6, the EC50 values for R3b and R3e were 2.97 ± 0.62 µM and 1.99 ± 0.42 µM, respectively, while in Calu-3, concentrations of 3.82 ± 1.42 µM and 1.92 ± 0.43 µM (24 h treatment) and 1.33 ± 0.35 µM and 2.31 ± 0.54 µM (48 h) were observed, respectively. The molecular docking calculations were carried out to main protease (Mpro), papain-like protease (PLpro), and RdRp following non-competitive, competitive, and allosteric inhibitory approaches. The in silico results suggested that the organoselenium is a potential non-competitive inhibitor of RdRp, interacting in the allosteric cavity located in the palm region. Overall, the cell-based results indicated that the chalcogen-zidovudine derivatives were more potent than AZT in inhibiting SARS-CoV-2 replication and that the compounds R3b and R3e play an important inhibitory role, expanding the knowledge about the promising therapeutic capacity of organoselenium against COVID-19.

Anti-SARS-CoV-2 Activity of Ampelozizyphus amazonicus (Saracura-Mirá): Focus on the Modulation of the Spike-ACE2 Interaction by Chemically Characterized Bark Extracts by LC-DAD-APCI-MS/MS.

Traditional medicine shows several treatment protocols for COVID-19 based on natural products, revealing its potential as a possible source of anti-SARS-CoV-2 agents. Ampelozizyphus amazonicus is popularly used in the Brazilian Amazon as a fortifier and tonic, and recently, it has been reported to relieve COVID-19 symptoms. This work aimed to investigate the antiviral potential of A. amazonicus, focusing on the inhibition of spike and ACE2 receptor interaction, a key step in successful infection. Although saponins are the major compounds of this plant and often reported as its active principles, a polyphenol-rich extract was the best inhibitor of the spike and ACE2 interaction. Chemical characterization of A. amazonicus bark extracts by LC-DAD-APCI-MS/MS before and after clean-up steps for polyphenol removal showed that the latter play an essential role in maintaining this activity. The effects of the extracts on viral replication were also assessed, and all samples (aqueous and ethanol extracts) demonstrated in vitro activity, inhibiting viral titers in the supernatant of Calu-3 cells after 24 hpi. By acting both in the SARS-CoV-2 cell entry process and its replication, A. amazonicus bark extracts stand out as a multitarget agent, highlighting the species as a promising candidate in the development of anti-SARS-CoV-2 drugs.

MLL2/KMT2D and MLL3/KMT2C expression correlates with disease progression and response to imatinib mesylate in chronic myeloid leukemia.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C. METHODS: Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. RESULTS: In patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway. CONCLUSION: Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.

Mechanisms of Vesicular Stomatitis Virus Inactivation by Protoporphyrin IX, Zinc-Protoporphyrin IX, and Mesoporphyrin IX.

Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.

Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV­2 Mpro).

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causative agent of the COVID-19 pandemic, a global public health problem. Despite the numerous studies for drug repurposing, there are only two FDA-approved antiviral agents (Remdesivir and Nirmatrelvir) for non-hospitalized patients with mild-to-moderate COVID-19 symptoms. Consequently, it is pivotal to search for new molecules with anti-SARS-CoV-2 activity and to study their effects in the human immune system. Ebselen (Eb) is an organoselenium compound that is safe for humans and has antioxidant, anti-inflammatory, and antimicrobial properties. Diphenyl diselenide ((PhSe)2) shares several pharmacological properties with Eb and is of low toxicity to mammals. Herein, we investigated Eb and (PhSe)2 anti-SARS-CoV-2 activity in a human pneumocytes cell model (Calu-3) and analyzed their toxic effects on human peripheral blood mononuclear cells (PBMCs). Both compounds significantly inhibited the SARS-CoV-2 replication in Calu-3 cells. The EC50 values for Eb and (PhSe)2 after 24 h post-infection (hpi) were 3.8 µM and 3.9 µM, respectively, and after 48 hpi were 2.6 µM and 3.4 µM. These concentrations are safe for non-infected cells, since the CC50 values found for Eb and (PhSe)2 on Calu-3 were greater than 200 µM. Importantly, the concentration rates tested on viral replication were not toxic to human PBMCs. Therefore, our findings reinforce the efficacy of Eb and demonstrate (PhSe)2 as a new candidate to be tested in future trials against SARS-CoV-2 infection/inflammation conditions.

Yellow fever vaccine protects mice against Zika virus infection.

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

BACKGROUND According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Gastrointestinal helminths of two populations of wild pigeons (Columba livia) in Brazil.

The present study analyzed gastrointestinal helminth communities in 265 wild pigeons (Columba livia) living in the municipalities of São Paulo and Tatuí, state of São Paulo, Brazil, over a one-year period. The birds were caught next to grain storage warehouses and were necropsied. A total of 790 parasites comprising one nematode species and one cestode genus were recovered from 110 pigeons, thus yielding an overall prevalence of 41.5%, mean intensity of infection of 7.2 ± 1.6 (range 1-144) and discrepancy index of 0.855. Only 15 pigeons (5.7%) presented mixed infection. The helminths isolated from the birds were Ascaridia columbae (Ascaridiidae) and Raillietina sp. (Davaineidae). The birds' weights differed according to sex but this did not influence the intensity of infection. The overall prevalence and intensity of infection did not differ between the sexes, but the prevalence was higher among the birds from Tatuí (47.8%). The gastrointestinal helminth community of C. livia was characterized in the two areas studied and parasite homogeneity was observed over the 12 months analyzed at both locations. These results make contributions to the current literature on health aspects of wild C. livia populations.

Effect of Intermediate Flush Using Different Devices to Prevent Chemical Smear Layer Formation.

This study compared the effect of intermediate flush with distilled water delivered by conventional irrigation, EndoVac microcannula or Self-Adjusting File (SAF) system in the prevention of chemical smear layer (CSL) formation. Thirty human premolars were used. Canals were prepared with Reciproc system and 5.25% NaOCl. After chemomechanical preparation, samples were divided in 3 groups (n=10) according to the intermediate irrigation protocol with distilled water using: conventional irrigation, EndoVac microcannula or SAF. A final flush with 2% chlorhexidine solution was used and scanning electron microscopy was performed to assess protocol effectiveness. Two calibrated evaluators attributed scores according the presence or absence of CSL on the surface of the root canal walls at the coronal, middle and apical thirds, as follows: (1) no CSL; (2) small amounts of CSL; (3) moderate CSL; and (4) heavy CSL. Differences between protocols were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between each root canal third. SAF resulted in less formation of CSL when compared with the conventional irrigation and EndoVac microcannula (p<0.05). When root canal thirds were analyzed, conventional irrigation and EndoVac groups showed less CSL formation at coronal and middle thirds in comparison to the apical third (p<0.05). In SAF group, there was no difference among the thirds (p>0.05). It may be concluded that an intermediate flush of distilled water, delivered by the SAF system resulted in a better reduction of CSL formation during chemomechanical preparation.

Hematologic reference intervals for wild Black vultures (Coragyps atratus).

BACKGROUND: The Black vulture (Coragyps atratus) is the most common species of vulture and is widespread in all America. The species feeds on rotting carcasses, and large groups are frequently seen in urban areas, concentrating especially on rubbish dumps. Although C atratus is a very common species in some areas, little is known about its health in the wild. OBJECTIVES: The aim of this study was to determine hematologic RIs of wild adult Black vultures. METHODS: Blood samples were obtained from 70 wild Black vultures captured in São Paulo, Brazil. Hematologic values were determined using conventional techniques applicable to birds. Reference intervals were determined using an Excel program with Reference Value Adviser (version 2.0). RESULTS: After statistical analysis, the following RIs were determined: HGB 8.5-12.5 g/dL, PCV 42.3-54.5%, MCV 203.2-402.6 fL, MCHC 17.4-26.2 g/dL, total solids 2.4-4.8 g/dL, RBC count 1.16-2.48 × 106 /µL, WBC count 5.93-27.14 × 103 /µL, heterophils 3.40-21.58 × 103 /µL, lymphocytes 0.19-5.16 × 103 /µL, eosinophils 0.0-3.07 × 103 /µL, monocytes 0.0-1.49 × 103 /µL, basophils 0.0-0.25 × 103 /µL, and heterophil:lymphocyte ratio 1.3-36.9. Thrombocyte mean was 14.14 × 103 /µL. CONCLUSIONS: Baseline hematologic data obtained in this study provide RIs that will be useful given that few studies have been carried out on the health of New World vultures.

Cytotoxic effect of the debris apically extruded during three different retreatment procedures.

We evaluated the cytotoxic effects of the debris apically extruded during root canal retreatment on primary human osteoblast (HOb) cells in vitro. TNF-α and IL-1ß levels were also measured. We examined three different techniques: conventional hand-files, and Mtwo and Reciproc retreatments. Filled mandibular incisors were prepared for a cytotoxicity assay in an experimental root model. The material was divided into three groups according to the technique used. Ten teeth were used as control. HOb cells were exposed to the extruded content and cytotoxicity was evaluated using the MTT test (assessing cell metabolic activity). TNF-α and IL-1ß production was also analyzed using enzyme-linked immunosorbent assay. Then, all the teeth were radiographed and the residual filling material was quantified. Data were subjected to analysis of variance (ANOVA) and Tukey's test (P < 0.05). The conventional hand-file technique was significantly more cytotoxic than the other methods (P < 0.05). Reciproc was less cytotoxic than Mtwo retreatment (P < 0.05). All endodontic retreatment techniques led to a significant upregulation of IL-1ß levels (P < 0.05). However, only the conventional hand-file technique caused a significant increase in TNF-α levels (P < 0.05). Root-filling removal did not affect the levels of these proteins (P > 0.05). The Reciproc system required less time than the other two methods to remove the root-filling materials (P < 0.01). The endodontic retreatment with Reciproc was the least cytotoxic and the least time-consuming method of gutta-percha and sealer removal. (J Oral Sci 58, 211-217, 2016).

Modulação autonômica da frequência cardíaca em crianças e jovens com paralisia cerebral / Autonomic heart rate modulation in children and youths with cerebral palsy

Introdução: A paralisia cerebral é uma doença que causa distúrbios neuromusculares, doenças cardiovasculares, além de distúrbios autonômicos e homeostáticos. Objetivo: Avaliar a modulação autonômica cardíaca de crianças e jovens com PC. Métodos: Estudo de delineamento transversal e observacional de carácter quantitativo. Foram coletados os intervalos R-R (IRR) do eletrocardiograma de 05 voluntários cadeirantes, com idade de 06 a 18 anos com PC através do cardiofrequencimetro Polar modelo V800®. Resultados: Idade média foi de 12,4 anos, ambos os gêneros, massa corpórea de 35,1 kg, estatura de 1,39 m e índice de massa corpórea 16,8 kg/m2 equivalente à desnutrição. Os dados apresentam a modulação autonômica cardíaca no domínio do tempo, as variáveis do desvio-padrão de todos os intervalos RR normais 60,0 ms, raiz-quadrada da média da soma dos quadrados das diferenças entre os IRR normais 60,3 ms, porcentagem dos IRR adjacentes maiores que 50 ms 16,1 % e no domínio da frequência a baixa frequência cardíaca (un) 55,7, a alta frequência cardíaca (un) 44,2, a razão da alta com a baixa frequência cardíaca 2,3. Conclusão: As crianças e adolescentes com paralisia cerebral têm uma baixa variabilidade da frequência cardíaca, com alta probabilidade de desenvolver doenças cardiovasculares, devido ao sedentarismo. (AU)

Introduction: Cerebral Palsy is a disease that causes neuromuscular disorders, cardiovascular diseases, in addition to disorders autonomic and homeostatic. Objective: To measure the cardiac autonomic modulation of children and young people with CP. Methods: Quantitative crosssectional and observational study. The electrocardiogram R-R intervals (IRR) were collected from 05 volunteers in wheelchairs, aged 6 to 18 years with CP through the cardiofrequencimeter Polar model V800®. Results: The average age is 12.4 years, both genders, body mass 35.1 kg, height 1.39 m and body mass index of 16.8 kg/m2 equivalent to malnutrition. The data show cardiac autonomic modulation in the time domain, the standard deviation variables of all normal RR intervals 60.0 ms, square root of the mean of the sum of squares of the differences between normal IRR 60.3 ms, percentage of adjacent IRR greater than 50 ms 16.1% and in the frequency domain, low heart rate (un) 55.7, high heart rate (un) 44.2, the ratio of discharge to low heart rate 2,3. Conclusion: Children and adolescents with cerebral palsy have a low heart rate variability, with a greater probability of developing cardiovascular diseases, due to sedentary lifestyle. (AU)

Effect of Intermediate Flush Using Different Devices to Prevent Chemical Smear Layer Formation

Abstract This study compared the effect of intermediate flush with distilled water delivered by conventional irrigation, EndoVac microcannula or Self-Adjusting File (SAF) system in the prevention of chemical smear layer (CSL) formation. Thirty human premolars were used. Canals were prepared with Reciproc system and 5.25% NaOCl. After chemomechanical preparation, samples were divided in 3 groups (n=10) according to the intermediate irrigation protocol with distilled water using: conventional irrigation, EndoVac microcannula or SAF. A final flush with 2% chlorhexidine solution was used and scanning electron microscopy was performed to assess protocol effectiveness. Two calibrated evaluators attributed scores according the presence or absence of CSL on the surface of the root canal walls at the coronal, middle and apical thirds, as follows: (1) no CSL; (2) small amounts of CSL; (3) moderate CSL; and (4) heavy CSL. Differences between protocols were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between each root canal third. SAF resulted in less formation of CSL when compared with the conventional irrigation and EndoVac microcannula (p<0.05). When root canal thirds were analyzed, conventional irrigation and EndoVac groups showed less CSL formation at coronal and middle thirds in comparison to the apical third (p<0.05). In SAF group, there was no difference among the thirds (p>0.05). It may be concluded that an intermediate flush of distilled water, delivered by the SAF system resulted in a better reduction of CSL formation during chemomechanical preparation.

Resumo O presente estudo comparou o efeito da utilização de uma irrigação intermediária com água destilada usando a irrigação convencional, a microcânula EndoVac ou o sistema Self-Adjusting File (SAF) na prevenção de formação da smear-layer química (SLQ). Trinta pré-molares humanos foram utilizados. Os canais foram preparados com sistema Reciproc e irrigados com NaOCl a 5,25%. Após o preparo químico mecânico, as amostras foram divididas em 3 grupos (n=10) de acordo com o protocolo de irrigação intermediária com água destilada utilizado: irrigação convencional, a microcânula EndoVac ou SAF. Utilizou-se uma lavagem final com solução de clorexidina a 2% e a microscopia electrónica de varredura foi utilizada para avaliar a eficácia dos protocolos. Dois avaliadores calibrados atribuíram escores de acordo com a presença ou ausência de SLQ nas paredes do canal radicular nos terços coronal, médio e apical, como a seguir: (1) sem SLQ; (2) pequenas quantidades de SLQ; (3) SLQ moderada e (4) muita SLQ. As diferenças entre protocolos foram analisadas com testes de Kruskal-Wallis e Mann-Whitney U. Os testes Friedman e Wilcoxon foram utilizados para comparação entre cada terço do canal radicular. SAF resultou em menor formação de SLQ quando comparado com a irrigação convencional e a microcânula EndoVac (p<0,05). Quando os terços dos canais radiculares foram analisados, os grupos irrigação convencional e microcânula EndoVac apresentaram menor formação de SLQ nos terços coronal e médio em relação ao terço apical (p<0,05). No grupo SAF, não houve diferença entre os terços (p>0,05). Dentro dos resultados do presente estudo, pode-se concluir que um fluxo intermediário de água destilada, administrado pelo sistema SAF resultou em melhor redução da formação de SLQ durante o preparo químico mecânico.

Gastrointestinal helminths of two populations of wild pigeons (Columba livia) in Brazil / Helmintos gastrointestinais de duas populações de pombos de vida livre (Columba livia) no Brasil

Abstract The present study analyzed gastrointestinal helminth communities in 265 wild pigeons (Columba livia) living in the municipalities of São Paulo and Tatuí, state of São Paulo, Brazil, over a one-year period. The birds were caught next to grain storage warehouses and were necropsied. A total of 790 parasites comprising one nematode species and one cestode genus were recovered from 110 pigeons, thus yielding an overall prevalence of 41.5%, mean intensity of infection of 7.2 ± 1.6 (range 1-144) and discrepancy index of 0.855. Only 15 pigeons (5.7%) presented mixed infection. The helminths isolated from the birds were Ascaridia columbae (Ascaridiidae) and Raillietina sp. (Davaineidae). The birds' weights differed according to sex but this did not influence the intensity of infection. The overall prevalence and intensity of infection did not differ between the sexes, but the prevalence was higher among the birds from Tatuí (47.8%). The gastrointestinal helminth community of C. livia was characterized in the two areas studied and parasite homogeneity was observed over the 12 months analyzed at both locations. These results make contributions to the current literature on health aspects of wild C. livia populations.

Resumo O presente estudo analisou comunidades gastrointestinais de helmintos em 265 indivíduos de Columba livia de vida livre nos municípios de São Paulo e Tatuí, estado de São Paulo, Brasil, durante um ano. As aves foram capturadas em áreas de armazenamento de grãos e sementes e necropsiadas. Um total de 790 parasitos representando uma espécie de nematódeo e um gênero de cestoide foram recuperados de 110 pombos com uma prevalência geral de 41,5%, intensidade média de infecção de 7,2 ± 1,6 (amplitude 1-144) e índice de discrepância de 0,855. Somente 15 (5,7%) pombos tiveram uma infecção mista. Os helmintos isolados das aves foram Ascaridia columbae (Ascaridiidae) e Raillietina sp. (Davaineidae). O peso das aves foi diferente entre os sexos, mas não influenciou a intensidade de infecção. A prevalência geral e a intensidade de infecção não foram diferentes entre sexo, mas a prevalência foi maior nas aves de Tatuí (47,8%). A comunidade gastrointestinal de helmintos de C. livia foi caracterizada nas duas áreas estudadas e uma homogeneidade de parasitos foi observada nos 12 meses analisados, em ambas localidades. Os resultados contribuem para a literatura atual sobre aspectos sanitários de populações de C. livia em vida livre.