Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Abstract\ud \ud Background\ud Phospholipases A2 (PLA2s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA2 isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines.\ud \ud \ud Methods\ud The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I.\ud \ud \ud Results\ud It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %).\ud \ud \ud Conclusions\ud These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.The authors would like to thank the financial support provided by the State\ud of São Paulo Research Foundation (FAPESP, grants n. 2010/03243-43 and\ud 2011/23236-4), the Coordination for the Improvement of Higher Education\ud Personnel (CAPES) and the National Council for Scientific and Technological\ud Development (CNPq process n. 476932/2012-2). We are also grateful to\ud Fabiana Rosseto Morais, from FCFRP-USP, for the technical assistance in the\ud flow cytometry analyses. Thanks are also due to the Center for the Study of\ud Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication\ud of this special collection (CNPq process 469660/2014-7)

Genetic diversity and structure of two species of Enyalius (Squamata: Leiosauridae) from neotropical biodiversity hotspots

Diversidade e estrutura genética de duas espécies de Enyalius (Squamata: Leiosauridae) em hotspots neotropicais de biodiversidade. Os lagartos do gênero Enyalius são endêmicos da América do Sul, sendo predominantemente encontrados no Cerrado e em fragmentos da Floresta Atlântica. Este é um gênero pouco estudado, e não foram encontrados dados relacionados à diversidade e à estrutura genética das espécies do gênero. Neste trabalho, investigamos a diversidade genética de populações de E. bilineatus (N = 20) e E. perditus (N = 28), usando um fragmento de 234-pb do citocromo b, e comparamos as sequências geradas com outras publicadas. Dezenove haplótipos distintos foram encontrados (11 de E. perditus e oito de E. bilineatus), sendo oito deles novos registros. Os valores de diversidade haplotípica foram muito similares para as duas espécies (0.684 para E. perditus e 0.647 para E. bilineatus). A distância genética entre as duas espécies foi de 20.3%, e as distâncias intraespecíficas foram 2.0% para E. perditus e 5.6% para E. bilineatus. Nossos dados sugerem que as populações de E. bilineatus são altamente divergentes e que a espécie deve apresentar diversidade críptica. Este é o primeiro estudo medindo a diversidade genética de espécies do gênero Enyalius oriundas de regiões consideradas hotspots da biodiversidade neotropical e apresenta dados relevantes para um melhor entendimento das relações inter e intrapopulacionais, assim como a distribuição das linhagens genéticas desse gênero endêmico.Genetic diversity and structure of two species of Enyalius (Squamata: Leiosauridae) from neotropical biodiversity hotspots. Enyalius, a lizard genus endemic to South America, is mostly distributed in the remains of the Atlantic Forest and in the Cerrado. The genus has been the topic of a few studies but none has quantified the genetic diversity and structure within and among populations of Enyalius. The genetic diversity and structure of populations of E. bilineatus (N = 20) and E. perditus (N = 28) are examined using a 234-bp fragment of the cytochrome b gene and compared with the sequences reported in other published data. Nineteen distinct haplotypes (eleven for E. perditus and eight for E. bilineatus) were found, eight of which were recorded for the first time. The haplotype diversities are highly similar for both species (0.684 for E. perditus and 0.647 for E. bilineatus). The genetic distance between the two species is 20.3% and the distance within species is 2.0% and 5.6% for E. perditus and E. bilineatus, respectively. Our data suggest that populations of E. bilineatus are genetically divergent and may reveal cryptic diversity. This is the first study to quantify the genetic diversity of species of Enyalius from Neotropical biodiversity hotspots. These data facilitate a better understanding of both within and among population variation, and highlight the distribution of genetic lineages of an endemic and poorly studied genus

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34(+) samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34(+) samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans

<div><p>Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence <i>in situ</i> hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R² = 0.60; p<0.0001) and patients (R² = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R² = 0.35; p<0.0001) and low correlation for patients (R² = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R² = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R² = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.</p></div

Variability analysis of flow-FISH and qPCR.

<p>(<b>A</b>) Intra-assay variation ±95% confidence interval. Flow-FISH, CV = 10.8%; qPCR, CV = 9.5%; p = 0.35. (<b>B</b>) Inter-assay variation ±95% confidence interval. Flow-FISH, CV = 9.5%; qPCR, CV = 16%; p = 0.02. (<b>C</b>) Bland-Altman plot for flow-FISH: mean difference between two independent measurements of 23 samples by their average; the bias was −0.08±1.07 and limits of agreement ranged from −2.23 to 2.05. (<b>D</b>) Bland-Altman plot for qPCR: mean difference between two independent measurements of 57 samples by their average; the bias was −0.37±1.7 and limits of agreement ranged from −3.02 to 3.78. For Bland-Altman analysis, telomere length measurements were represented in kilobases.</p

Comparison between flow-FISH and TRF analysis of leukocyte telomere length in healthy individuals and patients with bone marrow failure or idiopathic pulmonary fibrosis: linear correlation and Bland-Altman agreement.

<p>Telomere length of 70 healthy individuals was measured by flow-FISH and Southern blot: (<b>A</b>) Linear regression plots; solid line represents the data best fit (R² = 0.60); and (<b>B</b>) Bland-Altman plot for agreement analysis of flow-FISH (kb) and TRF analysis (kb). The bias±SD was 0.17±1.03 and limits of agreement (LoA) ranged from −1.88 to 2.24 kb. Telomere length of 51 patients was measured by both methods: (<b>C</b>) Linear regression plots; solid line represents the data best fit (R² = 0.51); and (<b>D</b>) Bland-Altman plot for agreement analysis of flow-FISH (kb) and TRF analysis (kb). The bias±SD was zero ±1.21 and the LoA ranged from −2.41 to 2.41. Measurements were represented in kilobases.</p

Demographic and clinical characteristics of participants of this study.

<p>Demographic and clinical characteristics of participants of this study.</p