Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p>Chagas disease is a neglected illness, with limited treatments, caused by the parasite <it>Trypanosoma cruzi</it>. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by <it>T. cruzi</it>. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.</p> <p>Results</p> <p>The differential genes expression of a sensitive <it>Trypanosoma cruzi </it>strain and its induced <it>in vitro </it>benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC₅₀ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.</p> <p>Conclusion</p> <p>Here, we propose NTR as the major marker of the appearance of resistance to BZ.</p

High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate

Early and sustained increase in time in range 1 year after initiation of hybrid close loop therapy via telemedicine in type 1 diabetes patients

La evidencia respalda la eficacia y seguridad del sistema Hybrid Close Loop (HCL) en pacientes con diabetes tipo 1 (DT1). Sin embargo, hay datos limitados disponibles sobre los resultados a largo plazo de los pacientes que reciben HCL con seguimiento por telemedicina. Métodos Un estudio de cohorte observacional prospectivo que incluyó pacientes con diabetes Tipo 1 que estaban actualizando al sistema HCL. La capacitación virtual y el seguimiento se realizaron a través de telemedicina. Los datos de MCG se analizaron para comparar el tiempo inicial dentro del rango (TIR), el tiempo por debajo del rango (TBR), la variabilidad glucémica y el modo automático (AM), con mediciones realizadas a los 3, 6 y 12 meses. Resultados Se incluyeron 134 pacientes con A1c basal 7,6% ± 1,1. El 40,5% tuvo algún evento de hipoglucemia grave en el último año. La TIR inicial, medida dos semanas después de comenzar AM, fue de 78,6 ± 9,94 %. No se observaron cambios evidentes a los tres (diferencia de medias − 0,15; IC-2,47; 2,17; p  = 0,96), seis (MD-1,09; IC-3,42, 1,24; p  = 0,12) y 12 meses (MD-1,30; IC-3,64). <1,04; p  = 0,08). No se encontraron cambios significativos en la TBR ni en la variabilidad glucémica a lo largo del seguimiento. El uso de AM fue de 85,6 ± 17,5% y el porcentaje de uso de sensor fue de 88,75 ± 9,5% a los 12 meses. No se informaron eventos de hipoglucemia (SH) graves. Conclusiones Los sistemas HCL permiten mejorar TIR, TBR y variabilidad glucémica de forma segura, temprana y sostenida hasta 1 año de seguimiento en pacientes con DM1 y alto riesgo de hipoglucemia seguidos a través de telemedicina.Q1Background and Aims Evidence supports the efficacy and safety of the Hybrid Close loop (HCL) system in patients with type 1 diabetes (T1D). However, limited data are available on the long-term outcomes of patients on HCL with telemedicine follow-up. Methods A prospective observational cohort study including T1D patients, who were upgrading to HCL system. Virtual training and follow-up were done through telemedicine. CGM data were analyzed to compare the baseline time in range (TIR), time below range (TBR), glycemic variability and auto mode (AM), with measurements performed at 3, 6 and 12 months. Results 134 patients were included with baseline A1c 7.6% ± 1.1. 40.5% had a severe hypoglycemia event in the last year. Baseline TIR, measured two weeks after starting AM was 78.6 ± 9.94%. No changes were evident at three (Mean difference − 0.15;CI-2.47,2.17;p = 0.96), six (MD-1.09;CI-3.42,1.24;p = 0.12) and 12 months (MD-1.30;CI-3.64,1.04;p = 0.08). No significant changes were found in TBR or glycemic variability throughout the follow-up. Use of AM was 85.6 ± 17.5% and percentage of use of sensor was 88.75 ± 9.5% at 12 months. No severe hypoglycemic (SH) events were reported. Conclusions HCL systems allow to improve TIR, TBR and glycemic variability safely, early and sustained up to 1 year of follow-up in patients with T1D and high risk of hypoglycemia followed through telemedicine.Revista Internacional - IndexadaS

Saponins from edible spears of wild asparagus inhibit AKT, p70S6K, and ERK signalling, and induce apoptosis through G0/G1 cell cycle arrest in human colon cancer HCT-116 cells

41 Páginas, 6 Figuras, 1 TablaThe effects of steroidal saponins from edible spears of wild triguero Huetor-Tajar asparagus on some of the oncogenic molecular pathways that are affected in human colon cancer cells were investigated. Reverse-phase chromatography and a new HPLC-MS method were used to respectively isolate and analyse the composition of the steroidal saponins. They were resistant to simulated digestion and, when in contact with HTC-116 human colon carcinoma cells, interfered with extracellular signal-regulated kinase (ERK), S6 kinase (p70S6K, mTOR), and RAC-alpha serine/threonine-protein kinase (AKT) pathways by a downregulation of these proteins. The expressions of cyclins D, E, and A were also decreased, leading to G0/G1 cell cycle arrest. In addition, these steroidal saponins induced typical features of apoptosis by the promotion of caspase-3 activity, poly(ADP-ribose) polymerase 1 (PARP-1) cleavage, and DNA fragmentation. These results offer potential dietary intervention strategy against human colon cancer cells.This study was supported by grants AGL2011-29632 and AGL2011-29008 funded by MICINN. S.L. acknowledges financial support from the Spanish MINECO (JCI-2012-13084, Juan de la Cierva) and the Spanish Research Council (CSIC)/JAE-doc Program (JAEDOC089), a contract cofounded by the European Social Fund (ESF).Peer reviewe

A New Perspective on Huntington's Disease: How a Neurological Disorder Influences the Peripheral Tissues

Huntington’s disease (HD) is a neurodegenerative disorder caused by a toxic, aggregationprone expansion of CAG repeats in the HTT gene with an age-dependent progression that leads to behavioral, cognitive and motor symptoms. Principally affecting the frontal cortex and the striatum, mHTT disrupts many cellular functions. In fact, increasing evidence shows that peripheral tissues are affected by neurodegenerative diseases. It establishes an active crosstalk between peripheral tissues and the brain in different neurodegenerative diseases. This review focuses on the current knowledge of peripheral tissue effects in HD animal and cell experimental models and identifies biomarkers and mechanisms involved or affected in the progression of the disease as new therapeutic or early diagnostic options. The particular changes in serum/plasma, blood cells such as lymphocytes, immune blood cells, the pancreas, the heart, the retina, the liver, the kidney and pericytes as a part of the blood–brain barrier are described. It is important to note that several changes in different mouse models of HD present differences between them and between the different ages analyzed. The understanding of the impact of peripheral organ inflammation in HD may open new avenues for the development of novel therapeutic targets

Diterpenoids from the Brown Alga Rugulopteryx okamurae and Their Anti-Inflammatory Activity

Brown algae of the Family Dictyotaceae produce an array of structurally diverse terpenoids, whose biomedical potential in the anti-inflammatory area has been scarcely explored. Herein, the chemical study of the alga Rugulopteryx okamurae has led to the isolation of ten new diterpenoids: rugukadiol A (1), rugukamurals A–C (2–4), and ruguloptones A–F (6–10). The structures of the new compounds were established by spectroscopic means. Compound 1 exhibits an unprecedented diterpenoid skeleton featuring a bridged tricyclic undecane system. Compounds 2–10 belong to the secospatane class of diterpenoids and differ by the oxygenated functions that they contain. In antiinflammatory assays, the new diterpenoid 1 and the secospatanes 5 and 10 significantly inhibited the production of the inflammatory mediator NO in LPS-stimulated microglial cells Bv.2 and macrophage cells RAW264.7. Moreover, compounds 1 and 5 were found to strongly inhibit the expression of Nos2 and the pro-inflammatory cytokine Il1b in both immune cell lines

Anti-inflammatory effect of the medicinal herbal mixture infusion, Horchata, from southern Ecuador against LPS-induced cytotoxic damage in RAW 264.7 macrophages

[EN] The phytochemical composition and the antioxidant and anti-inflammatory activities of a mixture of 23 plants, named Horchata, traditionally consumed in Ecuador, have been evaluated. The study was carried out using the hydroalcoholic extract (HHext) and infusion (IHext) of the horchata plant mixture. It was verified that thermal treatment affected the contents of vitamin C and carotenoids, but hardly those of polyphenols, which would be the main bioactive compounds in the infusion, the common form of preparation of horchata for consumption. Among phenolic compounds, caffeoylquinic acids, flavones and flavonols (mostly quercetin glycosides) were prominent. Both HHext and IHext extracts managed to protect RAW 264.7 macrophages against LPS-induced cytotoxic damage, increasing the levels of endogenous antioxidant enzymes and modulating the production of pro-inflammatory and anti-inflammatory cytokines. Greater protective effects were obtained for HHext compared to IHext, which was in agreement with its higher content of phenolic compounds favoured by a more efficient extraction in the hydroalcoholic medium. Nonetheless, the infusion still maintained a significant antioxidant and anti-inflammatory activity, which would support the protective effects on health traditionally attributed to its consumption by the population

Clinical factors associated with high glycemic variability defined by coefficient of variation in patients with type 2 diabetes

Antecedentes: La Variabilidad Glucémica Alta (VHG) ha convertirse en un predictor más fuerte de hipoglucemia. Sin embargo, aún se desconocen los factores clínicos asociados con el VHG. Objetivo:Determinar las variables clínicas que se asociaron con un coeficiente de variación (CV) superior al 36% evaluado mediante monitorización continua de glucosa (MCG) en un grupo de pacientes con diabetes mellitus. Métodos: Se evaluó una cohorte de pacientes con diabetes tipo 2 (T2D). Se evaluaron variables demográficas, HbA1c, tasa de filtración glomerular (TFG) y régimen de tratamiento. Se realizó un análisis bivariado, para evaluar la asociación entre la variable resultado (CV > 36%) y cada una de las variables independientes. Se construyó un modelo multivariado para evaluar las asociaciones después de controlar las variables de confusión. Resultados:Se analizaron los datos de MCG de 274 pacientes. CV> 36% estuvo presente en 56 pacientes (20,4%). En el análisis bivariado se incluyeron variables demográficas y clínicas, como tiempo desde el diagnóstico, antecedente de hipoglucemia, A1c, FG y tratamiento instaurado. En el análisis multivariante, FG 9% (OR 2,81; IC 1,05,7,51; p:0,04) y antecedentes de hipoglucemia (OR 2,09; IC 1,02, 4,32; p: 0,04) se asociaron con VHG. El tratamiento con iDPP4 (OR 0,39; IC 0,19, 0,82; p: 0,01) y AGLP1 (OR 0,08; IC 0,01, 0,68; p: 0,02) se asoció inversamente con la VG. Conclusión:Variables clínicas como FG 9% y antecedentes de hipoglucemia se asocian a un VG alto. Nuestros datos sugieren que el uso de tecnología y tratamientos capaces de reducir la variabilidad glucémica podría ser útil en esta población para reducir el riesgo de hipoglucemia y mejorar el control glucémico.Q3Background: High glycemic Variability (HGV) has become a stronger predictor of hypoglycemia. However, clinical factors associate with HGV still are unknown. Objective: To determine clinical variables that were associated with a coefficient of variation (CV) above 36% evaluated by continuous glucose monitoring (CGM) in a group of patients with diabetes mellitus. Methods: A cohort of patients with type 2 diabetes (T2D) was evaluated. Demographic variables, HbA1c, glomerular filtration rate (GFR) and treatment regimen were assessed. A bivariate analysis was performed, to evaluate the association between the outcome variable (CV> 36%) and each of the independent variables. A multivariate model was constructed to evaluate associations after controlling for confounding variables. Results: CGM data from 274 patients were analyzed. CV> 36% was present in 56 patients (20.4%). In the bivariate analysis, demographic and clinical variables were included, such as time since diagnosis, hypoglycemia history, A1c, GFR and treatment established. In the multivariate analysis, GFR 9% (OR 2.81; CI 1.05,7.51; p:0.04) and hypoglycemia history (OR 2.09; CI 1.02,4.32; p:0.04) were associated with HGV. Treatment with iDPP4 (OR 0.39; CI 0.19,0.82; p:0.01) and AGLP1 (OR 0.08; CI 0.01,0.68; p:0.02) was inversely associated with GV. Conclusion: Clinical variables such as GFR 9% and a history of hypoglycemia are associated with a high GV. Our data suggest that the use of technology and treatments able to reduce glycemic variability could be useful in this population to reduce the risk of hypoglycemia and to improve glycemic control.Revista Internacional - Indexad

Epithelial immunomodulation by aerosolized Toll-like receptor agonists prevents allergic inflammation in airway mucosa in mice

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand), when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy