Metabolic Syndrome as a Cardiovascular Disease Risk Factor: Patients Evaluated in Primary Care

To estimate the prevalence of metabolic syndrome (MS) in a population receiving attention in primary care centers (PCC) we selected a random cohort of ostensibly normal subjects from the registers of 5 basic-health area (BHA) PCC. Diagnosis of MS was with the WHO, NCEP and IDF criteria. Variables recorded were: socio-demographic data, CVD risk factors including lipids, obesity, diabetes, blood pressure and smoking habit and a glucose tolerance test outcome. Of the 720 individuals selected (age 60.3 ± 11.5 years), 431 were female, 352 hypertensive, 142 diabetic, 233 pre-diabetic, 285 obese, 209 dyslipemic and 106 smokers. CVD risk according to the Framingham and REGICOR calculation was 13.8 ± 10% and 8.8 ± 9.8%, respectively. Using the WHO, NCEP and IDF criteria, MS was diagnosed in 166, 210 and 252 subjects, respectively and the relative risk of CVD complications in MS subjects was 2.56. Logistic regression analysis indicated that the MS components (WHO set), the MS components (IDF set) and the female gender had an increased odds ratio for CVD of 3.48 (95CI%: 2.26–5.37), 2.28 (95%CI: 1.84–4.90) and 2.26 (95%CI: 1.48–3.47), respectively. We conclude that MS and concomitant CVD risk is high in ostensibly normal population attending primary care clinics, and this would necessarily impinge on resource allocation in primary care

The Piwi pathway in Blattella germanica ovaries

Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain).Oogenesis, the formation and maturation of an oocyte, is a crucial process in the female reproductive physiology in animals. It encompasses a series of events that must occur in the correct order and at the right time. Hormonal signals are fundamental in the oogenesis processes, for oocyte differentiation and growth, to determine changes in cell program or for chorion synthesis. However, the maintenance of genome stability in insect germ cells during the proliferation and differentiation is of paramount importance, and it is the Piwi pathway which plays this role. Using Blattella germanica as a model, a species representative of panoistic ovaries, we studied three of the core proteins of the Piwi pathway in the ovaries, in order to illuminate their function in the maintenance of germ cells stability. B. germanica transcriptomes and bioinformatic analyses were used to identify the main proteins involved in the biogenesis of piRNAs and its regulation. By quantitative real-time PCR, the expression of Piwi, Argonaute 3 (Ago3) and Yb (fs(1)Yb or Tudor 12) was analyzed in B. germanica ovaries, and their functions were studied by RNAi. Piwi, Ago3 and Yb, are mainly expressed in ovaries of last nymphal instar when the differentiation of germinal cells is usually arrested. The expression of these genes is maintained during the first gonadotrophic cycle. Depletion of Yb and Ago3 allowed us to demonstrate their involvement in the primary and secondary piRNAs pathway, respectively

Argonaute-2 role in dsRNA processing in cockroaches

Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain).Interference of RNA (RNAi) is a cellular process that allows the cell to protect itself against harmful agents, especially alien RNA. Through the treatment with double-stranded RNA (dsRNA), this mechanism is being used to perform specific knockdown of the expression of a given gene, in order to do laboratory functional genetic studies or even to control insect pests. The aim of the present work is to study an aspect of the functioning of RNAi in insects, in particular, to determine the functions the protein Argonaute-2 (Ago2). Using the cockroach Blattella germanica as a model, specimens treated with dsRNA against Ago2 (dsAgo2), and control, were subsequently treated with a heterologous dsRNA (dsPolyh). Four days after the second treatment, libraries of small RNAs were obtained from control and dsAgo2 depleted specimens. The small interfering RNAs (siRNAs) corresponding to dsPolyh were analyzed. siRNAs found in the libraries were not equally distributed along the whole length of both RNA strands from the dsRNA injected, but enriched in certain areas. This unequal distribution was similar both in Control- and dsAgo2-treated specimens. Nevertheless, dsAgo2 samples contained 4 times more 22 nt siRNAs (the most usual size in the libraries) than Control samples. The unequal distribution of siRNAs along a heterologous dsRNA sequence indicates that there is a selection of siRNAs independent of their use in the process of degradation of the target RNA. The similar distribution of siRNAs in control and dsAgo2 samples indicates that Ago2 is not the protein responsible for doing siRNA selection. Nevertheless, the higher amount of 22 nt siRNAs in dsAgo2-treated specimens shows Ago2 nuclease activity on ¿mature¿ siRNAs

Blattella germanica piRNAs in materno-zygotic transition

Poster presented at the Eight International Symposium on Molecular Insect Science, held 7-10th July 2019 in Barcelona (Spain).The Piwi-interacting RNAs (piRNAs) are a small non-coding RNAs involved in the maintenance of genome stability. piRNAs were described as expressed exclusively in germinal cells, but their presence in somatic cells has been taking relevance in the last years. We have identified the Blattella germanica piRNAs from 24 small RNA libraries, corresponding to different developmental stages (Llonga et al 2018 DOI: 10.1002/jez.b.22815). Now we focus on the identification of piRNAs from a maternal origin and the characterization of the piRNAs involved in the transition to the zygotic stage. The role of such piRNAs in the protection of the embryo genome and the relationship with their putative target RNAs (transposable elements and mRNAs) are key to the understanding of the general functions of these regulatory elements but also their particularities in hemimetabolous insects. The 24 small RNA libraries were aligned against the genome with Bowtie and R Statistical Software was used for expression analysis. Finally, ovary and early embryonic piRNA expressions were validated by qRT-PCR. The analysis of B. germanica individual small RNA libraries, corresponding to ovaries from 7-day-old adult females (OvD7), non-fertilized eggs (NFE), the zygote (ED0), and the first day of embryogenesis (ED1), show that piRNAs in these stages agree with the current model of two piRNA biogenesis pathways. The most expressed piRNAs in each library of this transition have been analyzed in order to identify specific functions for each stage. By mapping the sequence to the genome, we have identified the origin of these piRNAs and also a number of potential mRNAs targets

siRNA enrichment in Argonaute 2-depleted Blattella germanica

[Background] RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation.[Methods] Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced.[Results] Depleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals.[Conclusions/general significance] The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies.This work was supported by Spanish Ministry of Economy and Competitiveness and FEDER (grant number CGL2016-76011-R), Secretaria d'Universitats i Recerca, Catalan Goverment (grant number 2017 SGR 1030), Agencia Estatal de Investigación (grant number PID2019-104483GB-I00/AEI/10.13039/501100011033), and Agencia Estatal Consejo Superior de Investigaciones Científicas (grant numbers 2019AEP028 and 2019AEP029).Peer reviewe

Release of Ropinirole in Acrylate Transdermal Patches: Mutual Interactions Between Formulation Variables

The aim of this study is to evaluate the cooperative interactions between formulation variables of ropinirole transdermal patches and characterize the effects of drug loading and crystallinity, degree of ionization and drug-polymer solubilization, functionalization of acrylate polymeric basis, and the addition of permeation enhancers over the release profiles. Several series of transdermal films based on carboxylic or hydroxylic acrylates (DuroTak®) and containing 1 to 10% ropinirole hydrochloride were laminated by mold-casting and evaporation. Formulations were characterized for crystallinity, drug particle size, drug assay, and residual solvents. Release profiles were obtained at different drug ionization state using paddle over disk apparatus. Mechanisms were elucidated with nonlinear data fitting of relevant release equations. Fickian and erosion processes were evaluated with the Peppas-Sahlin equation, and burst release risks were estimated as an independent term added to Higuchi kinetics. X-ray diffraction and microscopy evidenced differences in drug-polymer solubilization and density of drug crystals. Concerning drug release, area under the curve of dissolved quantities and release percentage were discriminant variables in mutual influence. Peppas-Shalin equation was the majority descriptor of release suggesting a combination of Fickian and erosion processes, revealing a decrease in the Fickian component as drug loading increased. Major burst release risks were evidenced mostly with Higuchi kinetics with vinylacetate acrylates. The carboxylic polymer without vinylacetate provided the best release extent, being more highly efficient as lower the drug loading was. Permeation enhancers with carboxylic or aliphatic radicals have, additionally, modified the release properties of ropinirole. Chemical interactions between the drug and acrylic polymers have been demonstrated. Only the effect with carboxylic polymer is pH dependent. The vinyl acetate comonomer reduces the drug release rate most effectively in formulations with low drug loads. The acrylic polymers without vinylacetate achieved the highest drug solubilization and thus the highest degree of release, providing a release of approximately 15% of the drug load. Keywords: Acrylate-vinylacetate copolymer; Drug loading; Release; Ropinirole; Transdermal