There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

The aim was to evaluate the potential of mucus-permeating nanoparticles for the oral administration of insulin. These nanocarriers, based on the coating of zein nanoparticles with a polymer conjugate containing PEG, displayed a size of 260 nm with a negative surface charge and an insulin payload of 77 µg/mg. In intestinal pig mucus, the diffusivity of these nanoparticles (PPA-NP) was found to be 20-fold higher than bare nanoparticles (NP). These results were in line with the biodistribution study in rats, in which NP remained trapped in the mucus, whereas PPA-NP were able to cross this layer and reach the epithelium surface. The therapeutic efficacy was evaluated in Caenorhabditis elegans grown under high glucose conditions. In this model, worms treated with insulin-loaded in PPA-NP displayed a longer lifespan than those treated with insulin free or nanoencapsulated in NP. This finding was associated with a significant reduction in the formation of ROS as well as an important decrease in the glucose and fat content in worms. These effects would be related with the mucus-permeating ability of PPA-NP that would facilitate the passage through the intestinal peritrophic-like dense layer of worms (similar to mucus) and, thus, the absorption of insulin.

The aim was to evaluate the potential of nanocarriers, based on the coating of zein nanoparticles (ZNP) with a Gantrez (R) AN-PEG conjugate (GP), for the oral delivery of insulin. ZNP-GP displayed less negative surface charge and a 14-fold higher diffusion coefficient in pig intestinal mucus than ZNP. Both nanoparticles showed a spherical shape and an insulin load of 77.5 mu g/mg. Under simulated gastric conditions, ZNP-GP released significantly lower amount of insulin than ZNP, while under simulated intestinal conditions, both types of nanoparticles displayed similar behaviour. In Caenorhabditis elegans wild-type N2, grown under high glucose conditions, insulin treatments reduced glucose and fat accumulation without altering the growth rate, the worm length, or the pumping rate. The effect was significantly greater (p < 0.001) when insulin was nanoencapsulated in ZNP-GP compared with that encapsulated in ZNP or formulated in solution. This would be related to the highest capability of ZNP-GP to diffuse in the dense peritrophic-like layer covering intestinal cells in worms. In daf-2 mutants, the effect on fat and glucose reduction by insulin treatment was suppressed, indicating a DAF-2 dependent mechanism. In summary, ZNP-GP is a promising platform that may offer new opportunities for the oral delivery of insulin and other therapeutic proteins.

The metabolic properties of omega-6 fatty acid consumption are being increasingly accepted. We had previously observed that supplementation with a borage seed oil (BSO), as a source of linoleic (18:2n-6; LA) and gamma-linolenic (18:3n-6; GLA) acids, reduces body weight and visceral adiposity and improves insulin sensitivity in a diet-induced obesity model of Wistar rats. Here, it was investigated whether the anti-obesogenic properties of BSO could be maintained in a pre-obese model of rats, and if these effects are enhanced by a combination with low doses of quercetin, together with its potential role in the regulation of the adipocyte biology. The combination of BSO and quercetin during 8 weeks was able to ameliorate glucose intolerance and insulin resistance, and to improve liver steatosis. Although no effects were observed on body weight, animals supplemented with this combination exhibited a lower proportion of visceral adiposity. In addition, in vitro differentiation of epididymal adipose-precursor cells of the BSO-treated animals exhibited a down-regulation of Fasn, Glut4, Pparg and Srebp1 genes, in comparison with the control group. Finally, in vitro evaluation of the components of BSO demonstrated that the anti-adipogenic activity of quercetin was significantly potentiated by the combination with both LA and GLA through the down-regulation of different adipogenesis-key genes in 3T3-L1 cells. All these data suggest that omega-6 fatty acids LA and GLA, and their natural sources such as BSO, could be combined with quercetin to potentiate their effects in the prevention of the excess of adiposity and the insulin resistance.

Aging-related diseases can be triggered by multiple factors such as oxidative stress. Oxidative stress is an imbalance between free radicals and antioxidants, so today, compounds capable of reducing or neutralizing free radicals are being studied for a therapeutic use. Origanum vulgare L. is a traditional medicinal plant used for a wide number of health problems due to its antimicrobial, carminative and antioxidant activities. However, when administered orally, gastrointestinal digestion can modify some of therapeutical properties. To avoid this, two different solid oral formulations have been designed for an O. vulgare extract evaluating their antioxidant behaviours in vitro and in vivo after a simulation of gastrointestinal digestion. The results showed that the divided powder has a lower antioxidant activity both in vitro and in vivo than the encapsulated extract. The quantitative difference of polyphenols found on HPLC-DAD (especially luteolin, apigenin and caffeic acid) may explain the differences in pharmacological activity. Thus, we propose that the best form to administrate O. vulgare extracts to maintain the antioxidant properties is the encapsulated form, that is, two capsules of 250mg of a hydroalcoholic extract of O. vulgare with a minimum of 33 % of rosmarinic acid as a daily dose.

Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds (myricetin, kaempferol, naringin, hesperidin, apigenin, luteolin, resveratrol, curcumin and epicatechin), and phenolic acids (p-coumaric, ellagic, ferulic, gallic and vanillic acids) regulating C. elegans fat accumulation. Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress and ageing, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPRER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, ageing and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.

The characterization of compounds with antioxidant activity is of great interest due to their ability to reduce reactive oxygen species production and, therefore, prevent some age-related diseases. Its antioxidant capacity can be analyzed by different methods both in vitro and in vivo. Caenorhabditis elegans is an in vivo model widely used in ageing research. Until now, available tests analyze functional effects in the worms, so the antioxidant activity of the compound is indirectly monitored. We have developed a simple and a reliable method to quantify internal antioxidant activity in vivo. To validate this method, we analyzed an aqueous green tea extract and two other compounds with a well-known antioxidant activity and without this activity. The results obtained (EC50 green tea = 21.76 ± 1.28 µg/mL; EC50 positive control = 8.50 ± 0.33 µg/mL; negative control EC50 > 500 µg/mL) can help in the design of further in vivo experiments. Thus, our method can be used as a previous screening capable of reducing the gap between in vitro and in vivo assays.

In recent years it has been shown that the causes of chronic myeloproliferative neoplasms (MPNs) are more complex than a simple signaling aberration and many other mutated genes affecting different cell processes have been described. For instance, mutations in genes encoding epigenetic regulators are more frequent than expected. One of the latest genes described as mutated is SET binding protein 1 (SETBP1). In silico tools have revealed that there are several human SETBP1 paralogous to nuclear receptor binding SET domain protein 1 (NSD1), NSD2 and NSD3, for example, which are also involved in the development of other hematological malignancies. Therefore, the present study analyzed the mutational status of NSD1, NSD2, NSD3 and SETBP1 in BCR-ABL1 negative MPNs with or without Janus kinase 2 (JAK2) p.V617F mutation. The present study revealed that the NSD genes are not frequently mutated in MPNs. However, a novel SETBP1 mutation was identified in a patient with p.V617F JAK2 positive primary myelofibrosis. These results provide further insight into the genetic complexity of MPNs.

Brassicaceae contain bioactive compounds with potential positive effects on metabolic syndrome. Here, we evaluated the eventual anti-obesity properties of an ethanolic broccoli extract (BE), selected by a tested ability to reduce Caenorhabditis elegans fat content. Two doses (14 and 140 mg/kg animal) of BE were evaluated in a diet-induced obesity (DIO) Wistar rat model. After 10 weeks of BE supplementation, animals exhibited reduced body weight gain and food efficiency, decreased atherogenic index of plasma and improved glucose tolerance in comparison with non-supplemented rats. BE also reduced the retroperitoneal fat mass and adipocyte size, all associated to down-regulation of Cebpa, Srebp1, Fasn and Adipoq expression in adipocytes. Finally, BE significantly decreased liver steatosis, accompanied by the up-regulation of Acot8 and Acox1, and the down-regulation of Fasn, Fatp4 and Srebf1 expression in hepatocytes. Our data provides new knowledge about the potential role of broccoli components in the prevention of metabolic syndrome.

Cocoa polyphenols exhibit high antioxidant activity and have been proposed as a potential adjuvant for the treatment of metabolic disturbances. Here, we demonstrate that supplementation with low doses (14 and 140 mg per kg per rat) of a complete cocoa extract induces metabolic benefits in a diet-induced obesity (DIO) model of Wistar rats. After 10 weeks, cocoa extract-supplemented animals exhibited significantly lower body weight gain and food efficiency, with no differences in energy intake. Cocoa significantly reduced visceral (epididymal and retroperitoneal) and subcutaneous fat accumulation accompanied by a significant reduction in the adipocyte size, which was mediated by downregulation of the adipocyte-specific genes Cebpa, Fasn and Adipoq. Additionally, cocoa extract supplementation reduced the triacylglycerol/high density lipoprotein (TAG/HDL) ratio, decreased hepatic triglyceride accumulation, improved insulin sensitivity by reducing HOMA-IR, and significantly ameliorated glucose tolerance after an intraperitoneal glucose tolerance test. Finally, no adverse effect was observed in an in vivo toxicity evaluation of our cocoa extract at doses up to 500 mg kg -1 day -1. Our data demonstrate that low doses of cocoa extract supplementation (14 and 140 mg kg -1 day -1) are safe and sufficient to counteract obesity and type-2 diabetes in rats and provide new insights into the potential application of cocoa supplements in the management of the metabolic syndrome.

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