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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-NPs) was found to be 20-fold higher than bare nanoparticles (NPs). These results were in line with the biodistribution study in rats, in which NPs remained trapped in the mucus, whereas PPA-NPs 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-NPs displayed a longer lifespan than those treated with insulin free or nanoencapsulated in NPs. This finding was associated with a significant reduction in the formation of reactive oxygen species (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-NPs that would facilitate the passage through the intestinal peritrophic-like dense layer of worms (similar to mucus) and, thus, the absorption of insulin.

In this work, a bioactive persimmon extract was produced from discarded fruits. A central composite design was used to evaluate the effect of different extraction parameters and ripeness stages of persimmon fruits on the total phenolic content and antioxidant activity of the resulting extracts. Significantly greater phenolic contents were obtained from immature persimmon (IP) fruits. The optimum IP extract with the conditions set by the experimental design was industrially up-scaled and its composition and functional properties were evaluated and compared with those obtained under lab-scale conditions. Both extracts contained significant protein (>20%) and phenolic contents (∼11–27 mg GA/g dry extract) and displayed significant antiviral activity against murine norovirus and hepatitis A virus. Moreover, the extract showed no toxicity and significantly reduced the fat content and the cellular ageing of Caenorhabditis elegans (C. elegans) without affecting the worm development. These effects were mediated by down-regulation of fat-7, suggesting an anti-lipogenic activity of this extract., This work was financially supported by the grant RTI-2018-094268-B-C22 (MCI/AEI/FEDER, EU). Mendez D. A. is supported by the Ministry of Science, Technology and Innovation (Minciencias) of the Colombian Government (783-2017). M. J. Fabra and A. Martinez-Abad are recipients of Ramon y Cajal (RYC-2014-158) and Juan de la Cierva (IJDC-2017-31255), respectively, from the Spanish Ministry of Economy, Industry and Competitiveness. We also appreciate the funding of the CERCA program (Centres de Recerca de Catalunya) of the Generalitat de Catalunya and from the CIEN project “BIOPRO” from CDTI.

EZH2, a component of the polycomb repressive complex 2, catalyses the trimethylation of histone H3 lysine 27, a chromatin mark associated with transcriptional repression. EZH2 loss-of-function mutations are seen in myeloid neoplasms and are associated with an adverse prognosis. Missense mutations in the SET/CXC domain abrogate catalytic activity as assessed by in vitro histone methylation assays, but missense mutations clustering in the conserved DI and DII regions retain activity. To understand the role of DI and DII mutations, we initially developed a cell-based histone methylation assay to test activity in a cellular context. Murine induced pluripotent stem cells lacking EZH2 were transiently transfected with wild type or mutant EZH2 (n = 15) and any resulting histone methylation was measured by flow cytometry. All DI mutations (n = 5) resulted in complete or partial loss of methylation activity whilst 5/6 DII mutations retained activity. Next, we assessed the possibility of splicing abnormalities induced by exon 8 mutations (encoding DII) using RT-PCR from primary patient samples and mini-gene assays. Exon 8 mutations resulted in skipping of exon 8 and an out-of-frame transcript. We have therefore shown that mutations within regions encoding EZH2 domains DI and DII are pathogenic by loss of function and exon skipping, respectively.

Ultra-processed foods (UPFs) consumption could affect gut microbiota diversity and profile. We aimed to evaluate the effects of UPFs on microbiota, considering the role of sex. The consumption of UPFs (using NOVA criteria) was assessed with a validated 137-item food-frequency questionnaire. Participants (n = 359) were classified into less than three servings per day (n = 96) of UPFs and more than five (n = 90). Women and men were subclassified following the same criteria. 16S rRNA sequencing was performed from DNA fecal samples, and differences in microbiota were analyzed using EdgeR. The relationship between UPFs and bacteria was assessed by Spearman correlation and comparison of tertiles of consumption. Women who consumed more than five servings/day of UPFs presented an increase in Acidaminococcus, Butyrivibrio, Gemmiger, Shigella, Anaerofilum, Parabacteroides, Bifidobacterium, Enterobacteriales, Bifidobacteriales and Actinobacteria and a decrease in Melainabacter and Lachnospira. Bifidobacterium, Bifidobacteriales and Actinobacteria was positively associated with pizza and Actinobacteria with industrially processed dairy in women. Men who consumed more than five servings/day presented an increase of Granulicatella, Blautia, Carnobacteriaceae, Bacteroidaceae, Peptostreptococcaceae, Bacteroidia and Bacteroidetes and a decrease of Anaerostipes and Clostridiaceae. Bacteroidia and Bacteroidetes correlated positively with industrially processed meat. This study suggests that UPFs may affect microbiota composition differently in women and men.

The increasing prevalence of metabolic syndrome-related diseases, including type-2 diabetes and obesity, makes it urgent to develop new alternative therapies, such as probiotics. In this study, we have used Caenorhabditis elegans under a high-glucose condition as a model to examine the potential probiotic activities of Pediococcusacidilactici CECT9879 (pA1c). The supplementation with pA1c reduced C. elegans fat accumulation in a nematode growth medium (NGM) and in a high-glucose (10 mM) NGM medium. Moreover, treatment with pA1c counteracted the effect of the high glucose by reducing reactive oxygen species by 20%, retarding the aging process and extending the nematode median survival (>2 days in comparison with untreated control worms). Gene expression analyses demonstrated that the probiotic metabolic syndrome-alleviating activities were mediated by modulation of the insulin/IGF-1 signaling pathway (IIS) through the reversion of the glucose-nuclear-localization of daf-16 and the overexpression of ins-6 and daf-16 mediators, increased expression of fatty acid (FA) peroxisomal β-oxidation genes, and downregulation of FA biosynthesis key genes. Taken together, our data suggest that pA1c could be considered a potential probiotic strain for the prevention of the metabolic syndrome-related disturbances and highlight the use of C. elegans as an appropriate in vivo model for the study of the mechanisms underlying these diseases.

In recent years, food ingredients rich in bioactive compounds have emerged as candidates to prevent excess adiposity and other metabolic complications characteristic of obesity, such as low-grade inflammation and oxidative status. Among them, fungi have gained popularity for their high polysaccharide content and other bioactive components with beneficial activities. Here, we use the C. elegans model to investigate the potential activities of a Grifola frondosa extract (GE), together with the underlying mechanisms of action. Our study revealed that GE represents an important source of polysaccharides and phenolic compounds with in vitro antioxidant activity. Treatment with our GE extract, which was found to be nongenotoxic through a SOS/umu test, significantly reduced the fat content of C. elegans, decreased the production of intracellular ROS and aging-lipofuscin pigment, and increased the lifespan of nematodes. Gene expression and mutant analyses demonstrated that the in vivo anti-obesity and antioxidant activities of GE were mediated through the daf-2/daf-16 and skn-1/nrf-2 signalling pathways, respectively. Taken together, our results suggest that our GE extract could be considered a potential functional ingredient for the prevention of obesity-related disturbances., This work was performed with the financial support of the CIEN project BIOPRO from “Centro para el Desarrollo Tecnológico Industrial” (CDTI), Ministry of Science and Innovation, Government of Spain. The authors thank ARGAL ALIMENTACIÓN S.A., ANECOOP, ELMUBAS IBÉRICA S.L.U., Bodega Matarromera S.L., Kimitec Group, Mahou San Miguel, UVESA, and Entomo AgroIndustrial for their support of this project. We also thank Bolet Ben Fet (TEB Verd SCCL., Barcelona). Finally, the authors thank LABORATORIOS CINFA, S.A. and VISCOFAN S.A for the financial support of the Center for Nutrition Research, and the CERCA Programme (Generalitat de Catalunya) for the financial support of IRTA.

Phenolic compounds might modulate adiposity. Here, we report our observation that polyphenols and phenolic acids inhibit adipogenesis in 3T3-L1 with different intensity depending on the family and the stage of differentiation. While quercetin and resveratrol inhibited lipid accumulation along the whole process of differentiation, apigenin and myricetin were active during the early and latest stages, but not intermediate, contrary to hesperidin. The activity of phenolic acids was limited to the early stages of the differentiation process, except p-coumaric and ellagic acids. This anti-adipogenic effect was accompanied by down-regulation of Scd1 and Lpl. Molecular docking analysis revealed that the inhibitory activity of these phenolic compounds over the early stages of adipogenesis exhibits a significant correlation (r = 0.7034, p = 0.005) with their binding affinity to the ligand-binding domain of PPAR&gamma, Results show that polyphenols and phenolic acids would interact with specific residues of the receptor, which could determine their potential anti-adipogenic activity during the early stages of the differentiation. Residues Phe264, His266, Ile281, Cys285 and Met348 are the most frequently involved in these interactions, which might suggest a crucial role for these amino acids modulating the activity of the receptor. These data contribute to elucidate the possible mechanisms of phenolic compounds in the control of adipogenesis.

Bioinformatic analyses of CALR mutations in myeloproliferative neoplasms support a role in signaling

Summary: Whole exome sequencing was performed in a patient with myelodysplastic syndrome before and after progression to acute myeloid leukaemia. Mutations in several genes, including SETBP1, were identified following leukaemic transformation. Screening of 328 patients with myeloid disorders revealed SETBP1 mutations in 14 patients (4·3%), 7 of whom had -7/del(7q) and 3 had i(17)(q10), cytogenetic markers associated with shortened overall survival and increased risk of leukaemic evolution. SETBP1 mutations were frequently acquired at the time of leukaemic evolution, coinciding with increase of leukaemic blasts. These data suggest that SETBP1 mutations may play a role in MDS and chronic myelomonocytic leukaemia disease progression. © 2013 John Wiley and Sons Ltd.

The BCR-ABL negative chronic myeloproliferative disorders (CMPDs) are a group of stem cell clonal haematological malignancies characterised by abnormal proliferation and survival of one or more myeloid lineage cells. It has been proposed that they are caused by abnormalities in some signal transduction pathways mainly due to acquired somatic mutations, some of them in tyrosine kinase genes. One of the most prevalent mutations is V617F in the pseudokinase domain (JH2) coded by JAK2 exon 14. This mutation, reported in 2005, has been associated with nearly 95% of patients diagnosed of polycythaemia vera (PV) and near a half of the patients with essential thrombocythaemia (ET) and primary myelofibrosis (MF). However, the frequency of this mutation is below 20% for the remaining chronic myeloproliferative disorders and is absent in lymphoid neoplasms. JAK2V617F has been an important milestone in the knowledge of the molecular mechanisms leading to classic myeloproliferative disorders. However, there are still some patients with PV, ET and MF lacking V617F whose molecular defect is unknown. The disease in these cases could be due to other abnormalities in JAK2 or other related genes. In fact, in a few cases new point mutations have been reported, affecting also the JH2 domain, and one mutation affecting the JH1 domain [1] and reviewed in [2], some of them in other haematological malignancies. In addition, 10% of patients diagnosed of MF and some with ET show somatic activating mutations in the thrombopoietin receptor gene (MPL) inducing constitutive cytokine-independent activation of the JAK-STAT pathway.

Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation.

When studying mutations in DNA samples, determining whether novel sequence changes are somatic mutations or germline polymorphisms can be difficult. Here we describe a novel and very simple approach for identification of somatic mutations and loss of heterozygosity (LoH) events in DNA samples where no matched tissue sample is available. Our method makes use of heterozygous polymorphisms that are located near the putative mutation to trace both germinal alleles.

Review on 8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome), with data on clinics, and the genes involved.

Background Despite the discovery of the p.V617F in JAK2 , the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617F JAK2 -negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes ( CBL , CBLB and CBLC ) in a selected group of patients with myeloproliferative neoplasms. We also included V617F JAK2- positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. Design and Methods Using denaturing high performance liquid chromatography, we screened for mutations in CBL , CBLB and CBLC in a group of 172 V617F JAK2 -negative and 232 V617F JAK2 -positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. Results An initial screening of all coding exons of CBL , CBLB and CBLC in 44 V617F JAK2 -negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617F JAK2 -negative and 232 V617F JAK2 -positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617F JAK2 -positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. Conclusions Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617F JAK2 -positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.