New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses

This work was financially supported by CTQ2017-85685-R, CTQ2017-85454-C2-1-P, and CTQ2017-86125-P (MICIU/AEI/ERDF). J.M.P. and J.D.V. thank UEQ for funding. J.V.P. is supported by an FPU fellowship (FPU17/04749). D.A. was supported by the Italian Cystic Fibrosis Foundation (grant FFC#3/2019) and by Fondazione Cassa Rurale Trento Rovereto (ref 2018.256). The authors acknowledge the Universidad of Granada (Spain) cell culture and microscopy central facilities (CIC-UGR).A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via sitedirected mutagenesis (S94V and R205Y), we increased the chloride affinity by more than an order of magnitude (kd = 106 ± 6 mM) at physiological pH. In addition, cis−trans isomerization of the chromophore produces a dual emission band with different chloride sensitivities, which allowed us to develop a ratiometric methodology to measure intracellular chloride concentrations.MICIU/AEI/ERDF CTQ2017-85685-R CTQ2017-85454-C2-1-P CTQ2017-86125-PUEQSpanish Government FPU17/04749Ministry of Health, ItalyItalian Cystic Fibrosis Research Foundation 3/2019Fondazione Cassa Rurale Trento Rovereto 2018.25

KOMBINIRANO DJELOVANJE BITURATA I EKSTRAKTA YUCCA SCHIDIGERI NA GASTROINTESTINALNI SUSTAV SVINJA OKO ODBIJANJA

After the banning of antibiotics as growth promoters, animal production in the EU faced the problem of increased post-weaning disorders, and several solutions were proposed (acidifiers, probiotics, herb extracts, enzyme cocktails, etc.). Short chain fatty acids (SCFA) are the main end product of bacterial fermentation in the hind gut in monogastric animals and can be used as energy supply for the epithelial cells of gut mucosa. Thus the aim of the study was to test the usefulness of novel microencapsulated sodium butyrate and Yucca Schidigera extract (encourages friendly bacteria growth, reduces ammonia) preparation on pig performance and small intestine development in the critical weaning period. Microencapsulation helps to avoid stomach degradation and provides better distribution of the compounds along the small intestine. Suckling piglets, 14-day old, were divided into 2 groups, control and with butyrate addition. Control pigs received a standard feed supplemented with a microencapsulated acidifier (0.3%). Butyrate pigs received the same feed and acidifier, as controls, supplemented with butyrate/yucca preparation (0.3%). At days 28 (weaning), 35 and 56 of life, 6 pigs from each group were killed and the gastrointestinal tract was harvested and measured. The initial body weight (14 d) of pigs from the control group was higher than that of the experimental, but at day 56 it did not differ significantly (P=0.48). The relative stomach and pancreas weights in the experimental group showed a tendency toward higher values in all time points, and the small intestine relative weight in day 35 was higher as compared to the controls. The growth rate of the pigs at day 35 decreased significantly in the control (P=0.007), but did not in the experimental (P=0.058) group compared to day 31. Measurements of the small intestine sections revealed increased mucosa thickness in butyrate treated pigs. This was due to both villi and crypt enlargement. In conclusion, our results demonstrated the effectiveness sodium butyrate and Yucca Shidigera extract in the prevention of weaning problems in pigs, however further research on the novel preparation is needed.Nakon zabrane antibiotika kao promotora rasta, proizvodnja životinja u EU suočila se sa sve više poremećaja nakon odbijanja pa je predloženo nekoliko rješenja (acidifikatori, probiotici, biljni ekstrakti, kokteli enzima, itd.). Kratkolančane masne kiseline glavni su krajnji proizvod bakterijske fermentacije u stražnjem crijevu u monogastričnih životinja pa se mogu upotrijebiti za snabdijevanje energijom epitelnih stanica crijevne sluznice /mukoze. Stoga je cilj ovog rada bio testirati korisnost novog natrijevog biturata u mikrokapsuli i pripravka ekstrakta Yucca Schidigera (potiče rast dobročudnih bakterija, smanjuje amonijak) na performansu svinja i razvoj tankog crijeva u kritičnom razdoblju odbijanja. Mikrokapsule pomažu da se izbjegne oštećenje želuca i pruža bolju razdiobu spojeva duž tankog crijeva. Praščići na sisi, u dobi od 14 dana, bili su podijeljeni u dvije skupine, kontrolnu i skupinu s dodavanjem biturata. Kontrolni praščići dobivali su standardnu hranu s dodatkom acidifikatora/zakiseljivača u mikrokapsuli (0.03%). Praščići na bituratima dobivali su istu hranu i acidifikator/zakiseljivač s dodatkom biturata/yucca pripravka (0.03%). U dobi od 28 (odbijanje), 35 i 56 dana žrtvovano je 6 svinja iz svake skupine te je pregledan i izmjeren gastrointestinalni sustav. Početna tjelesna masa (14. dan) svinja iz kontrolne skupine bila je veća od mase svinja pokusne skupine ali 56. dan nije se značajno razlikovala (P=0.48). Relativne mase želuca i gušterače u pokusnoj skupini pokazale su tendenciju viših vrijednosti u svim razdobljima, a relativna masa tankog crijeva 35. dana bila je viša u usporedbi s kontrolnom skupinom. Stopa rasta svinja 35. dana znatno se smanjila u kontrolnoj skupini (P=0.007) ali ne u kontrolnoj skupini (P=0.058) u usporedbi s 31. danom. Mjere tankog crijeva pokazale su povećanu debljinu sluznice u svinja tretiranih bituratom. To se pripisuje povećanju dlačica i udubina (villi i crypt). U zaključku, naši su rezultati pokazali djelotvornost natrijevog biturata i ekstrakta Yucca Schidigeri u prevenciji problema odbijanja u svinja, međutim, potrebna su dalja istraživanja novih pripravaka

Influence of the oxidizing agent in the synthesis of graphite oxide

The oxidation capacity of several procedures described in the literature which use different oxidizing agents has been exhaustively studied in order to describe the best route for oxidation of this material. The oxidation capacities of different types of materials were evaluated in the synthesis of graphite oxide in an effort to obtain a product with similar characteristics to those provided by commonly employed methods. The results obtained show that graphite oxide structures are greatly influenced by the nature of the oxidizing agent used. It was concluded that it is possible not only to establish the number of oxygenated groups attached to the structure but also, and depending on the oxidizing agent used, to know the stability of graphite oxide. The different characteristics of each graphite oxide obtained could facilitate their use in multiple applications.La capacidad de oxidación de varios procedimientos descritos en la literatura que utilizan diferentes agentes oxidantes ha sido exhaustivamente estudiada con el fin de describir la mejor ruta de oxidación de este material. Se evaluaron las capacidades de oxidación de diferentes tipos de materiales en la síntesis de óxido de grafito en un esfuerzo por obtener un producto con características similares a las proporcionadas por los métodos comúnmente empleados. Los resultados obtenidos muestran que las estructuras del óxido de grafito están muy influenciadas por la naturaleza del agente oxidante utilizado. Se concluyó que es posible no solo establecer el número de grupos oxigenados adheridos a la estructura sino también, y dependiendo del agente oxidante utilizado, conocer la estabilidad del óxido de grafito

Eukaryotic elongation factor 2 controls TNF-alpha translation in LPS-induced hepatitis

Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-alpha. TNF-alpha is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-alpha expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38gamma/delta MAPK proteins is required for the elongation of nascent TNF-alpha protein in macrophages. The MKK3/6-p38gamma/delta pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-alpha elongation. These results identify a new signaling pathway that regulates TNF-alpha production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-alpha production is involved

Essential role of protein tyrosine phosphatase 1B in obesity-induced inflammation and peripheral insulin resistance during aging

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes (T2DM). In this study, we have evaluated the role of PTP1B in the development of aging-associated obesity, inflammation, and peripheral insulin resistance by assessing metabolic parameters at 3 and 16 months in PTP1B) ⁄ ) mice maintained on mixed genetic background (C57Bl ⁄ 6J · 129Sv ⁄ J). Whereas fat mass and adipocyte size were increased in wild-type control mice at 16 months, these parameters did not change with aging in PTP1B) ⁄ ) mice. Increased levels of pro-inflammatory cytokines, crown-like structures, and hypoxia-inducible factor (HIF)-1a wereobserved only in adipose tissue from 16-month-old wild-type mice. Similarly, islet hyperplasia and hyperinsulinemia were observed in wild-type mice with agingassociated obesity, but not in PTP1B) ⁄ ) animals. Leanness in 16- month-old PTP1B) ⁄ ) mice was associated with increased energy expenditure. Whole-body insulin sensitivity decreased in 16- month-old control mice; however, studies with the hyperinsulinemic– euglycemic clamp revealed that PTP1B deficiency prevented this obesity-related decreased peripheral insulin sensitivity. At a molecular level, PTP1B expression and enzymatic activity were upregulated in liver and muscle of 16-month-old wild-type mice as were the activation of stress kinases and the expression of p53. Conversely, insulin receptor-mediated Akt ⁄ Foxo1 signaling was attenuated in these aged control mice. Collectively, these data implicate PTP1B in the development of inflammation and insulin resistance associated with obesity during aging and suggest that inhibition of this phosphatase by therapeutic strategies might protect against age-dependentT2DMThis work was supported by grants from Ministerio de Ciencia e Innovación (Spain) SAF2009-08114 and (to A.M.V.), BFU2008- 04901-C03-02 and 03 (to M.R and J.M.C., respectively), BFU2008-01283 (to M.V), Comunidad de Madrid S2010/BMD- 2423 and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) (Instituto Salud Carlos III). CBMSO is recipient of institutional aid from Ramón Areces Foundation. We also acknowledge grants NIH-R01 DK080756, ADA 7-07-RA-80, and NIH U24-DK093000 (to J.K.K.) and UMass Mouse Phenotyping Center supported by UMass Diabetes and Endocrinology Research Center Grant (DK32520) and EFSD/Amylin Programme 2011 grant (to A.M.V.)

Benralizumab reduces blood basophil percentage and activation in vitro without eliciting degranulation

PI21/0089

Impact of global PTP1B deficiency on the gut barrier permeability during NASH in mice.

OBJECTIVE:Non-alcoholic steatohepatitis (NASH) is characterized by a robust pro-inflammatory component at both hepatic and systemic levels together with a disease-specific gut microbiome signature. Protein tyrosine phosphatase 1 B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory signaling cascades. In this study, we have explored the role of PTP1B in the composition of gut microbiota and gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. METHODS:Gut features and barrier permeability were characterized in wild-type (PTP1B WT) and PTP1B-deficient knockout (PTP1B KO) mice fed a chow or methionine/choline-deficient (MCD) diet for 4 weeks. The impact of inflammation was studied in intestinal epithelial and enteroendocrine cells. The secretion of GLP-1 was evaluated in primary colonic cultures and plasma of mice. RESULTS:We found that a shift in the gut microbiota shape, disruption of gut barrier function, higher levels of serum bile acids, and decreased circulating glucagon-like peptide (GLP)-1 are features during NASH. Surprisingly, despite the pro-inflammatory phenotype of global PTP1B-deficient mice, they were partly protected against the alterations in gut microbiota composition during NASH and presented better gut barrier integrity and less permeability under this pathological condition. These effects concurred with higher colonic mucosal inflammation, decreased serum bile acids, and protection against the decrease in circulating GLP-1 levels during NASH compared with their WT counterparts together with increased expression of GLP-2-sensitive genes in the gut. At the molecular level, stimulation of enteroendocrine STC-1 cells with a pro-inflammatory conditioned medium (CM) from lipopolysaccharide (LPS)-stimulated macrophages triggered pro-inflammatory signaling cascades that were further exacerbated by a PTP1B inhibitor. Likewise, the pro-inflammatory CM induced GLP-1 secretion in primary colonic cultures, an effect augmented by PTP1B inhibition. CONCLUSION:Altogether our results have unraveled a potential role of PTP1B in the gut-liver axis during NASH, likely mediated by increased sensitivity to GLPs, with potential therapeutic value

Pharmacological and genetic increases in liver NADPH levels ameliorate NASH progression in female mice

Non-alcoholic steatohepatitis (NASH) is one of the fastest growing liver diseases worldwide, and oxidative stress is one of NASH main key drivers. Nicotinamide adenine dinucleotide phosphate (NADPH) is the ultimate donor of reductive power to a number of antioxidant defences. Here, we explored the potential of increasing NADPH levels to prevent NASH progression. We used nicotinamide riboside (NR) supplementation or a G6PD-tg mouse line harbouring an additional copy of the human G6PD gene. In a NASH mouse model induced by feeding mice a methionine-choline deficient (MCD) diet for three weeks, both tools increased the hepatic levels of NADPH and ameliorated the NASH phenotype induced by the MCD intervention, but only in female mice. Boosting NADPH levels in females increased the liver expression of the antioxidant genes Gsta3, Sod1 and Txnrd1 in NR-treated mice, or of Gsr for G6PD-tg mice. Both strategies significantly reduced hepatic lipid peroxidation. NR-treated female mice showed a reduction of steatosis accompanied by a drop of the hepatic triglyceride levels, that was not observed in G6PD-tg mice. NR-treated mice tended to reduce their lobular inflammation, showed a reduction of the NK cell population and diminished transcription of the damage marker Lcn2. G6PD-tg female mice exhibited a reduction of their lobular inflammation and hepatocyte ballooning induced by the MCD diet, that was related to a reduction of the monocyte-derived macrophage population and the Tnfa, Ccl2 and Lcn2 gene expression. As conclusion, boosting hepatic NADPH levels attenuated the oxidative lipid damage and the exhausted antioxidant gene expression specifically in female mice in two different models of NASH, preventing the progression of the inflammatory process and hepatic injury.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No - 832741. (Acronym Food-PPP-NAFLD). I.R-R was recipient and funded by the Marie Skłodowska-Curie grant. AP-F was a recipient of a predoctoral fellowship from the Spanish association against cancer - AECC (PRDMA18011PAST). JLL-A was funded by the Spanish Ministry of Science and Innovation (MICINN) (PTA2017‐14689‐I). PJF-M was funded by a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335 financed by the MCIN/AEI/10.13039/501100011033 and by the ESF Investing in your future). Work at the laboratory of PJF-M was funded by the AECC (SIRTBIO- LABAE18008FERN) and the RETOS Program projects from the MICINN (SAF2017-85766-R and PID2020-114077RB-I00/AEI/10.13039/501100011033). E.G.-D. was a recipient of a predoctoral grant financed by the Spanish Ministry (FPU18/05350). A. M. V. is funded by Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem).Peer reviewe