A New Hyaluronic Emulgel of Hesperetin for Topical Application-An In Vitro Evaluation.

The present study aimed to formulate and characterize a hesperetin formulation to achieve adequate deposition and retention of hesperetin in the epidermis as a target for some cosmetic/dermatological actions. To derive the final emulgel, various formulations incorporating different proportions of Polysorbate 80 and hyaluronic acid underwent testing through a Box-Behnken experimental design. Nine formulations were created until the targeted emulgel properties were achieved. This systematic approach, following the principles of a design of experiment (DoE) methodology, adheres to a quality-by-design (QbD) paradigm, ensuring a robust and purposeful formulation and highlighting the commitment to a quality-driven design approach. The emulsions were developed using the phase inversion method, optimizing the emulgel with the incorporation of hyaluronic acid. Physically stable optimized emulgels were evaluated for their globule size, surface charge, viscosity, pH, electrical conductivity, and hesperetin content. These assays, along with the temperature swing test, were used to select the optimal formulation. It was characterized by a droplet size, d[4,3], of 4.02 µm, a Z-potential of -27.8 mV, an O/W sign, a pH of 5.2, and a creamy texture and proved to be stable for at least 2 months at room temperature. Additionally, in vitro release kinetics from the selected emulgel exhibited a sustained release profile of hesperetin. Skin assays revealed adequate retention of hesperetin in the human epidermis with minimum permeation. Altogether, these results corroborate the promising future of the proposed emulgel in cosmetic or dermatological use on healthy or diseased skin.

Intranasal Drug Administration in Alzheimer-Type Dementia: Towards Clinical Applications.

Alzheimer-type dementia (ATD) treatments face limitations in crossing the blood-brain barrier and systemic adverse effects. Intranasal administration offers a direct route to the brain via the nasal cavity's olfactory and trigeminal pathways. However, nasal physiology can hinder drug absorption and limit bioavailability. Therefore, the physicochemical characteristics of formulations must be optimized by means of technological strategies. Among the strategies that have been explored, lipid-based nanosystems, particularly nanostructured lipid carriers, are promising in preclinical investigations with minimal toxicity and therapeutic efficacy due to their ability to overcome challenges associated with other nanocarriers. We review the studies of nanostructured lipid carriers for intranasal administration in the treatment of ATD. Currently, no drugs for intranasal administration in ATD have marketing approval, with only three candidates, insulin, rivastigmine and APH-1105, being clinically investigated. Further studies with different candidates will eventually confirm the potential of the intranasal route of administration in the treatment of ATD.

New Vehiculation Systems of Mometasone Furoate for the Treatment of Inflammatory Skin Diseases.

A pre-formulation study was carried out to obtain liposomal formulations of mometasone furoate as an alternative system to marketed forms of corticosteroid for the treatment of inflammatory skin lesions. Mometasone furoate was loaded in glycerosomes and glyceroethosomes, which were also modified with hyaluronic acid (glyceroethohyalurosomes). Vesicles were designed, elaborated, and characterized, and their biocompatibility, efficacy against oxidative stress and skin lesions were assessed in vitro, in human epidermal cells, and in vivo, in a mouse skin epidermal hyperplasia model. All formulations tested showed great encapsulation efficiency, nanometric size, formed monodispersed systems and a highly negative Z potential. Similar values were obtained over nine months storage at 4 °C, which indicates the great stability of the three types of nanoliposomes at least during the time tested. Among them, 0.1% mometasone furoate glyceroethohyalurosomes were the best formulation to protect cells against oxidative stress and their anti-inflammatory efficacy was confirmed in vivo, being even more effective than the marketed form (Elocom®), as the reduction in the inflammation was even ~15% higher than that achieved with the commercial cream. Selected formulations could be potential candidates as new vehiculation systems for mometasone furoate. The presence of hyaluronic acid in glyceroethohyalurosomes makes them the best candidates in preventing/treating skin inflammatory lesions.

New scenario in the field of medical devices in the European Union: Switzerland and the United Kingdom become third countries.

OBJECTIVE: To compare the measures taken by the European Union,  Switzerland and the United Kingdom to ensure the continuity of the medical  devices market, complying with the requirements of Regulation  2017/745. METHOD: To carry out this work, a review was made of the official websites of  the European Commission, the Spanish Agency for Medicines and Health  Products, the Swiss Agency for Therapeutic Products and the Medicines and  Healthcare Products Regulatory Agency of the United Kingdom. Bibliographic  searches were also conducted on Pubmed and the internet (Google), using  terms such as "withdrawal of the Mutual Recognition Agreement of Swiss  European Union medical device conformity certificates, new UK medical device  regulation", for a period extending from January 2020 to December 2021. RESULTS: As a result of the disappearance of the legal framework  that supported free trade between Switzerland, the United Kingdom and  the European Union, products that used to be unrestrictedly distributed in Europe have become imports having to comply with the relevant legal  requirements. Distributors for their part have become importers, and  declarations of conformity and CE certificates have lost their validity.  Furthermore, notified bodies from Switzerland and the United Kingdom are no  longer recognized by the European Commission. Switzerland, the United Kingdom and the European Union have had to grant grace periods to allow  regulatory agencies and economic operators to adapt to the new situation. CONCLUSIONS: The transition period toward the new economic scenario has not  yet ended. Both Switzerland and the United Kingdom have had to take  stronger measures than the EU to adapt to the changes. Both Switzerland and  the United Kingdom are expected to finally incorporate the requirements of the  new Regulation in their internal legal systems.

OBJETIVO: Comparar las medidas que se han tomado por parte de la Unión  Europea, Suiza y Reino Unido para mantener la continuidad de mercado  cumpliendo con los requisitos regulatorios del Reglamento 745/2017 de   productos Sanitarios.Método: Para realizar este trabajo se han revisado las webs oficiales de la  omisión Europea, la Agencia Española del Medicamento y Productos Sanitarios,  la Swiss Agency for Therapeutic Products y la Medicines and  Healthcare Products Regulatory Agency del Reino Unido y se han realizado  búsquedas bibliográficas en PubMed y en internet (Google) con términos como  "withdrawal Mutual Recognition Agreement of certificates of conformity  European Union Switzerland medical devices, new regulation medical devices  UK" y similares para un periodo comprendido entre enero de 2020 y diciembre  de 2021. RESULTADOS: Como resultado del cese del marco legal que sostenía el libre  comercio entre Suiza y Reino Unido de la Unión Europea, la distribución de  productos sanitarios se ha convertido en una importación, teniendo que  cumplir con los requisitos legales pertinentes. Los distribuidores han pasado a  ser importadores, y las declaraciones de conformidad y certificados de  Conformidad Europea han perdido su validez. Además, los Organismos  Notificados ya no son reconocidos por la Comisión EuroAbstract  pea. En  consecuencia, Suiza, Reino Unido y la Unión Europea han tenido que conceder  periodos de gracia para permitir a las agencias reguladoras y operadores  conómicos adaptarse a las nuevas condiciones. CONCLUSIONES: El periodo de transición para la adaptación al nuevo escenario económico todavía no ha concluido. Además, el Reglamento acaba de entrar plenamente en vigor, por lo que se creará normativa de desarrollo que deberá implementarse también en estos países. Por tanto, será necesaria una nueva reglamentación que permita abordar estos aspectos.

American and European legislation on border medical devices.

INTRODUCTION: Borderline medical devices are products in a 'gray area,' this means due to their characteristics, they could belong to different 'legal products.' In addition, regulation is a controversial topic and may change depending on the country which may put public health at risk and distort the market. AREAS COVERED: This article analyzes how borderline medical devices are managed in the American and the European legislation. We compared the decisions made by both regulations on the devices of the Manual on Borderline and Classification Medical Devices of the European Commission for the first three sections, those which deal exclusively with medical devices. EXPERT OPINION: Borderline medical devices do not have to be understood as something specific to each country. The different classification of products creates international borders. It is necessary to create working groups in international organizations in which global consensus is reached. Although a priori it seems that the American system could be more efficient, studies with quantitative data from authorized devices are needed to show that. Until EUDAMED is not fully operational and open access, it will not be possible to develop them.

Borderline products do not have a simple product classification and can be managed differently depending on different countries. The different classification between countries has economic consequences for companies and patients. In this article, the American and European regulations system is compared, specifically borderline products, using as a tool the Borderline and Classification Manual of the European Commission. Results show the international consensus is necessary to avoid barriers to trade and contribute to innovation. Although both regulations have points of improvement, with the data from the Manual (EU), it seems that American regulatory system could be more efficient, although copying some of its strengths could be complicated due to the intrinsic characteristics of the European system. However, studies with quantitative data are needed to corroborate this statement.

Nuevo escenario en el ámbito de los productossanitarios en la Unión Europea: Suiza y Reino Unido se convierten en terceros países / New scenario in the field of medical devices in the European Union: Switzerland and the United Kingdom become third countries

Objetivo: Comparar las medidas que se han tomado por parte dela Unión Europea, Suiza y Reino Unido para mantener la continuidadde mercado cumpliendo con los requisitos regulatorios del Reglamento 745/2017 de Productos Sanitarios.Método: Para realizar este trabajo se han revisado las webs oficialesde la Comisión Europea, la Agencia Española del Medicamento y Productos Sanitarios, la Swiss Agency for Therapeutic Products y la Medicines and Healthcare Products Regulatory Agency del Reino Unido y se hanrealizado búsquedas bibliográficas en PubMed y en internet (Google)con términos como “withdrawal Mutual Recognition Agreement of certificates of conformity European Union Switzerland medical devices, newregulation medical devices UK” y similares para un periodo comprendidoentre enero de 2020 y diciembre de 2021.Resultados: Como resultado del cese del marco legal que sosteníael libre comercio entre Suiza y Reino Unido de la Unión Europea, ladistribución de productos sanitarios se ha convertido en una importación,teniendo que cumplir con los requisitos legales pertinentes. Los distribuidores han pasado a ser importadores, y las declaraciones de conformidady certificados de Conformidad Europea han perdido su validez. Además,los Organismos Notificados ya no son reconocidos por la Comisión Europea. En consecuencia, Suiza, Reino Unido y la Unión Europea han tenidoque conceder periodos de gracia para permitir a las agencias reguladoras y operadores económicos adaptarse a las nuevas condiciones. (AU)

Objective: To compare the measures taken by the European Union, Switzerland and the United Kingdom to ensure the continuity of the medicaldevices market, complying with the requirements of Regulation 2017/745.Method: To carry out this work, a review was made of the official websites of the European Commission, the Spanish Agency for Medicines andHealth Products, the Swiss Agency for Therapeutic Products and the Medicines and Healthcare Products Regulatory Agency of the United Kingdom.Bibliographic searches were also conducted on Pubmed and the internet(Google), using terms such as “withdrawal of the Mutual Recognition Agreement of Swiss European Union medical device conformity certificates,new UK medical device regulation”, for a period extending from January2020 to December 2021.Results: As a result of the disappearance of the legal framework thatsupported free trade between Switzerland, the United Kingdom and theEuropean Union, products that used to be unrestrictedly distributed inEurope have become imports having to comply with the relevant legalrequirements. Distributors for their part have become importers, and declarations of conformity and CE certificates have lost their validity. Furthermore, notified bodies from Switzerland and the United Kingdom are nolonger recognized by the European Commission. Switzerland, the United Kingdom and the European Union have had to grant grace periods toallow regulatory agencies and economic operators to adapt to the newsituation. (AU)

Nanoliposomes in Cancer Therapy: Marketed Products and Current Clinical Trials.

The drugs used for cancer treatment have many drawbacks, as they damage both tumor and healthy cells and, in addition, they tend to be poorly soluble drugs. Their transport in nanoparticles can solve these problems as these can release the drug into tumor tissues, as well as improve their solubility, bioavailability, and efficacy, reducing their adverse effects. This article focuses on the advantages that nanotechnology can bring to medicine, with special emphasis on nanoliposomes. For this, a review has been made of the nanoliposomal systems marketed for the treatment of cancer, as well as those that are in the research phase, highlighting the clinical trials being carried out. All marketed liposomes studied are intravenously administered, showing a reduced intensity of side-effects compared with the nonliposomal form. Doxorubicin is the active ingredient most frequently employed. Ongoing clinical trials expand the availability of liposomal medicines with new clinical indications. In conclusion, the introduction of drugs in nanoliposomes means an improvement in their efficacy and the quality of life of patients. The future focus of research could be directed to develop multifunctional targeted nanoliposomes using new anticancer drugs, different types of existing drugs, or new standardized methodologies easily translated into industrial scale.

Blockade of the trans-sulfuration pathway in acute pancreatitis due to nitration of cystathionine ß-synthase.

Acute pancreatitis is an inflammatory process of the pancreatic gland that may lead to dysregulation of the trans-sulfuration pathway. The aims of this work were firstly to study the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches identifying the causes of this dysregulation in an experimental model of acute pancreatitis; and secondly to reveal the effects of S-adenosylmethionine administration on these pathways. Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Cerulein-induced acute pancreatitis rapidly caused marked depletion of methionine, S-adenosylmethionine, 5'-methylthioadenosine, cystathionine, cysteine, and glutathione levels in pancreas, but S-adenosylhomocysteine and homocysteine remained unchanged. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis. Although cystathionine ß-synthase protein levels did not change with acute pancreatitis, Nos2 mRNA and protein levels were markedly up-regulated and caused tyrosine nitration of cystathionine ß-synthase in pancreas. S-adenosylmethionine administration enhanced Nos2 mRNA expression and cystathionine ß-synthase nitration and triggered homocysteine accumulation in acute pancreatitis. Furthermore, S-adenosylmethionine administration promoted enrichment of the euchromatin marker H3K4me3 in the promoters of Tnf-α, Il-6, and Nos2 and enhanced the mRNA up-regulation of these genes. Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. In conclusion, tyrosine-nitration of cystathionine ß-synthase blockades the trans-sulfuration pathway in acute pancreatitis promoting homocysteine accumulation upon S-adenosylmethionine treatment.

p38α deficiency restrains liver regeneration after partial hepatectomy triggering oxidative stress and liver injury.

p38α MAPK negatively regulates the G1/S and G2/M cell cycle transitions. However, liver-specific p38α deficiency impairs cytokinesis and reduces hepatocyte proliferation during cirrhosis and aging in mice. In this work, we have studied how p38α down-regulation affects hepatocyte proliferation after partial hepatectomy, focusing on mitotic progression, cytokinesis and oxidative stress. We found that p38α deficiency triggered up-regulation of cyclins A1, B1, B2, and D1 under basal conditions and after hepatectomy. Moreover, p38α-deficient hepatocytes showed enhanced binucleation and increased levels of phospho-histone H3 but impaired phosphorylation of MNK1 after hepatectomy. The recovery of liver mass was transiently delayed in mice with p38α-deficient hepatocytes vs wild type mice. We also found that p38α deficiency caused glutathione oxidation in the liver, increased plasma aminotransferases and lactate dehydrogenase activities, and decreased plasma protein levels after hepatectomy. Interestingly, p38α silencing in isolated hepatocytes markedly decreased phospho-MNK1 levels, and silencing of either p38α or Mnk1 enhanced binucleation of hepatocytes in culture. In conclusion, p38α deficiency impairs mitotic progression in hepatocytes and restrains the recovery of liver mass after partial hepatectomy. Our results also indicate that p38α regulates cytokinesis by activating MNK1 and redox modulation.

Age-dependent regulation of antioxidant genes by p38α MAPK in the liver.

p38α is a redox sensitive MAPK activated by pro-inflammatory cytokines and environmental, genotoxic and endoplasmic reticulum stresses. The aim of this work was to assess whether p38α controls the antioxidant defense in the liver, and if so, to elucidate the mechanism(s) involved and the age-related changes. For this purpose, we used liver-specific p38α-deficient mice at two different ages: young-mice (4 months-old) and old-mice (24 months-old). The liver of young p38α knock-out mice exhibited a decrease in GSH levels and an increase in GSSG/GSH ratio and malondialdehyde levels. However, old mice deficient in p38α had higher hepatic GSH levels and lower GSSG/GSH ratio than young p38α knock-out mice. Liver-specific p38α deficiency triggered a dramatic down-regulation of the mRNAs of the key antioxidant enzymes glutamate cysteine ligase, superoxide dismutase 1, superoxide dismutase 2, and catalase in young mice, which seems mediated by the lack of p65 recruitment to their promoters. Nrf-2 nuclear levels did not change significantly in the liver of young mice upon p38α deficiency, but nuclear levels of phospho-p65 and PGC-1α decreased in these mice. p38α-dependent activation of NF-κB seems to occur through classical IκB Kinase and via ribosomal S6 kinase1 and AKT in young mice. However, unexpectedly the long-term deficiency in p38α triggers a compensatory up-regulation of antioxidant enzymes via NF-κB activation and recruitment of p65 to their promoters. In conclusion, p38α MAPK maintains the expression of antioxidant genes in liver of young animals via NF-κΒ under basal conditions, whereas its long-term deficiency triggers compensatory up-regulation of antioxidant enzymes through NF-κΒ.

Targeted delivery of Cyclosporine A by polymeric nanocarriers improves the therapy of inflammatory bowel disease in a relevant mouse model.

The therapy of inflammatory bowel diseases is still rather inefficient, and about 80% of patients require surgery at some stage. Improving the treatments by more efficient medication is, therefore, an urgent medical need. The objective of this project was to demonstrate targeted delivery of Cyclosporine-A (CYA) to the inflamed areas of the intestinal mucosa after oral administration, enabling improved alleviation of the symptoms and, at the same time, reduced systemic drug absorption and associated adverse effects. As had already been demonstrated in previous studies, nano- to micrometer-sized drug particles will accumulate at inflamed mucosal areas, providing a platform for such purposes. CYA as incorporated in poly-(lactic-co-glycolic-Acid) (PLGA) nano- and mirocarriers, respectively, each homogeneous in size and providing controlled drug release over 24h at intestinal pH-value. For comparative reasons, a commercial formulation (Sandimmun Neoral®) was included in the study. In an acute model of DSS-induced inflammation in Balb/c mice, up to three doses were administered for each formulation: 50mg/kg, 25mg/kg and 12.5mg/kg. Drug-free particles were included as control. The following parameters were evaluated: body weight, colon length, colon weight/length ratio, cytokine expression and histological analysis. Plasma levels of CYA were analysed to compare systemic bioavailability. While disease parameters, such as, e.g. colon length, always improved with an optimum dose of 25mg/kg, the commercial and the microparticulate formulations led to measurable plasma levels and adverse effects in terms of body weight loss at the highest dose. In contrast, when administering the same doses as nanoparticles, plasma concentrations remained always below the detection limit, and the body weight of the animals remained unchanged. In conclusion, this study corroborates the potential of nanocarriers enabling an improved topical delivery of CYA to the inflamed gut mucosa after oral administration yielding the same improvement of disease parameters at only half the dose in comparison to microparticles and a commercial oral formulation, respectively, and at the same time minimizing systemic exposure and associated adverse effect.

p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

BACKGROUND: Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. METHODS: Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. RESULTS: We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. CONCLUSIONS: p38α MAPK is essential for actin dynamics with age in hepatocytes.

Redox signaling in the gastrointestinal tract.

Redox signaling regulates physiological self-renewal, proliferation, migration and differentiation in gastrointestinal epithelium by modulating Wnt/ß-catenin and Notch signaling pathways mainly through NADPH oxidases (NOXs). In the intestine, intracellular and extracellular thiol redox status modulates the proliferative potential of epithelial cells. Furthermore, commensal bacteria contribute to intestine epithelial homeostasis through NOX1- and dual oxidase 2-derived reactive oxygen species (ROS). The loss of redox homeostasis is involved in the pathogenesis and development of a wide diversity of gastrointestinal disorders, such as Barrett's esophagus, esophageal adenocarcinoma, peptic ulcer, gastric cancer, ischemic intestinal injury, celiac disease, inflammatory bowel disease and colorectal cancer. The overproduction of superoxide anion together with inactivation of superoxide dismutase are involved in the pathogenesis of Barrett's esophagus and its transformation to adenocarcinoma. In Helicobacter pylori-induced peptic ulcer, oxidative stress derived from the leukocyte infiltrate and NOX1 aggravates mucosal damage, especially in HspB+ strains that downregulate Nrf2. In celiac disease, oxidative stress mediates most of the cytotoxic effects induced by gluten peptides and increases transglutaminase levels, whereas nitrosative stress contributes to the impairment of tight junctions. Progression of inflammatory bowel disease relies on the balance between pro-inflammatory redox-sensitive pathways, such as NLRP3 inflammasome and NF-κB, and the adaptive up-regulation of Mn superoxide dismutase and glutathione peroxidase 2. In colorectal cancer, redox signaling exhibits two Janus faces: On the one hand, NOX1 up-regulation and derived hydrogen peroxide enhance Wnt/ß-catenin and Notch proliferating pathways; on the other hand, ROS may disrupt tumor progression through different pro-apoptotic mechanisms. In conclusion, redox signaling plays a critical role in the physiology and pathophysiology of gastrointestinal tract.

Regulation of cytokinesis and its clinical significance.

Dysregulation of the cell cycle leads to polyploid cells, which are classified into mononuclear or binuclear polyploid cells depending on the number of nuclei. Polyploidy is common in plants and in animals. Physiologically, polyploidy and binucleation are differentiation markers and also features of the aging process. In fact, although they provide multiple copies of genes required for survival, a negative correlation between growth capacity and polyploidy has been reported, and thus, suppression or reversal of this phenomenon may be a growth advantage. On the other hand, unscheduled polyploidization may cause genomic instability that might lead to neoplastic aneuploidy. The aim of this review is to analyze the mechanisms that lead to polyploidy, and particularly binucleation, and highlight the potential of ploidy as a marker of illness severity or the success of the adaptive response for an injury, with special emphasis in the liver under physiological and pathological conditions. Hepatocyte binucleation occurs in late fetal development and postnatal maturation, especially after weaning via phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt). It also increases upon aging of the liver as well as in liver cirrhosis and cancer. Liver binucleation mainly indicates the severity of the damage. Furthermore, the eventual increase in hepatocyte binucleation points out compensatory proliferation associated with liver injury. Ploidy conveyor would also permit hepatocyte adaptation to xenobiotic or nutritional injury. In contrast, polyploidy is a feature of many human cancers, and it may predispose to genomic instability and generation of aneuploidization that play a major role in carcinogenesis. Finally, a better understanding of the polyploidization process is needed in order to approach clinical research but also, to get deeper knowledge of cell cycle control. The fascinating regulation of cell cycle in liver and the generation and reversal of ploidies will provide more clues for the mystery of liver regeneration.

Hesperetin induces melanin production in adult human epidermal melanocytes.

One of the major sources of flavonoids for humans are citrus fruits, hesperidin being the predominant flavonoid. Hesperetin (HSP), the aglycon of hesperidin, has been reported to provide health benefits such as antioxidant, anti-inflammatory and anticarcinogenic effects. However, the effect of HSP on skin pigmentation is not clear. Some authors have found that HSP induces melanogenesis in murine B16-F10 melanoma cells, which, if extrapolated to in vivo conditions, might protect skin against photodamage. Since the effect of HSP on normal melanocytes could be different to that observed on melanoma cells, the described effect of HSP on murine melanoma cells has been compared to the effect obtained using normal human melanocytes. HSP concentrations of 25 and 50 µM induced melanin synthesis and tyrosinase activity in human melanocytes in a concentration-dependent manner. Compared to control melanocytes, 25 µM HSP increased melanin production and tyrosinase activity 1.4-fold (p < 0.01) and 1.1-fold (p < 0.01), respectively, and the corresponding increases in the case of 50 µM HSP were 1.9-fold (p < 0.001) and 1.3-fold (p < 0.001). Therefore, HSP could be considered a valuable photoprotective substance if its capacity to increase melanin production in human melanocyte cultures could be reproduced on human skin.

p38α deficiency and oxidative stress cause cytokinesis failure in hepatocytes.

Cytokinesis is the last step in mitosis and it implies re-organization of the actin cytoskeleton. Its failure is one of the major mechanisms of polyploidy and binucleation in mammals. Our aims were 1) to assess the role of redox-sensitive p38α MAPK in cytokinesis by studying the liver of wild type mice or liver-specific p38α knock-out mice; 2) to assess the role of oxidative stress associated with hepatocyte isolation on cytokinesis. When p38α was down-regulated in hepatocytes, MK2 phosphorylation on threonine 334 was completely abrogated. Activation of MNK-1, required for abscission of the intercellular bridge, was diminished. Key proteins of the RhoA pathway (phospho-PRK2, nuclear phosphorylated cofilin, and cytosolic p27) were assessed confirming the impairment of this pathway. The absence of p38α in aging liver also led to a decrease in HSP27 phosphorylation, which is required for actin polymerization. Indeed, a severe impairment in the F-actin filamentous structure was found in the liver of old mice upon p38α deficiency. Consequently, long-term p38α MAPK down-regulation markedly affects the RhoA pathway and actin cytoskeleton dynamics inducing actin disassembly and cytokinesis failure upon aging. On the other hand, hepatocyte isolation caused marked glutathione depletion, increased generation of reactive oxygen species, and activated cell cycle entry. Addition of N-acetyl cysteine to isolation media prevented glutathione depletion, restrained the cell cycle entry, and abrogated defective cytokinesis and the formation of binucleated hepatocytes during isolation. Our results show that hepatocytes do enter into S phase but they do not complete cell division with age upon p38α deficiency or upon oxidative stress associated with isolation leading in both cases to cytokinesis failure and binucleation.

Long term p38-a deficiency up-regulates antioxidant enzymes through compensatory NF-?B activation.

p38a MAPK may function as a mediator of reactive oxygen species signaling and thus p38a is considered a sensor of oxidative stress. In liver-specific p38a knock-out (KO) adult mice we previously found glutathione depletion and down-regulation of antioxidant enzymes. Our aim was to assess the influence of long-term p38a deficiency on oxidative stress and on the regulation of antioxidant enzymes in liver of old mice. To this end, wild type or liver-specific KO mice after weaning, at 4-6 months of age, or at 24 months of age were used. Reduced glutathione (GSH) and oxidized glutathione levels were determined by mass spectrometry, gene expression of antioxidant enzymes was determined by RT-PCR, and induction of NRF-2 and PGC-1a as well as activation of NF-?B were assessed by western blotting. We report that GSH levels decreased upon aging only in liver of wild-type mice, but not in p38a KO mice. The mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was up-regulated in adult wild-type in comparison with mice after weaning, but their gene expression was down-regulated in old wild-type mice. Although the mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was down-regulated in adult KO mice vs KO mice after weaning, their gene expression was up-regulated in old KO mice. This up-regulation was not associated with nuclear translocation of NRF-2, which decreased upon aging in KO mice, nor with up-regulation of PGC-1a. However, phosphorylation of p65 was markedly increased in old KO mice as an index of NF-?B activation. In conclusion, long term deficiency of p38a in the liver causes compensatory activation of NF?B that is likely to be responsible for the up-regulation of antioxidant enzymes upon aging, independently of Nrf-2 and PGC-1a.

Liver-specific p38α deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice.

UNLABELLED: p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor ß, tumor necrosis factor-α, interleukin-1ß, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. CONCLUSION: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.

Neural differentiation from human embryonic stem cells as a tool to study early brain development and the neuroteratogenic effects of ethanol.

The in vitro generation of neural cells from human embryonic stem cells is a powerful tool to acquire better knowledge of the cellular and molecular events involved in early human neural and brain development under physiological and pathological conditions. Prenatal alcohol exposure can induce important anomalies in the developing brain, the embryogenesis being an important critical period for the craniofacial defects and mental disabilities associated with fetal alcohol syndrome. Here, we report the generation of neural progenitors (NPs) from human embryonic stem cells. Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts and the proper timing of neurons and glia cells generation. Immunocytochemical and real time (RT)-polymerase chain reaction analyses reveal that cells appeared as clusters during neuroepithelial cell proliferation and that the genes associated with the neuroectodermal (Pax-6) and the endodermic (α-fetoprotein) lineages decreased in parallel to the upregulation of the genes of NPs (nestin and Tuj1), followed by their differentiation into neurons (MAP-2+, GABA+), oligodendrocytes [galactocerebroside (GalC+)], and astrocytes (GFAP+). We further demonstrate, for the first time, that human NPs express the endocannabinoid receptors (CB1 and CB2) and the enzymes involved in endocannabinoids synthesis (NAPE-PLD) and degradation (FAAH). Using this in vitro culture, we demonstrate that ethanol exposure impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton, and affects the expression of different genes associated with neural differentiation. The results provide new insights into the effects of ethanol on human embryogenesis and neuroprogenitors and offer an opportunity to delineate potential therapeutic strategies to restore early ethanol-induced brain damage.

RhoE stimulates neurite-like outgrowth in PC12 cells through inhibition of the RhoA/ROCK-I signalling.

Neurite formation involves coordinated changes between the actin cytoskeleton and the microtubule network. Rho GTPases are clearly implicated in several aspects of neuronal development and function. Indeed, RhoA is a negative regulator of neurite outgrowth and its effector Rho-kinase mediates the Rho-driven neurite retraction. Considering that RhoE/round protein (Rnd3) acts antagonistically to RhoA and it is also able to bind and inhibit rho kinase-I (p160ROCK) - ROCK-I, it is tempting to speculate a role of RhoE in neurite formation. We show for the first time that, in the absence of nerve growth factor (NGF), RhoE induces neurite-like outgrowth. Our results demonstrate that over-expression of RhoE decreases the activity of RhoA and reduces the expression of both ROCK-I and the phosphorylated myosin light chain phosphatase (MLCPp). Conversely, over-expression of either active RhoA or ROCK-I abolishes the RhoE-promoted neurite outgrowth, suggesting that RhoE induces neurite-like formation through inhibition of the RhoA/ROCK-I signalling. We also show that Rac and Cdc42 have a role in RhoE-induced neurite outgrowth. Finally, the present data further indicate that RhoE may be involved in the NGF-induced neurite outgrowth in PC12 cells, as depletion of RhoE by siRNA reduces the neurite formation induced by NGF. These findings provide new insights into the molecular mechanism implicated in neuronal development and may provide novel therapeutic targets in neurodegenerative disorders.