Search

La avena negra (Avena fatua L.) es una de las malezas más frecuentes de los cereales de invierno en Argentina. Su control se realiza mayoritariamente mediante la aplicación de herbicidas inhibidores de ACCasa y ALS. La eficacia de estos herbicidas se evalúa principalmente mediante el control visual y la supervivencia, sin considerar otros procesos demográficos tal como la fecundidad. Entre 2012 y 2015 se realizaron siete experimentos en la Facultad de Agronomía de la Universidad de Buenos Aires. El objetivo fue estudiar el efecto de tres inhibidores de la ALS (pyroxsulam, iodosulfuron + mesosulfuron, imazamox) y dos inhibidores de la ACCasa (fenoxaprop y pinoxaden) sobre la supervivencia, crecimiento y fecundidad de individuos de avena negra. Además, se estudió el efecto del origen de la población (con o sin aplicación previa de herbicidas). Cada herbicida se aplicó en cinco dosis (0x, 0,25x, 0,5x, 1x, 1,5x), siendo x la dosis recomendada en marbete. Se evaluó la supervivencia a los 30 y 60 días después del tratamiento y la biomasa individual a los 30 días después del tratamiento y en madurez. La fecundidad se evaluó cosechando las semillas de las plantas sobrevivientes. A los 30 días de aplicados los herbicidas, a la dosis de uso recomendada, pinoxadén mostró menor supervivencia que los otros herbicidas. Iodosulfuron + mesosulfuron e imazamox a la dosis recomendada fueron más efectivos que pyroxsulam y fenoxaprop. A 60 días después del tratamiento no hubo diferencias entre pinoxaden, iodosulfuron + mesosulfuron e imazamox a la dosis recomendada y fueron más efectivos que fenoxaprop y pyroxsulam. Los individuos que sobrevivieron produjeron menor biomasa que los no tratados. En promedio los individuos con o sin aplicación de herbicidas produjeron 31 semillas por gramo de biomasa individual. Wild oat (Avena fatua L.) is one of the most frequent weeds of winter cereals in Argentina. Its control is carried out mainly with the application of ACCase and ALS inhibitor herbicides. The efficacy of these herbicides is evaluated by visual control and survival, without considering other demographic processes such as fecundity. Between 2012 and 2015, seven experiments were carried out at the Faculty of Agronomy of the University of Buenos Aires. The objective was to study the effect of three ALS inhibitors (pyroxsulam, iodosulfuron + mesosufuron, imazamox) and two ACCase inhibitors (fenoxaprop and pinoxaden) on the survival, growth and fecundity of wild oat individuals. In addition, the effect of the origin of the population (with or without previous application of herbicides) was studied. Each herbicide was applied at five rates (0x, 0.25x, 0.5x, 1x, 1.5x), being x the recommended rate on the label. Survival at 30 and 60 days after treatment and individual biomass at 30 days after treatment and at maturity were evaluated. Fecundity was evaluated by harvesting the seeds of the surviving plants. Thirty days after the herbicides application, at the recommended rate, pinoxadén showed lower survival than the other herbicides. Iodosulfuron + mesosulfuron and imazamox at the recommended rate were more effective than pyroxsulam and fenoxaprop. At 60 days after treatment there were no differences between pinoxaden, iodosulfuron + mesosulfuron and imazamox at the recommended rate and were more effective than fenoxaprop and pyroxsulam. The survivors individuals produced less biomass than the untreated ones. On average, individuals with or without herbicide application produced 31 seeds per gram of individual biomass. Gerencia de Comunicación Institucional, DG SICyP, INTA Fil: Scursoni, Julio Alejandro. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Martin, Andrés Nelson. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Abbati, Paula. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Bastanchuri, María Mercedes. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Gueventter, Mateo. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Di Iorio, Estefania. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Pulido, Agustín. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Sherriff, Tomás. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Quistre, Juan Pablo. Universidad de Buenos Aires. Facultad de Agronomía; Argentina

Communication is becoming more important than ever for health care and health care professionals, as the recent COVID-19 pandemic has dramatically highlighted. The fast evolution of the mass and social media and the continuous development of new health-related platforms and applications are imposing new challenges that involve also laboratory medicine and that need to be carefully considered. In fact, these novel, fast and effective strategies of communication are inherently prone to the risk of publishing misleading, incorrect or fake information which can spread uncontrollably and diffuse all over the world in a very short time. However, social media are undoubtedly a great opportunity to communicate, in a responsible and credible way, health-related data and scientific updates and discoveries. As for the therapeutic alliance, it is now required to establish an "information alliance" between different healthcare professionals which, based on a trustworthy relationship, will allow the correct diffusion of health-related information and will contribute to citizens' education.

To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications.Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current "gold standard" scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and DeltaNp63alpha for stemness, 14-3-3sigma for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and alpha6, beta1, and beta4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (alpha-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed "hemicornea" were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed.Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and DeltaNp63alpha, while expression of 14-3-3sigma, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and alpha6, beta1, and beta4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and alpha-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation.Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered "hemicornea" might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring.

In plasma a real "antioxidant barrier” is identifiable, and to constitute it both inside (eg. carotenoids, ascorbate, vitamin E, etc.) and outside (eg. GSH, protein, bilirubin, uric acid, etc.) substances contribute. Each of these components blocks the potential damaging action of free radicals, whose reactivity is precisely linked to the particular "shortage" of these small particles negative. Any "insult" against this barrier allows free radicals to attack and damage cell structures, setting the stage for lesions typical of oxidative stress and all its dangerous consequences (premature aging, disease, etc.) [1]. The aim of the present study was to measure two components of the plasma oxidative barrier to evaluate the animal welfare status: the Biological Antioxidant Potential (BAP) and the anti oxidant power (OXY). The BAP is used as an index to determine the biological antioxidant capacity and is measured on the basis of the capacity of the plasma sample to reduce ferric ions to ferrous ions. The OXY-adsorbent test measures the ability of a plasma or serum sample to oppose a massive oxidative insult induced by a solution of hypochlorous acid. The present study was performed on wild boars (Sus scrofa) bred in the wild of two areas (C1and D1) of a 820 hectares wood belonging to Campania Region, included in the “Cilento e Vallo di Diano” National Park. The wood is made up of Quercus cerris and Castano sativa, mixed mesoxerofil conifer. Blood samples, from the jugular vein, were collected from 15 and 18 wild boars of the C1 and D1 areas respectively, during the routine sanitary control. On the serum, BAP test and OXY-adsorbent test were evaluated (DIACRON, Grosseto, Italy). The results are shown in Table 1. The data show no significative difference between wild boars of C1 and D1 area, both in BAP test and OXY-adsorbent test. These preliminary values are valid for the definition of the normal range in healthy and, therefore, used as parameters to evaluate the stress oxidative status and then animal welfare [2].

Animal welfare can be assessed by measurements of relevant stress markers. The adrenal cortex response does occur in diverse difficult situations and is useful in welfare assessment, in particular the production of cortisol acts in providing extra energy for forthcoming activity. Furthermore, a high level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress, an emerging health risk factor involved in the aging and in a lot of diseases. The reactive oxygen metabolites (ROMs) are used as an index to determine the level of oxidative stress (oxidant capacity) by measuring the amount of organic hydroperoxide (ROOH) converted into radicals that oxidize N,N-diethyl-p-phenylenediamine [1, 2]. The present study was performed on wild boars (Sus scrofa) bred in the wild of two areas (C1and D1) of a 820 hectares wood belonging to Campania Region, included in the “Cilento e Vallo di Diano” National Park. The wood is made up of Quercus cerris and Castano sativa, mixed mesoxerofil conifer. Blood samples, from the jugular vein, were collected from 15 and 18 wild boars of the C1 and D1 areas respectively, during the routine sanitary control. On the serum, cortisol and ROMs (DIACRON, Grosseto, Italy) were evaluated. The results are shown in Table 1. Cortisol was higher in D1 than in C1 area, but there was not any significative difference; ROMs in D1 were significantly higher than C1 area, with P

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision(4,5,6). The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina(5,6). The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve(5,6). The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

Cockayne syndrome (CS) is a rare hereditary disorder in which infants suffer severe developmental and neurological alterations and early death. Two genes encoding RNA polymerase II cofactors, CSA and CSB, are mutated in this syndrome. CSA and CSB proteins are known to be involved in the transcriptioncoupled DNA repair pathway but the sensitivity of mutant cells to a number of physical/chemical agents besides UV radiation, such as ionizing radiation, hydrogen peroxide and bioenergetic inhibitors indicate that these proteins play a pivotal role in additional pathways. In this review we will discuss the evidence that implicate CS proteins in the control of oxidative stress response with special emphasis on recent findings that show an altered redox balance and dysfunctional mitochondria in cells derived from patients. Working models of how these new functions might be key to developmental and neurological disease in CS will be discussed.

Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS-A and CS-B present an altered redox balance with increased steady-state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na / K-ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild-type genes. High-resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS-A and CS-B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an anti antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the 'managing' of the redox state in the cell. Our studies show that sodium 2-propenyl-thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 μm on HuT 78 cells was analyzed, showing activation of the p38-MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic-metabolizing enzymes, inducing down-activation or detoxification of the carcinogens. Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved in the cellular detoxification system, such as glutathione S-transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water-soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol-proteins involved in fundamental cellular processes.

Natural organic matter, including humic substances (HS), comprises complex secondary structures with no defined covalent chemical bonds and stabilized by inter- and intra-molecular interactions, such as hydrogen bonding, Van der Waal’s forces, and pi-pi interactions. The latest view describes HS aggregates as a hydrogel-like structure comprised by a hydrophobic core of aromatic residues surrounded by polar and amphiphilic molecules akin a self-assembled soft material. A different view is based on the classification of this material as either mass or surface fractals. The former is intended as made by the clustering of macromolecules generating dendritic networks, while the latter have been modelled in terms of a solvent-impenetrable core surrounded by a layer of lyophilic material. This study reviews the evolution of the increasingly refined models that appeared in the literature, all capable to describing the physicochemical properties of HS. All the models are critically examined and revisited in terms of their ability to provide key information on the structural organization of HS. Understanding how the molecular association pathway influences aggregation of HS also provides a key acknowledgment of their role in the environment.

The assessment of Technosols quality in urban environments is pivotal for the maintenance of ecosystems impacted by human activities. The study was performed on Technosols constructed in experimental mesocosms in the suburban area of Naples (Southern Italy) to highlight changes in the main soil properties over eight years and to identify the most suitable indices at quality monitoring. In this study, several chemical, biological, and integrated indices were analysed to evaluate the mineral accumulation, potential ecological risk, edaphon activity, fertility, and the overall soil quality. The Technosols showed alkaline pH, nitrogen ranged from 24.5 to 39.5 g kg−1, high organic matter contents above 40 g kg−1, and there were no evident processes of soil compaction. Heavy metals (Cr, Cu, Fe, Mg, Mn, Ni, Pb, and Zn) did not exceed the thresholds defined by the Italian law for urban soils, despite their volcanic components. During eight years, the chemical indices depicted changes in the elements balance and increase in ecological risk, the biological indices indicated a reduction in the fungal fraction (fivefold) and in the resources utilisation and carbon storage. The soil quality index with all parameters highlighted the reduction in the soil quality (from 0.78 to 0.65) due to the decrease of the chemical quality, the increase of microbial stress conditions, and changes of the microbial composition, underlining the importance of integrating chemical and biological information for monitoring Technosols.

This study demonstrated that it is possible to differentiate wild boars living in habitats with different animal densities by the measurement of oxidative stress markers. Therefore, reactive oxygen metabolites, the antioxidant barrier, i.e., the biological antioxidant potential and the antioxidative power (OXY-Adsorbent), as well as cortisol were measured in freely ranging wild boars. In two different areas of a State Forest in the Campania Region (Italy), 42 freely ranging, managed wild boars were captured with a corral trap, and blood samples were collected. The wild boars were divided by age (>1 year old and

Objective To evaluate the prognostic significance of oxidative stress (OS) and antioxidant defense status measurement in patients with chronic lymphocytic leukemia (CLL). Method d-ROMs test and BAP test were evaluated at diagnosis of 165 patients with CLL and correlated with clinical-biological features and prognosis. Results An increased oxidative damage (d-ROMs test) and a reduced antioxidant potential (BAP test), were found in CLL patients than normal controls (p

In the Network Society the use of hashtags has become a daily routine for the participation on the Big Conversation Iorio and Ruoto (Nessun tempo, 2015). Designated by a ‘hash’ symbol (#), a hashtag is a keyword assigned to information that describes it and aides in searching. Hashtags are now central to organize information on Social Networks. Hashtags organize discussion around specific topics or events and they are becoming an integrated part of the Infosphere, the whole informational environment constituted by all informational entities. The sentiment analysis of Hashtags shared on the Big Conversation can return a possible snapshot about the sentiment shared by users. Scope of this work is to present an application of sentiment analysis on the Italian hashtags of mainly social networks as part of the ‘Infosphere’. This analysis returns a semantic sentiment report about the hashtags shared by the users of the social networks, that can produce a semantic sentiment trend about users. This approach could be applied to every language simply changing the sentiment thesaurus used.

Oxidative energy production is inevitably associated with the generation of reactive oxygen species (ROS), excessive concentrations of which can lead to cellular pathol‐ ogy. A free radical may be defined as any molecule that has one or more unpaired electrons. The superoxide anion, the hydroxyl radical, and the hypochlorite radical are some of the highest reactive radicals of oxygen. Owing to their high reactivity and to their capability of initiating an uncontrolled cascade of chain reactions, ROS produce extensive protein damage and cytoskeletal modifications and inhibit cellular mechanisms. Aerobic organisms are equipped with a powerful battery of mechanisms that protect them from the adverse effects of lipid peroxidation (LPO) and other manifestations of oxygen toxicity. Defective sperm function frequently causes male infertility, due to abnormal flagella movement, failure to recognize the zona, and inhibition of sperm-oocyte fusion. ROS are fundamental mediators of physiological sperm function, such as signal transduction mechanisms that have an effect on fertility. ROS can have positive effects on sperm and the concentration functions depending on the nature and the concentration of the ROS involved. They are necessary in regulating the hyperactivation and the ability of the spermatozoa to undergo acrosome reaction. An increased amount of superoxide anion (O2) is one of the first steps required by the spermatozoa for induction and development of hyperactivation and capacitation. Numerous studies have shown that oxidative stress plays an important role in the pathophysiology of infertility and assisted fertility. The paternal genome is of primary importance in the normal embryo and fetal develop‐ ment. ROS-induced sperm damage during sperm translation, such as signal trans‐ duction through the seminiferous tubules and epididymis, is one of the most important mechanisms leading to sperm DNA damage. Male germ cells are extremely © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. vulnerable to oxidative stress as the sperm membrane is rich in unsaturated fatty acids and lacks the capacity for DNA repair. Spermatozoa are particularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA) and their cytoplasm contains low concentrations of the scavenging enzymes. Many clinical and research institutes are investigating the usefulness of antioxidant supplementation and their role in prevention of the infertility problems. Incubation under oxygen in vitro was detrimental to human spermatozoa, decreasing motility and viability. Since then, many reports have associated ROS with impaired sperm function, including decreased motility, abnormal morphology, and decreased sperm-egg penetration. Increasing knowledge of the mechanisms whereby ROS and endogenous antioxidant systems influence reproductive processes can assist to optimize the application of exogenous antioxi‐ dants to fertility treatment.

Hearing loss is the most common sensory disorder in humans, and genetic factors are a major cause. Approximately 15-20% of genetic cases exhibit an autosomal dominant pattern of transmission. So far, 41 autosomal dominant loci have been mapped and 17 genes have been identified. Here we report the mapping of a novel locus for autosomal dominant non-syndromic hearing loss, DFNA47, to chromosome 9p21-22 in a large multigenerational Italian family with progressive hearing impairment. Most affected individuals noticed hearing impairment after their teens with subsequent gradual progression to a moderate-severe loss. There were no obvious vestibular dysfunction and other associated abnormalities. A maximum lod score of 3.14 was obtained with marker D9S157 (at theta=0) after a genome wide search. The study of additional markers allowed us to confirm this region with positive lod scores of 3.58 (at theta=0 from D9S285) and of 3.67 (at theta=0 from D9S162). Recombinants define a region of approximately 9 cM flanked by markers D9S268 and D9S942. Multipoint linkage analysis showed a Lod score of 4.26. Few known genes map to the region, and those possibly related by function to hearing are being screened for disease-causing mutations.

Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles(1,2). Cultured keratinocyte stem cells, known as holoclones(3-6), generate sheets of epithelium used to restore severe skin, mucosal and corneal defects(7-9). Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder(10). Epidermal stem cells from an adult patient affected by LAM5-beta 3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA ( encoding LAM5-beta 3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up ( 1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease. The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

Mutations in the GJB2 gene, encoding the gap-junction channel protein connexin 26, account for the majority of recessive forms and some of the dominant cases of deafness. Here, we report the frequency of GJB2 alleles in the Italian population affected by hearing loss and the functional analysis of six missense mutations. Genetic studies indicate that, apart from the common 35delG, only few additional mutations can be detected with a significant frequency in our population. Transfection of communication-incompetent HeLa cells with Cx26 missense mutations revealed three distinct classes of functional deficits in terms of protein expression, subcellular localisation and/or functional activity. Moreover, the M34T mutant acted as a dominant inhibitor of wild-type Cx26 channel activity when the two proteins were co-expressed in a manner mimicking a heterozygous genotype. These data support the hypothesis of a functional role for M34T as a dominant allele and represent a further step towards a complete understanding of the role of GJB2 in causing hearing loss.