Comparative analysis of immobilized biocatalyst: study of process variables in trans-esterification reaction.

This study deals with an experimental investigation into the trans-esterification behavior of two biocatalysts produced by different immobilization techniques of the same lipase from Pseudomonas cepacia. Biocatalysts catalyzed trans-esterification of commercial frying oil. It was verified that no enzyme leaching occurs and stepwise addition of ethanol is needed to improve the process performance. When stoichiometric ethanol has been added completely at the beginning of the reaction, percent mass fraction of esters reached 33% after 9 h and 52% after 30 h. Instead, when ethanol has been added in steps, ester production gradually increased at any time. Moreover, different amounts of biocatalyst were added to the reaction system and it was found that it is not necessary to add more than 3% of biocatalyst with respect to the oil mass to avoid inhibition. The immobilization method influences the reaction progress and the intermediate-glyceride profiles were analyzed. Results showed that the two biocatalysts have the same affinity towards triglycerides, but the covalently immobilized lipase (epobond P. cepacia) has a lower affinity towards diglycerides and monoglycerides and, in general, a lower activity than the absorbed lipase (lipo P. cepacia).

p14ARF interacts with the focal adhesion kinase and protects cells from anoikis.

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing.

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Steam pretreatment of Saccharum officinarum L. bagasse by adding of impregnating agents for advanced bioethanol production.

The main byproduct of the sugarcane industry, Saccharum officinarum L. bagasse (sugarcane bagasse, SCB), is widely used as lignocellulose biomass for bio-ethanol (EtOH) production. In this research study, SCB was pretreated by steam explosion (SE) method using two different impregnating agents: sulfur dioxide (SD) and hydrogen peroxide (HP). As matter of fact, the use of impregnating agents improves the performance of SE method, increasing the concentrations of fermentable sugars after enzymatic saccharification, and decreasing the inhibitor compounds produced during the steam pretreatment step. The aim of this study was to investigate and compare the use of the two impregnating agents in various SE-conditions in order to optimize pretreatment parameters. For every pretreatment condition, it has been evaluated: concentration of fermentable sugars, glucose and xylose yields, and the effects of the inhibitor compounds on enzymatic hydrolysis step. The obtained results allow to improve the efficiency of the whole process of bio-EtOH synthesis enhancing the amount of fermentable sugars produced and the eco-sustainability of the whole process. Indeed, the optimization of steam pretreatment leads to a reduction of energy requirements and to a lower environmental impact.

Regulation of Stearoyl Coenzyme A Desaturase 1 Gene Promoter in Bovine Mammary Cells.

Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Bovine mammary epithelial cells (BME-UV1), cultured in vitro, have been proposed as a model to reproduce the biology of the mammary gland. The present study was designed to investigate the responsiveness of bovine SCD1 promoter to serum, insulin, oleic acid, and NFY transcription factor in BME-UV1 cells. A luciferase-based reporter assay was used to monitor the transcriptional activity of the SCD1 promoter region in BME-UV1 cells treated or not with insulin and/or oleic acid. The level of endogenous SCD1 mRNA was evaluated by Real time PCR. Insulin (20 ng/mL) induced a 2.0 to 2.5-fold increase of SCD1 promoter activity. Additionally, the effect of insulin was inhibited by oleic acid, serum components, and NFY enforced expression. Serum and NFY showed no synergistic or additive effect on SCD1 promoter activity suggesting that they repress SCD1 transcription through the same responsive element.

Eggshell: A green adsorbent for heavy metal removal in an MBR system.

Presence of heavy metals as well as different metal ions in treated wastewater is a problem for the environment as well as human health. This paper aims to investigate the possibility to combine an MBR (membrane biological reactor) with an adsorption process onto powdered eggshell and eggshell membrane in order to improve metal removal from wastewater. The first step of the experimental analysis consists of the evaluation of the compatibility between the two processes. Then, a study about sorbent concentration and size effect on fouling was conducted, because the use of this kind of sorbent could affect membrane performance. The second step of the work concerns the check up of eggshell removal capacity as a function of sorbent size, achieved treating an aqueous solution containing Al(3+), Fe(2+) and Zn(2+) as water pollutants. Finally, synthetic wastewater, containing the metal species, was treated by two alternative process schemes: one of them performs the metal uptake in a dedicated adsorption unit, before the MBR. In the second, the two processes take place in the same unit. Results demonstrate that the optimization of the first option could be a solution to MBR upgrading.

Enzymatic transesterification of waste vegetable oil to produce biodiesel.

An experimental study on enzymatic transesterification was performed to produce biodiesel from waste vegetable oils. Lipase from Pseudomonas cepacia was covalently immobilized on a epoxy-acrylic resin support. The immobilized enzyme exhibited high catalytic specific surface and allowed an easy recovery, regeneration and reutilisation of biocatalyst. Waste vegetable oils - such as frying oils, considered not competitive with food applications and wastes to be treated - were used as a source of glycerides. Ethanol was used as a short chain alcohol and was added in three steps with the aim to reduce its inhibitory effect on lipase activity. The effect of biocatalyst/substrate feed mass ratios and the waste oil quality have been investigated in order to estimate the process performances. Biocatalyst recovery and reuse have been also studied with the aim to verify the stability of the biocatalyst for its application in industrial scale.

A biochemically structured model for ethanol fermentation by Kluyveromyces marxianus: A batch fermentation and kinetic study.

Anaerobic batch fermentations of ricotta cheese whey (i.e. containing lactose) were performed under different operating conditions. Ethanol concentrations of ca. 22g L(-1) were found from whey containing ca. 44g L(-1) lactose, which corresponded to up to 95% of the theoretical ethanol yield within 15h. The experimental data could be explained by means of a simple knowledge-driven biochemically structured model that was built on bioenergetics principles applied to the metabolic pathways through which lactose is converted into major products. Use of the model showed that the observed concentrations of ethanol, lactose, biomass and glycerol during batch fermentation could be described within a ca. 6% deviation, as could the yield coefficients for biomass and ethanol produced on lactose. The model structure confirmed that the thermodynamics considerations on the stoichiometry of the system constrain the metabolic coefficients within a physically meaningful range thereby providing valuable and reliable insight into fermentation processes.

Understanding polycystic ovarian syndrome pathogenesis: an updated of its genetic aspects.

Polycystic ovary syndrome (PCOS) is the most frequent cause of female infertility. It is also characterized by metabolic defects that raise the risk for cardiovascular disease. Despite the progress in the definition of the clinical aspects of the syndrome, only very few definite data are available about the ethiopathogenetic mechanisms that subtend PCOS. It is likely that the PCOS phenotype derives from the interaction between environmental and genetic factors. While environmental factors have easily been investigated, the individuation of the genetic factors seem to be more complex. Indeed, PCOS appears to be inherited as a complex, polygenic trait. Several family studies have been conducted with the aim to clarify the genetic aspects of PCOS, but their findings are often conflicting and not conclusive.Moreover, it is difficult to establish with certainty which genes are involved and their effective role in the development of the syndrome because in PCOS, genetic analysis is hampered by low fecundity, lack of a male phenotype, absence of an animal model, and dissimilarity of the diagnostic criteria used to select the patients. Since multiple biochemical pathways are implicated in PCOS pathogenesis, genes of steroid hormone metabolism, gonadotropin release and action, insulin secretion and action, adipose tissue metabolism and others have been investigated. Nevertheless, none of them seems to play a key role in the ethiopathogenesis of PCOS. This article reviews the large body of literature generated to support the presence of genetic abnormalities in PCOS women by taking in consideration the most important studies regarding PCOS candidate genes.

Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V(NAR) shares many of the properties of the well-characterised single-domain camelid V(H)H but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V(NAR) 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V(NAR) domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K(D) of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K(D) yet reported for V(NAR)-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V(NAR) paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V(NAR) database. This study allowed us to identify, for the first time, both V(NAR)-specific and V(NAR)/Ig V(L)/TCR V(alpha) overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V(NAR)-specific hallmarks align precisely with previously defined mutational 'cold spots' in natural nurse shark cDNA sequences. These findings will aid future V(NAR) engineering and optimisation studies towards the development of V(NAR) single-domain proteins as viable biotherapeutics.

Factor analysis of transesterification reaction of waste oil for biodiesel production.

In the present paper a factor analysis is presented for the enzymatic transesterification of waste oil for biodiesel production. The experimental data on batch reactor evidence two key variables: enzyme loading and mixing conditions. These variables were subjected to a factor analysis and their combined effect on the reaction performance was determined. Response surface methodology (RSM) was used based on a linear first order model (steepest ascent method) and on a second order one in proximity of the optimal solution. The result was a model able to predict reaction performance within the range of mixing rates and enzyme amount considered for model formulation and outside of it, as shown in the final validation. Best performances were obtained at high stirring and high enzyme loading.

[Mesothelial cyst of the kidney. A rare case report of this neoformation on the left kidney].

The authors are hereby presenting a rare case of neoformation developing on the left kidney in a 80-year-old patient affected by left lumbar backache. The neoformation appears doubtful in nature, on ultrasonography, CT scan and MRI. The lesion is roundish and contiguous at the kidney convex edge; due to its clinical aspects, it requires a surgical management and is, therefore, easily excised. The histopathologic examination confirms it is a mesothelial cyst. The case is presented for its absolute rarity and for the preoperative diagnostic doubts it may generate.

[Angiomyofibroblastoma of the spermatic funiculus. A case report of this lesion of the spermatic funiculus].

The authors are hereby presenting a rare case of angiomyofibroblastoma of the funiculus in a 20-year-old patient, having a non-aching tumefaction at the left region of the scrotum. This tumefaction was solid and non homogeneous, both on ultrasonography and MRI, of about 5cm in diameter, fully separated from the testicle. The markers were all negative. We proceeded with surgical treatment of the neoformation. It is mandatory to include this lesion within the differential diagnosis of testicle masses. The case is presented for its absolute histopathologic rarity and for the difficulty in diagnosis.

TBP-1 protects the human oncosuppressor p14ARF from proteasomal degradation.

The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARF's interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

Implementation of HaemScreen, a workplace-based genetic screening program for hemochromatosis.

There is debate as to whether community genetic screening for the mutation(s) causing hereditary hemochromatosis (HH) should be implemented, due to issues including disease penetrance, health economic outcomes, and concerns about community acceptance. Hemochromatosis is a common preventable iron overload disease, due in over 90% of cases to C282Y homozygosity in the HFE gene. We are, therefore, piloting C282Y screening to assess understanding of genetic information and screening acceptability in the workplace setting. In this program, HaemScreen, education was by oral or video presentation in a group setting. C282Y status was assessed by polymerase chain reaction (PCR) and melt-curve analysis on DNA obtained by cheek-brush sampling. Of eligible participants, 5.8% (1.5-15.8%) attended information and screening sessions, of whom 97.7% (5571 individuals) chose to be tested. Twenty-two C282Y (1 : 253) homozygotes were identified and offered clinical follow-up. There were 638 heterozygotes (1 : 8.7). The determinants for participation have been analyzed in terms of the principles outlined in the Health Belief Model. Widespread screening for HH is readily accepted in a workplace setting, and a one-to-many education program is effective. The level of participation varies greatly and the advertizing and session logistics should be adapted to the specific features of each workplace.

[Gastric diverticulum. A case report with long-term follow-up and a review of the literature]. / Il diverticolo dello stomaco. Descrizione di un caso clinico con follow-up a lungo termine e revisione della letteratura.

A rare case of symptomatic gastric diverticulum surgically managed is reported. The pathogenetic and clinical aspects of the disease are discussed and the usefulness of endoscopy as a means for differentiation between medical and surgical cases is stressed. A review of the literature is made and the conclusion is drawn that this uncommon pathology is often asymptomatic and requires surgical management only in complicated cases and in symptomatic patients that are not responding to medical treatment, as showed by the long-term follow-up (333 months) of the reported case.

Design of RNA-binding proteins and ligands.

The rapidly expanding database of RNA structures and protein complexes is beginning to lead to the successful design of specific RNA-binding molecules. Recent combinatorial and structure-based approaches have utilized known nucleic-acid-binding scaffolds from both proteins and small molecules to display a relatively small set of functional groups often used in protein--RNA recognition. Several studies have shown that the tethering of multiple binding modules can enhance RNA-binding affinity and specificity, a strategy also commonly used in DNA recognition.

The human tumor suppressor arf interacts with spinophilin/neurabin II, a type 1 protein-phosphatase-binding protein.

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.