Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Optimization of a multielectrode array (MEA)-based approach to study the impact of Aβ on the SH-SY5Y cell line

The human brain stores, integrates, and transmits information recurring to millions of neurons, interconnected by countless synapses. Though neurons communicate through chemical signaling, information is coded and conducted in the form of electrical signals. Neuroelectrophysiology focus on the study of this type of signaling. Both intra and extracellular approaches are used in research, but none holds as much potential in high-throughput screening and drug discovery, as extracellular recordings using multielectrode arrays (MEAs). MEAs measure neuronal activity, both in vitro and in vivo. Their key advantage is the capability to record electrical activity at multiple sites simultaneously. Alzheimer’s disease (AD) is the most common neurodegenerative disease and one of the leading causes of death worldwide. It is characterized by neurofibrillar tangles and aggregates of amyloid-β (Aβ) peptides, which lead to the loss of synapses and ultimately neuronal death. Currently, there is no cure and the drugs available can only delay its progression. In vitro MEA assays enable rapid screening of neuroprotective and neuroharming compounds. Therefore, MEA recordings are of great use in both AD basic and clinical research. The main aim of this thesis was to optimize the formation of SH-SY5Y neuronal networks on MEAs. These can be extremely useful for facilities that do not have access to primary neuronal cultures, but can also save resources and facilitate obtaining faster high-throughput results to those that do. Adhesion-mediating compounds proved to impact cell morphology, viability and exhibition of spontaneous electrical activity. Moreover, SH-SY5Y cells were successfully differentiated and demonstrated acute effects on neuronal function after Aβ addition. This effect on electrical signaling was dependent on Aβ oligomers concentration. The results here presented allow us to conclude that the SH-SY5Y cell line can be successfully differentiated in properly coated MEAs and be used for assessing acute Aβ effects on neuronal signaling.

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca2+ response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 A degrees C and changes in intracellular calcium concentration ([Ca2+](i)) following KCl stimulation were measured using Fura-2, at 12 or 22 A degrees C-test. The increase in [Ca2+](i) resulted primarily from extracellular Ca2+ entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca2+ response was observed between 12- and 22 A degrees C-acclimated fish. In particular, a greater increase in [Ca2+](i) at a high level of adrenaline was observed in 22 A degrees C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

Genetic Suppressors of mrp-5 Lethality in C. elegans

Heme is an essential cofactor in numerous proteins, but is also cytotoxic. Thus, directed pathways must exist for regulating heme homeostasis. C. elegans is a powerful genetic animal model for elucidating these pathways because it is a heme auxotroph. Worms acquire dietary heme though HRG-1-related importers, and intestinal export was demonstrated to be mediated by the ABC transporter MRP-5. Loss of mrp-5 results in embryonic lethality. Although heme transporters have been identified, there are significant gaps in our understanding for the heme trafficking beyond HRG-1 and MRP-5. To identify additional components, we conducted a forward genetic screen utilizing the null allele mrp-5(ok2067). Screening of 160,000 haploid genomes yielded thirty-two mrp-5(ok2067) suppressor mutants. Deep-sequencing variant analysis revealed three of the suppressors subunits of adapter protein complex 3 (AP-3). We now seek to identify mechanisms for how adaptor protein deficiencies bypass a defect in MRP-5-mediated heme export.

Proton-pump inhibitors adverse effects: a review of the evidence and position statement by the Sociedad Española de Patología Digestiva

Introduction: In the last few years a significant number of papers have related the use of proton-pump inhibitors (PPIs) to potential serious adverse effects that have resulted in social unrest. Objective: The goal of this paper was to provide a literature review for the development of an institutional position statement by Sociedad Española de Patología Digestiva (SEPD) regarding the safety of long-term PPI use. Material and methods: A comprehensive review of the literature was performed to draw conclusions based on a critical assessment of the following: a) current PPI indications; b) vitamin B12 deficiency and neurological disorders; c) magnesium deficiency; d) bone fractures; e) enteric infection and pneumonia; f) interactions with thienopyridine derivatives; e) complications in cirrhotic patients. Results: Current PPI indications have remained unchanged for years now, and are well established. A general screening of vitamin B12 levels is not recommended for all patients on a PPI; however, it does seem necessary that magnesium levels be measured at therapy onset, and then monitored in subjects on other drugs that may induce hypomagnesemia. A higher risk for bone fractures is present, even though causality cannot be concluded for this association. The association between PPIs and infection with Clostridium difficile is mild to moderate, and the risk for pneumonia is low. In patients with cardiovascular risk receiving thienopyridines derivatives it is prudent to adequately consider gastrointestinal and cardiovascular risks, given the absence of definitive evidence regardin potential drug-drug interactions; if gastrointestinal risk is found to be moderate or high, effective prevention should be in place with a PPI. PPIs should be cautiously indicated in patients with decompensated cirrhosis. Conclusions: PPIs are safe drugs whose benefits outweigh their potential side effects both short-term and long-term, provided their indication, dosage, and duration are appropriate.

SSR genetic diversity assessment of popular pigeonpea varieties in Malawi reveals unique fingerprints

Background: Pigeonpea ( Cajanus cajan L. Millsp.) is a drought tolerant legume of the Fabaceae family and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, it is grown as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant quality reduction and yield losses. In this study, 48 polymorphic SSR markers were used to assess the diversity among all pigeonpea varieties cultivated in Malawi to determine if a genetic fingerprint could be identified to distinguish the popular varieties. Results: A total of 212 alleles were observed with an average of 5.58 alleles per marker and a maximum of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC), ranged from 0.03 to 0.89 with an average of 0.30. A neighbor-joining tree produced 4 clusters. The most commonly cultivated varieties, which include released varieties and cultivated land races, were well-spread across all the clusters observed, indicating that they generally represented the genetic diversity available in Malawi, although substantial variation was evident that can still be exploited through further breeding. Conclusion: Screening of the allelic data associated with the five most popular cultivated varieties, revealed 6 markers – CCB1, CCB7, Ccac035, CCttc003, Ccac026 and CCttc019 – which displayed unique allelic profiles for each of the five varieties. This genetic fingerprint can potentially be applied for seed certification to confirm the genetic purity of seeds that are delivered to Malawi farmers.

A study of asymptomatic bacteriuria in Egyptian school-going children.

Background: Urinary tract infections (UTI) are a common and important clinical problem in childhood. Upper urinary tract infections (i.e., acute pyelonephritis) may lead to renal scarring, hypertension, and end-stage renal disease. Despite the presence of simple and reliable methods of preliminary screening of children's urine, urinary tract infection continues to be under diagnosed. Objectives: The aim of this study was to establish prevalence rates of significant bacteriuria in asymptomatic school children by simple urine tests in comparison to standard urine culture techniques in Giza, Egypt. Patients and methods: A total of 1000 apparently healthy school going children (6-12) years, 552 boys (55.2%) and 448 girls (44.8%), were enrolled in this cross-sectional prevalence survey. Results: Overall prevalence of significant bacteriuria was 6%. Higher prevalence occurred in girls (11.4%) than boys (1.6%). Escherichia coli was isolated in 35(58%) cases (3 boys and 32 girls), Staph. aureus in 13 (22%) cases (3 boys and 10 girls), Enterobacter in 6 girls (10%), Kelbsiella pneumoniae in 3 boys (5%) and Proteus vulgaris in 3 girls (5%) Conclusion: Asymptomatic bacteriurea could be detected by urine screening program at school age. Overall prevalence of significant bacteriuria was 6%, with predominance in girls than boys.

Chemical composition of Boletus pinophilus and Clitocybe subconnexa: preservation with gamma irradiation

The short shelf life of mushrooms is a barrier for their distribution and, therefore, there has been extensive research to find technologies that ensure the preservation of mushrooms, maintaining their organoleptic and nutritional properties (1]. Irradiation has proved its technological feasibility to be safely used in the reduction of food losses, being recognized by international organizations as a valid conservation alternative in extending shelflife of many foods. The aim of the present work was to validate the use of 2 kGy dose of gamma radiation to maintain chemical composition of wild mushrooms. Boletus pinophilus Pihit & Dermek and Clitocybe subconnexa Murrill wild samples were obtained in Tnis-os-Montes; subsequently, the samples were divided in two groups: control (non-irradiated, 0 kGy) and irradiated (2 kGy). The irradiation of the samples was performed in a 6°Co experimental chamber. Moisture, protein, fat, carbohydrates and ash were determined following the standard procedures [2]. Free sugars and tocopherols were determined by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI) and a fluorescence detector, respectively; fatty acids were determined by gas-liquid chromatography with flame ionization detection (GC-FID) [3]. The protein and ash content was preserved in both groups, although the sugars and tocopherols decreased in the irradiated samples. Sugars and fatty acids showed significant changes after irradiation treatment, particularly in B. pinophillus, nevertheless, the magnitude of the obtained differences did not seem to be sufficient to affect the chemical profiles of the assayed mushrooms. Overall, the detected chemical changes might be considered as allowable, in view of the high advantages offered by gamma irradiation at decontamination and/or disinfestation level.

Effects of electron-beam irradiation on chemical and antioxidation parameters of wild Macrolepiota procera dried samples

Mushrooms are very perishable foods due to their high susceptibility to moisture loss, changes in color and texture, or microbiological spoilage. Drying is considered as the most appropriate method to prevent these alterations, but it has some limitations, such as shrinkage, enzymatic and non-enzymatic browning reactions, and oxidation of lipids and vitamins. Irradiation might effectively attenuate the undesirable changes caused by drying process, ensuring also higher shelf-life of mushrooms and their decontamination [I]. In the present work, the combined effects of electron-beam irradiation (at 0, 0.5, 1 and 6 kGy doses) and storage time (at 0, 6 and 12 months) were evaluated and compared. Macrolepiota procera (Scop.) Singer wild samples were obtained in Tnis-os-Montes, in the Northeast of Portugal, and dried at 30 •c in an oven. Subsequently, the samples were divided in four groups: control (non-irradiated, 0 kGy); sample 1 (0.5 kGy); sample 2 (1 kGy) and sample 3 (6 kGy). The irradiation was performed at the lNCTInstitute of Nuclear Chemistry and Technology (lNCT), in Warsaw, Poland. Moisture, protein, fat, carbohydrates and ash were determined following standard procedures. Free sugars and tocopherols were determined by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI) and a fluorescence detector, respectively; fatty acids were determined by gas-liquid chromatography with flame ionization detection (GC-FID). Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2- picrylhydrazyl) radical scavenging activity, reducing power, inhibition of ~-carotene bleaching and inhibition oflipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. All the parameters showed a decrease tendency with storage time. Trehalose and y-tocopherol were preserved with 1 kGy dose. Electron-beam irradiation did not impart additional changes to most of the chemical and antioxidant parameters of M. procera dried samples. This is a very promising result, since electron-beam irradiation might attenuate most unwanted changes caused by drying, maintaining its long-term effectiveness.

Ultrasound and microwave assisted extraction of ergosterol from Agaricus bisporus L.: optimization through response surface methodology

There is scientific evidence demonstrating the benefits of mushrooms ingestion due to their richness in bioactive compounds such as mycosterols, in particular ergosterol [I]. Agaricus bisporus L. is the most consumed mushroom worldwide presenting 90% of ergosterol in its sterol fraction [2]. Thus, it is an interesting matrix to obtain ergosterol, a molecule with a high commercial value. According to literature, ergosterol concentration can vary between 3 to 9 mg per g of dried mushroom. Nowadays, traditional methods such as maceration and Soxhlet extraction are being replaced by emerging methodologies such as ultrasound (UAE) and microwave assisted extraction (MAE) in order to decrease the used solvent amount, extraction time and, of course, increasing the extraction yield [2]. In the present work, A. bisporus was extracted varying several parameters relevant to UAE and MAE: UAE: solvent type (hexane and ethanol), ultrasound amplitude (50 - 100 %) and sonication time (5 min-15 min); MAE: solvent was fixed as ethanol, time (0-20 min), temperature (60-210 •c) and solid-liquid ratio (1-20 g!L). Moreover, in order to decrease the process complexity, the pertinence to apply a saponification step was evaluated. Response surface methodology was applied to generate mathematical models which allow maximizing and optimizing the response variables that influence the extraction of ergosterol. Concerning the UAE, ethanol proved to be the best solvent to achieve higher levels of ergosterol (671.5 ± 0.5 mg/100 g dw, at 75% amplitude for 15 min), once hexane was only able to extract 152.2 ± 0.2 mg/100 g dw, in the same conditions. Nevertheless, the hexane extract showed higher purity (11%) when compared with the ethanol counterpart ( 4% ). Furthermore, in the case of the ethanolic extract, the saponification step increased its purity to 21%, while for the hexane extract the purity was similar; in fact, hexane presents higher selectivity for the lipophilic compounds comparatively with ethanol. Regarding the MAE technique, the results showed that the optimal conditions (19 ± 3 min, 133 ± 12 •c and 1.6 ± 0.5 g!L) allowed higher ergosterol extraction levels (556 ± 26 mg/100 g dw). The values obtained with MAE are close to the ones obtained with conventional Soxhlet extraction (676 ± 3 mg/100 g dw) and UAE. Overall, UAE and MAE proved to he efficient technologies to maximize ergosterol extraction yields.

Internationalization of IT company: Case study of a Finnish MOOC platform

The interest to small and media size enterprises’ (SMEs) internationalization process is increasing with a growth of SMEs’ contribution to GDP. Internet gives an opportunity to provide variety of services online and reach market niche worldwide. The overlapping of SMEs’ internationalization and online services is the main issue of the research. The most SMEs internationalize according to intuitive decisions of CEO of the company and lose limited resources to worthless attempts. The purpose of this research is to define effective approaches to online service internationalization and selection of the first international market. The research represents single holistic case study of local massive open online courses (MOOCs) platform going global. It considers internationalization costs and internationalization theories applicable to online services. The research includes preliminary screening of the markets and in-depth analysis based on macro parameters of the market and specific characteristics of the customers and expert evaluation of the results. The specific issues as GILT (Globalization, Internationalization, Localization and Translation) approach and Internet-enabled internationalization are considered. The research results include recommendations on international market selection methodology for online services and for effective internationalization strategy development.

Chromatographic analysis of antioxidant and related compounds in Polyporus squamosus from different origins

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Development of procedures for the triazole fungicides determination in fruits and liquid samples using microextraction techniques and chromatographic separation

Agricultural crops can be damaged by funguses, insects, worms and other organisms that cause diseases and decrease the yield of production. The effect of these damaging agents can be reduced using pesticides. Among them, triazole compounds are effective substances against fungus; for example, Oidium. Nevertheless, it has been detected that the residues of these fungicides in foods as well as in derivate products can affect the health of the consumers. Therefore, the European Union has established several regulations fixing the maximum residue of pesticide levels in a wide range of foods trying to assure the consumer safety. Hence, it is very important to develop adequate methods to determine these pesticide compounds. In most cases, gas or liquid chromatographic (GC, LC) separations are used in the analysis of the samples. But firstly, it is necessary to use proper sample treatments in order to preconcentrate and isolate the target analytes. To reach this aim, microextraction techniques are very effective tools; because allow to do both preconcentration and extraction of the analytes in one simple step that considerably reduces the source of errors. With these objectives, two remarkable techniques have been widely used during the last years: solid phase microextraction (SPME) and liquid phase microextraction (LPME) with its different options. Both techniques that avoid the use or reduce the amount of toxic solvents are convenient coupled to chromatographic equipments providing good quantitative results in a wide number of matrices and compounds. In this work simple and reliable methods have been developed using SPME and ultrasound assisted emulsification microextraction (USAEME) coupled to GC or LC for triazole fungicides determination. The proposed methods allow confidently determine triazole concentrations of μg L‐1 order in different fruit samples. Chemometric tools have been used to accomplish successful determinations. Firstly, in the selection and optimization of the variables involved in the microextraction processes; and secondly, to overcome the problems related to the overlapping peaks. Different fractional factorial designs have been used for the screening of the experimental variables; and central composite designs have been carried out to get the best experimental conditions. Trying to solve the overlapping peak problems multivariate calibration methods have been used. Parallel Factor Analysis 2 (PARAFAC2), Multivariate Curve Resolution (MCR) and Parallel Factor Analysis with Linear Dependencies (PARALIND) have been proposed, the adequate algorithms have been used according to data characteristics, and the results have been compared. Because its occurrence in Basque Country and its relevance in the production of cider and txakoli regional wines the grape and apple samples were selected. These crops are often treated with triazole compounds trying to solve the problems caused by the funguses. The peel and pulp from grape and apple, their juices and some commercial products such as musts, juice and cider have been analysed showing the adequacy of the developed methods for the triazole determination in this kind of fruit samples.

Biotechnological, nutritional and therapeutic uses of Pleurotus spp. (Oyster mushroom) related with its chemical composition: A review on the past decade findings

The particular characteristics of growth and development of mushrooms in nature result in the accumulation of a variety of secondary metabolites, several of them with biological activities. The genus Pleurotus is a cosmopolitan group of mushrooms with high nutritional value and therapeutic properties, besides a wide array of biotechnological and environmental applications. Scope and approach: The present report aims to provide a critical review on aspects related to chemical compounds isolated from the genus Pleurotus with possible biotechnological, nutritional and therapeutic uses. Investigations on the genus have immensely accelerated during the last ten years, so that only reports published after 2005 have been considered. Key findings and conclusions: The most important Pleurotus species cultivated in large scale are P. ostreatus and P. pulmonarius. However, more than 200 species have already been investigated to various degrees. Both basidiomata and mycelia of Pleurotus are a great renewable and easily accessible source of functional foods/nutraceuticals and pharmaceuticals with antioxidant, antimicrobial, anti-inflammatory, antitumor and immunomodulatory effects. A series of compounds have already been precisely defined including several polysaccharides, phenolics, terpenes and sterols. However, intensification of structure determination is highly desirable and demands considerable efforts. Further studies including clinical trials need to be carried out to ascertain the safety of these compounds as adequate alternatives to conventional drugs. Not less important is to extend the search for novel bioactives to less explored Pleurotus species.