Sequence-specific modification of a β-thalassemia locus by small DNA fragments in human erythroid progenitor cells

Gene therapy has been proposed as a definitive cure of beta-thalassemia. We applied a gene targeting approach, based on the introduction of small DNA fragments (SDF) into erythroid progenitor cells, to specifically modify the beta-globin gene sequence at codon 39. The strategy was first tested in normal individuals by delivering mutant SDF that were able to produce the beta-39 (C-T) mutation. Secondly, wild-type SDF were electroporated into target cells of beta-3i9/beta-39 b-thalassemic patients to correct the endogenous mutation. In both cases, gene modification was assayed by allele-specific polymerase chain reaction of DNA and mRNA, by restriction fragment length polymorphism analysis and by direct sequencing

Conjugation with Methylsulfonylmethane improves Hyaluronic Acid anti-inflammatory activity in a hydrogen peroxide-exposed tenocyte culture in vitro model

Abstract: Rotator cuff tears (RCTs) and rotator cuff disease (RCD) are important causes of disability in middle-aged individuals affected by nontraumatic shoulder dysfunctions. Our previous studies have demonstrated that four different hyaluronic acid preparations (HAPs), including Artrosulfur® hyaluronic acid (HA), may exert a protective effect in human RCT-derived tendon cells undergoing oxidative stress damage. Recently, methylsulfonylmethane (MSM) has proven to have anti-inflammatory properties and to cause pain relief in patients affected by tendinopathies. This study aims at evaluating three preparations (Artrosulfur® HA, MSM, and Artrosulfur® MSM + HA) in the recovery from hydrogen peroxide-induced oxidative stress damage in human tenocyte. Cell proliferation, Lactate Dehydrogenase (LDH) release, and inducible nitric oxide synthases (iNOS) and prostaglandin E2 (PGE2) modulation were investigated. In parallel, expression of metalloproteinases 2 (MMP2) and 14 (MMP14) and collagen types I and III were also examined. Results demonstrate that Artrosulfur® MSM + HA improves cell escape from oxidative stress by decreasing cytotoxicity and by reducing iNOS and PGE2 secretion. Furthermore, it differentially modulates MMP2 and MMP14 levels and enhances collagen III expression after 24 h, proteins globally related to rapid acceleration of the extracellular matrix (ECM) remodelling and thus tendon healing. By improving the anti-cytotoxic effect of HA, the supplementation of MSM may represent a feasible strategy to ameliorate cuff tendinopathies

Conjugation with Methylsulfonylmethane Improves Hyaluronic Acid Anti-Inflammatory Activity in a Hydrogen Peroxide-Exposed Tenocyte Culture In Vitro Model

Rotator cuff tears (RCTs) and rotator cuff disease (RCD) are important causes of disability in middle-aged individuals affected by nontraumatic shoulder dysfunctions. Our previous studies have demonstrated that four different hyaluronic acid preparations (HAPs), including Artrosulfur\uae hyaluronic acid (HA) (Alfakjn S.r.l., Garlasco, Italy), may exert a protective effect in human RCT-derived tendon cells undergoing oxidative stress damage. Recently, methylsulfonylmethane (MSM) (Barentz, Paderno Dugnano, Italy) has proven to have anti-inflammatory properties and to cause pain relief in patients affected by tendinopathies. This study aims at evaluating three preparations (Artrosulfur\uae HA, MSM, and Artrosulfur\uae MSM + HA) in the recovery from hydrogen peroxide-induced oxidative stress damage in human tenocyte. Cell proliferation, Lactate Dehydrogenase (LDH) release, and inducible nitric oxide synthases (iNOS) and prostaglandin E2 (PGE2) modulation were investigated. In parallel, expression of metalloproteinases 2 (MMP2) and 14 (MMP14) and collagen types I and III were also examined. Results demonstrate that Artrosulfur\uae MSM + HA improves cell escape from oxidative stress by decreasing cytotoxicity and by reducing iNOS and PGE2 secretion. Furthermore, it differentially modulates MMP2 and MMP14 levels and enhances collagen III expression after 24 h, proteins globally related to rapid acceleration of the extracellular matrix (ECM) remodelling and thus tendon healing. By improving the anti-cytotoxic effect of HA, the supplementation of MSM may represent a feasible strategy to ameliorate cuff tendinopathies

EEG-based cognitive control behaviour assessment: an ecological study with professional air traffic controllers

Several models defining different types of cognitive human behaviour are available. For this work, we have selected the Skill, Rule and Knowledge (SRK) model proposed by Rasmussen in 1983. This model is currently broadly used in safety critical domains, such as the aviation. Nowadays, there are no tools able to assess at which level of cognitive control the operator is dealing with the considered task, that is if he/she is performing the task as an automated routine (skill level), as procedures-based activity (rule level), or as a problem-solving process (knowledge level). Several studies tried to model the SRK behaviours from a Human Factor perspective. Despite such studies, there are no evidences in which such behaviours have been evaluated from a neurophysiological point of view, for example, by considering brain activity variations across the different SRK levels. Therefore, the proposed study aimed to investigate the use of neurophysiological signals to assess the cognitive control behaviours accordingly to the SRK taxonomy. The results of the study, performed on 37 professional Air Traffic Controllers, demonstrated that specific brain features could characterize and discriminate the different SRK levels, therefore enabling an objective assessment of the degree of cognitive control behaviours in realistic setting

Current Genetic Epidemiology of β-Thalassemias and Structural Hemoglobin Variants in the Lazio Region (Central Italy) Following Recent Migration Movements

The aim of this study was to describe the changing pattern of mutational spectrum of β-thalassemia (β-thal) in the Lazio region (Central Italy), as consequence of recent demographic variations. From 1994 until present, 256 immigrant subjects with hemoglobin disorders (including 191 heterozygotes and 65 homozygotes or compound heterozygotes) coming from 44 different foreign countries, have been molecularly characterized. 14 β-globin gene mutations were identified and their frequencies reflect different ethnic origins: 8 of these mutations account for 76.97% of all molecular defects, while 6 of them are much rare, representing less than 2% of the total. These data differ, both in type and percentage, from the mutational spectrum detected in the native population in 1995. Since a few defects are prevalent in each country, a proper strategy for the identification of mutations in immigrant individuals relies on the prior knowledge of their frequency in native ethnic group

Combined supplementation of ascorbic acid and thyroid hormone T3 affects tenocyte proliferation. The effect of ascorbic acid in the production of nitric oxide

Background: Tissue engineering is now increasingly focusing on cell-based treatments as pro-mising tools to improve tendon repair. However, many crucial aspects of tendon biology remain to be understood before adopting the best experimental approach for cell-tissue engineering. Methods: The role played by Ascorbic Acid (AA) alone and in combination with thyroid hormone T3 in the viability and proliferation of primary human tendon-derived cells was investigated. Human tenocyte viability was detected by Trypan blue exclusion test and cellular proliferation rate was evaluated by CFSE CellTraceâ¢. In addition, the potential role of the AA in the production of Nitric Oxide (NO) was also examined. Results: In this in vitro model, an increase in tenocyte proliferation rate was observed as a consequence of progressively increased concentrations of AA (from 10 to 50 μg/ml). The addition of the T3 hormone to the culture further increased tenocyte proliferation rate. In detail, the most evident effect on cellular growth was achieved using the combined supplementation of 50 μg/ml AA and 10-7 M T3. Conclusion: We showed that the highest concentration of AA (100 and 500 μg/ml) caused cytotoxicity to human tenocytes. Moreover, it was shown that AA reduces NO synthesis. These results show that AA is a cell proliferation inducer that triggers tenocyte growth, while it reduces NO synthesis

Heat-Driven Iontronic Nanotransistors

Thermoelectric polyelectrolytes are emerging as ideal material platform for self-powered bio-compatible electronic devices and sensors. However, despite the nanoscale nature of the ionic thermodiffusion processes underlying thermoelectric efficiency boost in polyelectrolytes, to date no evidence for direct probing of ionic diffusion on its relevant length and time scale has been reported. This gap is bridged by developing heat-driven hybrid nanotransistors based on InAs nanowires embedded in thermally biased Na+-functionalized (poly)ethyleneoxide, where the semiconducting nanostructure acts as a nanoscale probe sensitive to the local arrangement of the ionic species. The impact of ionic thermoelectric gating on the nanodevice electrical response is addressed, investigating the effect of device architecture, bias configuration and frequency of the heat stimulus, and inferring optimal conditions for the heat-driven nanotransistor operation. Microscopic quantities of the polyelectrolyte such as the ionic diffusion coefficient are extracted from the analysis of hysteretic behaviors rising in the nanodevices. The reported experimental platform enables simultaneously the ionic thermodiffusion and nanoscale resolution, providing a framework for direct estimation of polyelectrolytes microscopic parameters. This may open new routes for heat-driven nanoelectronic applications and boost the rational design of next-generation polymer-based thermoelectric materials

Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells' doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p < 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems

In Vitro Grown Sheep Preantral Follicles Yield Oocytes with Normal Nuclear-Epigenetic Maturation

BACKGROUND: Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes. CONCLUSIONS/SIGNIFICANCE: In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation