Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis

Skeletal myogenesis is orchestrated by distinct regulatory signaling pathways, including PI3K/AKT, that ultimately control muscle gene expression. Recently discovered myogenic micro-RNAs (miRNAs) are deeply implicated in muscle biology. Processing of miRNAs from their primary transcripts is emerging as a major step in the control of miRNA levels and might be well suited to be regulated by extracellular signals. Here we report that the RNA binding protein KSRP is required for the correct processing of primary myogenic miRNAs upon PI3K/AKT activation in myoblasts C2C12 and in the course of injury-induced muscle regeneration, as revealed by Ksrp knock-out mice analysis. PI3K/AKT activation regulates in opposite ways two distinct KSRP functions inhibiting its ability to promote decay of myogenin mRNA and activating its ability to favor maturation of myogenic miRNAs. This dynamic regulatory switch eventually contributes to the activation of the myogenic program

Surface Plasmon Resonance Reveals a Different Pattern of Proinsulin Autoantibodies Concentration and Affinity in Diabetic Patients

Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10−9 M and 3.50×107 M−1, respectively) than the adults (median 167.4×10−9 M and 0.84×107 M−1, respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction

PHAT XX. AGB Stars and Other Cool Giants in M31 Star Clusters

The presence of asymptotic giant branch (AGB) stars in clusters provides key constraints for stellar models, as has been demonstrated with historical data from the Magellanic Clouds. In this work, we look for candidate AGB stars in M31 star clusters from the Panchromatic Hubble Andromeda Treasury survey. Our photometric criteria selects stars brighter than the tip of the red giant branch, which includes the bulk of the thermally pulsing AGB stars as well as early-AGB stars and other luminous cool giants expected in young stellar populations (e.g., massive red supergiants, and intermediate-mass red helium-burning stars). The AGB stars can be differentiated, a posteriori, using the ages already estimated for our cluster sample. 937 candidates are found within the cluster aperture radii, half ( 3c450) of which are very likely cluster members. Cross-matching with additional databases reveals two carbon stars and 10 secure variables among them. The field-corrected age distribution reveals the presence of young supergiants peaking at ages smaller than 108 yr, followed by a long tail of AGB stars extending up to the oldest possible ages. This long tail reveals the general decrease in the numbers of AGB stars from initial values of 3c 50 7 10-6 M 99-1 at 108 yr down to 3c 5 7 10-6 M 99-1 at 1010 yr. Theoretical models of near-solar metallicity reproduce this general trend, although with localized discrepancies over some age intervals, whose origin is not yet identified. The entire catalog is released together with finding charts to facilitate follow-up studies

A novel application of entropy analysis for assessing changes in movement variability during cumulative tackles in young elite rugby league players.

The aim of this study was to identify between-position (forwards vs. backs) differences in movement variability in cumulative tackle events training during both attacking and defensive roles. Eleven elite adolescent male rugby league players volunteered to participate in this study (mean ± SD, age; 18.5 ± 0.5 years, height; 179.5 ± 5.0 cm, body mass; 88.3 ± 13.0 kg). Participants performed a drill encompassing four blocks of six tackling (i.e. tackling an opponent) and six tackled (i.e. being tackled by an opponent while carrying a ball) events (i.e. 48 total tackles) while wearing a micro-technological inertial measurement unit (WIMU, Realtrack Systems, Spain). The acceleration data were used to calculate sample entropy (SampEn) to analyse the movement variability during tackles performance. In tackling actions SampEn showed significant between-position differences in block 1 (p = 0.0001) and block 2 (p = 0.0003). Significant between-block differences were observed in backs (block 1 vs 3, p = 0,0021; and block 1 vs 4, p = 0,0001) but not in forwards. When being tackled, SampEn showed significant between-position differences in block 1 (p = 0.0007) and block 3 (p = 0.0118). Significant between-block differences were only observed for backs in block 1 vs 4 (p = 0,0025). Movement variability shows a progressive reduction with cumulative tackle events, especially in backs and when in the defensive role (tackling). Forwards present lower movement variability values in all blocks, particularly in the first block, both in the attacking and defensive role. Entropy measures can be used by practitioners as an alternative tool to analyse the temporal structure of variability of tackle actions and quantify the load of these actions according to playing position

MicroRNAs in pulmonary arterial remodeling

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH

The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis

Medico-legal assessment of personal damage in older people: report from a multidisciplinary consensus conference

Ageing of the global population represents a challenge for national healthcare systems and healthcare professionals, including medico-legal experts, who assess personal damage in an increasing number of older people. Personal damage evaluation in older people is complex, and the scarcity of evidence is hindering the development of formal guidelines on the subject. The main objectives of the first multidisciplinary Consensus Conference on Medico-Legal Assessment of Personal Damage in Older People were to increase knowledge on the subject and establish standard procedures in this field. The conference, organized according to the guidelines issued by the Italian National Institute of Health (ISS), was held in Bologna (Italy) on June 8, 2019 with the support of national scientific societies, professional organizations, and stakeholders. The Scientific Technical Committee prepared 16 questions on 4 thematic areas: (1) differences in injury outcomes in older people compared to younger people and their relevance in personal damage assessment; (2) pre-existing status reconstruction and evaluation; (3) medico-legal examination procedures; (4) multidimensional assessment and scales. The Scientific Secretariat reviewed relevant literature and documents, rated their quality, and summarized evidence. During conference plenary public sessions, 4 pairs of experts reported on each thematic area. After the last session, a multidisciplinary Jury Panel (15 members) drafted the consensus statements. The present report describes Conference methods and results, including a summary of evidence supporting each statement, and areas requiring further investigation. The methodological recommendations issued during the Conference may be useful in several contexts of damage assessment, or to other medico-legal evaluation fields

Cholinergic Modulation of Narcoleptic Attacks in Double Orexin Receptor Knockout Mice

To investigate how cholinergic systems regulate aspects of the sleep disorder narcolepsy, we video-monitored mice lacking both orexin (hypocretin) receptors (double knockout; DKO mice) while pharmacologically altering cholinergic transmission. Spontaneous behavioral arrests in DKO mice were highly similar to those reported in orexin-deficient mice and were never observed in wild-type (WT) mice. A survival analysis revealed that arrest lifetimes were exponentially distributed indicating that random, Markovian processes determine arrest lifetime. Low doses (0.01, 0.03 mg/kg, IP), but not a high dose (0.08 mg/kg, IP) of the cholinesterase inhibitor physostigmine increased the number of arrests but did not alter arrest lifetimes. The muscarinic antagonist atropine (0.5 mg/kg, IP) decreased the number of arrests, also without altering arrest lifetimes. To determine if muscarinic transmission in pontine areas linked to REM sleep control also influences behavioral arrests, we microinjected neostigmine (50 nl, 62.5 µM) or neostigmine + atropine (62.5 µM and 111 µM respectively) into the nucleus pontis oralis and caudalis. Neostigmine increased the number of arrests in DKO mice without altering arrest lifetimes but did not provoke arrests in WT mice. Co-injection of atropine abolished this effect. Collectively, our findings establish that behavioral arrests in DKO mice are similar to those in orexin deficient mice and that arrests have exponentially distributed lifetimes. We also show, for the first time in a rodent narcolepsy model, that cholinergic systems can regulate arrest dynamics. Since perturbations of muscarinic transmission altered arrest frequency but not lifetime, our findings suggest cholinergic systems influence arrest initiation without influencing circuits that determine arrest duration