Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus

Background: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. Results: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 μM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 μM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. Conclusions: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.Fil: 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: Bombicino, Silvina Sonia. 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentin

Rheological Characterisation of Dairy Emulsions For Cold Foam Applications

Dairy foams are complex aerated materials where the liquid matrix is an emulsion made by oil droplets dispersed in a water system. An innovative application of these systems leads to an interesting product derived from instant whipped creams that are stored and consumed at low temperatures (typically between −4 and −18°C) like an ice cream. This novel product requires a specific texture due to the particular conditions related to its consumption. In the present work, the effects of some relevant ingredients (emulsifiers, sugars, and fats) on rheological properties and freezing temperature of dairy emulsions were investigated. Samples were prepared on lab scale and it was found that structure extension is affected strongly by stabilizers (carrageenan and guar gum) and in a lower measure by fat content. As far as freezing point is concerned a significant effect only of sugars (type and amount) and fats was measured. A formulation having interesting properties for low temperature applications was obtained an..

Factores socioeconómicos que generan obesidad en mujeres peruanas: un modelo logístico ordenado - 2021

En el Perú la obesidad es un gran problema de salud, debido a ello, el objetivo de la investigación es determinar los factores socioeconómicos que generan obesidad en las mujeres peruanas. Para ello se utilizó los datos de ENDES del 2021, a través de un modelo logístico ordenado, contando como variable dependiente la obesidad caracterizada por el Índice de Masa Corporal – IMC, así mismo, las variables independientes son el estado civil, consumo de frutas, número de hijos, edad, consumo de bebidas alcohólicas, índice de riqueza, lugar de residencia y zona geográfica. Entre los principales resultados obtenidos tenemos que si la mujer no consume frutas y consume bebidas alcohólicas incrementara su IMC, también obtuvimos que las diferencias económicas influyen en gran medida en el incremento del IMC, teniendo así que si la mujer es de nivel pobre la probabilidad de padecer de obesidad aumenta, mientras que si es de nivel económico alto disminuye. Otra de las características que impacta es que si la mujer es casada o conviviente este es un factor que incrementa la posibilidad de tener obesidad, caso contrario cuando la mujer se encuentra sola. Llegamos a la conclusión, que cada una de nuestras variables de estudio tienen un alto nivel de significancia en la obesidad, sin embargo, la obesidad no es un problema sin solución, ya que se pueden tomar medidas de concientización para las mujeres peruanas y sobre todo que tomen la iniciativa de un cambio en su estilo de vida.Trabado de investigacio

A combined clinical and biomarker approach to predict diuretic response in acute heart failure

Background: Poor diuretic response in acute heart failure is related to poor clinical outcome. The underlying mechanisms and pathophysiology behind diuretic resistance are incompletely understood. We evaluated a combined approach using clinical characteristics and biomarkers to predict diuretic response in acute heart failure (AHF). Methods and results: We investigated explanatory and predictive models for diuretic response—weight loss at day 4 per 40 mg of furosemide—in 974 patients with AHF included in the PROTECT trial. Biomarkers, addressing multiple pathophysiological pathways, were determined at baseline and after 24 h. An explanatory baseline biomarker model of a poor diuretic response included low potassium, chloride, hemoglobin, myeloperoxidase, and high blood urea nitrogen, albumin, triglycerides, ST2 and neutrophil gelatinase-associated lipocalin (r2 = 0.086). Diuretic response after 24 h (early diuretic response) was a strong predictor of diuretic response (β = 0.467, P < 0.001; r2 = 0.523). Addition of diuretic response after 24 h to biomarkers and clinical characteristics significantly improved the predictive model (r2 = 0.586, P < 0.001). Conclusions: Biomarkers indicate that diuretic unresponsiveness is associated with an atherosclerotic profile with abnormal renal function and electrolytes. However, predicting diuretic response is difficult and biomarkers have limited additive value. Patients at risk of poor diuretic response can be identified by measuring early diuretic response after 24 h

The prognostic value of multiparameter flow cytometry minimal residual disease assessment in relapsed multiple myeloma

Letter to the editor.-- et al.This study was supported by the Cooperative Research Thematic Network grants RD12/0036/0058 of the Red de Cancer (Cancer Network of Excellence); Instituto de Salud Carlos III, Spain, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339; 06/1354; 02/0905; 01/0089/01-02; PS09/01897/01370; G03/136; Sara Borrell: CD13/00340); and Asociación Española Contra el Cáncer (GCB120981SAN), Spain. The study was also supported internationally by the International Myeloma Foundation Junior Grant Proposal and the Multiple Myeloma Research Foundation research fellow award.Peer Reviewe

Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma

In recent years, several studies on large series of multiple myeloma (MM) patients have demonstrated the clinical utility of flow cytometry monitoring of minimal residual disease (flow-MRD) in bone marrow (BM), for improved assessment of response to therapy and prognostication. However, disturbing levels of variability exist regarding the specific protocols and antibody panels used in individual laboratories. Overall, consensus exists about the utility of combined assessment of CD38 and CD138 for the identification of BM plasma cells (PC); in contrast, more heterogeneous lists of markers are used to further distinguish between normal/reactive PCs and myeloma PCs in the MRD settings. Among the later markers, CD19, CD45, CD27, and CD81, together with CD56, CD117, CD200, and CD307, have emerged as particularly informative; however, no single marker provides enough specificity for clear discrimination between clonal PCs and normal PCs. Accordingly, multivariate analyses of single PCs from large series of normal/reactive vs. myeloma BM samples have shown that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient. However, the specific antibody clones, fluorochrome conjugates and sources of the individual markers determines its optimal (vs. suboptimal or poor) performance in an eight-color staining. Assessment of clonality, via additional cytoplasmic immunoglobulin (CyIg) κ vs. CyIgλ evaluation, may contribute to further establish the normal/reactive vs. clonal nature of small suspicious PC populations at high sensitivity levels, provided that enough cells are evaluatedGrant sponsor: Red Tematica de Investigacion Cooperativa en Cancer (RTICC) of the Instituto de Salud Carlos III (Ministry of Economy and Competitivity, Madrid, Spain) – FEDER; Grant number: RD12/0036/0048; Grant sponsors: EuroFlow Consortium; the International Myeloma Foundation-Black Swan Research Initiative.Peer Reviewe

Executive functions and borderline personality features in adolescents with major depressive disorder

BackgroundExecutive functions (EF) consolidate during adolescence and are impaired in various emerging psychiatric disorders, such as pediatric Major Depressive Disorder (pMDD) and Borderline Personality Disorder. Previous studies point to a marked heterogeneity of deficits in EF in pMDD. We examined the hypothesis that deficits in EF in adolescents with pMDD might be related to comorbid Borderline Personality features (BPF).MethodsWe examined a sample of 144 adolescents (15.86 ± 1.32) diagnosed with pMDD. Parents rated their child’s EF in everyday life with the Behavior Rating Inventory of Executive Function (BRIEF) and BPF with the Impulsivity and Emotion Dysregulation Scale (IED-27). The adolescents completed equivalent self-rating measures. Self- and parent-ratings of the BRIEF scores were compared with paired t-Tests. Correlation and parallel mediation analyses, ICC, and multiple regression analyses were used to assess symptom overlap, parent-child agreement, and the influence of depression severity.ResultsOver the whole sample, none of the self- or parent-rated BRIEF scales reached a mean score above T > 65, which would indicate clinically impaired functioning. Adolescents tended to report higher impairment in EF than their parents. Depression severity was the strongest predictor for BPF scores, with Emotional Control predicting parent-rated BPF and Inhibit predicting self-rated BPF. Furthermore, the Behavioral Regulation Index, which includes EF closely related to behavioral control, significantly mediated the relationship between depression severity and IED-27 factors emotional dysregulation and relationship difficulties but not non-suicidal self-injuries.ConclusionOn average, adolescents with depression show only subtle deficits in executive functioning. However, increased EF deficits are associated with the occurrence of comorbid borderline personality features, contributing to a more severe overall psychopathology. Therefore, training of executive functioning might have a positive effect on psychosocial functioning in severely depressed adolescents, as it might also improve comorbid BPF.Clinical trial registrationwww.ClinicalTrials.gov, identifier NCT03167307

Decoupling astrocytes in adult mice impairs synaptic plasticity and spatial learning

The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition