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Brescia's museums network has adopted and developed in the last years a wide number of ways to make its heritage inclusive and accessible to everyone. Via the creation of different tools and initiatives, Brescia Museums Foundation, that manages the network, is at constant work to ensure the possibility to all members of the public to fully experience the cultural heritage.

Urinary extracellular vesicles (UEV) mainly derive from cells of the urogenital tract and their cargo (proteins, nucleic acids, lipids, etc.) reflects their cells of origin. Na chloride cotransporter (NCC) is expressed at the kidney level in the distal convoluted tubule, is involved in salt reabsorption, and is the target of the diuretic thiazides. NCC protein has been recognized and quantified in UEV in previous studies; however, UEV NCC mRNA has never been studied. This study aimed to identify and analyze NCC mRNA levels in primary aldosteronism (PA). The rationale for this investigation stems from previous observations regarding NCC (protein) as a possible biomarker for the diagnosis of PA. To evaluate modulations in the expression of NCC, we analyzed NCC mRNA levels in UEV in PA and essential hypertensive (EH) patients under different conditions, that is, before and after saline infusion, anti-aldosterone pharmacological treatment, and adrenal surgery. NCC mRNA was measured by RT-qPCR in all the samples and was regulated by volume expansion. Its response to mineralocorticoid receptor antagonist was correlated with renin, and it was increased in PA patients after adrenalectomy. NCC mRNA is evaluable in UEV and it can provide insights into the pathophysiology of distal convolute tubule in different clinical conditions including PA.

ObjectiveApparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH).Aim of the StudyTo detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension.MethodsIn this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.ResultsAmong family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118–220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92–124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8–38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5–14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.ConclusionsHSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Aldosterone and renin measurement is a cornerstone for primary aldosteronism (PA) diagnosis, but different thresholds are used according to different assays. A fully automated chemiluminescence (CL) immunoassay for renin and aldosterone was recently proposed, showing good performance for PA screening by aldosterone to renin ratio (ARR). This study aimed to define the accuracy of this assay in the screening and in the most popular confirmatory test of autonomous aldosterone production, the intravenous saline loading test (ivSLT). We compared aldosterone results obtained by CL vs radioimmunoassay (RIA) in hypertensive patients investigated for PA (102 baseline and 85 after ivSLT). An excellent correlation was observed between RIA and CL in the entire population for aldosterone (r=0.922) and ARR (r=0.977). For ARR, Deming regression proved a good accordance between methods and, consistent with the fit model, our previous institutional ARR cut-off of 32 (pg ml−1)/(pg ml−1) corresponded to 20 pg ml−1 mU−1 l−1 in CL assay. However, the correlation was weaker in the low end of aldosterone concentrations (r=0.676 for aldosterone

Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients.A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA.Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism.The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay.

Purpose The current clinical investigation for primary aldosteronism (PA) diagnosis requires complex expensive tests from the initial suspicion to the final subtype classification, including invasive approaches; therefore, appropriate markers for subtype definition are greatly desirable. The present study performs a metabolomics analysis to further examine specific molecular signatures of PA urines EXPERIMENTAL DESIGN: The study considered PA subtype and gender-related differences using two orthogonal advanced UHPLC-MS metabolomics approaches. Patients with essential hypertension (n = 36) and PA (n = 50) who were referred to the outpatient hypertension clinic and matched healthy subjects (n = 10) are investigated. Results Statistically significant changes (p 1.5) of metabolites involved in central carbon, energy, and nitrogen metabolism are identified, especially purine and pyrimidine nucleosides and precursors, and free amino acids. PLS-DA interpretation provides strong evidence of a disease-specific metabolic pattern with dAMP, diiodothyronine, and 5-methoxytryptophan as leading factors, and a sex-specific metabolic pattern associated with orotidine 5-phosphate, N-acetylalanine, hydroxyproline, and cysteine. The results are verified using an independent sample set, which confirms the identification of specific signatures. Conclusions and clinical relevance Metabolomics is used to identify low molecular weight molecular markers of PA, which paves the way for follow-up validation studies in larger cohorts.

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

Purpose A circadian timing system is involved in the maintenance of fluid and electrolyte balance and blood pressure control. Aldosterone and vasopressin modulate ion transporters and channels crucial in sodium (Na) and water reabsorption such as the epithelium Na channel and the renal thiazide-sensitive NaCl cotransporter (NCC). We analyzed in urinary exosomes the intraday variations of NCC and prostasin expression and the association with electrolytes and water balance parameters. Experimental design Blood and urine samples were collected at five time points during the day from five healthy subjects. Blood renin, aldosterone, cortisol, ACTH, and plasmatic and urinary Na, potassium, creatinine, adiuretin (ADH), NCC, and prostasin were evaluated. Results ACTH and cortisol showed a circadian pattern, similarly to aldosterone, while exosomal NCC and prostasin pattern were similar to urinary ADH, decreased in the morning and subsequently increased in the afternoon and evening. Conclusions and clinical relevance In urinary exosomes, NCC and prostasin had a diurnal pattern parallel to ADH and aquaporin 2, confirming that, in healthy subjects, both prostasin and NCC relate to water balance. These results provide suggestions for a possible chronotherapeutic approach in patients treated with thiazides, diuretic drugs acting as specific inhibitors of NCC-mediated Na reabsorption.

Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disease resulting from mutations within the hydroxysteroid (11β-dehydrogenase2 [HSD11B2]) gene causing a prominent mineralocorticoid receptor activation by cortisol and hypokalemic low renin hypertension as the main clinical feature.The objective of the study was to characterize AME for possible novel HSD11B2 mutations and to define the role of HSD11B2 promoter methylation in the phenotypic expression of the disease.Two proband brothers and 10 relatives participated in the study.Peripheral blood mononuclear cell DNA was used for HSD11B2 exon sequencing, and a new predicted structure of 11β-hydroxysteroid dehydrogenase type 2 was generated by an in silico three-dimensional modeling. Promoter methylation was determined by bisulfite pyrosequencing. Urinary tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone ratio, a surrogate marker of 11β-hydroxysteroid dehydrogenase type 2 activity, was measured by gas chromatography-mass spectrometry.A novel homozygous variant at HSD11B2 exon 3 site (c.C662G) resulting in an alanine-to-glycine change at position 221 was discovered by sequencing the DNA of the probands. A monoallelic mutation was found in the DNA of the parents and other four relatives. In silico three-dimensional modeling showed that the Ala221Gly substitution could perturb a hydrophobic interaction by reducing the enzymatic affinity for the substrate. The HSD11B2 promoter methylation of normotensive heterozygous relatives was similar to that of wild types, whereas the hypertensive heterozygous subjects showed higher methylation than wild types, consistently with a transcriptional repressive effect of promoter hypermethylation.A novel HSD11B2 functional mutation accounting for an Ala221Gly substitution causes AME. The hypertension phenotype is also epigenetically modulated by HSD11B2 methylation in subjects heterozygous for the mutation.

The albumin and globulin seed storage proteins present in all plants accumulate in storage vacuoles. Prolamins, which are the major proteins in cereals seeds and are present only there, instead accumulate within the endoplasmic reticulum (ER) lumen as very large insoluble polymers termed protein bodies. Inter-chain disulfide bonds play a major role in polymerization and insolubility of many prolamins. The N-terminal domain of the maize prolamin 27 kD gamma-zein is able to promote protein body formation when fused to other proteins and contains seven cysteine residues involved in interchain bonds. We show that progressive substitution of these amino acids with serine residues in full length gamma-zein leads to similarly progressive increase in solubility and availability to traffic from the ER along the secretory pathway. Total substitution results in very efficient secretion, whereas the presence of a single cysteine is sufficient to promote partial sorting to the vacuole via a wortmannin-sensitive pathway, similar to the traffic pathway of vacuolar storage proteins. We propose that the mechanism leading to accumulation of prolamins in the ER is a further evolutionary step of the one responsible for accumulation in storage vacuoles.

27 kDa gamma-zein is a major storage prolamin of Zea mays seeds. Wild type and mutated forms were expressed in tobacco protoplasts, to identify signals that allow its assembly into endoplasmic reticulum-located protein bodies.

A correct diagnosis of primary aldosteronism (PA) requires adrenal venous sampling (AVS) for the classification of subtypes (bilateral hyperplasia, BAH, or adenoma, APA). Since such testing is not easily practicable, appropriate markers for the definition of subtypes are desirable. We hypothesized that an aldosterone excess was associated with abnormalities in urinary proteome, specific for PA subtypes. The project work was divided into 3 phases: (1) screening/identification by proteomic analysis and further characterization by RT-PCR and immunohistochemistry of the candidate protein; (2) clinical validation by quantitative ELISA assay of 57 (33 M, 24 F) PA patients and 50 normotensive controls (21 M, 29 F); (3) analysis of adrenal tissue of 8 individuals who had undergone adrenalectomy for APA or other adrenal tumors. The proteomic analysis showed a different expression of Serpin B3 Inhibitor-SCCA1 (SB3) in APA and BAH patients. Urine SB3 concentrations in normotensive controls, quantified by ELISA assay and normalized by urinary creatinine, resulted much lower in males (6.72 ng SB3 per mg creatinine, C.I. 4.43–10.19) than in females (20.56 ng SB3 per mg creatinine, C.I. 12.43–33.99, p < 0.00001). SB3 concentrations were not significantly different in males affected by different PA subtypes (BAH, n = 19 and APA, n = 14) compared with normotensive subjects (n = 21). In contrast, in PA females, SB3 was significantly higher in APA (n = 13) than in BAH patients (n = 11) or in normotensive controls (n = 29) (P < 0.01 and

Background: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbor unique subset of proteins, reflecting their cellular source. Methods: With the aim of establishing the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR. Results: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. Conclusions: The selection of appropriate urinary exosomal miRNA isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert-deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.