The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity

Tecnologia social: uma estratégia para o desenvolvimento

Esta publicação apresenta reflexões de diversos representantes de instituições governamentais, do terceiro setor, da sociedade civil e de universidades sobre o tema da Tecnologia Social

A transcriptomic study of Hereditary Angioedema attacks

Background: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. Objective: We sought to explore the involvement of other putative genes in edema formation. Methods: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. Results: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. Conclusions: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE

Antidiabetic Drug Prescription Pattern in Hospitalized Older Patients with Diabetes

Objective: To describe the prescription pattern of antidiabetic and cardiovascular drugs in a cohort of hospitalized older patients with diabetes. Methods: Patients with diabetes aged 65 years or older hospitalized in internal medicine and/or geriatric wards throughout Italy and enrolled in the REPOSI (REgistro POliterapuie SIMI—Società Italiana di Medicina Interna) registry from 2010 to 2019 and discharged alive were included. Results: Among 1703 patients with diabetes, 1433 (84.2%) were on treatment with at least one antidiabetic drug at hospital admission, mainly prescribed as monotherapy with insulin (28.3%) or metformin (19.2%). The proportion of treated patients decreased at discharge (N = 1309, 76.9%), with a significant reduction over time. Among those prescribed, the proportion of those with insulin alone increased over time (p = 0.0066), while the proportion of those prescribed sulfonylureas decreased (p < 0.0001). Among patients receiving antidiabetic therapy at discharge, 1063 (81.2%) were also prescribed cardiovascular drugs, mainly with an antihypertensive drug alone or in combination (N = 777, 73.1%). Conclusion: The management of older patients with diabetes in a hospital setting is often sub-optimal, as shown by the increasing trend in insulin at discharge, even if an overall improvement has been highlighted by the prevalent decrease in sulfonylureas prescription

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Human neuroserpin: structure and time-dependent inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA). Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS–tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin–cognate protease complexes

A case of remittent C1-inhibitor deficiency

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and kinin/contactsystem activation. Mutations in C1-inhibitor gene almost consistently result in reduced C1-inhibitor functional level in plasma causing hereditary angioedema, a life-threatening autosomal dominant disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlyingth is variability remains undisclosed. We report a case of a patient suffering from abdominal pain and presenting markedly reduced C1-inhibitor plasma levels, episodically undergoing spontaneous normalization, car- rying the Arg378Cys missense mutation in the serpin domain. Immunostaining analysis of patient plasma revealed the presence of C1-inhibitor oligomers, together with the occurrence of a SDS stable band that disappeared in reducing conditions, suitable for a disulphide bridged Arg378Cys homodimer. Expression studies in eukaryotic cell lines resulted in a drop in mutant C1-innhibitor secretion compared to wild type and confirmed the plasma observations. Notwithstanding, the purified recombinant proteins behave similarly. Both proteins formed stable covalent complexes with target proteases, and the kinetic of inhibition of the mutant was just slightly diminished, although this reduction increased with temperature.Thus,our findings suggest that the Arg378Cys C1-inhibitor mutant once correctly folded should maintain the wild type functional and structural features and instead it should bear a folding defect, abnormally susceptible to environmental factors, which may occasionally promote protein oligomerization. Moreover it can form a disulphide linked homodimer. Both these processes could account for its variability in plasma levels

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress

As tecnologias leves como geradoras de satisfação em usuários de uma unidade de saúde da família

O objetivo desta investigação foi avaliar a qualidade da assistência prestada em um núcleo de saúde da família (NSF), enfocando a satisfação dos usuários, com base nas tecnologias leves, bem como analisar aspectos dessa assistência, produtores de satisfação e de insatisfação, no que diz respeito ao vínculo, à responsabilização, à resolubilidade, às expectativas, aos relacionamentos, ao conforto e acesso, e identificar recomendações para intervenções locais. Após realizarmos uma caracterização geral da população atendida no serviço em estudo, selecionamos os sujeitos participantes da pesquisa. A coleta de dados deu-se por meio da entrevista semi-estruturada. A abordagem foi de natureza qualitativa, e os dados foram ordenados pelo método do Discurso do Sujeito Coletivo (DSC). A análise revela a importância atribuída, pelos usuários do serviço, às tecnologias leves, mas também a necessidade de diminuir o tempo de espera para as consultas e os encaminhamentos e obter o acesso a medicamentos e à atenção odontológica no próprio serviço, fatores que levam à grande insatisfação por parte dos usuários