Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells

H1069Q substitution represents the most frequent mutation of the copper transporter ATP7B causing Wilson disease in Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes but moves in response to copper overload to the endo-lysosomal compartment to support copper excretion via bile canaliculi. In heterologous or hepatoma-derived cell lines, overexpressed ATP7B-H1069Q is strongly retained in the ER and fails to move to the post-Golgi sites, resulting in toxic copper accumulation. However, this pathogenic mechanism has never been tested in patients' hepatocytes, while animal models recapitulating this form of WD are still lacking. To reach this goal, we have reprogrammed skin fibroblasts of homozygous ATP7B-H1069Q patients into induced pluripotent stem cells and differentiated them into hepatocyte-like cells. Surprisingly, in HLCs we found one third of ATP7B-H1069Q localized in the Golgi complex and able to move to the endo-lysosomal compartment upon copper stimulation. However, despite normal mRNA levels, the expression of the mutant protein was only 20% compared to the control because of endoplasmic reticulum-associated degradation. These results pinpoint rapid degradation as the major cause for loss of ATP7B function in H1069Q patients, and thus as the primary target for designing therapeutic strategies to rescue ATP7B-H1069Q function

Clinical characteristics of vulnerable populations hospitalized and diagnosed with COVID-19 in Buenos Aires, Argentina

There is not in Argentina publications regarding the presentation of patients with COVID-19 requiring hospitalized and emergency care in vulnerable populations (lower incomes and less education tend at greater risk for poor health status and healthcare access), and it has few reports in developing countries. The objective is to determine whether in the care of vulnerable patients, to succeed against COVID-19, multiple public health tools and interventions will be needed to minimize morbidity and mortality. The study is a prospective cohort investigation of patients with lab-confirmed COVID-19, who required to any of the Health Centers response from April 8, 2020, to August 18, 2020. In Buenos Aires Metropolitan Area (AMBA), April 8, 2020 the virus was identified in patients hospitalized in the "Southeast Network" (SN), AMBA. SN covering an area of 661 square kilometers, with 1.8 million inhabitants residing in urban, and rural areas. A total of 14 health centers with different levels of care complexity provide care to patients in the region. The information of each patient with COVID-19 evaluated by SN, was incorporated in an Epidemiological Dashboard. The investigation was designed and reported with consideration of observational studies in epidemiology. We describe the hospitals presentation and care of persons who required SN response and were ultimately diagnosed with COVID-19. From April 8, 2020, to August 18, 2020, were included 1495 patients with lab-confirmed COVID-19 in SN. A total of 58% patients were men, and the mean age (SD) was 48.9 (15.59) years. Eighty one percent patients with pre-existing diseases, most frequent hypertension and diabetes, but hypertension, chronic lung disease, and cardiovascular disease presented higher risk. A total of 13% were hospitalized in Intensive Therapy Unit. The mortality of the cohort was 9.77%. Mortality was higher for patients aged 65 or more (OR 5.09), and for those had some pre-existing disease (OR 2.61). Our observations are consistent with reports demonstrating older persons, and those with comorbidities have the highest risk of mortality related to COVID-19. However, unlike other reports from developed or some developing countries, the mortality in our study is lower. This finding may be related to age of our cohort is younger than other published. Also, the health system was able to respond to the demand.Fil: Yacobitti, A.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Otero, L.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Doldan Arrubarrena, V.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Arano, J.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Lage, S.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Silberman, M.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Zubieta, M.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Erbetta, I.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Danei, P.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Baeck, G.. Hospital Mi Pueblo; ArgentinaFil: Vallejos, V.. No especifíca;Fil: Cavalli, F.. No especifíca;Fil: Calderón, N.. Gobierno de la Provincia de Buenos Aires. Hospital Zonal General de Agudos Doctor Lucio Melendez.; ArgentinaFil: Di Gregorio, M.. Gobierno de la Provincia de Buenos Aires. Hospital Zonal General de Agudos Doctor Lucio Melendez.; ArgentinaFil: Hernandez, V.. Hospital Dr. Arturo Oñativia - Salta Capital.; ArgentinaFil: Bruno, D.. Hospital Dr. Arturo Oñativia - Salta Capital.; ArgentinaFil: Rodera, B.. Municipalidad de Quilmes (buenos Aires). Hospital Zonal General de Agudos Doctor Isidoro Iriarte.; ArgentinaFil: Macherett, I.. Municipalidad de Quilmes (buenos Aires). Hospital Zonal General de Agudos Doctor Isidoro Iriarte.; ArgentinaFil: Parisi, M.. Municipalidad de Quilmes (buenos Aires). Hospital Zonal General de Agudos Doctor Isidoro Iriarte.; ArgentinaFil: Gallastegui, M.. Municipalidad de Quilmes (buenos Aires). Hospital Zonal General de Agudos Doctor Isidoro Iriarte.; ArgentinaFil: Paz, A.. Municipalidad de Quilmes (buenos Aires). Hospital Sub Zonal Materno Infantil Doctor Eduardo Oller.; ArgentinaFil: Bernardi, R.. No especifíca;Fil: Azcárate, S.. Gobierno de la Provincia de Buenos Aires. Hospital Provincial Evita Pueblo.; ArgentinaFil: Hraste, A.. Gobierno de la Provincia de Buenos Aires. Hospital Provincial Evita Pueblo.; ArgentinaFil: Caridi, Délida Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Salgado, P.. Universidad de Buenos Aires. Rectorado. Instituto de Investigaciones en Salud Pública; ArgentinaFil: Kochen, Sara Silvia. Gobierno de la Provincia de Buenos Aires. Hospital de Alta Complejidad Cuenca Alta Doctor Nestor Carlos Kirchner.; Argentina. Universidad Nacional Arturo Jauretche; Argentina. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state

<p>Abstract</p> <p>Background</p> <p>A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.</p> <p>Results</p> <p>By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.</p> <p>Conclusions</p> <p>Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.</p> <p>See: <url>http://www.biomedcental.com/1741-7007/8/125</url></p

Alteration of endosomal trafficking is associated with early-onset parkinsonism caused by SYNJ1 mutations

Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease

Influenza vaccination coverage among medical residents: An Italian multicenter survey

Although influenza vaccination is recognized to be safe and effective, recent studies have confirmed that immunization coverage among health care workers remain generally low, especially among medical residents (MRs). Aim of the present multicenter study was to investigate attitudes and determinants associated with acceptance of influenza vaccination among Italian MRs. A survey was performed in 2012 on MRs attending post-graduate schools of 18 Italian Universities. Each participant was interviewed via an anonymous, self-administered, web-based questionnaire including questions on attitudes regarding influenza vaccination. A total of 2506 MRs were recruited in the survey and 299 (11.9%) of these stated they had accepted influenza vaccination in 2011-2012 season. Vaccinated MRs were older (P = 0.006), working in clinical settings (P = 0.048), and vaccinated in the 2 previous seasons (P < 0.001 in both seasons). Moreover, MRs who had recommended influenza vaccination to their patients were significantly more compliant with influenza vaccination uptake in 2011-2012 season (P < 0.001). "To avoid spreading influenza among patients" was recognized as the main reason for accepting vaccination by less than 15% of vaccinated MRs. Italian MRs seem to have a very low compliance with influenza vaccination and they seem to accept influenza vaccination as a habit that is unrelated to professional and ethical responsibility. Otherwise, residents who refuse vaccination in the previous seasons usually maintain their behaviors. Promoting correct attitudes and good practice in order to improve the influenza immunization rates of MRs could represent a decisive goal for increasing immunization coverage among health care workers of the future. © 2014 Landes Bioscience

Geographical heterogeneity of clinical and serological phenotypes of systemic sclerosis observed at tertiary referral centres. The experience of the Italian SIR-SPRING registry and review of the world literature

Introduction: Systemic sclerosis (SSc) is characterized by a complex etiopathogenesis encompassing both host genetic and environmental -infectious/toxic- factors responsible for altered fibrogenesis and diffuse microangiopathy. A wide spectrum of clinical phenotypes may be observed in patients' populations from different geographical areas. We investigated the prevalence of specific clinical and serological phenotypes in patients with definite SSc enrolled at tertiary referral centres in different Italian geographical macro-areas. The observed findings were compared with those reported in the world literature.Materials and methods: The clinical features of 1538 patients (161 M, 10.5%; mean age 59.8 +/- 26.9 yrs.; mean disease duration 8.9 +/- 7.7 yrs) with definite SSc recruited in 38 tertiary referral centres of the SPRING (Systemic sclerosis Progression INvestiGation Group) registry promoted by Italian Society of Rheumatology (SIR) were obtained and clustered according to Italian geographical macroareas.Results: Patients living in Southern Italy were characterized by more severe clinical and/or serological SSc phenotypes compared to those in Northern and Central Italy; namely, they show increased percentages of diffuse cutaneous SSc, digital ulcers, sicca syndrome, muscle involvement, arthritis, cardiopulmonary symptoms, interstitial lung involvement at HRCT, as well increased prevalence of serum anti-Scl70 autoantibodies. In the same SSc population immunusppressive drugs were frequently employed. The review of the literature underlined the geographical heterogeneity of SSc phenotypes, even if the observed findings are scarcely comparable due to the variability of methodological approaches.Conclusion: The phenotypical differences among SSc patients' subgroups from Italian macro-areas might be correlated to genetic/environmental co-factors, and possibly to a not equally distributed national network of information and healthcare facilities

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice