Glucosylceramidase Mass and Subcellular Localization Are Modulated by Cholesterol in Niemann-Pick Disease Type C

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability

The secretion and maturation of prosaposin and procathepsin D are blocked in embryonic neural progenitor cells

The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin-cathepsin D (proCath-Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired. Furthermore, neither Prosap nor proCath D were secreted from eHNPCs. The block of the processing and secretion shared by Prosap and proCath D was overcome during the course of differentiation of eHNPCs into a mixed population of astrocytes and neuronal cells. Upon differentiation, large amounts of Prosap and proCath D were secreted from the cells, while saposins and mature Cath D were produced inside the cells. The dramatic accumulation of Prosap (an antiapoptotic factor) and reduction of mature Cath D (a proapoptotic factor) in the undifferentiated eHNPCs most likely play a role in the molecular mechanisms regulating the resistance to apoptotic signals of these cells and might represent a critically important issue in HNPCs biology. © 2008 Elsevier B.V. All rights reserved

Ripensare i depositi archeologici, promuovere l’infomobilità. Presentazione del progetto e risultati preliminari

Nell’ambito del Dottorato in Scienze e Tecnologie per l’Archeologia e i Beni Culturali dell’Università degli Studi di Ferrara, è stata attivata una linea di ricerca il cui focus è l’individuazione di una gamma significativa di parametri adottabile nella gestione dei depositi museali. La ricerca è rafforzata dalla messa a disposizione da parte del MIUR di una borsa di dottorato sul Fondo ministeriale per il sostegno dei giovani (Fondo Giovani) - Anno Finanziario 2012. La collaborazione con gli organismi preposti alla salvaguardia e alla valorizzazione del patrimonio culturale, in particolare con il riferimento al MIBACT e con l’Associazione Nazionale Musei Locali e Istituzionali (ANMLI), ha consentito la stesura condivisa di un questionario informatizzato in grado di rilevare lo stato dell’arte nell’ambito della gestione e movimentazione delle collezioni museali. Una prima applicazione del questionario è stata condotta in importanti complessi internazionali tra i quali il Museo del Oro di Bogotà o il Muséum national d’Histoire naturelle di Parigi. Oggetto del presente lavoro sarà pertanto la presentazione/analisi dei primi risultati della ricerca. Gli Autori, sebbene l’indagine, per complessità e numero di soggetti coinvolti, sia ancora in itinere, ritengono utile, già a questo stadio, confrontarsi con la comunità scientifica di riferimento anche al fine di ottimizzare il proprio operato

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice

KMT5B gene; Neurodevelopment; MiceGen KMT5B; Neurodesenvolupament; RatolinsGen KMT5B; Neurodesarrollo; RatonesPathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.This work was supported by LB692 Nebraska Tobacco Settlement Biomedical Research Development Program (to H.A.F.S.); The Simons Foundation Autism Research Initiative–Bridge to Independence Award SFARI 381192 (to H.A.F.S.); The A*STAR, Singapore, IAF-PP Program H17/01/a0/004 (to C.Y.L.); The Wong Boon Hock Society research program Yong Loo Lin School of Medicine (to Z.X.C.); NIH training grant 2T32GM008638-25 (L.B.); The Intramural Research Program of the National Human Genome Research Institute (to W.G.); The National Center for Advancing Translational Sciences of the NIH award number TL1TR001880 (to S.E.S.); The Eunice Kennedy Shriver National Institute of Child Health and Human Development award number HD009003-01 (to S.E.S.); Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania (to S.E.S.); and Swiss National Science Foundation (SNSF) grant 320020_179547 and funds from the University of Zurich Research Priority Programs (URPP) AdaBD: Adaptive Brain Circuits in Developments (to A.Rau.). F.J.K. was funded by the Deutsche Forschungsgemeinschaft grant number FOR 2488. In silico modeling was supported by the Spanish Ministerio de Ciencia e Innovación grant number PID2019-111217RB-I00 (to X.d.l.C.). This study used data from the DDD study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). This study makes use of DECIPHER (www.deciphergenomics.org), which is funded by Wellcome (grant number 223718/Z/21/Z). See Nature PMID: 25533962 or www.ddduk.org/access.html for full acknowledgement

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance

SGCD Missense Variant in a Lagotto Romagnolo Dog with Autosomal Recessively Inherited Limb-Girdle Muscular Dystrophy

An 8-month-old female Lagotto Romagnolo dog was presented for a 1-month history of an initial severe reluctance to move, rapidly progressing to a marked stiff gait and progressive muscular weakness and evolving to tetraparesis, which persuaded the owner to request euthanasia. A primary muscle pathology was supported by necropsy and histopathological findings. Macroscopically, the muscles were moderately atrophic, except for the diaphragm and the neck muscles, which were markedly thickened. Histologically, all the skeletal muscles examined showed atrophy, hypertrophy, necrosis with calcification of the fibers, and mild fibrosis and inflammation. On immunohistochemistry, all three dystrophin domains and sarcoglycan proteins were absent. On Western blot analysis, no band was present for delta sarcoglycan. We sequenced the genome of the affected dog and compared the data to more than 900 control genomes of different dog breeds. Genetic analysis revealed a homozygous private protein-changing variant in the SGCD gene encoding delta- sarcoglycan in the affected dog. The variant was predicted to induce a SGCD:p.(Leu242Pro) change in the protein. In silico tools predicted the change to be deleterious. Other 770 Lagotto Romagnolo dogs were genotyped for the variant and all found to be homozygous wild type. Based on current knowledge of gene function in other mammalian species, including humans, hamsters, and dogs, we propose the SGCD missense variant as the causative variant of the observed form of muscular dystrophy in the index case. The absence of the variant allele in the Lagotto Romagnolo breeding population indicates a rare allele that has appeared recently

Impact of a mediterranean dietary pattern and its components on cardiovascular risk factors, glucose control, and body weight in people with type 2 diabetes: A real-life study

This study evaluates the relation of a Mediterranean dietary pattern and its individual components with the cardiovascular risk factors profile, plasma glucose and body mass index (BMI) in people with type 2 diabetes. We studied 2568 participants at 57 diabetes clinics. Diet was assessed with the EPIC (European Prospective Investigation into Cancer and Nutrition) questionnaire, adherence to the Mediterranean diet was evaluated with the relative Mediterranean diet score (rMED). A high compared to a low score was associated with a better quality of diet and a greater adherence to the nutritional recommendations for diabetes. However, even in the group achieving a high score, only a small proportion of participants met the recommendations for fiber and saturated fat (respectively 17% and 30%). Nonetheless, a high score was associated with lower values of plasma lipids, blood pressure, glycated hemoglobin, and BMI. The relationship of the single food items components of the rMED score with the achievement of treatment targets for plasma lipids, blood pressure, glucose, and BMI were also explored. The study findings support the Mediterranean dietary model as a suitable model for type 2 diabetes and the concept that the beneficial health effects of the Mediterranean diet lie primarily in its synergy among various nutrients and foods rather than on any individual component

Mono- and biallelic germline variants of DNA glycosylase genes in colon adenomatous polyposis families from two continents

Recently, biallelic germline variants of the DNA glycosylase genes MUTYH and NTHL1 were linked to polyposis susceptibility. Significant fractions remain without a molecular explanation, warranting searches for underlying causes. We used exome sequencing to investigate clinically well-defined adenomatous polyposis cases and families from Finland (N=34), Chile (N=21), and Argentina (N=12), all with known susceptibility genes excluded. Nine index cases (13%) revealed germline variants with proven or possible pathogenicity in the DNA glycosylase genes, involving NEIL1 (mono- or biallelic) in 3 cases, MUTYH (monoallelic) in 3 cases, NTHL1 (biallelic) in 1 case, and OGG1 (monoallelic) in 2 cases. NTHL1 was affected with the well-established, pathogenic c.268C>T, p.(Gln90Ter) variant. A recurrent heterozygous NEIL1 c.506G>A, p.(Gly169Asp) variant was observed in two families. In a Finnish family, the variant occurred in trans with a truncating NEIL1 variant (c.821delT). In an Argentine family, the variant co-occurred with a genomic deletion of exons 2 - 11 of PMS2. Mutational signatures in tumor tissues complied with biological functions reported for NEIL1. Our results suggest that germline variants in DNA glycosylase genes may occur in a non-negligible proportion of unexplained colon polyposis cases and may predispose to tumor development.Peer reviewe

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

The Italian National Project of Astrobiology-Life in Space-Origin, Presence, Persistence of Life in Space, from Molecules to Extremophiles

The \u2018\u2018Life in Space\u2019\u2019 project was funded in the wake of the Italian Space Agency\u2019s proposal for the development of a network of institutions and laboratories conceived to implement Italian participation in space astrobiology experiments