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IntroductionThis study aims to define the phenotypic and molecular spectrum of the two clinical forms of β-galactosidase (β-GAL) deficiency, GM1-gangliosidosis and mucopolysaccharidosis IVB (Morquio disease type B, MPSIVB).MethodsClinical and genetic data of 52 probands, 47 patients with GM1-gangliosidosis and 5 patients with MPSIVB were analysed.ResultsThe clinical presentations in patients with GM1-gangliosidosis are consistent with a phenotypic continuum ranging from a severe antenatal form with hydrops fetalis to an adult form with an extrapyramidal syndrome. Molecular studies evidenced 47 variants located throughout the sequence of the GLB1 gene, in all exons except 7, 11 and 12. Eighteen novel variants (15 substitutions and 3 deletions) were identified. Several variants were linked specifically to early-onset GM1-gangliosidosis, late-onset GM1-gangliosidosis or MPSIVB phenotypes. This integrative molecular and clinical stratification suggests a variant-driven patient assignment to a given clinical and severity group.ConclusionThis study reports one of the largest series of b-GAL deficiency with an integrative patient stratification combining molecular and clinical features. This work contributes to expand the community knowledge regarding the molecular and clinical landscapes of b-GAL deficiency for a better patient management.

Bacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell-cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production. Staphylococcus epidermidis is a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly-(1→6)-β- N -acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of live S. epidermidis biofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present in S. epidermidis biofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.

The human GLB1 gene encodes a lysosomal β-galactosidase (β-Gal) and an elastinbinding protein(EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex amongneuraminidase-1 (NEU1) and protective protein/ cathepsin A(PPCA)is suggested responsible for impaired elastogenesis in autosomal recessive β-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects ofGLB1, PPCA and NEU1gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H andW273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, theW273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder that predominantly affects the skeletal, cardiovascular, and ocular systems. The primary cause of premature death in untreated patients is related to thoracic aortic aneurysms and dissections (TAA). MFS is caused by mutations in the gene for fibrillin-1, FBN1, and multiple factors such as halpoinsufficiency, fibrillin-1 proteolysis, abnormal TGF-ß signaling, increased matrix metalloproteinase (MMP) expression, and changes in cell-matrix interaction contribute to the complex pathogeneisis of this disorder. In the initial part of this study, we investigated whether recombinant fibrillin-1 polypeptides and aortic extracts from a fibrillin-1 underexpressing mouse model (mgR/mgR) and human Marfan patients can stimulate macrophage chemotaxis. Recombinant fibrillin-1 fragments containing a GxxPG motif and the corresponding synthetic peptides could induce macrophage migration mediated by elastin binding protein (EBP). Both aortic extrtacts from the mgR/mgR mice as well as aortic extracts from Marfan patients significantly increased macrophage chemotaxis compared with extracts from wild type mice and healthy controls. Because of the fact that the chemotaxis inductive effect of Marfan aortic extracts could be inhibited by pretreatment of the cells with lactose or pretreatment of the samples with monoclonal anti-elastin antibody (BA4), we infer that the induction of chemotaxis of Marfan aortic extracts is at least partially associated with EBP. Additionally, aortic extracts from patients with MFS showed a statistically significant increase in reactivity to BA4 in ELISA. BA4 recognizes GxxPG containing peptides or fragments which are ligands of EBP. We additionally performed a therapeutic study on the mgR/mgR mice to test the effects of treatment with BA4 in an attempt to antagonize the secondary deleterious effects of elastin and fibrillin fragmentation. We investigated whether a monoclonal anti-elastin antibody (BA4) or treatment with the anti- inflammatory medication indomethacin could decrease the development of TAA in the mgR/mgR mice. Both treatments were started at 3 weeks of age and continued for 8 weeks. The mgR/mgR mice received BA4 through intraperitoneal injection once a week. In a separate group, indomethacin was added to drinking water continuously for 8 weeks. Here we show that both BA4 and indomethacin treatment significantly improves elastin integrity in the aortic wall of mgR/mgR mice. Treatment with BA4 significantly decreased MMP-2, MMP-9, MMP-12 expression and pSMAD2 activity. Compared to the untreated group, there were significantly less macrophages in the aortic adventitia after BA4 treatment. Indomethacin treatment led to similar results. The results of this study further confirm the hypothesis that abnormal secondary cellular events caused by GxxPG containing fragments contribute to the development of TAA in MFS. Chemotactic activity observed in Marfan aortic extracts and efficient anti- inflammatory treatment suggest an important role of matrix-fragment induced inflammatory activity in the aortic wall in the pathogenesis of TAA. Furthermore, this study might provide new impact on therapeutic approaches and could contribute to new strategies for the long-term management of aortic aneurysm in MFS., Das Marfan-Syndrom (MFS) ist eine relativ häufige autosomal dominant vererbte Störung des Bindegewebes mit zahlreichen Symptomen im Skelettsystem, am Auge, und im Herzkreislaufsystem. Die Haupttodesursache bei unbehandelten Patienten ist die akute Dissektion der aufsteigenden Aorta (TAA), das in der Regel nach einer jahrelangen, langsam fortschreitenden Erweiterung der aufsteigenden Aorta auftritt. Das MFS wird durch Mutationen im Gen für Fibrillin-1, FBN1, verursacht, und mehrere Faktoren wie Halpoinsuffizienz, Fibrillin-1 Proteolyse, abnormale TGF-ß Aktivität, Erhöhung der Expression von Matrixmetalloproteinase (MMP) und Veränderungen in der Zell-Matrix-Interaktion können zur Pathogenese des MFS beitragen. Im ersten Teil dieser Studie untersuchten wir, ob rekombinante Fibrillin-1-Polypeptide und Aortenextrakte aus einem Fibrillin-1-hypomorphen Mausmodell (mgR/mgR) und Marfan Patienten die Makrophagenchemotaxis stimulieren können. Rekombinante Fibrillin-1-Fragmente mit einem GxxPG (Glyzin-X-X-Prolin-Glyzin) Motiv und den entsprechenden synthetischen Peptiden konnten die Migration von Makrophagen durch Interaktion mit dem Elastin-bindendes-Protein (EBP) stimulieren. Sowohl die mgR/mgR-Aortenextrakte als auch die Aortenextrakte von MFS-Patienten erhöhten signifikant die Makrophagenchemotaxis im Vergleich zum Extrakten ausWildtyp-Mäusen und gesunde Kontrollen. Aufgrund der Tatsache, dass die chemotaktische Aktivität von MFSAortenextrakten durch Vorbehandlung der Makrophagen mit Laktose (EBP-Hemmung) bzw. durch den monoklonalen Anti- Elastin-Antikörper (BA4) signifikant gehemmt werden, schliessen wir, dass die durch MFS-Aortenextrakte stimulierte Makrophagenchemotaxis zumindest teilweise durch das EBP vermittelt ist. Darüber hinaus zeigten Aortenextrakte von MFS- Patienten eine statistisch signifikante Zunahme der BA4-Reaktivität im ELISA. BA4 erkennt GxxPG Peptide oder Fragmente, die EBP-Liganden sind. Zusätzlich führten wir eine Therapiestudie an mgR/mgR Mäusen durch, um die Wirkungen einer gezielten Blockade der sekundären schädlichen Auswirkung von Elastin und Fibrillin-1 Fragmente durch Behandlung mit BA4 zu prüfen. Wir untersuchten, ob Antikörper BA4 oder antiinflammatorische Therapie mit dem Medikament Indomethacin die Entstehung der Aortendissektion in dem Fibrillin-1-hypomorphen Mausmodell (mgR/mgR) reduzieren konnte. Beide Behandlungen begannen in der dritten Lebenswoche und dauerten 8 Wochen. Die mgR/mgR Mäuse erhielten BA4 durch intraperitoneale Injektion einmal proWoche. In einer separaten Gruppe wurde Indomethacin im Trinkwasser kontinuierlich für 8 Wochen verabreicht. Wir könnten zeigen, dass die Behandlung mit BA4 sowie durch Indomethacin die Elastinintegrität in Aortenwand in mgR/mgR Mäusen signifikant verbesserten. Die BA4 Behandlung verminderte signifikant die MMP-2, MMP-9, MMP-12 Expression und pSMAD2 Aktivität. Im Vergleich zu der unbehandelten Gruppe gab es signifikant weniger Makrophagen in der Aortenadventitia nach BA4 Behandlung. Indomethacin-Behandlung führte zu ähnlichen Ergebnissen. Die Ergebnisse dieser Studie bestätigen die Hypothese, dass GxxPG-Fragmente abnormale sekundäre zelluläre Reaktionen hervorrufen und somit zur Entwicklung von TAA in MFS beitragen können. Die chemotaktische Aktivität bei Marfan Aortenextrakten und die Effizienz der Antiinflammatorische Therapie zeigen eine wichtige Rolle der durch Matrix- Fragment stimulierte entzündliche Aktivität in der Aortenwand bei der Pathogenese des TAA in MFS. Darüber hinaus zeigt diese Studie potentiell neue therapeutische Optionen auf.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

In order to study the cortisol-binding factors in blood, human plasma was applied to a 11 beta-hydroxy-3-oxo-4-androstene-17 beta-carboxyaminoethylamine-(HACA) 1,4-butanediol diglycidyl ether-Sepharose column. Elution of the column with cortisol buffer produced two protein peaks, the minor peak yielded a protein complex of molecular weight approximately 200 kDa, subsequently termed hucolin. SDS-PAGE analysis under reducing and non-reducing conditions revealed hucolin was a disulphide-linked complex of 35-kDa and 75-kDa subunits. Twenty-five amino acid residues of the N-terminus of the 35-kDa subunit were determined and homology searches revealed an 88% sequence identity with the N-terminal region of beta-ficolin, a transforming growth factor-beta 1 (TGF-beta 1)-binding protein purified from porcine uterus.