Identification of Cellular Pathogenicity Markers for SIL1 Mutations Linked to Marinesco-Sjögren Syndrome.

Background and objective: Recessive mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), a rare neuropediatric disorder. MSS-patients typically present with congenital cataracts, intellectual disability, cerebellar ataxia and progressive vacuolar myopathy. However, atypical clinical presentations associated with SIL1 mutations have been described over the last years; compound heterozygosity of SIL1 missense mutations even resulted in a phenotype not fulfilling the clinical diagnostic criteria of MSS. Thus, a read-out system to evaluate reliably the pathogenicity of amino acid changes in SIL1 is needed. Here, we aim to provide suitable cellular biomarkers enabling the robust evaluation of pathogenicity of SIL1 mutations. Methods: Five SIL1 variants including one polymorphism (p.K132Q), three known pathogenic mutations (p.V231_I232del, p.G312R, and p.L457P) and one ambiguous missense variant (p.R92W) were studied along with the wild-type proteins in Hek293 in vitro models by cell biological assays, immunoprecipitation, immunoblotting, and immunofluorescence as well as electron microscopy. Moreover, the SIL1-interactomes were interrogated by tandem-affinity-purification and subsequent mass spectrometry. Results: Our combined studies confirmed the pathogenicity of p.V231_I232del, p.G312R, and p.L457P by showing instability of the proteins as well as tendency to form aggregates. This observation is in line with altered structure of the ER-Golgi system and vacuole formation upon expression of these pathogenic SIL1-mutants as well as the presence of oxidative or ER-stress. Reduced cellular fitness along with abnormal mitochondrial architecture could also be observed. Notably, both the polymorphic p.K132Q and the ambiguous p.R92W variants did not elicit such alterations. Study of the SIL1-interactome identified POC1A as a novel binding partner of wild-type SIL1; the interaction is disrupted upon the presence of pathogenic mutants but not influenced by the presence of benign variants. Disrupted SIL1-POC1A interaction is associated with centrosome disintegration. Conclusions: We developed a combination of cellular outcome measures to evaluate the pathogenicity of SIL1 variants in suitable in vitro models and demonstrated that the p. R92W missense variant is a polymorphism rather than a pathogenic mutation leading to MSS

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3.

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation

Homozygous WASHC4 variant in two sisters causes a syndromic phenotype defined by dysmorphisms, intellectual disability, profound developmental disorder, and skeletal muscle involvement.

Funder: European Regional Development Fund; Id: http://dx.doi.org/10.13039/501100008530Recessive variants in WASHC4 are linked to intellectual disability complicated by poor language skills, short stature, and dysmorphic features. The protein encoded by WASHC4 is part of the Wiskott-Aldrich syndrome protein and SCAR homolog family, co-localizes with actin in cells, and promotes Arp2/3-dependent actin polymerization in vitro. Functional studies in a zebrafish model suggested that WASHC4 knockdown may also affect skeletal muscles by perturbing protein clearance. However, skeletal muscle involvement has not been reported so far in patients, and precise biochemical studies allowing a deeper understanding of the molecular etiology of the disease are still lacking. Here, we report two siblings with a homozygous WASHC4 variant expanding the clinical spectrum of the disease and provide a phenotypical comparison with cases reported in the literature. Proteomic profiling of fibroblasts of the WASHC4-deficient patient revealed dysregulation of proteins relevant for the maintenance of the neuromuscular axis. Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4. The results of histological and coherent anti-Stokes Raman scattering microscopic studies support the concept of a functional role of the WASHC4 protein in humans by altering protein processing and clearance. The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland

Untersuchung der Auswirkung verschiedener Umwelteinflüsse auf Mikroorganismen mittels ortsaufgelöster Raman-Spektroskopie

In der vorliegenden Arbeit werden die Einflüsse von zwei verschiedenen Umweltfaktoren auf Bakterien oder Pilze mittels ortsaufgelöster Raman-Spektroskopie untersucht. Im ersten Teil wird der Einfluss antibiotischer Behandlung von Bakterien auf deren Raman-spektroskopische Identifizierbarkeit analysiert. Dazu wurden je zwei Vertreter der Gattungen Pseudomonas und Escherichia sowohl ohne als auch mit vier unterschiedlichen Antibiotika behandelt. Unter Verwendung linearer Diskriminanz-Modelle lassen sich für unabhängige Proben trotz Behandlung mit unterschiedlichen Antibiotika bis zu 97% der Bakterien anhand ihrer Raman-Spektren korrekt identifizieren. Ein Einfluss der Antibiotika-Konzentration ist dabei nicht zu beobachten. Im zweiten Teil der Arbeit werden olaginöse Pilze der Gattung Mortierella unter verschiedenen Bedingungen kultiviert, die dabei gebildeten Lipid-Tropfen Raman-spektroskopisch analysiert und anhand der Spektren der Sättigungsgrad der Lipide bestimmt. Die ortsaufgelöste Analyse der Lipidtropfen in intakten Hyphen zeigt, dass der Sättigungsgrad in weiten Teilen des Myzels gleich ist. Lediglich in einem Bereich von etwa 600 µm hinter der Hyphenspitze wird eine hohe Variabilität des Sättigungsgrades beobachtet. Andere morphologische Strukturen wie Verzweigungspunkte oder Anastomosen haben dagegen keinen Einfluss. Für sechs Spezies wurde der Sättigungsgrad quantitativ bestimmt und die Ergebnisse anhand von Gaschromatographie-Messungen validiert. Beide Methoden liefern auch für verschiedene Wachstumsbedingungen jeweils vergleichbare Werte. Es zeigte sich, dass die Spezies auf Glyerin-Medium Lipide mit größerem Anteil an ungesättigten Fettsäuren produzieren als auf Glukose-Medium. Somit wird in dieser Arbeit gezeigt, dass die ortsaufgelöste Raman-Spektroskopie geeignet ist, um die Auswirkungen verschiedener Umwelteinflüsse auf Mikroorganismen qualitativ und quantitativ zu untersuchen

SERS Background Imaging – a Versatile Tool Towards More Reliable SERS Analytics

Surface-enhanced Raman scattering (SERS) is a highly selective and sensitive straightforward analytical method, which is however not yet established in routine analysis due to a lack of reliability and reproducibility. Here we utilise the broad SERS continuum background (SERS-BG) accompanying every SERS measurement as a versatile tool towards more reliable SERS analytics. We apply a heterogeneous gold SERS substrate immersed with an adenosine triphosphate solution to show that the integrated SERS-BG distinctly correlates with the intensity of the analyte signals in the SERS spectrum. Based on this relationship we introduce an easy-to-handle, automatable and more reliable SERS measurement procedure starting with fast and high-contrast imaging of the SERS substrate followed by hot spot localisation and recording of highly enhanced SERS spectra at the centre of the diffraction-limited spot. We further demonstrate the applicability of SERS-BG imaging by combining it with other optical modalities and electron microscopy to assess structure-property relationships. Additionally, we perform Monte-Carlo simulations to evaluate the sampling error in SERS experiments highlighting the advantages of our method over conventional SERS experiments.</div

Multiplexed Online-Monitoring of Microfluidic Free-Flow Electrophoresis via Mass Spectrometry

Free-flow electrophoresis is a tool for the continuous fractionation of electrically charged analytes. In this study we introduce a novel method to couple microchip-based free-flow electrophoresis with mass spectrometry. The successive connection of multiple microchip outlets to the electrospray ionization source of a mass spectrometer is automated using a multiposition valve. With this novel setup it is possible to continuously fractionate and collect compounds while simultaneously monitoring the process online with mass spectrometry. The functionality of the method is demonstrated by the successful separation and identification of the biomolecules AMP, ATP and CoA, which are fundamental for numerous biochemical processes in every organism