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Recessive 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.

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INTRODUCTION: Label-free Raman-based imaging techniques create the possibility of bringing chemical and histologic data into the operation room. Relying on the intrinsic biochemical properties of tissues to generate image contrast and optical tissue sectioning, Raman-based imaging methods can be used to detect microscopic tumor infiltration and diagnose brain tumor subtypes. METHODS: Here, we review the application of three Raman-based imaging methods to neurosurgical oncology: Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, and stimulated Raman histology (SRH). RESULTS: Raman spectroscopy allows for chemical characterization of tissue and can differentiate normal and tumor-infiltrated tissue based on variations in macromolecule content, both ex vivo and in vivo. To improve signal-to-noise ratio compared to conventional Raman spectroscopy, a second pulsed excitation laser can be used to coherently drive the vibrational frequency of specific Raman active chemical bonds (i.e. symmetric stretching of -CH2 bonds). Coherent Raman imaging, including CARS and stimulated Raman scattering microscopy, has been shown to detect microscopic brain tumor infiltration in fresh brain tumor specimens with submicron image resolution. Advances in fiber-laser technology have allowed for the development of intraoperative SRH as well as artificial intelligence algorithms to facilitate interpretation of SRH images. With molecular diagnostics becoming an essential part of brain tumor classification, preliminary studies have demonstrated that Raman-based methods can be used to diagnose glioma molecular classes intraoperatively. CONCLUSIONS: These results demonstrate how label-free Raman-based imaging methods can be used to improve the management of brain tumor patients by detecting tumor infiltration, guiding tumor biopsy/resection, and providing images for histopathologic and molecular diagnosis.

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Lipid storage provides energy for cell survival, growth, and reproduction and is closely related to the organismal response to stress imposed by toxic chemicals. However, the effects of toxicants on energy storage as it impacts certain life-history traits have rarely been investigated. Here, we used the nematode Caenorhabditis elegans as a test species for a chronic exposure to copper (Cu) at EC20 (0.50 mg Cu/l). Effects on the fatty acid distribution in C. elegans body were determined using coherent anti-Stokes Raman spectroscopy (CARS) to link population fitness responses with individual ecophysiological responses. Cu inhibited nematode reproductive capacity and offspring growth in addition to shortening the lifespan of exposed individuals. In adult nematodes, Cu exposure led to significant reduction of lipid storage compared to the Cu-free control: Under Cu, lipids filled only 0.5% of the nematode body volume vs. 7.5% in control nematodes, lipid droplets were on average 74% smaller and the number of tiny lipids (0-10 µm2) was increased. These results suggest that (1) Cu has an important effect on the life-history traits of nematodes; (2) the quantification of lipid storage can provide important information on the response of organisms to toxic stress; and (3) CARS microscopy is a promising tool for non-invasive quantitative and qualitative analyses of lipids as a measure of nematode fitness.

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We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver.

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Clinical translation of coherent anti-Stokes Raman scattering microscopy is of great interest because of the advantages of noninvasive label-free imaging, high sensitivity, and chemical specificity. For this to happen, we have identified and review the technical barriers that must be overcome. Prior investigations have developed advanced techniques (features), each of which can be used to effectively overcome one particular technical barrier. However, the implementation of one or a small number of these advanced features in previous attempts for clinical translation has often introduced more tradeoffs than benefits. In this review, we outline a strategy that would integrate multiple advanced features to overcome all the technical barriers simultaneously, effectively reduce tradeoffs, and synergistically optimize CARS microscopy for clinical translation. The operation of the envisioned system incorporates coherent Raman micro-spectroscopy for identifying vibrational biomolecular markers of disease and single-frequency (or hyperspectral) Raman imaging of these specific biomarkers for real-time in vivo diagnostics and monitoring.

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During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs.

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We present a label-free, chemically-selective, quantitative imaging strategy to identify breast cancer and differentiate its subtypes using coherent anti-Stokes Raman scattering (CARS) microscopy. Human normal breast tissue, benign proliferative, as well as in situ and invasive carcinomas, were imaged ex vivo. Simply by visualizing cellular and tissue features appearing on CARS images, cancerous lesions can be readily separated from normal tissue and benign proliferative lesion. To further distinguish cancer subtypes, quantitative disease-related features, describing the geometry and distribution of cancer cell nuclei, were extracted and applied to a computerized classification system. The results show that in situ carcinoma was successfully distinguished from invasive carcinoma, while invasive ductal carcinoma (IDC) and invasive lobular carcinoma were also distinguished from each other. Furthermore, 80% of intermediate-grade IDC and 85% of high-grade IDC were correctly distinguished from each other. The proposed quantitative CARS imaging method has the potential to enable rapid diagnosis of breast cancer.

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BACKGROUND: Multiple sclerosis (MS) is a chronic disabling disorder histopathologically characterized by inflammation, demyelination and axonal loss. Conventional MRI has made most contributions to the diagnosis of MS. However, it is not sufficiently sensitive and specific to reveal the extent and severity of the damage in the disease. Other nuclear magnetic resonance (NMR) techniques including magnetic resonance spectroscopy, magnetization transfer imaging, diffusion weighted and diffusion tensor imaging, and functional MRI have provided additional information that improves the diagnosis and understanding of MS. Optical techniques including optical coherence tomography (OCT) and coherent anti-Stokes Raman scattering (CARS) microscopy have shown promise in diagnosis and mechanistic study of myelin diseases. OBJECTIVE: To review new imaging techniques and their potential in diagnosis of MS. METHOD: The principles of three imaging techniques (MRI, OCT and CARS) and their applications to MS studies are described. Their advantages and disadvantages are compared. CONCLUSION: Conventional MRI remains a critical tool in the diagnosis of MS. Alternative NMR/MRI techniques have improved specificity for the detection of lesions and provided more quantitative information about MS. Optical techniques including OCT and CARS microscopy are opening up new ways for diagnosis and mechanistic study of myelin diseases.