Abstract Background The pro-hormone chromogranin A (CgA) and its bioactive cleavage product catestatin (CST) are both associated with inflammatory bowel disease (IBD) and dysregulated barrier functions, but their exact role has remained elusive. Here, we demonstrate that CST regulates the colonic mucus layer. Methods CST levels were measured in feces of IBD patients. The mucus layer, goblet cells, and immune cell infiltration were analyzed by histology and electron microscopy in colon tissue from IBD patients and mice with selective deletion of the CST-coding region of the CgA gene. Results CST levels were elevated in feces of IBD patients compared to healthy controls. The thickness of the mucus layer was increased in non-affected, but not in inflamed, regions of the colon in IBD patients. The thickness of the mucus layer and concomitant mucus production were also increased in the CST-KO mouse. This mucus phenotype in CST-KO mice could be reversed by bone marrow transplantation from wildtype mice. Conclusions CST produced by bone-marrow derived immune cells reduces production of the mucus layer in the intestine. This might contribute to the reduced mucus layer in inflamed colon regions of IBD patients. Additionally, CST feces levels might be a biomarker for IBD.
Animals, plants, and many other organisms are highly complex biological systems, the functions as well as the underlying cellular processes of which can be best studied in vivo only using non-invasive techniques. Among these crucial tools, fluorescence microspectroscopy allows visualizing biological processes in these complex systems with great spatiotemporal resolution. Fluorescence microspectroscopy is the application of fluorescence spectroscopy on microscopic objects, which opens the door to functional imaging approaches. In the past decades, technical developments in the field of fluorescence microscopy have accelerated the application of functional imaging tools, resulting in a plethora of very sensitive imaging platforms. The opportunity to simultaneously monitor multiple fluorophores greatly improved image deconvolution procedures and now enables the detection of very small structures containing fluorescently tagged endogenous proteins in their natural context. The introduction of pulsed laser systems has paved the way for the use of fluorescence lifetime imaging (FLIM) for visualizing and quantifying protein–protein interactions in vivo. These developments are incorporated into many cutting-edge imaging platforms and are also made available for non-expert microscopists. Functional imaging applications encompass spatiotemporal imaging of many fluorophores using confocal or multiphoton microscopes, quantification of dynamic processes with the use of fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP), visualization of (macro)molecular complexes with Förster resonance energy transfer (FRET) and quantification thereof by FLIM. Since 2006, the imaging portfolio has expanded even further with the introduction of imaging devices that can break the diffraction limit of light, the so-called super-resolution microscopes. Imaging biological structures with a resolution down to ±30 nm is now possible by, for example, photoactivated localization microscopy, stimulated emission depletion, stochastic optical reconstruction microscopy or correlated light and electron microscopy (CLEM). This ‘Focus on' entitled ‘Microspectroscopy: functional imaging of biological systems' is coupled with the eponymous FEBS Practical Course, which will take place in Wageningen in September 2022. The course was conceived by the late Prof Antonie Visser and by Jan Willem Borst two decades ago. Since 2010, the course format has changed to cover functional imaging approaches used not only in plant sciences but also in (bio)medical research, the latter thanks to the contribution of Jack Fransen. Together with S. Weidtkamp-Peters and Y. Stahl from Düsseldorf, who joined the organizing team in 2016, we have been able to put together a 9-day program that highlights several state-of-the-art functional imaging modes. The course combines lectures by experts in the field in the morning with practical hands-on sessions in the afternoon, which makes the learning very efficient and allows participants to directly translate the theory into practice. Participants aim at becoming familiar with sensitive techniques and methods to image biochemical processes in living cells. To this end, microspectroscopic techniques are the method of choice as they can provide direct information on molecular interactions and dynamic events involving biomolecules with minimal perturbation of cellular integrity and function. Thus, the wealth of information and resources generated by efforts in genomics can be directly translated into understanding of the inner workings of both animal and plant cells and tissues in vivo. This can be achieved by using multimodal microscope systems and techniques and by implementing novel contrast schemes to monitor and quantify bio-molecular interactions and states in living cells in a multidimensional manner. Studying cellular biochemistry with these imaging tools results in time- and spectrally resolved data and their correlations over the temporal, spatial, and spectral dimensions. This practical course covers several microscopic and spectroscopic techniques to study molecular processes in living cells: (multiphoton) confocal microscopy, FRET-FLIM, ratio-imaging microscopy, total internal reflection fluorescence microscopy, FCS, and single-molecule detected fluorescence. Recent developments in super-resolution and correlative microscopy are also addressed during the course as well as the application of fluorescent bioprobes, which are crucial for cell-biology studies. Among them are novel (switchable) probes (with increased photostability and/or exploiting different photo-excited states), novel genetically encoded probes (such as visible fluorescent proteins, FlAsH-related tags, SNAP- and Halo-tag for super-resolution applications, and specific single-chain antibodies), functionalized magnetic beads, quantum dots and other increasingly used nanoparticles. Together, these microspectroscopic techniques make it now cellular biochemistry possible. Furthermore, the course provides an introduction to image analysis, to open-source image analysis tools, and to the analysis of spectroscopic data. This ‘Focus On' consists of three publications that showcase state-of-the-art imaging approaches and image analysis tools. The group of Ben Giepmans discusses the potential of larval zebrafish pancreas as a simple microscopy-friendly model system for studying the islets of Langerhans and type 1 diabetes. The beauty of zebrafish larvae is the possibility of exploring the fundamentals of organ development and malfunction in vivo. This model system also allows easy genetic modifications ranging from transient knockdowns to transgenic models or the integration of genetically encoded fluorescent proteins in knock-in models [[1]]. The review also illustrates two microscopic approaches, confocal microscopy, and light sheet imaging, useful to visualize the subcellular localization of specific fluorescent markers. The authors show that the zebrafish larvae are an extremely powerful tool for translational research and that they may also serve as a useful alternative to cell line–based studies. The wealth of different imaging systems is accompanied by an increasing number of software packages for the analysis of acquired images. Haase et al. [[2]] have summarized a long list of currently available software tools for image analysis. image j (or fiji) applications are discussed and tools to quantify the number of cells, and many other parameters are described. This comprehensive compilation of software for image analyses includes tips for choosing the best approach to analyze the results of any given type of imaging experiments, which, given the more and more increasing complexity of the acquired imaging data, is no trivial task. Another fast-advancing and highly relevant area in microscopy is the combination of electron microscopy and fluorescence microscopy, the so-called correlative imaging. Here, the gap between in vivo light microscopy and electron microscopy is closed. Cambi and co-workers present a historical overview of CLEM combined with fluorescence microscopy [[3]]. In particular, they describe how the engineering of fluorescent proteins and the development of super-resolution fluorescence microscopy have significantly renewed the interest in CLEM resulting in the present application of fluorescence CLEM in many different areas of cellular and molecular biology. In summary, this ‘Focus on’ provides FEBS Letters readers and the participants in the FEBS Practical Course ‘Microspectroscopy: functional imaging of biological systems' with an overview of some of the most important state-of-the-art microspectroscopy techniques and germane analysis tools. Jan Willem Borst is Assistant Professor in the Laboratory of Biochemistry at Wageningen University and Research, in Wageningen, The Netherlands. Furthermore, he has been involved in the Microspectroscopy Research Facility at Wageningen University since 1996. Jan Willem has a long-standing experience in the application of fluorescence spectroscopic methods to study protein localization, dynamics, and interactions in biological systems. Currently, research in the Borst lab focuses on the nuclear auxin signaling network in the liverwort Marchantia polymorpha and applies biochemical and fluorescence microspectroscopic approaches to understand the function of the auxin response and protein phase separation in vivo. Jack Fransen is Associate Professor at the Cell Biology Department of the Radboud University Medical Center in Nijmegen, The Netherlands. His background is in cell biology/electron microscopy. His group worked on intracellular transport in polarized cells focussing on the function of Rab proteins in the regulation of vesicular traffic at the Golgi and post-Golgi level using immunoelectron microscopical and fluorescence techniques. Later, the topic was switched to imaging in space and time of proteins and small molecules involved in localized energy supply in cells. In 2008, the group started to implement 3D (LM and EM) and 4D imaging within the department and established a microscopy facility at RadboudUMC. He has become the Operational Manager and is in-charge of the daily supervision of equipment and technologies within this RadboudUMC Technology Center Microscopy. Stefanie Weidtkamp-Peters is Adjunct Professor and Head of the Center for Advanced Imaging at Heinrich Heine University Düsseldorf, Germany. Since March 2021 she is the chair of GermanBioImaging, the German Society for Microscopy and Image analysis. She is a cell biologist by training and earned her PhD at the Fritz-Lipmann Institute of Age Research in Jena for applying advanced imaging methods like FRAP and FCS to unravel the dynamic behavior of proteins within the mammalian cell nucleus. As a postdoctoral research fellow at Heinrich Heine University Düsseldorf she used fluorescence spectroscopy and FRET to study protein interactions and oligomerization in living cells. Since 2015, she is engaged in establishing information infrastructure for FAIR image data management at both local and international levels. Yvonne Stahl is Adjunct Professor in the Institute for Developmental Genetics at the Heinrich-Heine University in Düsseldorf, Germany. She finished her PhD at Heriot-Watt University, Edinburgh, UK, in 2003 about barley starch synthesis enzymes. Thereafter, she worked as a Research Fellow and as an Academic Senior Councilor at the Institute for Genetics at the Heinrich Heine University, Düsseldorf, Germany, on root stem cell maintenance in Arabidopsis thaliana. She habilitated in developmental genetics in 2016 and became adjunct professor in 2021. Her current research focusses on root development and architecture in Arabidopsis thaliana and barley. She uses genetics and in vivo fluorescence spectroscopic methods to investigate how subcellular localizations, dynamic interactions and differential complexes of information molecules regulate plant root stem cell homeostasis.
The BRAF-MDQ1,2 and RAID3 are patient-reported outcome measures derived from the patient perspective. The 20 item BRAF-MDQ has 4 factors (physical, cognitive, emotional fatigue, living with fatigue) and the 7 item RAID is uni-dimensional. The BRAF-MDQ properties have not been tested outside the UK, nor the validity of the new Swedish RAID examined.
Objectives
To test the structure, construct and criterion validity of the BRAF-MDQ and the RAID in 6 EU countries.
Methods
Survey of RA clinic patients in clinics in France, Germany, Netherlands, Spain, Sweden and UK (F, G, N, Sp, Sw, UK), using BRAF-MDQ, RAID and SF-36. Factor structure examined by Confirmatory Factor Analysis and internal consistency by Cronbach9s Alpha. Criterion validity for the BRAF-MDQ evaluated by internal Spearman9s correlations, and for BRAF-MDQ and RAID fatigue item by correlation with each other and SF-36 Vitality subscale. Construct validity of BRAF-MDQ and RAID evaluated by Spearman9s Correlation with each other, HAQ, and SF-36 domains. Analysis conducted overall and by individual country and for BRAF-MDQ by total and subscales.
Results
1276 patients participated: F 206, G 216, N 317, Sp 157, Sw 170, UK 210. Disease duration <5 yrs for 25%, 76% female, median HAQ 1.0 (IQR 0.375–1.5). Mean total BRAF-MDQ was 26/70 (SD 26) with subscales physical fatigue 11.38/22 (SD 5.77), living with fatigue 6.46/21 (SD 5.4), cognitive 4.28/15 (SD 8.7) and emotional fatigue 3.41/12 (SD 3.22). Mean RAID was 4.15/10 (SD 2.31). Confirmatory factor analysis: BRAF-MDQ original UK 4 factor structure was confirmed in each country, demonstrating one factor for each set of variables, with high factor loadings (0.71–0.96). Bootstrapping (4 sets of analyses on 20 random samples of 50% of patients/country), found the structure held true in at least 19/20 sets per country. RAID single factor structure was confirmed in all countries (0.75–0.95). Internal consistency ranged from 0.75–0.96 for the BRAF-MDQ (total and subscales) and the RAID ranged from 0.93–0.96 (overall and by country). Criterion validity overall and by country: The BRAF-MDQ correlated internally and with the SF-36 Vitality and RAID fatigue items (0.6–0.93). Construct validity: BRAF-MDQ and RAID correlatied with each other, the HAQ, and remaining 8 SF36 domains (0.46–0.86).
Conclusions
The BRAF-MDQ and RAID demonstrate strong, consistent factor structure and internal consistency, with moderate-good criterion and construct validity across 6 EU countries, reflecting their robust methods of development. This indicates no specific country scoring is necessary, and strengthens the case for measuring both fatigue and impact in multi-country studies.
References
Nicklin et al, Arth Care Res 2010:62:1552–8. Nicklin et al, Arth Care Res 2010:62:1559–6. Gossec et al, Ann Rheum Dis 2009;68:1680–5
Animal and clinical studies have shown respiratory muscle dysfunction caused by treatment with glucocorticoids. The present study was designed to investigate whether anabolic steroids are able to antagonize the loss of diaphragm force induced by long-term low-dose methylprednisolone (MP) administration. Male adult rats were randomized to receive saline or MP (0.2 mg . kg-1 .day-1 sc) during 9 mo, with or without nandrolone decanoate (ND; 1 mg . kg-1 . wm -1 im) during the last 3 mo. The approximately 10% reduction in force generation of isolated diaphragm bundles induced by MP was completely abolished by addition of ND. The MP-induced decrease in number of fibers expressing type IIb myosin heavy chains was not reversed by ND. MP slightly reduced type I, IIa, and IIx fiber cross-sectional areas (CSA), but not type IIb fiber CSA. Addition of ND abolished the reduction in IIa and IIx fiber CSA. The MP-induced alterations in glycogenolytic activity and fatty acid oxidation capacity were not reversed by ND. In conclusion, the marked reduction in diaphragm force caused by long-term low-dose MP was completely abolished by addition of ND. ND in part also antagonized the effects of MP on diaphragm morphology but showed no beneficial effects on biochemical changes.
Systemic sclerosis (SSc) patients are at risk for organ involvement and premature death. The occurrence of organ involvement that is reported differs widely between various long term cohort studies; ILD 25–90%, PAH 8–32%, CI 5–30%, and SRC 4–12%. Differences in findings also apply to survival, the 5- and 10-year survival rates between studies vary from 80% to 90% and from 60% to 85% respectively (1–3).
Objectives
To assess the occurrence of organ involvement and death in a large, unselected cohort of Dutch SSc patients at the moment of diagnosis and during 5 years of follow-up, stratified by disease subtype and auto-antibodies.
Methods
Up to 2015, 690 SSc patients were included in the Nijmegen SSc cohort. Occurrence of interstitial lung disease (ILD), pulmonary arterial hypertension (PAH), cardiac involvement (CI), scleroderma renal crisis (SRC) and occurrence of death were determined using survival analysis, stratified by disease subtype (limited cutaneous SSc and diffuse cutaneous SSc) and auto-antibodies (ACA, ATA, anti-RNP).
Results
Organ involvement was already present at SSc diagnosis in 32% of patients. In 25%, organ involvement developed during follow-up, mostly ILD (22%). Significant differences between lcSSc and dcSSc were found in SRC at baseline and ILD, PAH and SRC during follow-up. Between the autoantibody subgroups, the occurrence of ILD, PAH and SRC at baseline and ILD during follow-up differed. There were no differences in survival between subtypes and auto-antibodies. The overall 5-year survival rate was 89%. Patients without organ involvement at SSc diagnosis had a better 5-year survival rate than patients with organ involvement at SSc diagnosis: 95% versus 73% respectively (p<0.001). (figure 1)
Conclusions
In many SSc patients, organ involvement is already present at diagnosis or develops in the first 5 years after diagnosis. Survival is significantly worse in patients who already have involvement at the moment of SSc diagnosis.
References
Muangchan C, Canadian Scleroderma Research G, Baron M, Pope J. The 15% rule in scleroderma: the frequency of severe organ complications in systemic sclerosis. A systematic review. The Journal of rheumatology. 2013;40(9):1545–56. Vonk MC, Broers BM, Heijdra YF, Ton E, Snijders R, van Dijk AP, et al. Systemic sclerosis and its pulmonary complications in the Netherlands An epidemiological study. AnnRheumDis. 2008. Nihtyanova SI, Schreiber BE, Ong VH, Rosenberg D, Moinzadeh P, Coghlan JG, et al. Prediction of pulmonary complications and long-term survival in systemic sclerosis. Arthritis & rheumatology. 2014;66(6):1625–35.
Protein tyrosine phosphatases (PTPs) are central players in many different cellular processes and their aberrant activity is associated with multiple human pathologies. In this review, we present current knowledge on the PTPRR subfamily of classical PTPs that is expressed in neuronal cells and comprises receptor-type (PTPBR7, PTP-SL) as well as cytosolic (PTPPBSgamma-37, PTPPBSgamma-42) isoforms. The two receptor-type isoforms PTPBR7 and PTP-SL both localize in late endosomes and the Golgi area. PTPBR7, however, is additionally localized at the cell surface and on early endosomes. During cerebellar maturation, PTPBR7 expression in developing Purkinje cells ceases and is replaced by PTP-SL expression in the mature Purkinje cells. All PTPRR isoforms contain a kinase interacting motif that makes them mitogen-activated protein kinase phosphatases. The distinct subcellular localization of the different PTPRR isoforms may reflect differential roles in growth-factor-induced MAPK-mediated retrograde signaling cascades. Studies in PTPRR-deficient mice established that PTPRR isoforms are physiological regulators of MAPK phosphorylation levels. Surprisingly, PTPRR-deficient mice display defects in motor coordination and balancing skills, while cerebellar morphological abnormalities, which are often encountered in ataxic mouse models, are absent. This is reminiscent of the phenotype observed in a handful of mouse mutants that have alterations in cerebellar calcium ion homeostasis. Elucidation of the molecular mechanisms by which PTPRR deficiency imposes impairment of cerebellar neurons and motor coordination may provide candidate molecules for hereditary cerebellar ataxias that still await identification of the corresponding disease genes.
Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple protein isoforms carrying distinct C termini. Here, we demonstrate by expressing individual DMPKs in various cell types, including C2C12 and DMPK−/− myoblast cells, that unique sequence arrangements in these tails control the specificity of anchoring into intracellular membranes. Mouse DMPK A and C were found to associate specifically with either the endoplasmic reticulum (ER) or the mitochondrial outer membrane, whereas the corresponding human DMPK A and C proteins both localized to mitochondria. Expression of mouse and human DMPK A—but not C—isoforms in mammalian cells caused clustering of ER or mitochondria. Membrane association of DMPK isoforms was resistant to alkaline conditions, and mutagenesis analysis showed that proper anchoring was differentially dependent on basic residues flanking putative transmembrane domains, demonstrating that DMPK tails form unique tail anchors. This work identifies DMPK as the first kinase in the class of tail-anchored proteins, with a possible role in organelle distribution and dynamics.
