Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

BACKGROUND AND AIMS: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. CONCLUSIONS: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

Host-device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling.

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.

Loss of MYO5B expression deregulates late endosome size which hinders mitotic spindle orientation.

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.

Pancreatic beta cell autophagy is impaired in type 1 diabetes.

AIMS/HYPOTHESIS: Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. METHODS: Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). RESULTS: We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3-LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3-LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002). CONCLUSIONS/INTERPRETATION: We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.

Correction to: Neodymium as an alternative contrast for uranium in electron microscopy.

After publication of our article, it has come to our attention that the legend.

Neodymium as an alternative contrast for uranium in electron microscopy.

Uranyl acetate is the standard contrasting agent in electron microscopy (EM), but it is toxic and radioactive. We reasoned neodymium acetate might substitute uranyl acetate as a contrasting agent, and we find that neodymium acetate indeed can replace uranyl acetate in several routine applications. Since neodymium acetate is not toxic, not radioactive and easy to use, we foresee neodymium will replace uranyl in many EM sample preparation applications worldwide.

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells.

AIMS/HYPOTHESIS: The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. METHODS: We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). RESULTS: ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. CONCLUSIONS/INTERPRETATION: The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

Supramolecular Packing and Macroscopic Alignment Controls Actuation Speed in Macroscopic Strings of Molecular Motor Amphiphiles.

Three-dimensional organized unidirectionally aligned and responsive supramolecular structures have much potential in adaptive materials ranging from biomedical components to soft actuator systems. However, to control the supramolecular structure of these stimuli responsive, for example photoactive, materials and control their actuation remains a major challenge. Toward the design of "artificial muscles", herein, we demonstrate an approach that allows hierarchical control of the supramolecular structure, and as a consequence its photoactuation function, by electrostatic interaction between motor amphiphiles (MA) and counterions. Detailed insight into the effect of various ions on structural parameters for self-assembly from nano- to micrometer scale in water including nanofiber formation and nanofiber aggregation as well as the packing structure, degree of alignment, and actuation speed of the macroscopic MA strings prepared from various metal chlorides solution, as determined by electronic microscopy, X-ray diffraction, and actuation speed measurements, is presented. Macroscopic MA strings prepared from calcium and magnesium ions provide a high degree of alignment and fast response photoactuation. By the selection of metal ions and chain length of MAs, the macroscopic MA string structure and function can be controlled, demonstrating the potential of generating multiple photoresponsive supramolecular systems from an identical molecular structure.

A small protein probe for correlated microscopy of endogenous proteins.

Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of components to change its target or color. A general FLIPPER-body targeting GFP highlights histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for other targets, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current approaches.

Guanine quadruplex structures localize to heterochromatin.

Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation.

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.

Normothermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor livers.

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non-DCD livers. DCD and non-DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma-glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP-preserved livers compared to SCS-preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2-fold higher in NMP-preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP-preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non-DCD and DCD livers, respectively) compared with SCS-preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non-DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP-preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994-1005 2016 AASLD.

Scanning EM of non-heavy metal stained biosamples: Large-field of view, high contrast and highly efficient immunolabeling.

Scanning electron microscopy (SEM) is increasing its application in life sciences for electron density measurements of ultrathin sections. These are traditionally analyzed with transmission electron microscopy (TEM); by most labs, SEM analysis still is associated with surface imaging only. Here we report several advantages of SEM for thin sections over TEM, both for structural inspection, as well as analyzing immuno-targeted labels such as quantum dots (QDs) and gold, where we find that QD-labeling is ten times more efficient than gold-labeling. Furthermore, we find that omitting post-staining with uranyl and lead leads to QDs readily detectable over the ultrastructure, but under these conditions ultrastructural contrast was even almost invisible in TEM examination. Importantly, imaging in SEM with STEM detection leads to both outstanding QDs and ultrastructural contrast. STEM imaging is superior over back-scattered electron imaging of these non-contrasted samples, whereas secondary electron detection cannot be used at all. We conclude that examination of ultrathin sections by SEM, which may be immunolabeled with QDs, will allow rapid and straightforward analysis of large fields with more efficient labeling than can be achieved with immunogold. The large fields of view routinely achieved with SEM, but not with TEM, allows straightforward raw data sharing using virtual microscopy, also known as nanotomy when this concerns EM data in the life sciences.

Absence of cell-surface EpCAM in congenital tufting enteropathy.

Mutations in the epithelial cell adhesion molecule (EpCAM; CD326) gene are causal for congenital tufting enteropathy (CTE), a disease characterized by intestinal abnormalities resulting in lethal diarrhea in newborns. Why the different mutations all lead to the same disease is not clear. Here, we report that most mutations, including a novel intronic variant, will result in lack of EpCAM's transmembrane domain, whereas two mutations allow transmembrane localization. We find that these mutants are not routed to the plasma membrane, and that truncated mutants are secreted or degraded. Thus, all epcam mutations lead to loss of cell-surface EpCAM, resulting in CTE.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Identification of stem cells in small intestine and colon by marker gene Lgr5.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Here, using two knock-in alleles, we reveal exclusive expression of Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.

Brilliant camouflage: photonic crystals in the diamond weevil, Entimus imperialis.

The neotropical diamond weevil, Entimus imperialis, is marked by rows of brilliant spots on the overall black elytra. The spots are concave pits with intricate patterns of structural-coloured scales, consisting of large domains of three-dimensional photonic crystals that have a diamond-type structure. Reflectance spectra measured from individual scale domains perfectly match model spectra, calculated with anatomical data and finite-difference time-domain methods. The reflections of single domains are extremely directional (observed with a point source less than 5°), but the special arrangement of the scales in the concave pits significantly broadens the angular distribution of the reflections. The resulting virtually angle-independent green coloration of the weevil closely approximates the colour of a foliaceous background. While the close-distance colourful shininess of E. imperialis may facilitate intersexual recognition, the diffuse green reflectance of the elytra when seen at long-distance provides cryptic camouflage.

Activation of a passive, mesoporous silica nanoparticle layer through attachment of bacterially-derived carbon-quantum-dots for protection and functional enhancement of probiotics.

Probiotic bacteria employed for food supplementation or probiotic-assisted antibiotic treatment suffer from passage through the acidic gastro-intestinal tract and unintended killing by antibiotics. Carbon-quantum-dots (CQDs) derived from bacteria can inherit different chemical groups and associated functionalities from their source bacteria. In order to yield simultaneous, passive protection and enhanced, active functionality, we attached CQDs pyrolytically carbonized at 220 â€‹°C from Lactobacillus acidophilus or Escherichia coli to a probiotic strain (Bifidobacterium infantis) using boron hydroxyl-modified, mesoporous silica nanoparticles as an intermediate encapsulating layer. Fourier-transform-infrared-spectroscopy, X-ray-photoelectron-spectroscopy and scanning-electron-microscopy were employed to demonstrate successful encapsulation of B. infantis by silica nanoparticles and subsequent attachment of bacterially-derived CQDs. Thus encapsulated B. infantis possessed a negative surface charge and survived exposure to simulated gastric fluid and antibiotics better than unencapsulated B. infantis. During B. infantis assisted antibiotic treatment of intestinal epithelial layers colonized by E. coli, encapsulated B. infantis adhered and survived in higher numbers on epithelial layers than B. infantis without encapsulation or encapsulated with only silica nanoparticles. Moreover, higher E. coli killing due to increased reactive-oxygen-species generation was observed. In conclusion, the active, protective encapsulation described enhanced the probiotic functionality of B. infantis, which might be considered as a first step towards a fully engineered, probiotic nanoparticle.