Changes in the Cell Wall Proteome of Leaves in Response to High Temperature Stress in Brachypodium distachyon

High temperature stress leads to complex changes to plant functionality, which affects, i.a., the cell wall structure and the cell wall protein composition. In this study, the qualitative and quantitative changes in the cell wall proteome of Brachypodium distachyon leaves in response to high (40 °C) temperature stress were characterised. Using a proteomic analysis, 1533 non-redundant proteins were identified from which 338 cell wall proteins were distinguished. At a high temperature, we identified 46 differentially abundant proteins, and of these, 4 were over-accumulated and 42 were under-accumulated. The most significant changes were observed in the proteins acting on the cell wall polysaccharides, specifically, 2 over- and 12 under-accumulated proteins. Based on the qualitative analysis, one cell wall protein was identified that was uniquely present at 40 °C but was absent in the control and 24 proteins that were present in the control but were absent at 40 °C. Overall, the changes in the cell wall proteome at 40 °C suggest a lower protease activity, lignification and an expansion of the cell wall. These results offer a new insight into the changes in the cell wall proteome in response to high temperature.     

Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis

Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.     

Synchronized Cell Cycle Arrest Promotes Osteoclast Differentiation

Osteoclast progenitors undergo cell cycle arrest before differentiation into osteoclasts, induced by exposure to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The role of such cell cycle arrest in osteoclast differentiation has remained unclear, however. We here examined the effect of synchronized cell cycle arrest on osteoclast formation. Osteoclast progenitors deprived of M-CSF in culture adopted a uniform morphology and exhibited cell cycle arrest at the G₀–G₁ phase in association with both down-regulation of cyclins A and D1 as well as up-regulation of the cyclin-dependent kinase inhibitor p27^Kip1. Such M-CSF deprivation also promoted the differentiation of osteoclast progenitors into multinucleated osteoclasts expressing high levels of osteoclast marker proteins such as NFATc1, c-Fos, Atp6v0d2, cathepsin K, and integrin β3 on subsequent exposure to M-CSF and RANKL. Our results suggest that synchronized arrest and reprogramming of osteoclast progenitors renders them poised to respond to inducers of osteoclast formation. Further characterization of such effects may facilitate induction of the differentiation of heterogeneous and multipotent cells into desired cell lineages.     

The Role of Insulin-like Peptide in Maintaining Hemolymph Glucose Homeostasis in the Pacific White Shrimp Litopenaeus vannamei

Insulin-like peptide (ILP) has been identified in various crustaceans, but whether it has a similar function in regulating hemolymph glucose as vertebrate insulin is unclear. We analyzed the components of hemolymph sugar in the Pacific white shrimp, Litopenaeus vannamei, and investigated the changes of hemolymph glucose concentration and the expressions of ILP and glucose metabolism genes under different treatments. We found glucose was a major component of hemolymph sugar in shrimp. Starvation caused hemolymph glucose to rise first and then decline, and the raised hemolymph glucose after exogenous glucose injection returned to basal levels within a short time, indicating that shrimp have a regulatory mechanism to maintain hemolymph glucose homeostasis. In addition, injections of bovine insulin and recombinant LvILP protein both resulted in a fast decline in hemolymph glucose. Notably, RNA interference of LvILP did not significantly affect hemolymph glucose levels, but it inhibited exogenous glucose clearance. Based on the detection of glucose metabolism genes, we found LvILP might maintain hemolymph glucose stability by regulating the expression of these genes. These results suggest that ILP has a conserved function in shrimp similar to insulin in vertebrates and plays an important role in maintaining hemolymph glucose homeostasis.     

Stem Cells behind the Barrier

Epidermal stem cells sustain the adult skin for a lifetime through self-renewal and the production of committed progenitors. These stem cells generate progeny that will undergo terminal differentiation leading to the development of a protective epidermal barrier. Whereas the molecular mechanisms that govern epidermal barrier repair and renewal have been extensively studied, pathways controlling stem cell differentiation remain poorly understood. Asymmetric cell divisions, small non-coding RNAs (microRNAs), chromatin remodeling complexes, and multiple differentiation factors tightly control the balance of stem and progenitor cell proliferation and differentiation, and disruption of this balance leads to skin diseases. In this review, we summarize and discuss current advances in our understanding of the mechanisms regulating epidermal stem and progenitor cell differentiation, and explore new relationships for maintenance of skin barrier function.     

IKKα Induces Epithelial–Mesenchymal Changes in Mouse Skin Carcinoma Cells That Can Be Partially Reversed by Apigenin

NMSC (non-melanoma skin cancer) is a common tumor in the Caucasian population, accounting for 90% of skin cancers. Among them, squamous cell carcinomas (SCCs) can metastasize and, due to its high incidence, constitute a severe health problem. It has been suggested that cutaneous SCCs with more risk to metastasize express high levels of nuclear IKKα. However, the molecular mechanisms that lead to this enhanced aggressiveness are largely unknown. To understand in depth the influence of nuclear IKKα in skin SCC progression, we have generated murine PDVC57 skin carcinoma cells expressing exogenous IKKα either in the nucleus or in the cytoplasm to further distinguish the tumor properties of IKKα in both localizations. Our results show that IKKα promotes changes in both subcellular compartments, resembling EMT (epithelial–mesenchymal transition), which are more pronounced when IKKα is in the nucleus of these tumor cells. These EMT-related changes include a shift toward a migratory phenotype and induction of the expression of proteins involved in cell matrix degradation, cell survival and resistance to apoptosis. Additionally, we have found that apigenin, a flavonoid with anti-cancer properties, inhibits the expression of IKKα and attenuates most of the pro-tumoral EMT changes induced by IKKα in mouse tumor keratinocytes. Nevertheless, we have found that apigenin only inhibits the expression of the IKKα protein when it is localized in the cytoplasm.     

New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW) on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State

It is known that increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW) produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H₂O₂-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H₂O₂ on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation.     

Exogenous Sphingosine 1-Phosphate Protects Murine Splenocytes Against Hypoxia-Induced Injury

Sphingosine-1-phosphate (S1P), a biologically active pleiotropic lipid, is involved in several physiological processes especially in the area of vascular biology and immunology encompassing cell survival, angiogenesis, vascular tone, immune response etc. by interacting with specific cell surface receptors. Hypoxia, a condition common to innumerable pathologies, is known to lethally affect cell survival by throwing off balance global gene expression, redox homeostasis, bioenergetics etc. Several molecular events of cellular adaptations to hypoxia have been closely linked to stabilization of hypoxia inducible factor-1α (HIF-1α). Signalling functions of S1P in physiological events central to hypoxia-induced pathologies led us to investigate efficacy of exogenous S1P in preconditioning murine splenocytes to sustain during cellular stress associated with sub-optimal oxygen. The present study recapitulated the pro-survival benefits of exogenous S1P under normobaric hypoxia. Results indicate a direct effect of S1P supplementation on boosting cellular adaptive responses via HIF-1α stabilization and, activation of pro-survival mediators ERK and Akt. Overwhelming anti-oxidative and anti-inflammatory benefits of S1P preconditioning could also be captured in the present study, as indicated by improved redox homeostasis, reduced oxidative damage, balanced anti/pro-inflammatory cytokine profiles and temporal regulation of nitric oxide secretion and intra-cellular calcium release. Hypoxia induced cell death and the associated stress in cellular milieu in terms of oxidative damage and inflammation could be alleviated with exogenous S1P preconditioning.     

High Glucose Exposure Impairs L-Cell Differentiation in Intestinal Organoids: Molecular Mechanisms and Clinical Implications

Intestinal organoids are used to analyze the differentiation of enteroendocrine cells (EECs) and to manipulate their density for treating type 2 diabetes. EEC differentiation is a continuous process tightly regulated in the gut by a complex regulatory network. However, the effect of chronic hyperglycemia, in the modulation of regulatory networks controlling identity and differentiation of EECs, has not been analyzed. This study aimed to investigate the effect of glucotoxicity on EEC differentiation in small intestinal organoid platforms. Mouse intestinal organoids were cultured in the presence/absence of high glucose concentrations (35 mM) for 48 h to mimic glucotoxicity. Chronic hyperglycemia impaired the expression of markers related to the differentiation of EEC progenitors (Ngn3) and L-cells (NeuroD1), and it also reduced the expression of Gcg and GLP-1 positive cell number. In addition, the expression of intestinal stem cell markers was reduced in organoids exposed to high glucose concentrations. Our data indicate that glucotoxicity impairs L-cell differentiation, which could be associated with decreased intestinal stem cell proliferative capacity. This study provides the identification of new targets involved in new molecular signaling mechanisms impaired by glucotoxicity that could be a useful tool for the treatment of type 2 diabetes.     

Electron-transfer coupling in microbial fuel cells. 2. performance of fuel cells containing selected microorganism—mediator—substrate combinations

Various phenoxazine, phenothiazine, phenazine, indophenol and bipyridilium derivatives were tested for their effectiveness as redox mediators in microbial fuel cells containing Alcaligenes eutrophus, Bacillus subtilis, Escherichia coli, or Proteus vulgaris as the active biological agent, and glucose or succinate as the oxidisable substrate. A ferricyanide-Pt cathode was used. The open-circuit cell e.m.f.′s increased in the order of increasing negative formal redox potentials at pH 7(E⁷_m) of the redox compounds. Several of the redox agents worked well as mediators, maintaining steady currents over several hours, and thionine was found to be particularly effective in maintaining relatively high cell voltages when current was drawn from the cell. A number of the compounds tested did not function well, either because they were incompletely or slowly reduced by the microorganisms or because of their instability. P. vulgaris, with thionine as mediator and glucose as substrate, showed the best performance in a fuel cell. This system was examined in some detail under various conditions of external load to establish the effects of organism concentration, mediator concentration, and substrate addition. Coulombic outputs from these cells were calculated by integration of the current-time plots. Coulombic yields of 30–60% were obtained, on the basis of (theoretical) complete oxidation of added substrate to CO₂ and water.     

Regulation of Neural Stem Cell Competency and Commitment during Indirect Neurogenesis

Indirect neurogenesis, during which neural stem cells generate neurons through intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. The mechanisms that specify intermediate progenitor identity and that regulate stem cell competency to generate intermediate progenitors remain poorly understood despite their roles in indirect neurogenesis. Well-characterized lineage hierarchy and available powerful genetic tools for manipulating gene functions make fruit fly neural stem cell (neuroblast) lineages an excellent in vivo paradigm for investigating the mechanisms that regulate neurogenesis. Type II neuroblasts in fly larval brains repeatedly undergo asymmetric divisions to generate intermediate neural progenitors (INPs) that undergo limited proliferation to increase the number of neurons generated per stem cell division. Here, we review key regulatory genes and the mechanisms by which they promote the specification and generation of INPs, safeguarding the indirect generation of neurons during fly larval brain neurogenesis. Homologs of these regulators of INPs have been shown to play important roles in regulating brain development in vertebrates. Insight into the precise regulation of intermediate progenitors will likely improve our understanding of the control of indirect neurogenesis during brain development and brain evolution.     

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.     

Islet Regeneration: Endogenous and Exogenous Approaches

Both type 1 and type 2 diabetes are characterized by a progressive loss of beta cell mass that contributes to impaired glucose homeostasis. Although an optimal treatment option would be to simply replace the lost cells, it is now well established that unlike many other organs, the adult pancreas has limited regenerative potential. For this reason, significant research efforts are focusing on methods to induce beta cell proliferation (replication of existing beta cells), promote beta cell formation from alternative endogenous cell sources (neogenesis), and/or generate beta cells from pluripotent stem cells. In this article, we will review (i) endogenous mechanisms of beta cell regeneration during steady state, stress and disease; (ii) efforts to stimulate endogenous regeneration and transdifferentiation; and (iii) exogenous methods of beta cell generation and transplantation.     

Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice

Basal cell carcinoma (BCC) originate from Hedgehog/Patched signaling-activated epidermal stem cells. However, the chemically induced tumorigenesis of mice with a CD4Cre-mediated biallelic loss of the Hedgehog signaling repressor Patched also induces BCC formation. Here, we identified the cellular origin of CD4Cre-targeted BCC progenitors as rare Keratin 5⁺ epidermal cells and show that wildtype Patched offspring of these cells spread over the hair follicle/skin complex with increasing mouse age. Intriguingly, Patched mutant counterparts are undetectable in age-matched untreated skin but are getting traceable upon applying the chemical tumorigenesis protocol. Together, our data show that biallelic Patched depletion in rare Keratin 5⁺ epidermal cells is not sufficient to drive BCC development, because the spread of these cells is physiologically suppressed. However, bypassing the repression of Patched mutant cells, e.g., by exogenous stimuli, leads to an accumulation of BCC precursor cells and, finally, to tumor development.     

Surfaceome Profiling Identifies Basigin-Chaperoned Protein Clients

The surface proteome or “surfaceome” is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.     

Toxicity Induced by Cytokines,Glucose, and Lipids Increase Apoptosis and Hamper Insulin Secretion in the 1.1E7 Beta Cell-Line

Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2–5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.     

A laccase-glucose oxidase biofuel cell prototype operating in a physiological buffer

Here we report on the design and study of a biofuel cell consisting of a glucose oxidase-based anode (Aspergillus niger) and a laccase-based cathode (Trametes versicolor) using osmium-based redox polymers as mediators of the biocatalysts’ electron transfer at graphite electrode surfaces. The graphite electrodes of the device are modified with the deposition and immobilization of the appropriate enzyme and the osmium redox polymer mediator. A redox polymer [Os(4,4′-diamino-2,2′bipyridine)₂(poly{N-vinylimidazole})-(poly{N-vinylimidazole})₉Cl]Cl (E⁰′ = −0.110 V versus Ag/AgCl) of moderately low redox potential is used for the glucose oxidizing anode and a redox polymer [Os(phenanthroline)₂(poly{N-vinylimidazole})₂-(poly{N-vinylimidazole})₈]Cl₂ (E⁰′ = 0.49 V versus Ag/AgCl) of moderately high redox potential is used at the dioxygen reducing cathode. The enzyme and redox polymer are cross-linked with polyoxyethylene bis(glycidyl ether). The working biofuel cell was studied under air at 37 °C in a 0.1 M phosphate buffer solution of pH range 4.4-7.4, containing 0.1 M sodium chloride and 10 mM glucose. Under physiological conditions (pH 7.4) maximum power density, evaluated from the geometric area of the electrode, reached 16 μW/cm² at a cell voltage of 0.25 V. At lower pH values maximum power density was 40 μW/cm² at 0.4 V (pH 5.5) and 10 μW/cm² at 0.3 V (pH 4.4).