Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

Faecal microbiota and fatty acids in feline chronic enteropathy.

BACKGROUND: Feline chronic enteropathy is a set of disorders defined as the presence of clinical signs of gastrointestinal disease for at least three weeks. The most common final diagnoses are inflammatory bowel disease and alimentary small cell lymphoma. The etiopathogenesis of these diseases is incompletely understood; however, it is hypothesised that they involve a combination of factors, including altered composition and/or functionality of the intestinal microbiome. An important factor in the interplay of the microbiome and host is the production of short- and branched-chain fatty acids.  The aim of this study was to evaluate the possible differences in faecal microbiota diversity, composition and fatty acid production between cats suffering from chronic enteropathy and healthy cats. Sixteen cats suffering from chronic enteropathy and fourteen healthy control cats were enrolled in the study. The microbiota compositions of faecal samples were analysed by using next-generation amplicon sequencing of the V3V4 fragment of the 16S rRNA gene. Fatty acids were evaluated by high-performance liquid chromatography. RESULTS: Both the alpha and beta diversities were significantly lower in samples obtained from cats with chronic enteropathy. The relative abundance of the phylum Proteobacteria, orders Lactobacillales and Enterobacterales, family Enteriobacteriaceae and genus Escherichia Shigella were higher in diseased cats, whereas the abundance of the phylum Bacteroidota and order Peptococcales were higher in control cats. The faecal concentrations of short-chain fatty acids were higher in cats with chronic enteropathy, with lower propionate proportions and higher butyrate proportions. CONCLUSION: The study revealed alterations in microbiota compositions and short-chain fatty acid concentration in cats suffering from chronic enteropathy, which is an important finding both for research on the pathogenesis of the disease and for potential therapeutic interventions in the form of faecal microbiota transplantation and/or probiotic supplementation.

Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.

Insulin-producing ß cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between ß cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Multifunctionality of Nanosized Calcium Apatite Dual-Doped with Li+/Eu3+ Ions Related to Cell Culture Studies and Cytotoxicity Evaluation In Vitro.

Li+/Eu3+ dual-doped calcium apatite analogues were fabricated using a microwave stimulated hydrothermal technique. XRPD, FT-IR, micro-Raman spectroscopy, TEM and SAED measurements indicated that obtained apatites are single-phased, crystallize with a hexagonal structure, have similar morphology and nanometric size as well as show red luminescence. Lithium effectively modifies the local symmetry of optical active sites and, thus, affects the emission efficiency. Moreover, the hydrodynamic size and surface charge of the nanoparticles have been extensively studied. The protein adsorption (lysozyme, LSZ; bovine serum albumin, BSA) on the nanoparticle surface depended on the type of cationic dopant (Li+, Eu3+) and anionic group (OH-, Cl-, F-) of the apatite matrix. Interaction with LSZ resulted in a positive zeta potential, and the nanoparticles had the lowest hydrodynamic size in this protein medium. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS), murine macrophages (J774.E), as well as human red blood cells (RBCs). The studied apatites were not cytotoxic to RBCs and J774.E cells; however, at higher concentrations of nanoparticles, cytotoxicity was observed against the U2OS cell line. No antimicrobial activity was detected against Gram-negative bacteria with one exception for P. aeruginosa treated with Li+-doped fluorapatite.

Preparation and preliminary evaluation of bio-nanocomposites based on hydroxyapatites with antibacterial properties against anaerobic bacteria.

Hexagonal nanocrystalline powders of the non-doped Ca10(PO4)6(OH)2 as well as activated with Ag+ and Eu3+ ions were synthesized by using different wet chemistry methods. Moreover, the obtained hydroxyapatite was loaded with Ag0, as well as nitroimidazole antimicrobials: metronidazole and tinidazole. The structural properties of the products were analyzed by X-ray diffraction (XRD), scanning (SEM) and transmission (TEM) electron microscopy as well as infrared (IR) and Raman spectroscopy. The photoluminescence properties of the Eu3+ and Ag+ co-doped Ca10(PO4)6(OH)2 were characterized via the PL emission, excitation spectra and the luminescence decay curve. The antimicrobial activity of the obtained materials against Prevotella bivia and Parabacteroides distasonis was studied. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS) as well as human red blood cells (RBC). The choice of the in vitro model was based on the fact that U2OS is a cancer cell line derived from bone tissue which is rich in apatites that play a pivotal role in the extracellular matrix formation. RBCs are the most abundant blood cells and they are used as a cell model in the study of biocompatibility of new prepared biocompounds with potential medical applications. The obtained multifunctional materials do not exhibit the haemolytic activity, therefore, they could be used as a promising antimicrobial agent and for anaerobic bacteria.

Developing an obesity intervention in Micronesia: From needs assessment to planning.

Evidence-based obesity reduction programs in the Pacific are scarce to nonexistent. Using a community-based participatory research model (CBPR) we developed a collaboration between our university and a small Pacific Island nation. We established an advisory council of local stakeholders and then conducted an extensive needs assessment with youth, parents, professionals, and lay public. Only 9% of participants had tried a weight loss program. There was a strong tendency to engage in binge-eating cycles; difficulty eating healthy during frequent community celebrations with few healthy food options available in general; and limited traditions that involved physical activity. Participants wanted to learn how to increase their physical activity, make healthier food choices, and learn to reward themselves for healthy behaviors. Diets were most frequently high in energy or protein foods, followed by starch, sugary and fried foods; they ate vegetables and fruits least often. Together, our team then created the culturally relevant Fit Kit Palau©. We highlight lessons learned including how to determine partnerships; manage multiple cultural and geographic barriers; build capacity; and balance program fidelity with responsivity. As the program moves forward, we hope to increase access, engagement, and adherence and provide a model for other PI countries.

Differential tumor biological role of the tumor suppressor KAI1 and its splice variant in human breast cancer cells.

The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells.

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrow-derived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCs and Th2 lymphocytes, (iii) proliferation of Th2 cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.

Adoptively transferred Tregs accumulate in a site-specific manner and ameliorate signs of less advanced collagen-induced arthritis progress in rats.

AIM: The aim of the study was to assess the therapeutic effect and migration of adoptively transferred Tregs in the course of collagen-induced arthritis (CIA) in rats. METHODS: Sorted CD4(+)CD25(+) cells were cultured in the presence of 17-ß-estradiol, stained with CellTracker and then administered into the articular capsule of ankle joint of animals in different stages of CIA progression. RESULTS: Tregs diminished CIA signs only in animals with less advanced disease progress. Moreover, migration of transferred cells into the LN in the near proximity of the injection site and with distal location was almost completely stopped in animals with fully developed CIA. CONCLUSION: Disease progression-related differences in migratory potential of in vitro induced Tregs may be responsible for the failure of cellular therapy during the advanced stages of CIA.

Alternative splicing of KAI1 abrogates its tumor-suppressive effects on integrin αvß3-mediated ovarian cancer biology.

Loss or downregulation of the tumor-suppressor KAI1 correlates with poor cancer patient prognosis. KAI1 functions by interacting with other proteins, including integrin cell adhesion and signaling receptors. We previously showed that KAI1 physically and functionally crosstalks with the tumor-biologically relevant integrin αvß3, thereby suppressing ovarian cancer cell migration and proliferation. Interestingly, in metastases, a KAI1 splice variant had been identified, indicating poor patient prognosis. Thus, we here characterized differential effects of the two KAI1 proteins upon their cellular restoration. Opposite to KAI1, KAI1-splice reduced αvß3-mediated cell adhesion, thereby inducing cell migration. This was accompanied by elevated αvß3 levels and drastically elevated focal adhesion kinase activation, however, without any obvious colocalization with αvß3, as observed for KAI1. Moreover, codistribution of KAI1 with the cell/cell-adhesion molecule E-cadherin was abrogated in KAI1-splice. Whereas KAI1 diminished cell proliferative activity, KAI1-splice prominently enhanced cell proliferation concomitant with elevated transcription and cell-surface expression of the epidermal growth factor receptor. Thus KAI1-splice does not only counteract the tumor-suppressive actions of KAI1, but - beyond that - promotes αvß3-mediated biological functions in favor of tumor progression and metastasis.

Communication adjustment of patients with a laryngectomy in Turkey: analysis by type of surgery and mode of speech.

Previous studies have routinely shown that individuals with a laryngectomy (IWL) have a decreased quality of life (QoL). However, the questionnaires frequently used are not specifically designed for persons with laryngeal cancer and may not reflect the issues of most concern to IWL. The purpose of this study was to investigate (1) the overall communication adjustment of IWL in Turkey, (2) the differences in communication adjustment among types of surgery for the treatment of laryngeal cancer, and (3) the differences in communication adjustment among types of speech following treatment for laryngeal cancer. Fifty-two patients (50 males and two females) who underwent surgical treatment for laryngeal cancer (i.e. total, supraglottic, frontolateral, vertical, supracricoid) and were being seen for follow-up care were recruited from an ENT clinic in Bursa, Turkey. Patients were provided with a written translation of the Self-Evaluation of Communication Experiences after Laryngectomy (SECEL) which yielded information on three subscales (General, Environment, and Attitude) and a Total score. Kruskall-Wallis and Wilcoxon signed ranks nonparametric tests of significance showed that overall adjustment was within the well-adjusted range for all types of surgery and modes of speech with two exceptions: Mean scores for supracricoid laryngectomy with cricohyoidopexy (28.5) and esophageal speech (29.3) were both within the poorly adjusted range on the Environment subscale [lowest mean score = 8.7 (supraglottic)]. Total mean scores ranged from 19.8 (supraglottic) to 49.9 (esophageal speech). Mean scores of the General subscale ranged from 4.0 (electrolaryngeal speech) to 7.7 (tracheoesophageal speech). Mean scores of the Attitude subscale ranged from 6.2 (supraglottic) to 19 (electrolaryngeal). Results of the Kruskall-Wallis test also showed a significant difference between type of surgery for the Environment subscale (p = 0.003), the Attitude subscale (p = 0.039), and the Total score (p = 0.007). The results suggest that overall, IWL in Turkey are well-adjusted to their new voice. However, results also showed that certain conservation surgeries (i.e. supraglottic, frontolateral, vertical) yielded more favorable outcomes than either supracricoid laryngectomy or speech following total laryngectomy. This finding is most likely related to the amount of tissue preserved following conservation surgery, in particular the preservation of the vocal folds.

Night vision disturbances after corneal refractive surgery.

A certain percentage of patients complain of "glare" at night after undergoing a refractive surgical procedure. When patients speak of glare they are, technically, describing a decrease in quality of vision secondary to glare disability, decreased contrast sensitivity, and image degradations, or more succinctly, "night vision disturbances." The definitions, differences, and methods of measurement of such vision disturbances after refractive surgery are described in our article. In most cases of corneal refractive surgery, there is a significant increase in vision disturbances immediately following the procedure. The majority of patients improve between 6 months to 1 year post-surgery. The relation between pupil size and the optical clear zone are most important in minimizing these disturbances in RK. In PRK and LASIK, pupil size and the ablation diameter size and location are the major factors involved. Treatment options for disabling glare are also discussed. With the exponential increase of patients having refractive surgery, the increase of patients complaining of scotopic or mesopic vision disturbances may become a major public health issue in the near future. Currently, however, there are no gold-standard clinical tests available to measure glare disability, contrast sensitivity, or image degradations. Standardization is essential for objective measurement and follow-up to further our understanding of the effects of these surgeries on the optical system and thus, hopefully, allow for modification of our techniques to decrease or eliminate post-refractive vision disturbances.