Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31–43 (but not peptide 57–68) uptake by cells, thereby impairing the ability of p31–43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31–43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.     

DNA electrophoretic migration patterns change after exposure of Jurkat cells to a single intense nanosecond electric pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.     

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.     

Focusing Narrowly or Broadly Attention When Judging Categorical and Coordinate Spatial Relations: A MEG Study

We measured activity in the dorsal system of the human cortex with magnetoencephalography (MEG) during a matching-to-sample plus cueing paradigm, where participants judged the occurrence of changes in either categorical or coordinate spatial relations (e.g., exchanges of left versus right positions or changes in the relative distances) between images of pairs of animals. The attention window was primed in each trial to be either small or large by using cues that immediately preceded the matching image. In this manner, we could assess the modulatory effects of the scope of attention on the activity of the dorsal system of the human cortex during spatial relations processing. The MEG measurements revealed that large spatial cues yielded greater activations and longer peak latencies in the right inferior parietal lobe for coordinate trials, whereas small cues yielded greater activations and longer peak latencies in the left inferior parietal lobe for categorical trials. The activity in the superior parietal lobe, middle frontal gyrus, and visual cortex, was also modulated by the size of the spatial cues and by the type of spatial relation change. The present results support the theory that the lateralization of each kind of spatial processing hinges on differences in the sizes of regions of space attended to by the two hemispheres. In addition, the present findings are inconsistent with the idea of a right-hemispheric dominance for all kinds of challenging spatial tasks, since response times and accuracy rates showed that the categorical spatial relation task was more difficult than the coordinate task and the cortical activations were overall greater in the left hemisphere than in the right hemisphere.     

Development of the digestive system of Argentine hake,Merluccius hubbsi,larvae

The Argentine hake Merluccius hubbsi is an important fishery resource of the Southwestern Atlantic Ocean and it is also a potential species for cultivation. In this work, the digestive system development in field-caught hake larvae was studied using histological and histochemical approaches. The digestive tract of larvae was divided into: oropharyngeal cavity (OPC), esophagus, stomach (that develops in the preflexion stage), and intestine. The annexed digestive glands consisted of the liver and the exocrine pancreas. At the beginning of the preflexion stage, teeth were developed in the OPC. There were mucous cells in the esophagus secreting different glycoconjugates from hatching. The enterocytes in the posterior intestine exhibited supranuclear vesicles associated with protein absorption. Mucous cells were observed in the posterior intestine in the preflexion stage and, in the anterior region, ending the flexion stage. Each type of glycoconjugates has a specific role. Acidic mucins lubricate and protect from mechanical damage, sialomucines protect from bacterial infections and neutral mucins regulate the acidity of mucus secretion, protect against abrasion and participate in the formation of the chyme, indicating a pregastric digestion. The liver was present since hatching with pancreatic tissue inside and increased in size acquiring the typical structure with hepatocyte cords, sinusoids, vacuoles, and hepatic duct. The hepatocytes vacuolization increased with larval development. The pancreas became extra-hepatic, with basophilic acinar cells and acidophilic zymogen granules. Throughout the ontogeny, the increased structural and functional complexity of the digestive system reflected the transition to exogenous feeding and nutritional increasing needs.     

Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.Subject terms: Nuclear organization, Cancer     

Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype

Background

Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.

Methodology/Principal Findings

In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.

Conclusions/Significance

Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.     

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin,Men10755

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials.The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4′ of the proximal sugar) with the hexanucleotides d(CGTACG)₂ and d(CGATCG)₂ was studied by a combined use of 2D-¹H and ³¹P NMR techniques. Both ¹H and ³¹P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (k_off) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of k_off , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)₂ sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues.A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.     

Nusinersen treatment and cerebrospinal fluid neurofilaments: An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.