Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

Gene delivery with low molecular weight linear polyethylenimines

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.     

Beta-glycosphingolipids improve glucose intolerance and hepatic steatosis of the Cohen diabetic rat

A link between altered levels of various gangliosides and the development of insulin resistance was described in transgenic mice. Naturally occurring glycosphingolipids were shown to exert immunomodulatory effects in a natural killer T (NKT) cell-dependent manner. This study examined whether glycosphingolipid-induced modulation of the immune system may reduce pancreatic and liver steatosis and stimulate insulin secretion in the Cohen diabetes-sensitive (CDS) rat, a lean model of non-insulin-resistant, nutritionally induced diabetes. Four groups of CDS rats fed a diabetogenic diet were treated with daily intraperitoneal injections of glycosphingolipids beta-glucosylceramide, beta-lactosylceramide, a combination of both (IGL), or vehicle (PBS) for up to 45 days. Immune modulation was assessed by fluorescence-activated cell sorting analysis of intrahepatic and intrasplenic lymphocytes. Steatosis was assessed by MRI imaging and histological examination of liver and pancreas, Blood glucose and plasma insulin concentrations were assessed during an oral glucose tolerance test. Administration of glycosphingolipids, particularly IGL, increased intrahepatic trapping of CD8 T and NKT lymphocytes. Pancreatic and liver histology were markedly improved and steatosis was reduced in all treated groups compared with vehicle-treated rats. Insulin secretion was restored after glycosphingolipid treatment, resulting in improved glucose tolerance. The immunomodulatory effect of beta-glycosphingolipids improved the beta-cell function of the hyperglycemic CDS rat. Thus our results suggest a role for the immune system in the pathogenesis of diabetes in this model.     

A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in streptomyces coelicolor A3(2)

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient limitation is likely to be a major signal for the onset of their development, resulting in spore formation by specialized aerial hyphae. Streptomycetes grow on numerous carbon sources, which they utilize in a preferential manner. The main signaling pathway underlying this phenomenon is carbon catabolite repression, which in streptomycetes is totally dependent on the glycolytic enzyme glucose kinase (Glk). How Glk exerts this fascinating dual role (metabolic and regulatory) is still largely a mystery. We show here that while Glk is made constitutively throughout the growth of Streptomyces coelicolor A3(2), its catalytic activity is modulated in a carbon source-dependent manner: while cultures growing exponentially on glucose exhibit high Glk activity, mannitol- grown cultures show negligible activity. Glk activity was directly proportional to the amount of two Glk isoforms observed by Western blot analysis. The activity profile of GlcP, the major glucose permease, correlated very well with that of Glk. Our data are consistent with a direct interaction between Glk and GlcP, suggesting that a Glk-GlcP permease complex is required for efficient glucose transport by metabolic trapping. This is supported by the strongly reduced accumulation of glucose in glucose kinase mutants. A model to explain our data is presented.     

Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes

Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid''s ability to detect novel plasmids.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.     

Raloxifene attenuates Gas6 and apoptosis in experimental aortic valve disease in renal failure

Renal failure is associated with aortic valve calcification. Using our rat model of uremia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in that disease. We also explored the effects of raloxifene, an estrogen receptor modulator, on valvular calcification. Gene array analysis was performed in aortic valves obtained from three groups of rats (n = 7 rats/group): calcified valves obtained from rats fed with uremic diet, valves after calcification resolution following diet cessation, and control. In addition, four groups of rats (n = 10 rats/group) were used to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet that also received daily raloxifene. Evaluation included imaging, histology, and antigen expression analysis. Gene array results showed that the majority of the altered expressed genes were in diet group valves. Most apoptosis-related genes were changed in a proapoptotic direction in calcified valves. Apoptosis and decreases in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of survival pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. In conclusion, downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure.