Microcinematographic Studies of Mycoplasma hominis Cells

Cells of two strains of Mycoplasma hominis growing in liquid medium on a glass surface were observed continuously, and cinematographic pictures were taken. Most of the observed structures showed reversible changes of their shape, suggesting the presence of contractile material in membrane or cytoplasma. The frequency and speed of such variations were measured. The deformations seem to be related to multiplication. The mechanisms of these phenomena are unknown.     

KCNE1 is an auxiliary subunit of two distinct ion channel superfamilies

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GM2 activator protein inhibits platelet activating factor signaling in rats

Platelet activating factor (PAF), an endogenous bioactive phospholipid, has been documented as a pivotal mediator in the inflammatory cascade underlying the pathogenesis of many diseases including necrotizing enterocolitis. Much effort has been directed towards finding an effective in vivo inhibitor of PAF signaling. Here, we report that a small, highly stable, lysosomal lipid transport protein, the GM2 activator protein (GM2AP) is able to inhibit the inflammatory processes otherwise initiated by PAF in a rat model of necrotizing enterocolitis. Based on behavioral observations, gross anatomical observations at necropsy, histopathology and immunocytochemistry, the administration of recombinant GM2AP inhibits the devastating gastrointestinal necrosis resulting from the injection of rats with LPS and PAF. Recombinant GM2AP treatment not only markedly decrease tissue destruction, but also helped to maintain tight junction integrity at the gastrointestinal level as judged by contiguous Zonula Occludens-1 staining of the epithelial layer lining the crypts.     

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors

The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

On the interpretation of twisted patterns in elongating plant cell wall: Informations obtained with ultracryotomy

Summary Ultracryotomy, which gives anin situ visualization of wall subunits, was used to study the texture of elongating wall of mung bean hypocotyl (Phaseolus aureus). The criss-crossed texture of the wall was confirmed. The controversed disposition of the subunits in twisted patterns was analyzed on the basis of observations of oblique sections on which a mild extraction may be performed. Despite the twisted appearance within one sectioning plane, no curved subunits were seen running between the criss-crossed layers. The results confirmed that the appearance of arcs is an illusion due to thin sectioning of successive strata in which the orientation of subunits rotates.     

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.