Comparative analysis of structural variations due to genome shuffling of Bacillus subtilis VS15 for improved cellulase production

Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N′ nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7.. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (K2Cr2O7) method

CD166(high) Uveal Melanoma Cells Represent a Subpopulation With Enhanced Migratory Capacity

Purpose: Cancer stem cells (CSCs) are a subpopulation of cells with the capacity to drive tumor growth. While there is evidence of the existence of CSCs in uveal melanoma (UM), there is no consensus on their defining markers. In this study, we examined putative CSC markers in UM cell lines, primary UM (PUM), and normal choroidal melanocytes (NCM). Methods: Nonadherent sphere assays were used to assess the tumorigenic potential of 15 PUMs, 8 high (M3) and 7 low (D3) metastatic risk. Flow cytometry was used to compare the expression of CSC markers between 10 PUMs and 4 NCMs, as well as in 8 UM cell lines grown under adherent and nonadherent conditions. Based on the data generated and from TCGA analyses, CD166 was investigated in detail, including its effect on cell migration using a tumor transendothelial migration assay. Results: M3 PUM had a greater melanosphere-forming efficiency than D3 PUM. CD166 and Nestin expression was upregulated in PUM compared to NCM by flow cytometry. UM cell lines resistant to anoikis had increased levels of CD271, Nestin, and CD166 compared with adherent cells. TCGA analysis showed that patients with higher CD166 expression had a poorer prognosis: this was supported by a Mel270 CD166high subpopulation that had enhanced migratory capabilities compared with CD166low cells. IHC showed that CD166 is expressed in the cytoplasm and cell membrane of PUM cells. Conclusions: UM contain a population of cells with characteristics of CSCs. In particular, CD166high UM cells appear to represent a subpopulation with enhanced migratory capacity

Effects of radiation and manganese oxide nanoparticles on human glioblastoma cell line U-87 MG glycolysis

Gliomas are the most common type of malignant brain tumors. Standard treatment of gliomas consists of surgical excision of the tumor with subsequent chemotherapy and radiotherapy. Tumor cells are characterized by rapid division with an increased uptake of glucose and its catabolism during glycolysis. To maintain rapid division, the level of glycolysis of the tumor cell is significantly increased, compared with normal cells. It is known that some nanoparticles (NP) have the property of accumulating in tumors. In particular, NPs of manganese oxide can penetrate into the brain and, with considerable accumulation, cause toxic effects. These facts served as a prerequisite for studying the effects of manganese oxide NPs on the viability of glioma cells. The purpose of this work was to study the effects of manganese oxide NPs, as well as their combination with gamma irradiation on the glycolysis of glioma cells. The cells were irradiated using the research radiobiological gamma-installation IGUR-1 based on 137Cs. The level of cell glycolysis was determined using the standard glycolytic stress test on a Seahorse XFp platform. Cell viability was determined using the ViaCount reagent staining of living and dead cells. Their count was performed using flow cytometry. We showed that the glycolysis of U-87 MG glioma cells was significantly reduced when incubated for 48 hours with manganese oxide NPs. Irradiation in combination with NPs or alone did not have significant effects on glycolysis of gliomas. Glioma incubation with manganese oxide NPs for 72 hours led to a significant reduction in cell viability. This study may be useful for the development of new therapies and diagnosis of gliomas

Paragraph: A graph-based structural variant genotyper for short-read sequence data

Accurate detection and genotyping of structural variations (SVs) from short-read data is a long-standing area of development in genomics research and clinical sequencing pipelines. We introduce Paragraph, an accurate genotyper that models SVs using sequence graphs and SV annotations. We demonstrate the accuracy of Paragraph on whole-genome sequence data from three samples using long-read SV calls as the truth set, and then apply Paragraph at scale to a cohort of 100 short-read sequenced samples of diverse ancestry. Our analysis shows that Paragraph has better accuracy than other existing genotypers and can be applied to population-scale studies. © 2019 The Author(s)

Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]

To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two-step, peel-and-digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC-coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT-qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO-1, Na/K-ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed

Study of the neuronal response to olfactory stimuli in control and LPS-stimulated mice by functional magnetic resonance imaging

Olfactory perception plays the key role in the inter­action of animals with biotic factors of the species-specific econiche. Identification of odorants informs nocturnal animals about social environment, presence of predators, or infected food. Olfactory efficiency depends on physiological conditions; in particular, odor sensitivity can be changed by infection. This work considers use of fMRI in the study of the influence of innate immunity activation on neuronal response during perception and differentiation of socially significant (2.5-dimethylpyrazine, 2-heptanon) and socially insignificant (1-hexanol and isoprene) olfactory stimuli by CD-1 mice. We stimulated innate immunity by intraperitoneal injection of bacterial lipopolysaccharide (LPS) at the dose 500 µg/kg three hours before tomography. Urethane anesthesia was used during MRI trail. Odor stimulation was done with a lab-made metering unit for supplying standard doses of volatile organic compounds. The supply of olfactory stimuli induced activation of neurons in the primary perceptual center and the centers of secondary processing of olfactory information. Olfactory stimulus type affected neuronal response rate in an olfactory bulb but did not affect response parameters in other brain regions studied. This increase in neuronal activity is likely to be of adaptive significance as a mechanism supporting olfactory sensitivity increase, which plays the key role in the identification of potential sources of infection

Synthesis and characterization of nanocomposites based on PANI and carbon nanostructures prepared by electropolymerization

Nanocomposites based on polyaniline (PANI) and carbon nanostructures (CNSs) (graphene (G) and multiwall carbon nanotubes (MWCNTs)) were prepared by in situ electrochemical polymerization. CNSs were inserted into the PANI matrix by dispersing them into the electrolyte before the electropolymerization. Electrochemical characterization by means of cyclic voltammetry and steady state polarization were performed in order to determine conditions for electro- polymerization. Electro-polymerization of the PANI based nanocomposites was carried out at 0.75 V vs. saturated calomel electrode (SCE) for 40 and 60 minutes. The morphology and structural characteristics of the obtained nanocomposites were studied by scanning electron microscopy (SEM) and Raman spectroscopy, while thermal stability was determined using thermal gravimetric analysis (TGA). According to the morphological and structural study, fibrous and porous structure of PANI based nanocomposites was detected well embedding both G and MWCNTs. Also, strong interaction between quinoidal structure of PANI with carbon nanostructures via π–π stacking was detected by Raman spectroscopy. TGA showed the increased thermal stability of composites reinforced with CNSs, especially those reinforced with graphene

ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P2X7 purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses