Differentiation of serum-free embryoid bodies from human induced pluripotent stem cells into networks

Three-dimensional aggregation cultures allow for complex development of differentiated human induced pluripotent stem cells. However, this approach is not easily amenable to live-cell imaging and electrophysiological applications due to the thickness and the geometry of the tissue. Here, we present an improvement on the traditional aggregation method by combining the use of cell culture inserts with serum-free embryoid bodies (SFEBs). The use of this technique allows the structures to maintain their three-dimensional structure while thinning substantially. We demonstrate that this technique can be used for electrophysiological recodings as well as live-cell calcium imaging combined with electrical stimulation, akin to organotypic slice preparations. This provides an important experimental tool that can be used to bridge 3-D structures with traditional monolayer approaches used in stem cell applications.     

Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival‐promoting kinases

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post‐natal rat NPs express low levels of pro‐apoptotic molecules and resist phosphatidylinositol 3′OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage‐restricted precursors as revealed by enrichment for Nestin⁺/SOX‐2⁺ cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin⁺/SOX‐2⁺ and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3‐times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.

     

Reactive Oxygen Species Mediated Bacterial Biofilm Inhibition via Zinc Oxide Nanoparticles and Their Statistical Determination

The formation of bacterial biofilm is a major challenge in clinical applications. The main aim of this study is to describe the synthesis, characterization and biocidal potential of zinc oxide nanoparticles (NPs) against bacterial strain Pseudomonas aeruginosa. These nanoparticles were synthesized via soft chemical solution process in a very short time and their structural properties have been investigated in detail by using X-ray diffraction and transmission electron microscopy measurements. In this work, the potential of synthesized ZnO-NPs (∼10–15 nm) has been assessed in-vitro inhibition of bacteria and the formation of their biofilms was observed using the tissue culture plate assays. The crystal violet staining on biofilm formation and its optical density revealed the effect on biofilm inhibition. The NPs at a concentration of 100 µg/mL significantly inhibited the growth of bacteria and biofilm formation. The biofilm inhibition by ZnO-NPs was also confirmed via bio-transmission electron microscopy (Bio-TEM). The Bio-TEM analysis of ZnO-NPs treated bacteria confirmed the deformation and damage of cells. The bacterial growth in presence of NPs concluded the bactericidal ability of NPs in a concentration dependent manner. It has been speculated that the antibacterial activity of NPs as a surface coating material, could be a feasible approach for controlling the pathogens. Additionally, the obtained bacterial solution data is also in agreement with the results from statistical analytical methods.     

Recessive <Emphasis Type="Italic">VARS2</Emphasis> mutation underlies a novel syndrome with epilepsy,mental retardation,short stature,growth hormone deficiency,and hypogonadism

Background

Most mitochondrial and cytoplasmic aminoacyl-tRNA synthetases (aaRSs) are encoded by nuclear genes. Syndromic disorders resulting from mutation of aaRSs genes display significant phenotypic heterogeneity. We expand aaRSs-related phenotypes through characterization of the clinical and molecular basis of a novel autosomal-recessive syndrome manifesting severe mental retardation, ataxia, speech impairment, epilepsy, short stature, microcephaly, hypogonadism, and growth hormone deficiency.

Results

A G>A variant in exon 29 of VARS2 (c.3650G>A) (NM_006295) was identified in the index case. This homozygous variant was confirmed by Sanger sequencing and segregated with disease in the family studied. The c.3650G>A change results in alteration of arginine to histidine at residue 1217 (R1217H) of the mature protein and is predicted to be pathogenic.

Conclusions

These findings contribute to a growing list of aaRSs disorders, broadens the spectrum of phenotypes attributable to VARS2 mutations, and provides new insight into genotype-phenotype correlations among the mitochondrial synthetase genes.

     

Zinc oxide and titanium dioxide nanoparticles induce oxidative stress,inhibit growth,and attenuate biofilm formation activity of <Emphasis Type="Italic">Streptococcus mitis</Emphasis>

Streptococcus mitis from the oral cavity causes endocarditis and other systemic infections. Rising resistance against traditional antibiotics amongst oral bacteria further aggravates the problem. Therefore, antimicrobial and antibiofilm activities of zinc oxide and titanium dioxide nanoparticles (NPs) synthesized and characterized during this study against S. mitis ATCC 6249 and Ora-20 were evaluated in search of alternative antimicrobial agents. ZnO and TiO₂-NPs exhibited an average size of 35 and 13 nm, respectively. The IC₅₀ values of ZnO and TiO₂-NPs against S. mitis ATCC 6249 were 37 and 77 µg ml^?1, respectively, while the IC₅₀ values against S. mitis Ora-20 isolate were 31 and 53 µg ml^?1, respectively. Live and dead staining, biofilm formation on the surface of polystyrene plates, and extracellular polysaccharide production show the same pattern. Exposure to these nanoparticles also shows an increase (26–83 %) in super oxide dismutase (SOD) activity. Three genes, namely bapA1, sodA, and gtfB like genes from these bacteria were identified and sequenced for quantitative real-time PCR analysis. An increase in sodA gene (1.4- to 2.4-folds) levels and a decrease in gtfB gene (0.5- to 0.9-folds) levels in both bacteria following exposure to ZnO and TiO₂-NPs were observed. Results presented in this study verify that ZnO-NPs and TiO₂-NPs can control the growth and biofilm formation activities of these strains at very low concentration and hence can be used as alternative antimicrobial agents for oral hygiene.     

Biomedical and therapeutic potential of exopolysaccharides by Lactobacillus paracasei isolated from sauerkraut: Screening and characterization

The intention of the study was evaluated for purification and characterization of exopolysaccharides from Lactobacillus paracasei; was isolated from homemade Sauerkraut sample collected from Sivakasi, Tamil Nadu, India, confirmed by biochemical and gene sequencing (16S rRNA). The purification and characterization of exopolysaccharides from candidate bacterium were studied on appearance, solubility of the EPS, carbohydrate estimation, emulsifying activity, sulphate, protein, uronic acid content, FTIR, HPLC and GC-MS analysis. The percentage of elemental carbon, (54.36%) hydrogen (21.74%), nitrogen (9.63%) and sulphur content (18.03%) were recorded in exopolysaccharides. The emulsification index (E24) of EPS was higher in toluene (79.20) and benzene (78.867) supplemented medium. FTIR spectrum of the candidate bacterial EPS confirmed presence of sulphate compounds, carboxyl group, and hydrogen bonded compounds etc. EPS exhibited 76.34% of Total Antioxidant Capacity (TAC), 71.15% of reducing power, 68.65% of Hydrogen Peroxide scavenging activity and also 60.31% DPPH radical scavenging activity. The potential antioxidant properties observed in exopolysaccharides from Lactobacillus paracasei is considered as valuable drugs.     

Decolorization and biotransformation pathway of textile dye by Cylindrocephalum aurelium

Due to environmental concern, the research to date has tended to focus on how textile dye removal can be carried out in a greener manner. Therefore, this study aims to evaluate the decolorization and biotransformation pathway of Mordant Orange-1 (MO-1) by Cylindrocephalum aurelium RY06 (C. aurelium RY06). Decolorization study was conducted in a batch experiment including the investigation of the effects of physio-chemical parameters. Enzymatic activity of C. aurelium RY06 during the decolorization was also investigated. Moreover, transformation and biodegradation of MO-1 by C. aurelium RY06 were observed using the gas chromatography–mass spectrometry. Manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase enzymes were detected during the decolorization. In general, the present work concluded that the MO-1 was successfully degraded by C. aurelium RY06 and transformed to be maleic acid and to be isophtalic acid.