Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer‐stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2‐dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non‐cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.      

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.     

Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase.

The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.     

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.     

Estimating Mammalian Abundance Using Camera Traps in the Tropical Forest of Similipal Tiger Reserve,Odisha, India

Knowledge on the occurrence and distribution of species is crucial for designing and evaluating conservation strategies within a geographical region. Similipal Tiger Reserve though confined to a small area needs information on the diversity and abundance of mammalian fauna to ensure conservation of tiger. Thus, we aimed to assess the diversity and abundance of medium to large sized mammals in Similipal Tiger Reserve by using remotely triggered camera traps. A total of 6413 camera trap days at 187 trap stations were deployed from November 2012 to July 2013 to estimate the status of mammal. We obtained 3763 independent photographs and detected 24 species of mammals. The relative abundance index of each mammalian species was calculated. Leopard (Panthera pardus) was the most abundant carnivore while barking deer (Muntiacus muntjac) was the most abundant prey. Anthropogenic activities like hunting, livestock grazing and free ranging domestic dogs were found to be the detrimental factors for the existing mammalian species. These activities should be addressed through conservation and development perception with an interdisciplinary approach, incorporating social and ecological components cautiously.     

Comparative Analysis of the Effects of Two Probiotic Bacterial Strains on Metabolism and Innate Immunity in the RAW 264.7 Murine Macrophage Cell Line

Probiotic and potential probiotic bacterial strains are routinely prescribed and used as supplementary therapy for a variety infectious diseases, including enteric disorders among a wide range of individuals. While there are an increasing number of studies defining the possible mechanisms of probiotic activity, a great deal remains unknown regarding the diverse modes of action attributed to these therapeutic agents. More precise information is required to support the appropriate application of probiotics. To address this objective, we selected two probiotics strains, Lactobacillus acidophilus MTCC-10307 (LA) and Bacillus clausii MTCC-8326 (BC) that are frequently prescribed for the treatment of intestinal disorders and investigated their effects on the RAW 264.7 murine macrophage cell line. Our results reveal that LA and BC are potent activators of both metabolic activity and innate immune responses in these cells. We also observed that LA and BC possessed similar activity in preventing infection simulated in vitro in murine macrophages by Salmonella typhimurium serovar enterica.     

Effect of procaine hydrochloride on DNA repair in Escherichia coli

Liquid-holding recovery and rejoining of γ-radiation-induced DNA singlestrand scissions in Escherichia coli could be effectively inhibited by procaine hydrochloride at the concentration of 20 m M. At this concentration, the drug also reversibly altered cellular permeability barrier as evidenced from the uptake of acriflavin by bacterial cells.     

Involvement of calcium and dipicolinic acid in the resistance of Bacillus cereus BIS-59 spores to u.v. and gamma radiations

The role of dipicolinic acid (DPA) in determining the resistance of Bacillus cereus spores to u.v. and gamma radiation was investigated. B. cereus BIS-59 spores containing varying amounts of DPA were prepared by appropriate compositional adjustments in the secondary media. Compared with spores containing 6 per cent DPA (dry weight) those containing 0.8 per cent DPA were far more sensitive to u.v. radiation. Similar u.v. radiation sensitivity was also found in respect of a DPA-less mutant of B. cereus T 6A 1. Pre-treatment of DPA deficient spores (of wild type or mutant B. cereus) with DPA or the presence of DPA during irradiation resulted in increased resistance of these spores to u.v. radiation. In the range 0.2 to 1 per cent DPA content of spores of B. cereus BIS-59, a striking inverse relationship could be discerned between the DPA content and the number of spore photo-products (5-thymidyl, 5,6-dihydrothymine) formed in DNA and spore viability. The resistance of B. cereus spores to gamma radiation did not seem to be influenced by their DPA content.