A Highlights from MBoC Selection: Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.     

Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCellhES1, under low serum concentration supplemented with bone morphogenetic protein-2

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is highly variable. So, developing generic differentiation protocols and their empirical testing on a range of independently derived hESC lines pose a daunting challenge due to considerable diversity in culture methods practiced between lines. Maintenance of BG01V and ReliCellhES1 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual passaging. We assessed cardiac differentiation from both the cell lines via embryoid body (EB) formation. Subsequent culture in low fetal bovine serum (5%)-containing medium produced spontaneously contracting EBs, in the presence of bone morphogenetic protein-2 (BMP-2; 25 ng/ml). Derived cardiomyocytes expressed cardiac genes and proteins and responded to functional assays. Further, the activation of the Smad signaling machinery evoked by BMP-2 has been confirmed through inhibitor studies. Therefore, in our hands, the same differentiation conditions functioned in two independently derived hESC lines. Similar studies in other lines may facilitate development of universal protocols. The present data may also provide valuable insights for testing of other factors that might promote cardiomyocyte differentiation in low-serum formulations.     

Effect of nitric oxide signaling in bacterial-treated soybean plant under salt stress

To understand protective roles of nitric oxide against salt stress, the effects of exogenous sodium nitroprusside on activities of lipoxygenase, peroxidase, phenylalanine ammonialyase, catalase, superoxide dismutase enzymes, proline accumulation, and distribution of sodium in soybean plants under salt were determined. Application of sodium nitroprusside + bacterium enhanced plant growth-promotion characteristics, activities of different enzymes, and proline accumulation in the presence of sodium nitroprusside under salt stress. Treatment with NaCl at 200 mM and sodium nitroprusside (0.1 mM) reduced Na⁺ levels but increased K⁺ levels in leaves in comparison with the NaCl-treated plants. Correspondingly, the plants treated with exogenous sodium nitroprusside and NaCl maintained a lower ratio of [Na⁺]/[K⁺] in NaCl-stressed plants.     

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6?h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5?mmHg in a sustained manner for at least 12?h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.     

Non-enzymatic glycation induces structural modifications of myoglobin

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25°C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased α-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.     

Suppression of cell proliferation, DNA protein cross-links, and induction of apoptosis by vanadium in chemical rat mammary carcinogenesis

Vanadium, a dietary micronutrient, has recently been considered as an important pharmacological agent. The present investigation was carried out to ascertain its anticarcinogenic potential against an experimental rat mammary carcinogenesis. Female Sprague-Dawley rats were treated with 7,12dimethylbenz(alpha)anthracene (DMBA) (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 ppm (4.27 micromol/L) was supplemented in drinking water and given ad libitum to the experimental group. The present study was an attempt to assess the effect of vanadium (ammonium monovanadate) on cell proliferation, apoptosis and histopathology in the mammary tissue. We also have examined DNA fragmentation and DNA protein cross-links (DPC) in the liver of rats as well. Immunohistochemical analysis indicated that early neoplasia in mammary tissue proceeds by a decrease in apoptotic cell death (ACD), which was also examined with TUNEL assay, rather than an increase in cell proliferation (P<0.01). DPC in liver were reduced by vanadium treatment (ANOVA, F=13.7, P<0.01). Agarose gel electrophoresis revealed DNA fragmentation in the vanadium-treated group, confirming apoptosis further. Results of the study indicate that the mammary preneoplasia is sensitive to vanadium intervention whereas normal proliferating cells are not.