Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.     

Seasonal Dynamics of the Airborne Bacterial Community and Selected Viruses in a Children’s Daycare Center

Children’s daycare centers appear to be hubs of respiratory infectious disease transmission, yet there is only limited information about the airborne microbial communities that are present in daycare centers. We have investigated the microbial community of the air in a daycare center, including seasonal dynamics in the bacterial community and the presence of specific viral pathogens. We collected filters from the heating, ventilation, and air conditioning (HVAC) system of a daycare center every two weeks over the course of a year. Amplifying and sequencing the 16S rRNA gene revealed that the air was dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes that are commonly associated with the human skin flora. Clear seasonal differences in the microbial community were not evident; however, the community structure differed when the daycare center was closed and unoccupied for a 13-day period. These results suggest that human occupancy, rather than the environment, is the major driver in shaping the microbial community structure in the air of the daycare center. Using PCR for targeted viruses, we detected a seasonal pattern in the presence of respiratory syncytial virus that included the period of typical occurrence of the disease related to the virus; however, we did not detect the presence of adenovirus or rotavirus at any time.     

Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949^th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.     

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.     

Metabolic fingerprints of human primary endothelial and fibroblast cells

Introduction

Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders.

Objectives

In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles.

Methods

Biolog Phenotype MicroArray? technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF).

Results

Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides.

Conclusion

Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.

     

A microscopically motivated model for the remodeling of cardiomyocytes

Biomechanics and Modeling in Mechanobiology - We present a thermodynamically based model that captures the remodeling effects in cardiac muscle cells. This work begins with the formulation of the...     

Contact guidance as a consequence of coupled morphological evolution and motility of adherent cells

Biomechanics and Modeling in Mechanobiology - Adherent cells seeded on substrates spread and evolve their morphology while simultaneously displaying motility. Phenomena such as contact guidance,...     

Cinnamaldehyde regulates H2O 2-induced skeletal muscle atrophy by ameliorating the proteolytic and antioxidant defense systems

Skeletal muscle atrophy/wasting is associated with impaired protein metabolism in diverse physiological and pathophysiological conditions. Elevated levels of reactive oxygen species (ROS), disturbed redox status, and weakened antioxidant defense system are the major contributing factors toward atrophy. Regulation of protein metabolism by controlling ROS levels and its associated catabolic pathways may help in treating atrophy and related clinical conditions. Although cinnamaldehyde (CNA) enjoys the established status of antioxidant and its role in ROS management is reported, impact of CNA on skeletal muscle atrophy and related pathways is still unexplored. In the current study, the impact of CNA on C2C12 myotubes and the possible protection of cultured cells from H ₂O ₂-induced atrophy is examined. Myotubes were treated with H ₂O ₂ in the presence and absence of CNA and the changes in the antioxidative, proteolytic systems, and mitochondrial functions were scored. Morphological analysis showed significant protective effects of CNA on length, diameter, and nuclei fusion index of myotubes. The evaluation of biochemical markers of atrophy; creatine kinase, lactate dehydrogenase, succinate dehydrogenase along with the study of muscle-specific structural protein (i.e., myosin heavy chain-fast [MHCf] type) showed significant protection of proteins by CNA. CNA pretreatment not only checked the activation of proteolytic systems (ubiquitin-proteasome E3-ligases [MuRF1/Atrogin1]), autophagy [Beclin1/LC3B], cathepsin L, calpain, caspase), but also prevented any alteration in the activities of antioxidative defense enzymes (catalase, glutathione- S-transferase, glutathione-peroxidase, superoxide dismutase, glutathione reductase). The results suggest that CNA protects myotubes from H ₂O ₂-induced atrophy by inhibiting/resisting the amendments in proteolytic systems and maintains cellular redox-balance.     

Entropic Forces Drive Cellular Contact Guidance

Contact guidance—the widely known phenomenon of cell alignment induced by anisotropic environmental features—is an essential step in the organization of adherent cells, but the mechanisms by which cells achieve this orientational ordering remain unclear. Here, we seeded myofibroblasts on substrates micropatterned with stripes of fibronectin and observed that contact guidance emerges at stripe widths much greater than the cell size. To understand the origins of this surprising observation, we combined morphometric analysis of cells and their subcellular components with a, to our knowledge, novel statistical framework for modeling nonthermal fluctuations of living cells. This modeling framework is shown to predict not only the trends but also the statistical variability of a wide range of biological observables, including cell (and nucleus) shapes, sizes, and orientations, as well as stress-fiber arrangements within the cells with remarkable fidelity with a single set of cell parameters. By comparing observations and theory, we identified two regimes of contact guidance: 1) guidance on stripe widths smaller than the cell size (w ≤ 160 μm), which is accompanied by biochemical changes within the cells, including increasing stress-fiber polarization and cell elongation; and 2) entropic guidance on larger stripe widths, which is governed by fluctuations in the cell morphology. Overall, our findings suggest an entropy-mediated mechanism for contact guidance associated with the tendency of cells to maximize their morphological entropy through shape fluctuations.