A Proteasome Inhibitor-stimulated Nrf1 Protein-dependent Compensatory Increase in Proteasome Subunit Gene Expression Reduces Polycomb Group Protein Level

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G₂, and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.     

Cloning and characterization of a DNA repair gene inHaemophilus influenzae

Using a high-efficiency DNA cloning vector pJ1–8, a DNA repair geneuvr1 has been self-cloned in bacteriumHaemophilus influenzae. Chimeric plasmid pKuvrl, carrying wild type allele ofuvr1 gene and flanking DNA sequences, specifically complements auvr1 gene mutation in the bacterial chromosome. Auvr1_} mutation could be transferred from chromosome byin vivo recombination to pKuvr1 and isolated and designated as plasmid pKuvrl₋. Plasmid pKuvrl carries a 11.3 kb chromosomal DNA insert which was scanned for the presence of any other DNA repair genes by a novel method of directed mutagenesis. Preliminary analysis of the 3 new mutants isolated by this method supports the notion that the insert contains more than one gene concerned with ultraviolet radiation-sensitivity.     

Healthcare Worker Contact Networks and the Prevention of Hospital-Acquired Infections

We present a comprehensive approach to using electronic medical records (EMR) for constructing contact networks of healthcare workers in a hospital. This approach is applied at the University of Iowa Hospitals and Clinics (UIHC) – a 3.2 million square foot facility with 700 beds and about 8,000 healthcare workers – by obtaining 19.8 million EMR data points, spread over more than 21 months. We use these data to construct 9,000 different healthcare worker contact networks, which serve as proxies for patterns of actual healthcare worker contacts. Unlike earlier approaches, our methods are based on large-scale data and do not make any a priori assumptions about edges (contacts) between healthcare workers, degree distributions of healthcare workers, their assignment to wards, etc. Preliminary validation using data gathered from a 10-day long deployment of a wireless sensor network in the Medical Intensive Care Unit suggests that EMR logins can serve as realistic proxies for hospital-wide healthcare worker movement and contact patterns. Despite spatial and job-related constraints on healthcare worker movement and interactions, analysis reveals a strong structural similarity between the healthcare worker contact networks we generate and social networks that arise in other (e.g., online) settings. Furthermore, our analysis shows that disease can spread much more rapidly within the constructed contact networks as compared to random networks of similar size and density. Using the generated contact networks, we evaluate several alternate vaccination policies and conclude that a simple policy that vaccinates the most mobile healthcare workers first, is robust and quite effective relative to a random vaccination policy.     

Functional characterization of cytochrome P450 CYP81A subfamily to disclose the pattern of cross-resistance in Echinochloa phyllopogon

Plant Molecular Biology - CYP81A P450s armor Echinochloa phyllopogon against diverse and several herbicide chemistries. CYP81A substrate preferences can be a basis for cross-resistance prediction...     

An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells

Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell cultures systems to achieve complete automation.     

The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.     

Outdoor microalgae cultivation in airlift photobioreactor at high irradiance and temperature conditions: effect of batch and fed-batch strategies,photoinhibition, and temperature stress

The microalgae Scenedesmus abundans cultivated in five identical airlift photobioreactors (PBRs) in batch and fed-batch modes at the outdoor tropical condition. The microalgae strain S. abundans was found to tolerate high temperature (35–45 °C) and high light intensity (770–1690 µmol m^− 2 s^− 1). The highest biomass productivities were 152.5–162.5 mg L^− 1 day^− 1 for fed-batch strategy. The biomass productivity was drastically reduced due to photoinhibition effect at a culture temperature of > 45 °C. The lipid compositions showed fatty acids mainly in the form of saturated and monounsaturated fatty acids (> 80%) in all PBRs with Cetane number more than 51. The fed-batch strategies efficiently produced higher biomass and lipid productivities at harsh outdoor conditions. Furthermore, the microalgae also accumulated omega-3 fatty acid (C18:3) up to 14% (w/w) of total fatty acid at given outdoor condition.

     

High-resolution single-shot phase-shifting interference microscopy using deep neural network for quantitative phase imaging of biological samples

White light phase-shifting interference microscopy (WL-PSIM) is a prominent technique for high-resolution quantitative phase imaging (QPI) of industrial and biological specimens. However, multiple interferograms with accurate phase-shifts are essentially required in WL-PSIM for measuring the accurate phase of the object. Here, we present single-shot phase-shifting interferometric techniques for accurate phase measurement using filtered white light (520±36 nm) phase-shifting interference microscopy (F-WL-PSIM) and deep neural network (DNN). The methods are incorporated by training the DNN to generate (a) four phase-shifted frames and (b) direct phase from a single interferogram. The training of network is performed on two different samples i.e., optical waveguide and MG63 osteosarcoma cells. Further, performance of F-WL-PSIM+DNN framework is validated by comparing the phase map extracted from network generated and experimentally recorded interferograms. The current approach can further strengthen QPI techniques for high-resolution phase recovery using a single frame for different biomedical applications.     

Genetic transformation in bacteria

Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria likebiplococcus pneumoniae and Gram-negative bacteria represented byHaemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low inHaemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. InHaemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.