Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the Φ105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production. © 2003 Elsevier Inc. All rights reserved.postprin

High Throughput Screening of a GlaxoSmithKline Protein Kinase Inhibitor Set Identifies an Inhibitor of Human Cytomegalovirus Replication that Prevents CREB and Histone H3 Post-Translational Modification.

To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high throughput strategy to screen a GlaxoSmithKline (GSK) Published Kinase Inhibitor Set (PKIS). This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen we identified compounds within chemotypes that impede HCMV replication and identified kinase proteins associated with inhibition of HCMV replication that are potential novel anti-HCMV targets. We focused our study on a top "hit" in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibits polypharmacology and is an inhibitor of several proteins from the AGC and CMCG kinase groups. Using western blotting we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (CREB and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identify SB-734117 as a novel anti-HCMV compound and find that inhibition of AGC and CMCG kinase proteins during productive HCMV replication is associated with inhibition of viral protein production and prevents post-translational modification of cellular factors associated with viral protein production

Identification of lead anti-human cytomegalovirus compounds targeting MAP4K4 via machine learning analysis of kinase inhibitor screening data

Chemogenomic approaches involving highly annotated compound sets and cell based high throughput screening are emerging as a means to identify novel drug targets. We have previously screened a collection of highly characterized kinase inhibitors (Khan et al., Journal of General Virology, 2016) to identify compounds that increase or decrease expression of a human cytomegalovirus (HCMV) protein in infected cells. To identify potential novel anti-HCMV drug targets we used a machine learning approach to relate our phenotypic data from the aforementioned screen to kinase inhibition profiling of compounds used in this screen. Several of the potential targets had no previously reported role in HCMV replication. We focused on one potential anti-HCMV target, MAPK4K, and identified lead compounds inhibiting MAP4K4 that have anti-HCMV activity with little cellular cytotoxicity. We found that treatment of HCMV infected cells with inhibitors of MAP4K4, or an siRNA that inhibited MAP4K4 production, reduced HCMV replication and impaired detection of IE2-60, a viral protein necessary for efficient HCMV replication. Our findings demonstrate the potential of this machine learning approach to identify novel anti-viral drug targets, which can inform the discovery of novel anti-viral lead compounds

An integrated general practice and pharmacy-based intervention to promote the use of appropriate preventive medications among individuals at high cardiovascular disease risk: protocol for a cluster randomized controlled trial

Background: Cardiovascular diseases (CVD) are responsible for significant morbidity, premature mortality, and economic burden. Despite established evidence that supports the use of preventive medications among patients at high CVD risk, treatment gaps remain. Building on prior evidence and a theoretical framework, a complex intervention has been designed to address these gaps among high-risk, under-treated patients in the Australian primary care setting. This intervention comprises a general practice quality improvement tool incorporating clinical decision support and audit/feedback capabilities; availability of a range of CVD polypills (fixed-dose combinations of two blood pressure lowering agents, a statin ± aspirin) for prescription when appropriate; and access to a pharmacy-based program to support long-term medication adherence and lifestyle modification. Methods: Following a systematic development process, the intervention will be evaluated in a pragmatic cluster randomized controlled trial including 70 general practices for a median period of 18 months. The 35 general practices in the intervention group will work with a nominated partner pharmacy, whereas those in the control group will provide usual care without access to the intervention tools. The primary outcome is the proportion of patients at high CVD risk who were inadequately treated at baseline who achieve target blood pressure (BP) and low-density lipoprotein cholesterol (LDL-C) levels at the study end. The outcomes will be analyzed using data from electronic medical records, utilizing a validated extraction tool. Detailed process and economic evaluations will also be performed. Discussion: The study intends to establish evidence about an intervention that combines technological innovation with team collaboration between patients, pharmacists, and general practitioners (GPs) for CVD prevention. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN1261600023342

Micro-Electro-Mechanical-Systems (MEMS) and Fluid Flows

The micromachining technology that emerged in the late 1980s can provide micron-sized sensors and actuators. These micro transducers are able to be integrated with signal conditioning and processing circuitry to form micro-electro-mechanical-systems (MEMS) that can perform real-time distributed control. This capability opens up a new territory for flow control research. On the other hand, surface effects dominate the fluid flowing through these miniature mechanical devices because of the large surface-to-volume ratio in micron-scale configurations. We need to reexamine the surface forces in the momentum equation. Owing to their smallness, gas flows experience large Knudsen numbers, and therefore boundary conditions need to be modified. Besides being an enabling technology, MEMS also provide many challenges for fundamental flow-science research

Shot noise in mesoscopic systems

This is a review of shot noise, the time-dependent fluctuations in the electrical current due to the discreteness of the electron charge, in small conductors. The shot-noise power can be smaller than that of a Poisson process as a result of correlations in the electron transmission imposed by the Pauli principle. This suppression takes on simple universal values in a symmetric double-barrier junction (suppression factor 1/2), a disordered metal (factor 1/3), and a chaotic cavity (factor 1/4). Loss of phase coherence has no effect on this shot-noise suppression, while thermalization of the electrons due to electron-electron scattering increases the shot noise slightly. Sub-Poissonian shot noise has been observed experimentally. So far unobserved phenomena involve the interplay of shot noise with the Aharonov-Bohm effect, Andreev reflection, and the fractional quantum Hall effect.Comment: 37 pages, Latex, 10 figures (eps). To be published in "Mesoscopic Electron Transport," edited by L. P. Kouwenhoven, G. Schoen, and L. L. Sohn, NATO ASI Series E (Kluwer Academic Publishing, Dordrecht

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms

Retargeted adenoviruses for radiation-guided gene delivery

The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment

Local Difference Measures between Complex Networks for Dynamical System Model Evaluation

Acknowledgments We thank Reik V. Donner for inspiring suggestions that initialized the work presented herein. Jan H. Feldhoff is credited for providing us with the STARS simulation data and for his contributions to fruitful discussions. Comments by the anonymous reviewers are gratefully acknowledged as they led to substantial improvements of the manuscript.Peer reviewedPublisher PD

Long-term, high frequency in situ measurements of intertidal mussel bed temperatures using biomimetic sensors

At a proximal level, the physiological impacts of global climate change on ectothermic organisms are manifest as changes in body temperatures. Especially for plants and animals exposed to direct solar radiation, body temperatures can be substantially different from air temperatures. We deployed biomimetic sensors that approximate the thermal characteristics of intertidal mussels at 71 sites worldwide, from 1998-present. Loggers recorded temperatures at 10-30 min intervals nearly continuously at multiple intertidal elevations. Comparisons against direct measurements of mussel tissue temperature indicated errors of similar to 2.0-2.5 degrees C, during daily fluctuations that often exceeded 15 degrees-20 degrees C. Geographic patterns in thermal stress based on biomimetic logger measurements were generally far more complex than anticipated based only on 'habitat-level' measurements of air or sea surface temperature. This unique data set provides an opportunity to link physiological measurements with spatially-and temporally-explicit field observations of body temperature