Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus

<p>Abstract</p> <p>Background</p> <p>The transcription regulator PhoP has been shown to be important for <it>Y. pestis </it>survival in macrophages and under various <it>in vitro </it>stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in <it>Y. pestis</it>. A series of biochemical experiments were done herein to study members of the PhoP regulon of <it>Y. pestis </it>biovar <it>Microtus</it>.</p> <p>Results</p> <p>By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in <it>Y. pestis</it>. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation.</p> <p>Conclusion</p> <p>The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which <it>Y. pestis </it>survives the antibacterial strategies employed by host macrophages.</p

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fevers

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers

Distribution, degradation and dynamics of dissolved organic carbon and its major compound classes in the Pearl River estuary, China

We investigated the distribution, degradation and dynamics of organic carbon and its major compound classes, carbohydrates and amino acids, based upon a cruise in the Pearl River estuary in April 2007. Dissolved oxygen (DO), nutrients, particulate organic carbon (POC), chlorophyll a (Chl a), dissolved organic carbon (DOC), total dissolved carbohydrates (TCHO, including monosaccharides, MCHO, and polysaccharides, PCHO) as well as total dissolved amino acids (TAA, both dissolved free, DFAA, and combined components. DCAA) were measured along a salinity gradient. Community respiration and biodegradable DOC were also determined via both short term (within 3 days) and long term (lasting 30 days) incubation. DOC, MCHO, TCHO, DFAA and TAA concentrations were high in the upper reach of the Pearl River estuary and decreased rapidly downstream. Anthropogenic sewage input appeared to be an important source of the DOC pool in the upper estuary. DOC distribution was non-conservative during the estuarine mixing, showing a net consumption of DOC in the upper reach and in the low salinity (S<20) region of the Pearl River estuary. Changes in the relative compositions of carbohydrates (MCHO vs. PCHO) and amino acids (DFAA vs. DCAA) along the salinity gradient further indicated that different processes (biodegradation, flocculation, and phytoplankton production) had different influences on distributions of organic compound classes in this estuarine system. Our one-month incubation experiment further revealed that a substantial portion (15-45%) of DOC from the estuary was biodegradable. Bacterial respiration rates were much higher (0.12-5.8 mu mol O-2 L-1 h(-1)) than the DOC consumption rates, suggesting that there were other oxygen consumption processes, such as nitrification besides the aerobic respiration of organic matter in the Pearl River estuary, as inferred by the distribution of NH4+ and NO3-. We estimated that 5.3 x 10(8) g C d(-1) of DOC can be exported out from the Lingdingyang Bay (a major subestuary of the Pearl River estuary) to the continental shelf of the South China Sea during this low flow season. (C) 2009 Elsevier B.V. All rights reserved.Natural Science Foundation of China [40576036, 90711005, 40821063

Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005

Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens

Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

Plague, caused by Yersinia pestis, is one of the oldest and most dangerous diseases in human history, and has claimed millions of lives in the three major historical pandemics. Although panic caused by the Black Death is fading, the threat of the reemergence of plague pandemics still exists, with the additional potential of misuse in biowarfare or bioterrorism. Rapid on-site detection and identification of the pathogen is of paramount significance for timely implementation of effective countermeasures. TaqMan probe-based real-time PCR assays can give quick and accurate identification; however, the need for cold delivery and storage prevents its potential on-site application. The objective of this study was to develop a stable PCR system for easy delivery and storage under room temperature, which is vital for conventional plague surveillance and for preparedness in public health emergencies. We present a solution to this particular issue, hoping that it is helpful to future applications

5-Fluorouracil Treatment Alters the Efficiency of Translational Recoding

5-fluorouracil (5-FU) is a chemotherapeutic agent that has been extensively studied since its initial development in the 1950s. It has been suggested that the mechanism of action of 5-FU involves both DNA- and RNA-directed processes, but this has remained controversial. In this study, using a series of in vivo reporter constructs capable of measuring translational recoding, we demonstrate that cells exposed to 5-FU display a reduced capacity to engage in a variety of translational recoding events, including +1 programmed frameshifting (PRF) and −1 PRF. In addition, 5-FU-treated cells are much less accurate at stop codon recognition, resulting in a significant increase in stop codon-readthrough. Remarkably, while the efficiency of cap-dependent translation appears to be unaffected by 5-FU, 5-FU-treated cells display a decreased ability to initiate cap-independent translation. We further show that knockdown of thymidylate synthase, an enzyme believed to be at the center of 5-FU-induced DNA damage, has no effect on the observed alterations in translational recoding. On the other hand, ribosomal RNA (rRNA) pseudouridylation, which plays an important role in translational recoding, is significantly inhibited. Taken together, our results suggest that the observed effect of 5-FU on recoding is an RNA-directed effect. Our results are the first to show definitely and quantitatively that translational recoding is affected by exposure to 5-FU. Thus, it is possible that a substantial portion of 5-FU cytotoxicity might possibly be the result of alterations in translational recoding efficiency

Automated Denudation of Oocytes

Denudation is a technique for removal of the cumulus cell mass from oocytes in clinical intracytoplasmic sperm injection (ICSI). Manual oocyte denudation requires long training hours and stringent skills, but still suffers from low yield rate and denudation efficiency due to human fatigue and skill variations across operators. To address these limitations, this paper reports a robotic system for automated oocyte denudation. In this system, several key techniques are proposed, including a vision-based contact detection method for measuring the relative z position between the micropipette tip and the dish substrate, recognition of oocytes and the surrounding cumulus cells, automated calibration algorithm for eliminating the misalignment angle, and automated control of the flow rate based on the model of oocyte dynamics during micropipette aspiration and deposition. Experiments on mouse oocytes demonstrated that the robotic denudation system achieved a high yield rate of 97.0 ± 2.8% and denudation efficiency of 95.0 ± 0.8%. Additionally, oocytes denuded by the robotic system showed comparable fertilization rate and developmental competence compared with manual denudation. Our robotic denudation system represents one step towards the automation and standardization of ICSI procedures

A Novel Stick-Slip Nanopositioning Stage Integrated with a Flexure Hinge-Based Friction Force Adjusting Structure

The piezoelectrically-actuated stick-slip nanopositioning stage (PASSNS) has been applied extensively, and many designs of PASSNSs have been developed. The friction force between the stick-slip surfaces plays a critical role in successful movement of the stage, which influences the load capacity, dynamic performance, and positioning accuracy of the PASSNS. Toward solving the influence problems of friction force, this paper presents a novel stick-slip nanopositioning stage where the flexure hinge-based friction force adjusting unit was employed. Numerical analysis was conducted to estimate the static performance of the stage, a dynamic model was established, and simulation analysis was performed to study the dynamic performance of the stage. Further, a prototype was manufactured and a series of experiments were carried out to test the performance of the stage. The results show that the maximum forward and backward movement speeds of the stage are 1 and 0.7 mm/s, respectively, and the minimum forward and backward step displacements are approximately 11 and 12 nm, respectively. Compared to the step displacement under no working load, the forward and backward step displacements only increase by 6% and 8% with a working load of 20 g, respectively. And the load capacity of the PASSNS in the vertical direction is about 72 g. The experimental results confirm the feasibility of the proposed stage, and high accuracy, high speed, and good robustness to varying loads were achieved. These results demonstrate the great potential of the developed stage in many nanopositioning applications