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Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5’-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity, and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4 resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Inosine-5′-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.

Metabolic pathways are frequently transferred between bacterial strains in the environment through horizontal gene transfer (HGT), yet laboratory engineering to introduce new metabolic pathways often fails. Successful use of a pathway requires co-evolution of both pathway and host, and these interactions may be disrupted upon transfer to a new host. Here we show that two different pathways for catabolism of coumarate failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly-acquired pathways and the modifications required to overcome these limitations. Both pathways required mutations to the host to enable effective growth with coumarate, but the necessary mutations differed depending on the chemistry and intermediates of the pathways. In one case, an intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid mutations to IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is already resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. These discoveries will aid in our understanding of HGT and ability to predictably engineer metabolism.

A SpaceWire is a standard for on-board satellite networks as the basis for future data-handling architectures. However, it cannot meet the deterministic requirement for safety/time critical application in spacecraft, where the delay of real-time (RT) message streams must be guaranteed. Therefore, SpaceWire-D is developed that provides deterministic delivery over a SpaceWire network. Formal analysis and verification of real-time systems is critical to their development and safe implementation, and is a prerequisite for obtaining their safety certification. Failure to meet specified timing constraints such as deadlines in hard real-time systems may lead to catastrophic results. In this paper, a formal verification method, Real-Time Logic (RTL), has been proposed to specify and verify timing properties of SpaceWire-D network. Based on the principal of SpaceWire-D protocol, we firstly analyze the timing properties of fundamental transactions, such as RMAP WRITE, and RMAP READ. After that, the RMAP WRITE trans...

A SpaceWire is a standard for on-board satellite networks as the basis for future data-handling architectures. It is becoming more and more popular in space applications due to its technical advantages, including reliability, low power and fault protection, etc. High reliability is the vital issue for spacecraft. Therefore, it is very important to analyze and improve the reliability performance of the SpaceWire network. This paper deals with the problem of reliability modeling and analysis with SpaceWire network. According to the function division of distributed network, a reliability analysis method based on a task is proposed, the reliability analysis of every task can lead to the system reliability matrix, the reliability result of the network system can be deduced by integrating these entire reliability indexes in the matrix. With the method, we develop a reliability analysis tool for SpaceWire Network based on VC, where the computation schemes for reliability matrix and the multi-path-task reliabilit...

SpaceWire is a full-duplex, point-to-point, high-speed serial data transfer standard which is intended for use in further on-board spacecraft, space missions and applications. However, space environment is harsh and complex which requires a communication network with high reliability. It is inevitable that SpaceWire network link fault or SpaceWire equipment damage is able to change the SpaceWire network status. Therefore, traditional static routing configuration cannot efficiently solve the network fault recovery problem. In this paper, our research is divided into three parts. First of all, we design a simulation and test platform for SpaceWire network by using 7 SpaceWire communication nodes and 3 SpaceWire routers. This SpaceWire network platform is the foundation for the next work. Secondly, we introduce the SpaceWire routing protocol and Dijkstra algorithm principle. Thirdly, in order to improve the reliability of SpaceWire network and restore communication fault, we propose a SpaceWire network dynamic reconfiguration method based on Dijkstra algorithm. By analyzing the check information from SpaceWire router ports and communication nodes, it dynamically calculates optimal routing paths and reconfigures the SpaceWire network routers when there is a fault on SpaceWire network. Based on the simulation and test platform for SpaceWire network, we complete the software design and implementation of this method. Verification result demonstrates that the method can perform real-time network reconfiguration and path optimization when a SpaceWire network fault occurs, improve the ability of self-adaptive of SpaceWire network and enhance the reliability and transmit efficiency as well.

Understanding the driving factors of land-use spatio-temporal change is important for the guidance of rational land-use management. Based on land-use data, household surveys and social economic data in 2000, 2005, 2010, and 2015, this study adopted the Binary Logistic Regression Model (BLRM) to analyze the driving factors of land-use spatio-temporal change in a large artificial forest area in the Ximeng County, Yunnan province, in Southwest China. Seventeen factors were used to reflect the socio-economic and natural environment conditions in the study area. The results show a land use pattern composed of forestland, dry cropland, and rubber plantation in Ximeng County. Over the past fifteen years, the area of artificial forests increased rapidly due to the &ldquo, Grain for Green&rdquo, policy, which has led to increases in rubber plantations, tea gardens, eucalyptus forests, etc. In contrast, the area of natural forest and dry cropland decreased due to reclamations for farming and constructions. The BLRM approach helped to identify the main driving factors of land-use spatio-temporal change, which includes land-use policies (protection of basic farmlands and natural reserves), topography (elevation and slope), accessibility (distance to the human settlements), and potential productivity (fertility and irrigation). The study revealed the relationship between land-use spatio-temporal change and its driving factors in mountainous Southwest China, providing a decision-making basis for rational land-use management and optimal allocation of land resources.

Fast growth in information technology extends the means of data transfer. Especially in recent years, many more measures have been proposed in order to raise the data rate. SpaceWire is a serial, high-speed, bi-directional, full duplex, point-to-point data transfer protocol, which is defined by ESA (European Space Agency) in 2003. Wormhole routing adopted by SpaceWire reduces router buffer and makes SpaceWire routers suited to be designed as AISCs. However, blocking problem adhered to wormhole routing limits the network throughput seriously. This paper starts with an introduction to the SpaceWire standard. From the angle of ASIC design, the author deeply analyzes what SpaceWire does to improve router's performance in three aspects. After that, a VLSI design of Spacewire router compliant with the Spacewire standard is presented, and simulation result of this router is provided.

Mycoplasma pneumoniae (M. pneumoniae) is a major cause of respiratory tract infection. There is no specific and simple diagnosis test for the clinic treatment. In this study, we evaluated the effectiveness of a recently developed commercial rapid M. pneumoniae medium testing method.There were 132 patients with acute respiratory tract infection enrolled in this study. All of the patients' throat swabs were taken in the morning after admission and determined using the rapid identification culture test for M. pneumoniae. In addition the M. pneumoniae polymerase chain reaction (PCR) test and routine bacterial and fungal microorganism culture test were used to identify the organisms following culture. In addition, serum from each patient was tested for M. pneumoniae using the passive particle agglutination test.The rapid identification culture procedure indicated that 41 patients were M. pneumoniae positive, only 6 patients were M. pneumoniae positive based on PCR, and all of the positive cultures had fungus contamination. There were only 26 of the 41 patients whose serum M. pneumoniae tested positive. Passive particle agglutination test shows 38.6% of the patients were M. pneumoniae positive (51 out of 132). Compared with the passive particle agglutination test, the specificity, sensitivity of rapid M. pneumoniae identification culture method are 81.48% and 50.98%.The rapid identification of M. pneumoniae culture test is easy to perform and provides results fast, but has poor concordance with the M. pneumoniae PCR and serum M. pneumoniae antibody test due to the high fungus contamination. This commercial rapid M. pneumoniae medium method needs improvement for clinical use.