High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

Analysis of two authorization protocols using Colored Petri Nets

To prevent unauthorized access to protected trusted platform module (TPM) objects, authorization protocols, such as the object-specific authorization protocol (OSAP), have been introduced by the trusted computing group (TCG). By using OSAP, processes trying to gain access to the protected TPM objects need to prove their knowledge of relevant authorization data before access to the objects can be granted. Chen and Ryan’s 2009 analysis has demonstrated OSAP’s authentication vulnerability in sessions with shared authorization data. They also proposed the Session Key Authorization Protocol (SKAP) with fewer stages as an alternative to OSAP. Chen and Ryan’s analysis of SKAP using ProVerif proves the authentication property. The purpose of this paper was to examine the usefulness of Colored Petri Nets (CPN) and CPN Tools for security analysis. Using OSAP and SKAP as case studies, we construct intruder and authentication property models in CPN. CPN Tools is used to verify the authentication property using a Dolev–Yao-based model. Verification of the authentication property in both models using the state space tool produces results consistent with those of Chen and Ryan.

No genetic bottleneck in Plasmodium falciparum wild-type Pfcrt alleles reemerging in Hainan Island, China, following high-level chloroquine resistance

Chloroquine-resistant Plasmodium falciparum was highly prevalent in Hainan, China, in the 1970s. Twenty-five years after cessation of chloroquine therapy, the prevalence of P. falciparum wild-type Pfcrt alleles has risen to 36% (95% confidence interval, 22.1 to 52.4%). The diverse origins of wild-type alleles indicate that there was no genetic bottleneck caused by high chloroquine resistance.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Smart coordinated distribution system control to enable high level penetration of rooftop PVs

This thesis addresses voltage violation problem, the most critical issue associated with high level penetration of photovoltaic (PV) in electricity distribution network. A coordinated control algorithm using the reactive power from PV inverter and integrated battery energy storage has been developed and investigated in different network scenarios in the thesis. Probable variations associated with solar generation, end-user participation and network parameters are also considered. Furthermore, a unified data model and well-defined communication protocol to ensure the smooth coordination between all the components during the operation of the algorithm is described. Finally this thesis incorporated the uncertainties of solar generation using probabilistic load flow analysis.

Business process management as the 'killer app' for Petri nets

Since their inception in 1962, Petri nets have been used in a wide variety of application domains. Although Petri nets are graphical and easy to understand, they have formal semantics and allow for analysis techniques ranging from model checking and structural analysis to process mining and performance analysis. Over time Petri nets emerged as a solid foundation for Business Process Management (BPM) research. The BPM discipline develops methods, techniques, and tools to support the design, enactment, management, and analysis of operational business processes. Mainstream business process modeling notations and workflow management systems are using token-based semantics borrowed from Petri nets. Moreover, state-of-the-art BPM analysis techniques are using Petri nets as an internal representation. Users of BPM methods and tools are often not aware of this. This paper aims to unveil the seminal role of Petri nets in BPM.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Performance modelling of a fault-tolerant real-time multiprocessor using stochastic Petri nets

The fault-tolerant multiprocessor (ftmp) is a bus-based multiprocessor architecture with real-time and fault- tolerance features and is used in critical aerospace applications. A preliminary performance evaluation is of crucial importance in the design of such systems. In this paper, we review stochastic Petri nets (spn) and developspn-based performance models forftmp. These performance models enable efficient computation of important performance measures such as processing power, bus contention, bus utilization, and waiting times.

On the invariants of coloured Petri Nets

In this paper, we develop a theorem that enables computation of the place invariants of the union of a finite collection of coloured Petri Nets when the individual nets satisfy certain conditions and their invariants are known. We consider the illustrative examples of the Readers-Writers problem, a resource sharing system, and a network of databases and show how this theorem is a valuable tool in the analysis of concurrent systems.

High-level coordination and strategy in the 2007 equine influenza outbreak response.

The equine influenza (EI) outbreak presented many challenges that required high-level coordination and decision making, as well as the development of new approaches for satisfactory and consistent resolution. This paper outlines the elements of the national coordination arrangements, preparatory arrangements in place prior to the outbreak that facilitated national coordination, and some of the issues faced and resolved in the response.

The rph2 Gene Is Responsible for High Level Resistance to Phosphine in Independent Field Strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SRQLD), New South Wales (SRNSW) and South Australia (SRSA), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SRQLD and SRNSW. We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SRSA and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.

Implications of high level pseudogene transcription in Mycobacterium leprae

Background: The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. Results: Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational ``silencing'' of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple ``in-frame'' stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Conclusion: Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational ``silencing'' mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.

Graphical simulation of Petri Nets

An interactive graphics package for modeling with Petri Nets has been implemented. It uses the VT-11 graphics terminal supported on the PDP-11/35 computer to draw, execute, analyze, edit and redraw a Petri Net. Each of the above mentioned tasks can be performed by selecting appropriate items from a menu displayed on the screen. Petri Nets with a reasonably large number of nodes can be created and analyzed using this package. The number of nodes supported may be increased by making simple changes in the program. Being interactive, the program seeks information from the user after displaying appropriate messages on the terminal. After completing the Petri Net, it may be executed step by step and the changes in the number of tokens may be observed on the screen, at each place. Some properties of Petri Nets like safety, boundedness, conservation and redundancy can be checked using this package. This package can be used very effectively for modeling asynchronous (concurrent) systems with Petri Nets and simulating the model by “graphical execution.”

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Ordered Ports – a Language Concept for High-level Distributed Programming

A new language concept for high-level distributed programming is proposed. Programs are organised as a collection of concurrently executing processes. Some of these processes, referred to as liaison processes, have a monitor-like structure and contain ports which may be invoked by other processes for the purposes of synchronisation and communication. Synchronisation is achieved by conditional activation of ports and also through port control constructs which may directly specify the execution ordering of ports. These constructs implement a path-expression-like mechanism for synchronisation and are also equipped with options to provide conditional, non-deterministic and priority ordering of ports. The usefulness and expressive power of the proposed concepts are illustrated through solutions of several representative programming problems. Some implementation issues are also considered.