Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

A structural basis for integrin activation by the cytoplasmic tail of the αIIb-subunit

A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short α- and/or β-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the αIIb-subunit and of a mutant αIIb-cytoplasmic tail that renders platelet integrin αIIbβ3 constitutively active. The structure of the wild-type αIIb-cytoplasmic tail reveals a “closed” conformation where the highly conserved N-terminal membrane-proximal region forms an α-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an “open” conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed αIIbβ3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based “on/off switch” model for integrin activation.

Activation-enhanced αIIbβ3-Integrin–Cytoskeleton Interactions Outside of Focal Contacts Require the α-Subunit

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin–cytoskeleton interactions of β2 integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin–cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of αIIbβ3-integrin, in which activation results in a large conformational change. Direct activation of αIIbβ3 by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin–cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both α- and β-cytoplasmic domains were required for these links. This suggests that 1) both β2- and β3-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin–cytoskeleton interactions; and 3) the role of the α-subunit in integrin–cytoskeleton interactions in at least some circumstances is more direct than generally supposed.

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley1

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)8-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Electronic tendering: an industry perspective

The construction industry is categorised as being an information-intensive industry and described as one of the most important industries in any developed country, facing a period of rapid and unparalleled change (Industry Science Resources 1999) (Love P.E.D., Tucker S.N. et al. 1996). Project communications are becoming increasingly complex, with a growing need and fundamental drive to collaborate electronically at project level and beyond (Olesen K. and Myers M.D. 1999; Thorpe T. and Mead S. 2001; CITE 2003). Yet, the industry is also identified as having a considerable lack of knowledge and awareness about innovative information and communication technology (ICT) and web-based communication processes, systems and solutions which may prove beneficial in the procurement, delivery and life cycle of projects (NSW Government 1998; Kajewski S. and Weippert A. 2000). The Internet has debatably revolutionised the way in which information is stored, exchanged and viewed, opening new avenues for business, which only a decade ago were deemed almost inconceivable (DCITA 1998; IIB 2002). In an attempt to put these ‘new avenues of business’ into perspective, this report provides an overall ‘snapshot’ of current public and private construction industry sector opportunities and practices in the implementation and application of web-based ICT tools, systems and processes (e-Uptake). Research found that even with a reserved uptake, the construction industry and its participating organisations are making concerted efforts (fortunately with positive results) in taking up innovative forms of doing business via the internet, including e-Tendering (making it possible to manage the entire tender letting process electronically and online) (Anumba C.J. and Ruikar K. 2002; ITCBP 2003). Furthermore, Government (often a key client within the construction industry),and with its increased tendency to transact its business electronically, undoubtedly has an effect on how various private industry consultants, contractors, suppliers, etc. do business (Murray M. 2003) – by offering a wide range of (current and anticipated) e-facilities / services, including e-Tendering (Ecommerce 2002). Overall, doing business electronically is found to have a profound impact on the way today’s construction businesses operate - streamlining existing processes, with the growth in innovative tools, such as e-Tender, offering the construction industry new responsibilities and opportunities for all parties involved (ITCBP 2003). It is therefore important that these opportunities should be accessible to as many construction industry businesses as possible (The Construction Confederation 2001). Historically, there is a considerable exchange of information between various parties during a tendering process, where accuracy and efficiency of documentation is critical. Traditionally this process is either paper-based (involving large volumes of supporting tender documentation), or via a number of stand-alone, non-compatible computer systems, usually costly to both the client and contractor. As such, having a standard electronic exchange format that allows all parties involved in an electronic tender process to access one system only via the Internet, saves both time and money, eliminates transcription errors and increases speed of bid analysis (The Construction Confederation 2001). Supporting this research project’s aims and objectives, researchers set to determine today’s construction industry ‘current state-of-play’ in relation to e-Tendering opportunities. The report also provides brief introductions to several Australian and International e-Tender systems identified during this investigation. e-Tendering, in its simplest form, is described as the electronic publishing, communicating, accessing, receiving and submitting of all tender related information and documentation via the internet, thereby replacing the traditional paper-based tender processes, and achieving a more efficient and effective business process for all parties involved (NT Governement 2000; NT Government 2000; NSW Department of Commerce 2003; NSW Government 2003). Although most of the e-Tender websites investigated at the time, maintain their tendering processes and capabilities are ‘electronic’, research shows these ‘eTendering’ systems vary from being reasonably advanced to more ‘basic’ electronic tender notification and archiving services for various industry sectors. Research also indicates an e-Tender system should have a number of basic features and capabilities, including: • All tender documentation to be distributed via a secure web-based tender system – thereby avoiding the need for collating paperwork and couriers. • The client/purchaser should be able to upload a notice and/or invitation to tender onto the system. • Notification is sent out electronically (usually via email) for suppliers to download the information and return their responses electronically (online). • During the tender period, updates and queries are exchanged through the same e-Tender system. • The client/purchaser should only be able to access the tenders after the deadline has passed. • All tender related information is held in a central database, which should be easily searchable and fully audited, with all activities recorded. • It is essential that tender documents are not read or submitted by unauthorised parties. • Users of the e-Tender system are to be properly identified and registered via controlled access. In simple terms, security has to be as good as if not better than a manual tender process. Data is to be encrypted and users authenticated by means such as digital signatures, electronic certificates or smartcards. • All parties must be assured that no 'undetected' alterations can be made to any tender. • The tenderer should be able to amend the bid right up to the deadline – whilst the client/purchaser cannot obtain access until the submission deadline has passed. • The e-Tender system may also include features such as a database of service providers with spreadsheet-based pricing schedules, which can make it easier for a potential tenderer to electronically prepare and analyse a tender. Research indicates the efficiency of an e-Tender process is well supported internationally, with a significant number, yet similar, e-Tender benefits identified during this investigation. Both construction industry and Government participants generally agree that the implementation of an automated e-Tendering process or system enhances the overall quality, timeliness and cost-effectiveness of a tender process, and provides a more streamlined method of receiving, managing, and submitting tender documents than the traditional paper-based process. On the other hand, whilst there are undoubtedly many more barriers challenging the successful implementation and adoption of an e-Tendering system or process, researchers have also identified a range of challenges and perceptions that seem to hinder the uptake of this innovative approach to tendering electronically. A central concern seems to be that of security - when industry organisations have to use the Internet for electronic information transfer. As a result, when it comes to e-Tendering, industry participants insist these innovative tendering systems are developed to ensure the utmost security and integrity. Finally, if Australian organisations continue to explore the competitive ‘dynamics’ of the construction industry, without realising the current and future, trends and benefits of adopting innovative processes, such as e-Tendering, it will limit their globalising opportunities to expand into overseas markets and allow the continuation of international firms successfully entering local markets. As such, researchers believe increased knowledge, awareness and successful implementation of innovative systems and processes raises great expectations regarding their contribution towards ‘stimulating’ the globalisation of electronic procurement activities, and improving overall business and project performances throughout the construction industry sectors and overall marketplace (NSW Government 2002; Harty C. 2003; Murray M. 2003; Pietroforte R. 2003). Achieving the successful integration of an innovative e-Tender solution with an existing / traditional process can be a complex, and if not done correctly, could lead to failure (Bourn J. 2002).

Mining a data reasoning model for personalized text classification

It is a big challenge to acquire correct user profiles for personalized text classification since users may be unsure in providing their interests. Traditional approaches to user profiling adopt machine learning (ML) to automatically discover classification knowledge from explicit user feedback in describing personal interests. However, the accuracy of ML-based methods cannot be significantly improved in many cases due to the term independence assumption and uncertainties associated with them. This paper presents a novel relevance feedback approach for personalized text classification. It basically applies data mining to discover knowledge from relevant and non-relevant text and constraints specific knowledge by reasoning rules to eliminate some conflicting information. We also developed a Dempster-Shafer (DS) approach as the means to utilise the specific knowledge to build high-quality data models for classification. The experimental results conducted on Reuters Corpus Volume 1 and TREC topics support that the proposed technique achieves encouraging performance in comparing with the state-of-the-art relevance feedback models.

Vaccination strategies for the prevention of cervical cancer

Infection with high-risk human papillomaviruses (HPVs) is an essential step in the multistep process leading to cervical cancer. There are approximately 120 different types of HPV identified: of these, 18 are high-risk types associated with cervical cancer, with HPV-16 being the dominant type in most parts of the world. The major capsid protein of papillomavirus, produced in a number of expression systems, self assembles to form virus-like particles. Virus-like particles are the basis of the first generation of HPV vaccines presently being tested in clinical trials. Virus-like particles are highly immunogenic and afford protection from infection both in animal models and in Phase IIb clinical trials. A number of Phase III trials are in progress to determine if the vaccine will protect against cervical disease and, in some cases, genital warts. However, it is predicted that these vaccines will be too expensive for the developing world, where they are desperately needed. Another problem is that they will be type specific. Novel approaches to the production of virus-like particles in plants, second-generation vaccine approaches including viral and bacterial vaccine vectors and DNA vaccines, as well as different routes of immunization, are also reviewed. © 2005 Future Drugs Ltd.

Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other south pacific islands

BACKGROUND: Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP. METHODOLOGY/PRINCIPAL FINDINGS: Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009-2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III. CONCLUSIONS/SIGNIFICANCE: This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.

Overexpression of the mammalian target of rapamycin (mTOR) and angioinvasion are poor prognostic factors in early stage NSCLC: A verification study

Background: A recent study by Dhillon et al. [12], identified both angioinvasion and mTOR as prognostic biomarkers for poor survival in early stage NSCLC. The aim of this study was to verify the above study by examining the angioinvasion and mTOR expression profile in a cohort of early stage NSCLC patients and correlate the results to patient clinico-pathological data and survival. Methods: Angioinvasion was routinely recorded by the pathologist at the initial assessment of the tumor following resection. mTOR was evaluated in 141 early stage (IA-IIB) NSCLC patients (67 - squamous; 60 - adenocarcinoma; 14 - others) using immunohistochemistry (IHC) analysis with an immunohistochemical score (IHS) calculated (% positive cells × staining intensity). Intensity was scored as follows: 0 (negative); 1+ (weak); 2+ (moderate); 3+ (strong). The range of scores was 0-300. Based on the previous study a cut-off score of 30 was used to define positive versus negative patients. The impact of angioinvasion and mTOR expression on prognosis was then evaluated. Results: 101 of the 141 tumors studied expressed mTOR. There was no difference in mTOR expression between squamous cell carcinoma and adenocarcinoma. Angioinvasion (p= 0.024) and mTOR staining (p= 0.048) were significant univariate predictors of poor survival. Both remained significant after multivariate analysis (p= 0.037 and p= 0.020, respectively). Conclusions: Our findings verify angioinvasion and mTOR expression as new biomarkers for poor outcome in patients with early stage NSCLC. mTOR expressing patients may benefit from novel therapies targeting the mTOR survival pathway. © 2011 Elsevier Ireland Ltd.

Increased generation of platelet-derived microparticles following percutaneous transluminal coronary angioplasty

Platelet-derived microparticles (PMPs) which are produced during platelet activation contribute to coagulation1 and bind to traumatized endothelium in an animal model2. Such endothelial injury occurs during percutaneous transluminal coronary angioplasty (PTCA), a procedure which restores the diameter of occluded coronary arteries using balloon inflations. However, re-occlusions subsequently develop in 20-25% of patients3, although this is limited by treatment with anti-platelet glycoprotein IIb/IIIa receptor drugs such as abciximab4. However, abciximab only partially decreases the need for revascularisation5, and therefore other mechanisms appear to be involved. As platelet activation occurs during PTCA, it is likely that PMPs may be produced and contribute to restenosis. This study population consisted of 113 PTCA patients, of whom 38 received abciximab. Paired peripheral arterial blood samples were obtained from the PTCA sheath: 1) following heparinisation (baseline); and 2) subsequent to all vessel manipulation (post-PTCA). Blood was prepared with an anti-CD61 (glycoprotein IIIa) fluorescence conjugated antibody to identify PMPs using flow cytometry, and PMP results expressed as a percentage of all CD61 events. The level of PMPs increased significantly from baseline following PTCA in the without abciximab group (paired t test, P=0.019). However, there was no significant change in the level of PMPs following PTCA in patients who received abciximab. Baseline clinical characteristics between patient groups were similar, although patients administered abciximab had more complex PTCA procedures, such as increased balloon inflation pressures (ANOVA, P=0.0219). In this study, we have clearly demonstrated that the level of CD61-positive PMPs increased during PTCA. This trend has been demonstrated previously, although a low sample size prevented statistical significance being attained6. The results of our work also demonstrate that there was no increase in PMPs after PTCA with abiciximab treatment. The increased PMPs may adhere to traumatized endothelium, contributing to re-occlusion of the arteries, but this remains to be determined. References: (1) Holme PA, Brosstad F, Solum NO. Blood Coagulation and Fibrinolysis. 1995;6:302-310. (2) Merten M, Pakala R, Thiagarajan P, Benedict CR. Circulation. 1999;99:2577-2582. (3) Califf RM. American Heart Journal.1995;130:680-684. (4) Coller BS, Scudder LE. Blood. 1985;66:1456-1459. (5) Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT on behalf of the EPIC investigators. Lancet. 1994;343:881-886. (6) Scharf RE, Tomer A, Marzec UM, Teirstein PS, Ruggeri ZM, Harker LA. Arteriosclerosis and Thrombosis. 1992;12:1475-87.