Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

An Improved Synthetic Route to the Potent Angiogenesis Inhibitor Benzyl Manα(1→3)-Manα(1→3)-Manα(1→3)-Manα(1→2)-Man Hexadecasulfate

An improved synthetic route to α(1→3)/α(1→2)-linked mannooligosaccharides has been developed and applied to a more efficient preparation of the potent anti-angiogenic sulfated pentasaccharide, benzyl Manα(1→3)-Manα(1→3)-Manα(1→3)-Manα(1→2)-Man hexadecasulfate, using only two monosaccharide building blocks. Of particular note are improvements in the preparation of both building blocks and a simpler, final deprotection strategy. The route also provides common intermediates for the introduction of aglycones other than benzyl, either at the building block stage or after oligosaccharide assembly. The anti-angiogenic activity of the synthesized target compound was confirmed via the rat aortic assay.

Ticagrelor, a platelet aggregation inhibitor for the potential prevention and treatment of arterial thrombosis and acute coronary syndromes

Ticagrelor is an orally active ADP P2Y12 receptor antagonist in development by AstraZeneca plc for the reduction of recurrent ischemic events in patients with acute coronary syndromes (ACS). Prior to the development of ticagrelor, thienopyridine compounds, such as clopidogrel, were the focus of research into therapies for ACS. Although the thienopyridines are effective platelet aggregation inhibitors, they are prodrugs and, consequently, exert a slow onset of action. In addition, the variability in inter-individual metabolism of thienopyridine prodrugs has been associated with reduced efficacy in some patients. Ticagrelor is not a prodrug and exhibits a more rapid onset of action than the thienopyridine prodrugs. In clinical trials conducted to date, ticagrelor was a potent inhibitor of ADP-induced platelet aggregation and demonstrated effects that were comparable to clopidogrel. In a phase II, short-term trial, the bleeding profile of participants treated with ticagrelor was similar to that obtained with clopidogrel; however, an increased incidence of dyspnea was observed - an effect that has not been reported with the thienopyridines. Considering the occurrence of dyspnea, and the apparent non-superiority of ticagrelor to clopidogrel, it is difficult to justify a clear benefit to the continued development of ticagrelor. Outcomes from an ongoing phase III trial comparing ticagrelor with clopidogrel in 18,000 patients with ACS are likely to impact on the future development of ticagrelor.

A hydrogen tethered inhibitor of matrix metalloproteinases

During wound repair, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs) is crucial for the normal extra cellular matrix turnover. However, the over expression of several MMPs including MMP-1, 2, 3, 8, 9 and MMP-10, combined with abnormally high levels of activation or low expression of TIMPs, may contribute to excessive degradation of connective tissue and formation of chronic ulcers. There are many groups exploring strategies for promoting wound healing involving delivery of growth factors, cells, ECM components and small molecules. Our approach for improving the balance of MMPs is not to add anything more to the wound, but instead to neutralise the over-expressed MMPs using inhibitors tethered to a bandage-like hydrogel. Our in vitro experiments using designed synthetic pseudo peptide inhibitors have been demonstrated to inhibit MMP activity in standard solutions. These inhibitors have also been tethered to polyethylene glycol hydrogels using a facile reaction between the linker unit on the inhibitor and the gel. After tethering the inhibition of MMPs diminishes to some extent and we postulate that this arises due to poor diffusion of the MMPs into the gels. When the tethered inhibitors were tested against chronic wound fluid obtained against patients we observed over 40% inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

A peptidomimetic inhibitor of matrix metalloproteinases containing a tetherable linker group

Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs). When over-expression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor have demonstrated inhibition of MMP-1, -2, -3 and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC50s which was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid we observed significant inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

The hedgehog pathway inhibitor GDC-0449 shows potential in skin and other cancers

Background: The hedgehog signaling pathway is vital in early development, but then becomes dormant, except in some cancer tumours. Hedgehog inhibitors are being developed for potential use in cancer. Objectives/Methods: The objective of this evaluation is to review the initial clinical studies of the hedgehog inhibitor, GDC-0449, in subjects with cancer. Results: Phase I trials have shown that GDC-0449 has benefits in subjects with metastatic or locally advanced basal-cell carcinoma and in one subjects with medulloblastoma. GDC-0449 was well tolerated. Conclusions: Long term efficacy and safety studies of GDC-0449 in these conditions and other solid cancers are now underway. These clinical trials with GDC-0449, and trials with other hedgehog inhibitors, will reveal whether it is beneficial and safe to inhibit the hedgehog pathway, in a wide range of solid tumours or not.

Does the p38 MAP kinase inhibitor pamapimod have potential for the treatment of rheumatoid arthritis?

Background: Methotrexate alone or in combination with other agents is the standard treatment for moderate-to-severe rheumatoid arthritis. As the biological agents are expensive, they are not usually used until methotrexate has failed to give a good response. Thus, there is scope for the development of cheaper drugs that can be used instead of methotrexate or in addition to methotrexate. Objectives/methods: Pamapimod is a p38α inhibitor being developed for use in the treatment of rheumatoid arthritis. The objective was to evaluate the recent clinical trials of pamapimod in subjects with rheumatoid arthritis. Results: There is no clear cut evidence that pamapimod alone or in the presence of methotrexate is efficacious in subjects with rheumatoid arthritis, but it does cause adverse effects. Conclusion: It is unlikely that pamapimod will be useful in the treatment of rheumatoid arthritis.

Reverse immigration

This book (256 pages, written in Korean) is a critical essay that reviews, questions, and criticises Korean and Eastern immigrants’ thinking and behaviour styles in Australia from their cultural perspectives, and discuss and proposes a creative cultural dimension for their better life in a multicultural context. Multiculturalism is not supportive of Eastern cultures because of individualistic collection of cultures, while transculturalism facilitates nurture of their culture in a community-oriented way within multicultural circumstances. Korean and Eastern immigrants, sharing oriental cultural systems and values, should approach to the Australian multicultural context with transculturalism which allows creating new cultural values in collaboration with and by participation into local communities. ------------------------------------------------------------ Many Eastern immigrants live in their own ethnic communities without or less interacting with Australian (communities). The author defines this phenomenon as “reverse immigration”. Reverse immigration refers to re-immigrating to their ethnic community in Australia or to their birth country despite they did not anticipate that this would happen to them before immigration to Australia. The author argues that Easterners’ collectivistic culture often devalues individuality and vice versa. Cultural clash between West and East often forces the immigrants to choose reverse immigration because of their lack of understanding of Western culture and their cultural characteristics such as low individuality, high power distance, and high uncertainty avoidance. For example, a vague boundary between individualist and collectivist in a collectivistic context (within their ethnic group) often leads to maladjustment to local communities and enhancement of cultural conservatism. The author proposes that the cultural clash can be overcome by cross-cultural activities named “transculturalism”. To Eastern immigrants, transculturalism can be achieved by acculturation of their two predominant cultures, the third-person perspective and generalised others. In a multicultural context, the former refers to the ability to share another person's feelings and emotions as if they were your own, and the latter does the ability to manage community and public expectations. When both cultural values are used for quality interactions between East and West, they allow Eastern immigrants to be more creative and critical and Australian to be more socially inclusive and culturally tolerant. With these discussions, the author discusses cultural differences throughout the book with four topics (chapters) and proposes transculturalism as a solution to the reverse immigration. ------------------------------------------------------------ Chapter 1 criticises Koreans’ attitudes and methods towards learning English that is less pragmatic and practical, but more likely to be a scholarly study. The author explains that Koreans’ non-pragmatic towards learning English has been firmly built based on their traditional systems and values that Koreans view English as a discipline and an aim of academic achievements rather than a means of communication. Within their cultural context, English can be perceived as more than a language, but something like vastly superior to their language and culture. Their collectivistic culture regards English as an unreachable and heterogeneous one that may threaten their cultural identity, so that “scholarly studying” is only the way to achieve (not learn) it. This discourages the immigrants to engage and involve in daily dialogues by “using” English as a second language. The author further advises the readers to be aware of Eastern collectivistic culture in communication and interaction that sometimes completely reverses private and public topics in a Western context. This leads them to feel that they have no content to talk to natives. ------------------------------------------------------------ Chapter 2 compares between Korea and Australia in terms of their educational systems and values, and proposes how Eastern overseas students can achieve critical and creative thinking within a Western educational setting. Interestingly, this chapter includes an explanation of why Eastern overseas students easily fail assessments including essay writing, oral presentations and discussions. One of the reasons the author explains is that Eastern students are not familiar to criticise others and think creatively, especially when they recognise that their words and ideas may harm the collectivistic harmony. Western educational systems focuses on enhancement of individuality such as self-confidence, self-esteem, and self-expression, while Eastern educational systems foster group-oriented values such as interpersonal relationship, and strong moral and spiritual values. Yet, the author argues that the collectivistic approach to criticism and creativity is often more critical and creative than Western individuals when they know what they are supposed to do for a group (or a community). Therefore, Eastern students need to think their cultural merits and demerits by using an individual perspective rather than generalised others’ perspective. The latter often discourages individual participation in a community, and the generalised others in a Western culture is weaker than Eastern. Furthermore, Western educational systems do not educate students to transform (loose) their individuality to fit into a group or a community. Rather they cultivate individuality for community prosperity. ------------------------------------------------------------ Chapter 3 introduces various cases of reverse immigration in workplaces that many immigrants return to their country or their ethnic community after many trials for acculturation. Reverse immigration is unexpected and not planned before immigration, so that its emotional embarrassment increases such severe social loneliness. Most Eastern immigrant workers have tried to adjust themselves in this new cultural environment at the early stages of immigration. However, their cultural features of collectivism, high power distance, high uncertainty avoidance, and long-term oriented cultures suppress individual initiative and eliminate the space for experiments in ways of acculturation. The author argues that returning to their ethnic community (physically and psychologically) leads to two significant problems: their distorted parenting and becoming more conservatives. The former leads the first generation of immigrants to pressure their children to pursue extrinsic or materialist values, such as financial success, fame and physical appearance, rather than on intrinsic values, while the latter refers to their isolated conservative characters because of their remoteness from the changes of their own country. The author also warns that their ethnic and religious groups actively strengthens immigrants’ social loneliness and systematically discourages immigrants’ interests and desire to be involving into local communities. The ethnic communities and leaders have not been interacting with Australian local communities and, as a result, are eager to conserve outdated cultural systems values. Even they have a tendency to weed out those people who wish to settle down within Australian local communities. They believe that those people can threaten their community’s survival and continuity. ------------------------------------------------------------ Chapter 4 titled multiculturalism argues that Korean and Eastern immigrants should more precisely understand Australia as a multicultural society in a way of collaboratively creating new cultural values. The author introduces multiculturalism with its definitions and history in Australia and argues the limitations of multiculturalism from an Easterner’s perspective. With well known tragedies of the second generations of U.S. immigrants, Cho Seung-Hui, a university student, massacred 32 people on the Virginia Tech before committing suicide and Hidal Hassan, an Army psychiatrist, killed 13 people at Fort Hood and the responses of ethnic community, the author explains that their mental illness may be derived from their parents’ (or ethnic group) culturally isolated attitude and socially static viewpoint of U.S. (Western system and values). The author insists that multiculturalism may restrict Eastern immigrants’ engagement and involvement in local communities. Multiculturalism has been systematically and historically developed based on Western systems and cultural values. In other words, multiculturalism requires high self-confidence and self-esteem that Eastern immigrants less prioritise them. It has been generally known that Easterners put more weight on human relationship than Westerners, but the author claims that this is not true. Within an individualistic culture, Westerners are more interested in building person-to-person connections and relationships. While Easterners are more interested in how individuals can achieve a sense of belonging within a group and a community. Therefore, multiculturalism is an ideology which forces Eastern immigrants to discard their strong desire to be part of a group and does not give a sense of belonging. In a consequence, the author advises that Eastern immigrants should aim towards “transculturalism” which allows them to actively participate in and contribute to their multicultural community. Transculturalism does not ask Easterners to discard their cultural values, but enables them to be a collectivistic individualist (a community leader) who is capable of developing new cultural values in a more creative and productive way. Furthermore, transculturalism encourages Western Australians in a multicultural context to collaborate with ethnic minorities to build a better community.

Reverse engineering and finite element analysis of a 3-wheeler automotive structure

Auto rickshaws (3-wheelers) are the most sought after transport among the urban and rural poor in India. The assembly of the vehicle involves assemblies of several major components. The L-angle is the component that connects the front panel with the vehicle floor. Current L-angle part has been observed to experience permanent deformation failure over period of time. This paper studies the effect of the addition of stiffeners on the L-angle to increase the strength of the component. A physical model of the L-angle was reversed engineered and modelled in CAD before static loading analysis were carried out on the model using finite element analysis. The modified L-angle fitted with stiffeners was shown to be able to withstand more load compare to previous design.

Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.