Regenerative silver nanoparticles for SERRS investigation of metmyoglobin with conserved heme pocket

Shell isolated silver nanoparticles with an ultrathin silica layer (Ag@SiO2NPs) are used as a surface-enhanced resonance Raman scattering (SERRS) substrate for probing metmyoglobin (metMb) in aqueous solution. The ultrathin silica layer protects metMb from reaching the bare silver surface and conserves the heme pocket during SERRS analysis with a Raman enhancement factor (EFSERS) of 4.78 × 104. In spite of the good SERRS enhancement, the interaction between the protein and Ag@SiO2NPs is weak enough to separate them by centrifugation in such a way that both are regenerated in their original form and can be reused. Using Ag@SiO2NPs as the SERRS substrate, the lowest detection limit of 2 nM was achieved for metMb whilst conserving the native structure of the heme centre.

Out-of-pocket expenditures for hospital care in Iran : who is at risk of incurring catastrophic payments?

Since the beginning of 1980s, the Iranian health care system has undergone several reforms designed to increase accessibility of health services. Notwithstanding these reforms, out-of-pocket payments which create a barrier to access health services contribute almost half of total health are financing in Iran. This study aimed to provide a greater understanding about the inequality and determinants of the out-of-pocket expenditure (OOPE) and the related catastrophic expenditure (CE) for hospital services in Iran using a nationwide survey data, the 2003 Utilisation of Health Services Survey (UHSS). The concentration index and the Heckman selection model were used to assess inequality and factors associated with these expenditures. Inequality analysis suggests that the CE is concentrated among households in lower socioeconomic levels. The results of the Heckman selection model indicate that factors such as length of stay, admission to a hospital owned by private sector or Ministry of Health and Medical Education, and living in remote areas are positively associated with higher OOPE. Results of the ordered-probit selection model demonstrate that length of stay, lower household wealth index, and admission to a private hospital are major factors contributing to the increase in the probability of CE. Also, we find that households living in East Azarbaijan, Kordestan and Sistan and Balochestan face a higher level of CE. Based on our findings, the current employer-sponsored health insurance system does not offer equal protection against hospital expenditure in Iran. It seems that a single universal health insurance scheme that covers health services for all Iranian—regardless of their employment status—can better protect households from catastrophic health spending.

Polly Pocket

Comprised of ten 3 minute and two 12 minutes animated episodes, featuring Polly Pockets and her friend in numerous adventures.

A practical guide to terminology for kimberlite facies: A systematic progression from descriptive to genetic, including a pocket guide

Kimberlite terminology remains problematic because both descriptive and genetic terms are mixed together in most existing terminology schemes. In addition, many terms used in existing kimberlite terminology schemes are not used in mainstream volcanology, even though kimberlite bodies are commonly the remains of kimberlite volcanic vents and edifices. We build on our own recently published approach to kimberlite facies terminology, involving a systematic progression from descriptive to genetic. The scheme can be used for both coherent kimberlite (i.e. kimberlite that was emplaced without undergoing any fragmentation processes and therefore preserving coherent igneous textures) and fragmental kimberlites. The approach involves documentation of components, textures and assessing the degree and effects of alteration on both components and original emplacement textures. This allows a purely descriptive composite component, textural and compositional petrological rock or deposit name to be constructed first, free of any biases about emplacement setting and processes. Then important facies features such as depositional structures, contact relationships and setting are assessed, leading to a composite descriptive and genetic name for the facies or rock unit that summarises key descriptive characteristics, emplacement processes and setting. Flow charts summarising the key steps in developing a progressive descriptive to genetic terminology are provided for both coherent and fragmental facies/deposits/rock units. These can be copied and used in the field, or in conjunction with field (e.g. drill core observations) and petrographic data. Because the approach depends heavily on field scale observations, characteristics and process interpretations, only the first descriptive part is appropriate where only petrographic observations are being made. Where field scale observations are available the progression from developing descriptive to interpretative terminology can be used, especially where some petrographic data also becomes available.

AITPM Email Newsletter Volume 0911, November 2009 : President's Message

President’s Report Hello fellow AITPM members, It is interesting to follow the news at present, where transport costs are getting a significant airing. Treasury Secretary Dr Ken Henry has enunciated something Australians may have considered extremely radical just a few years back, but in the present time appears to quite a few to be a realistic alternative. That being a rethink of the way we are charged for using our vehicles. It appears that serious consideration is being given to congestion charging, perhaps in place at least to some extent, of fuel excise. As a transport professional I am pleased that the debate has elevated to the national level, and would look forward that AITPM might contribute appropriately to it. As a motorist though, I naturally have my concerns about being hit in the hip pocket. Not that I actually drive during congested periods very much. I am fortunate to live five minutes either side of two well serviced bus corridors, one of which will eventually become a busway, and work in the central business district, which is hub from all spokes in Brisbane. As such, bus and foot are my preferred commute modes. Ah but I should not gloat, as my smart card fare is about to increase by 20 percent in the New Year! And if the newspapers are to be believed, further substantial increments are proposed over the coming few years. This is reported to recoup some more of the costs of actually providing the quality public transport system that we enjoy in our region. So I expect it will be very interesting to see how transport economics will play out in reality in the coming few years, and how governments cater to Australians who either cannot afford substantial increases in transport costs or have no viable alternatives to those facilities whose costs will increase. The 2010 AITPM National Conference, “What’s New?”, still has the opportunity for authors to submit an abstract for consideration so please consider how you might contribute to the event. Best regards to all, Jon Bunker

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.

AITPM national newsletter President's report May 2010

We’ve had a bit of sticker shock in these parts. Well, apparently. Since my last missive, Brisbane’s Clem Jones Tunnel which was initially free now has a toll, at least partially, at the introductory rate of $2.95 for a one-way car ride between 5a.m. and midnight – free overnight. From 9 May 2010 the toll will be $4.28. Since the introductory toll was introduced, use of the tunnel appears to have declined somewhat – no surprise to transport professionals I suppose. An additional factor may have been that the “novelty value” of driving through the tunnel for free had worn off. This demonstrates to me that much of the community may still see the use of road infrastructure as a rite of passage, with only some actually weighing up the true value of their travel time and vehicle wear and tear against their out of pocket (or onto credit card) cost. Thus, we’re in pioneering times and the role of transport economics in the overall transport infrastructure planning realm is of considerable importance – especially as much of the new big ticket infrastructure is likely to be tolled into the future. The Queensland Premier, Anna Bligh, made poignant commentary about Brisbane City Council’s tunnel use in that such infrastructure is built for future times and not just as a quick fix for current traffic problems. My expectation is that once Airport Link, which is really the northern half of the corridor, opens in 2012, there will be a significant spike in Clem7 usage.

What are the real costs of major trauma?

Background This economic evaluation reports the results of a detailed study of the cost of major trauma treated at Princess Alexandra Hospital (PAH), Australia. Methods A bottom-up approach was used to collect and aggregate the direct and indirect costs generated by a sample of 30 inpatients treated for major trauma at PAH in 2004. Major trauma was defined as an admission for Multiple Significant Trauma with an Injury Severity Score >15. Direct and indirect costs were amalgamated from three sources, (1) PAH inpatient costs, (2) Medicare Australia, and (3) a survey instrument. Inpatient costs included the initial episode of inpatient care including clinical and outpatient services and any subsequent representations for ongoing-related medical treatment. Medicare Australia provided an itemized list of pharmaceutical and ambulatory goods and services. The survey instrument collected out-of-pocket expenses and opportunity cost of employment forgone. Inpatient data obtained from a publically funded trauma registry were used to control for any potential bias in our sample. Costs are reported in Australian dollars for 2004 and 2008. Results The average direct and indirect costs of major trauma incurred up to 1-year postdischarge were estimated to be A$78,577 and A$24,273, respectively. The aggregate costs, for the State of Queensland, were estimated to range from A$86.1 million to $106.4 million in 2004 and from A$135 million to A$166.4 million in 2008. Conclusion These results demonstrate that (1) the costs of major trauma are significantly higher than previously reported estimates and (2) the cost of readmissions increased inpatient costs by 38.1%.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Frames of Anime: Culture and Image-Building

With the rising popularity of anime amongst animation students, audiences and scholars around the world, it has become increasingly important to critically analyse anime as being more than a ‘limited’ form of animation, and thematically as encompassing more than super robots and pocket monsters. Frames of Anime: Culture and Image-Building charts the development of Japanese animation from its indigenous roots within a native culture, through Japan’s experience of modernity and the impact of the Second World War. This text is the result of a rigorous study that recognises the heterogeneous and polymorphous background of anime. As such, Tze-Yue has adopted an ‘interdisciplinary and transnational’ (p. 7) approach to her enquiry, drawing upon face-to-face interviews, on-site visits and biographical writings of animators. Tze-Yue delineates anime from other forms of animation by linking its visual style to pre-modern Japanese art forms and demonstrating the connection it shares with an indigenous folk system of beliefs. Via the identification of traditional Japanese art forms and their visual connectedness to Japanese animation, Tze-Yue shows that the Japanese were already heavily engaged in what was destined to become anime once technology had enabled its production. Tze-Yue’s efforts to connect traditional Japanese art forms, and their artistic elements, to contemporary anime reveals that the Japanese already had a rich culture of visual storytelling that pre-dates modern animation. She identifies the Japanese form of the magic lantern at the turn of the 19th century, utsushi-e, as the pre-modern ancestor of Japanese animation, describing it as ‘Edo anime’ (p. 43). Along with utsushi-e, the Edo period also saw the woodblock print, ukiyo-e, being produced for the rising middle class (p. 32). Highlighting the ‘resurfacing’ of ‘realist’ approaches to Japanese art in ukiyo-e, Tze-Yue demonstrates the visual connection of ukiyo-e and anime in the …

Adaptive evolution of color vision as seen through the eyes of butterflies

Butterflies and primates are interesting for comparative color vision studies, because both have evolved middle- (M) and long-wavelength- (L) sensitive photopigments with overlapping absorbance spectrum maxima (lambda(max) values). Although positive selection is important for the maintenance of spectral variation within the primate pigments, it remains an open question whether it contributes similarly to the diversification of butterfly pigments. To examine this issue, we performed epimicrospectrophotometry on the eyes of five Limenitis butterfly species and found a 31-nm range of variation in the lambda(max) values of the L-sensitive photopigments (514-545 nm). We cloned partial Limenitis L opsin gene sequences and found a significant excess of replacement substitutions relative to polymorphisms among species. Mapping of these L photopigment lambda(max) values onto a phylogeny revealed two instances within Lepidoptera of convergently evolved L photopigment lineages whose lambda(max) values were blue-shifted. A codon-based maximum-likelihood analysis indicated that, associated with the two blue spectral shifts, four amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) have evolved substitutions in parallel and exhibit significant d(N)/d(S) >1. Homology modeling of the full-length Limenitis arthemis astyanax L opsin placed all four substitutions within the chromophore-binding pocket. Strikingly, the Ser137Ala substitution is in the same position as a site that in primates is responsible for a 5- to 7-nm blue spectral shift. Our data show that some of the same amino acid sites are under positive selection in the photopigments of both butterflies and primates, spanning an evolutionary distance >500 million years.