3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Absence of intersystem crossing in 1,4-didehydrobenzene

The rate of singlet-to-triplet intersystem crossing in 1,4-didehydrobenzene (the biradical produced as a reactive intermediate in the thermal cycloaromatization of enediynes), cannot be increased by the application of an external magnetic field. The rate of product formation and the distribution of stable products of 2,3-di-n-propyl-1,4-didehydrobenzene thermolysis is unchanged at magnetic flux densities in the range 0–2000 G and at 66 000 G. Similarly, the rate of thermolysis of an unsymmetrical enediyne is insensitive to magnetic field flux in the same range. This finding precludes the modulation of enediyne reaction rates in pharmaceutical and synthetic pursuits.

Alkyl radical geometry controls geminate cage recombination in alkylcobalamins

Abstract: The radical pair that results from photolysis of adenosylcob(II1)alamin (AdoCbl"') undergoes primary geminate recombination with a first-order rate constant of 1 x lo9 s-l. In contrast, methylcob(II1)alamin (CH3Cbl"') and aristeromicylcob(II1)alamin (AriCblII', the carbocyclic analogue of AdoCbl"' in which the ribofuranose ring oxygen has been replaced with a methylene group) does not undergo primary geminate recombination. The ribofwanose group enables a high rate of geminate recombination in the [Ado' Cbl"'] radical pair. This may be due to a stereoelectronic (p-anomeric) effect that maintains a pyramidal geometry at the 5'-carbon of the 5'-deoxyadenosyl radical, or it may be due to hindered rotation about the C4t-C5, bond such that /?-elimination to the olefin is prevented. Recombination in the geminate singlet radical pair is in competition with diffusive escape to form a solvent-separated radical pair. Hyperfine coupling from Co" promotes intersystem crossing to the triplet radical pair (Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. SOC. 1993, 115, 12152). Recombination of the [CH3' Cbl"] radical pair is not prevented by a lack of intersystem crossing, as neither unlabeled or I3C-labeled CH3Cbl"' undergoes geminate recombination. There is only a small difference in the rate of diffusive recombination in the solvent cage for AdoCbl"', AriCbl"', and CH3Cbl"' following photolysis: 2.01 x 10" s-l, 2.20 x lo4 s-l, and 1.16 x lo4 s-l. The rate of diffusive recombination is limited by productive collisions and not by radical geometry or intersystem crossing. The CF3' radical that results from photolysis of (trifluoromethyl)cob(III)alamin (CF3Cbl"') maintains its pyramidal geometry and undergoes faster diffusive recombination in the solvent cage at 51 x lo4 s-l. The C-Co bond dissociation enthalpy in AriCbl"' is 37 f 1.4 kcaymol. The profound difference in geminate recombination rates for AdoCbl"' and CH3Cbl"' is consistent with their different biological roles as enzymatic cofactors: AdoCbl"' is an initiator of radical chain chemistry in the active site, whereas CH3Cbl"' is a methyl group donor in an S~2-type process.

The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C virus (HCV ) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site(IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

One-year delayed effect of fog on malaria transmission : a time-series analysis in the rain forest area of Mengla County, south-west China

Background: Malaria is a major public health burden in the tropics with the potential to significantly increase in response to climate change. Analyses of data from the recent past can elucidate how short-term variations in weather factors affect malaria transmission. This study explored the impact of climate variability on the transmission of malaria in the tropical rain forest area of Mengla County, south-west China. Methods: Ecological time-series analysis was performed on data collected between 1971 and 1999. Auto-regressive integrated moving average (ARIMA) models were used to evaluate the relationship between weather factors and malaria incidence. Results: At the time scale of months, the predictors for malaria incidence included: minimum temperature, maximum temperature, and fog day frequency. The effect of minimum temperature on malaria incidence was greater in the cool months than in the hot months. The fog day frequency in October had a positive effect on malaria incidence in May of the following year. At the time scale of years, the annual fog day frequency was the only weather predictor of the annual incidence of malaria. Conclusion: Fog day frequency was for the first time found to be a predictor of malaria incidence in a rain forest area. The one-year delayed effect of fog on malaria transmission may involve providing water input and maintaining aquatic breeding sites for mosquitoes in vulnerable times when there is little rainfall in the 6-month dry seasons. These findings should be considered in the prediction of future patterns of malaria for similar tropical rain forest areas worldwide.

Accountable-eHealth systems : the next step forward for privacy

EHealth systems promise enviable benefits and capabilities for healthcare. But, the technologies that make these capabilities possible brings with them undesirable drawback such as information security related threats which need to be appropriately addressed. Lurking in these threats are patient privacy concerns. Fulfilling these privacy concerns have proven to be difficult since they often conflict with information requirements of care providers. It is important to achieve a proper balance between these requirements. We believe that information accountability can achieve this balance. In this paper we introduce accountable-eHealth systems. We will discuss how our designed protocols can successfully address the aforementioned requirement. We will also compare characteristics of AeH systems with Australia’s PCEHR system and identify similarities and highlight the differences and the impact those differences would have to the eHealth domain.

Legal issues related to Accountable-eHealth systems in Australia

Information privacy requirements of patients and information requirements of healthcare providers (HCP) are competing concerns. Reaching a balance between these requirements have proven difficult but is crucial for the success of eHealth systems. The traditional approaches to information management have been preventive measures which either allow or deny access to information. We believe that this approach is inappropriate for a domain such as healthcare. We contend that introducing information accountability (IA) to eHealth systems can reach the aforementioned balance without the need for rigid information control. IA is a fairly new concept to computer science, hence; there are no unambiguously accepted principles as yet. But the concept delivers promising advantages to information management in a robust manner. Accountable-eHealth (AeH) systems are eHealth systems which use IA principles as the measure for privacy and information management. AeH systems face three main impediments; technological, social and ethical and legal. In this paper, we present the AeH model and focus on the legal aspects of AeH systems in Australia. We investigate current legislation available in Australia regarding health information management and identify future legal requirements if AeH systems are to be implemented in Australia.

A duty of care

Mass production of PhD training compromises graduate quality. As PhD quality becomes more stratified, industry will increasingly turn to quality-branded institutions and programs when distinguishing among job candidates.

The role of human factors when evaluating information accountability for eHealth systems

The availability of health information is rapidly increasing; its expansion and proliferation is inevitable. At the same time, breeding of health information silos is an unstoppable and relentless exercise. Information security and privacy concerns are therefore major barriers in the eHealth socio-eco system. We proposed Information Accountability as a measurable human factor that should eliminate and mitigate security concerns. Information accountability measures would be practicable and feasible if legislative requirements are also embedded. In this context, information accountability constitutes a key component for the development of effective information technology requirements for health information system. Our conceptual approach to measuring human factors related to information accountability in eHealth is presented in this paper with some limitations.

The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Do RNA viruses require genome cyclisation for replication?

Complementary sequences at the 5′ and 3′ ends of the dengue virus RNA genome are essential for viral replication, and are believed to cyclise the genome through long-range base pairing in cis. Although consistent with evidence in the literature, this view neglects possible biologically active multimeric forms that are equally consistent with the data. Here, we propose alternative multimeric structures, and suggest that multigenome noncovalent concatemers are more likely to exist under cellular conditions than single cyclised monomers. Concatemers provide a plausible mechanism for the dengue virus to overcome the single-stranded (+)-sense RNA virus dilemma, and can potentially assist genome transport from the virus-induced vesicles into the cytosol.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

Investigation of ultra trace detection in functionalised-surface enhanced Raman spectroscopy (SERS)

We have developed an explanation for ultra trace detection found when using Au/Ag SERS nanoparticles linked to biochemical affinity tags, e.g. antibodies. The nanoparticle structure is not as usually assumed and the aggregated nanoparticles constitute hot spots that are indispensable for these very low levels of analyte detection, even more so when using a direct detection method.

Accountable-eHealth systems : the next step forward for privacy

eHealth systems promise enviable benefits and capabilities for healthcare delivery. However, the technologies that make these capabilities possible introduce undesirable drawbacks such as information security related threats, which need to be appropriately addressed. Lurking in these threats are information privacy concerns. Addressing them has proven to be difficult because they often conflict with information access requirements of healthcare providers. Therefore, it is important to achieve an appropriate balance between these requirements. We contend that information accountability (IA) can achieve this balance. In this paper, we introduce accountable-eHealth (AeH) systems, which are eHealth systems that utilise IA as a measure of information privacy. We discuss how AeH system protocols can successfully achieve the aforementioned balance of requirements. As a means of implementation feasibility, we compare characteristics of AeH systems with Australia’s Personally Controlled Electronic Health Record (PCEHR) sys-tem and identify similarities and highlight the differences and the impact those differences would have to the eHealth domain.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.