The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

Background: The high intracellular salt concentration re quired to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å. Results: The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion: The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface wasunexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions

Cell patterning on photolithographically defined parylene-C:SiO2 substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO(2 )wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO(2) regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology

White coat hypertension is associated with increased small vessel disease in the brain

Objective: Small vessel disease, as measured by white matter hyperintensity (WMH) in the brain, is known to be associated with increased stroke risk and cognitive impairment. This study explored the relationship between WMH on computerised tomography (CT) and white coat hypertension/effect (WCH/E) in patients with recent transient ischaemic attack (TIA) or lacunar stroke (LS). Design and method: Ninety-six patients recruited for the ASIST trial (Arterial Stiffness in Lacunar Stroke and TIA) underwent measurement of clinic blood pressure (BP) and ambulatory BP monitoring (APBM) within two weeks of TIA or LS. Twenty-three patients had normotension (clinic BP / = 140/90mmHg and day-time ABPM < 135/85mmHg). Arterial stiffness was measured using carotid-femoral pulse wave velocity (PWV) (Complior®, ALAM Medical) and carotid-ankle vascular index (CAVI) (VaSera VS-1500N®, Fukuda Denshi). CT images were scored for WMH using the four-point Fazekas visual rating scale. Patients were grouped into no-mild WMH (scores 0–1) or moderate-severe (scores 2–3) groups. The relationship between BP, vascular stiffness and WMH was explored with t-tests, chi-square and logistic regression accounting for known cardiovascular risk factors. Results: Forty-four percent of patients with WCH/E had moderate-severe WMH compared to 17% of normotensives (p = 0.047). The regression model with WMH as the dependent factor, and WCH/E and cardiovascular risk factors as independent factors showed WCH/E and either CAVI or PWV to be the only independent significant factor contributing to WMH (CAVI:p = 0.038, PWV:p = 0.043)

Defining the chromatin signature of inducible genes in T cells

BACKGROUND Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation. RESULTS To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation. CONCLUSIONS These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation

Shock Tube Study of the Thermal Conductivity of Argon

Analysis of end-wall thermal boundary layer behind reflected shock to determine thermal conductivity of argon over temperatures 3150 to 9225

Physiotherapy in upper abdominal surgery - what is current practice in Australia?

Background: Upper abdominal surgery (UAS) has the potential to cause post-operative pulmonary complications (PPCs). In the absence of high-quality research regarding post-operative physiotherapy management, consensus-based best practice guidelines formulated by Hanekom et al. (2012) are available to clinicians providing recommendations for post-UAS treatment. Such best practice guidelines have recommended that physiotherapists should be using early mobilisation and respiratory intervention to minimise risk of PPCs. However, recent evidence supports the implementation of mobilisation as a standalone treatment in PPC prevention, though the diversity in literature poses questions regarding ideal current practice. This project aimed to document and report the assessment measures and interventions physiotherapists are utilising following UAS, establishing whether current management is reflective of best practice guidelines and recent evidence. Results: An online survey was completed by 57 experienced Australian physiotherapists working with patients following UAS (35% survey response rate, 63% completion rate). On day one following UAS, when a patient’s condition is not medically limited, most physiotherapists routinely mobilise. Additionally, routine chest treatment continues to be implemented, with only 23% (n = 11/47) of physiotherapists mobilising patients without accompanying specific respiratory intervention. Variability of screening tools used to identify post-operative patients at high risk of PPC development was evident. Patient-dependent factors such as ‘fatigue’ and ‘non-compliance’ were among those identified as barriers to treatment, all influencing the commencement of treatment. Conclusions: Physiotherapists indicated that early mobilisation away from the bedside was the preferred post-operative treatment within the UAS patient population. Many continue to perform routine respiratory interventions despite recent literature suggesting it may provide no additional benefit to preventing PPCs. Current intervention choice is reflective of guidelines [1], however, recent literature has called this into question and more research needs to be done to establish if these recommendations are the most effective at reducing PPCs. Continued research is necessary to promote translation of knowledge to ensure physiotherapists are mobilising patients day one post-UAS. Likewise, future work should focus on identification of barriers, the strategies used to overcome limitations and the creation of a reliable and validated screening tool to ensure appropriate prioritisation and allocation of physiotherapy resources within the UAS patient population

Nature As Ecology: Toward a More Constructive Ecocriticism

Cheryll Glotfelty's essay collection The Ecocriticism Reader: Landmarks in Literary Ecology was published in 1996, eighteen years after William Rueckert coined the term “ecocriticism,” and yet Glotfelty's main goal, through three hundred ninety-one pages and twenty-five essays, was still to answer the question “what is ecocriticism?”. Many of the essays included in the Reader are in direct conversation – if not outright argument – with one another and even today – sixteen years later still – “what is ecocriticism?” is a question with no easy answer. This is as it should be. As a discipline that is connected to literary theory by the dual bridges of culture and science – a culture that is becoming increasingly aware of its culpability in worldwide environmental destruction through the work of science – ecocriticism would be worse than useless if it was unable to reinvent itself in the face of this growing environmental awareness. It continues – appropriately – to adapt, not unlike the ecosystems that it discusses. But how does one define the parameters of a critical discourse that can't agree on its own tenets? I suggest that instead of searching for a definition of ecocriticism in the answers it provides, we look instead at the questions it asks. There are many, for certain, but a few stand out: the Big Questions that are the most repeated and that can often be glimpsed lurking behind many smaller inquiries. By enumerating these Big Questions and synthesizing some of the most important responses to them, I create an outline – a skeleton, if you will – upon which the muscles of ecocriticism can be seen to work. Extrapolating from this model, then, I critique the thesis of Timothy Morton's book Ecology Without Nature and through that critique suggest a constructive revision of Morton's idea of “ecology without nature,” a conceptual mode of responsible, ecological living that I label “nature as ecology.” Ultimately, this essay is both a critique of the current state of ecocritical discourse and an argument for a new, more constructive direction