Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation.

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory Ki value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.

The ageing human B cell repertoire: a failure of selection?

B cells undergo a number of different developmental stages, from initial formation of their B cell receptor (BCR) genes to differentiation into antibody-secreting plasma cells. Because the BCR is vital in these differentiation steps, autoreactive and exogenous antigen binding to the BCR exert critical selection pressures to shape the B cell repertoire. Older people are more prone to infectious disease, less able to respond well to vaccination and more likely to have autoreactive antibodies. Here we review evidence of changes in B cell repertoires in older people, which may be a reflection of age-related changes in B cell selection processes.

Substrate temperature controls molecular orientation in two-component vapor-deposited glasses.

Vapor-deposited glasses can be anisotropic and molecular orientation is important for organic electronics applications. In organic light emitting diodes (OLEDs), for example, the orientation of dye molecules in two-component emitting layers significantly influences emission efficiency. Here we investigate how substrate temperature during vapor deposition influences the orientation of dye molecules in a model two-component system. We determine the average orientation of a linear blue light emitter 1,4-di-[4-(N,N-diphenyl)amino]styryl-benzene (DSA-Ph) in mixtures with aluminum-tris(8-hydroxyquinoline) (Alq3) by spectroscopic ellipsometry and IR dichroism. We find that molecular orientation is controlled by the ratio of the substrate temperature during deposition and the glass transition temperature of the mixture. These findings extend recent results for single component vapor-deposited glasses and suggest that, during vapor deposition, surface mobility allows partial equilibration towards orientations preferred at the free surface of the equilibrium liquid.

Variation of the aryl substituent on the piperazine ring within the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold unveils potent, non-competitive inhibitors of the inflammatory caspases.

The inflammatory caspases (caspase-1, -4 and -5) are potential therapeutic targets for autoimmune and inflammatory diseases due to their involvement in the immune response upon inflammasome formation. A series of small molecules based on the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold were synthesized with varying substituents on the piperazine ring. Several compounds were pan-selective inhibitors of the inflammatory caspases, caspase-1, -4 and -5, with the ethylbenzene derivative CK-1-41 displaying low nanomolar Ki values across this family of caspases. Three analogs were nearly 10 fold selective for caspase-5 over caspase-1 and -4. The compounds display non-competitive, time dependent inhibition profiles. To our knowledge, this series is the first example of small molecule inhibitors of all three inflammatory caspases.

Trophic Magnification of Organic Chemicals: A Global Synthesis.

Production of organic chemicals (OCs) is increasing exponentially, and some OCs biomagnify through food webs to potentially toxic levels. Biomagnification under field conditions is best described by trophic magnification factors (TMFs; per trophic level change in log-concentration of a chemical) which have been measured for more than two decades. Syntheses of TMF behavior relative to chemical traits and ecosystem properties are lacking. We analyzed >1500 TMFs to identify OCs predisposed to biomagnify and to assess ecosystem vulnerability. The highest TMFs were for OCs that are slowly metabolized by animals (metabolic rate kM < 0.01 day(-1)) and are moderately hydrophobic (log KOW 6-8). TMFs were more variable in marine than freshwaters, unrelated to latitude, and highest in food webs containing endotherms. We modeled the probability that any OC would biomagnify as a combined function of KOW and kM. Probability is greatest (∼100%) for slowly metabolized compounds, regardless of KOW, and lowest for chemicals with rapid transformation rates (kM > 0.2 day(-1)). This probabilistic model provides a new global tool for screening existing and new OCs for their biomagnification potential.

Coronary computed tomography angiography and its increasing application in day to day cardiology practice.

Coronary artery disease (CAD) is the leading single cause of death in Australia affecting around 1.4 million people. Coronary computed tomography angiography has an established role in the assessment of patients with low to intermediate pretest probability for CAD who have chest pain and is typically used with the aim to rule out significant coronary artery stenosis. Use was initially limited because of concerns over radiation exposure, a Medicare rebate restricted to specialist referrals and an absence of data supporting its use as an alternative to functional testing in patients with chest pain. Recent advances in scanner technology and image sequencing, along with data from randomised control trials, have addressed these issues and indicate that coronary computed tomography angiography will play a greater role in the assessment of CAD in the coming years.

Outcomes of transcatheter aortic valve implantation in high surgical risk and inoperable patients with aortic stenosis: a single Australian Centre experience.

BACKGROUND: Degenerative aortic stenosis is the most common valvular heart disease in the elderly, and many patients are not suitable for aortic valve replacement surgery. Transcatheter aortic valve implantation (TAVI) is a new therapeutic option for selected patients at high risk for surgery. AIM: To evaluate the safety and efficacy of TAVI in Australian patients. METHODS: This is a prospective study of patients undergoing TAVI for severe symptomatic aortic stenosis at The Prince Charles Hospital, Brisbane, Australia between August 2008 and July 2013. Patients were at high risk of surgical aortic valve replacement, or inoperable, as deemed by a multidisciplinary 'heart team'. Outcomes include procedural success and complications, 30-day and 1-year mortality and stroke, combined end-points as outlined by the Valve Academic Research Consortium 2 consensus document. RESULTS: Two hundred and nine patients underwent TAVI during the study period. The mean age was 83.7 ± 6.7 years, and 101 (48%) were men. The valve systems utilised were as follows: Edwards-SAPIEN valve in 104 (49.5%), Medtronic CoreValve in 86 (41.2%) and Boston Scientific Lotus valve in 19 (9.3%) patients. Thirty-day and 1-year mortality rates were 5.7% and 11.5% respectively. Thirty-day and 1-year stroke rates were 4.3% and 6.2% respectively. The composite end-points of device success, early safety and clinical efficacy occurred in 80.4%, 27.3% and 68.4%. CONCLUSIONS: TAVI with various valve systems, delivered through several approaches, is feasible in high surgical risk and inoperable patients with severe aortic stenosis, with acceptable outcomes at short-term and intermediate-term follow up.

Oligomycin frames a common drug-binding site in the ATP synthase.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c(10) ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c(10) ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

Design and synthesis of potent macrocyclic HIV-1 protease inhibitors involving P1-P2 ligands.

A series of potent macrocyclic HIV-1 protease inhibitors have been designed and synthesized. The compounds incorporated 16- to 19-membered macrocyclic rings between a nelfinavir-like P2 ligand and a tyrosine side chain containing a hydroxyethylamine sulfonamide isostere. All cyclic inhibitors are more potent than their corresponding acyclic counterparts. Saturated derivatives showed slight reduction of potency compared to the respective unsaturated derivatives. Compound containing a 16-membered ring as the P1-P2 ligand showed the most potent enzyme inhibitory and antiviral activity.

Metamorphosis enhances the effects of metal exposure on the mayfly, Centroptilum triangulifer.

The response of larval aquatic insects to stressors such as metals is used to assess the ecological condition of streams worldwide. However, nearly all larval insects metamorphose from aquatic larvae to winged adults, and recent surveys indicate that adults may be a more sensitive indicator of stream metal toxicity than larvae. One hypothesis to explain this pattern is that insects exposed to elevated metal in their larval stages have a reduced ability to successfully complete metamorphosis. To test this hypothesis we exposed late-instar larvae of the mayfly, Centroptilum triangulifer, to an aqueous Zn gradient (32-476 µg/L) in the laboratory. After 6 days of exposure, when metamorphosis began, larval survival was unaffected by zinc. However, Zn reduced wingpad development at concentrations above 139 µg/L. In contrast, emergence of subimagos and imagos tended to decline with any increase in Zn. At Zn concentrations below 105 µg/L (hardness-adjusted aquatic life criterion), survival between the wingpad and subimago stages declined 5-fold across the Zn gradient. These results support the hypothesis that metamorphosis may be a survival bottleneck, particularly in contaminated streams. Thus, death during metamorphosis may be a key mechanism explaining how stream metal contamination can impact terrestrial communities by reducing aquatic insect emergence.

Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46.

Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional coupling induced by Cx46. In contrast, expression of D3Y or L11S failed to inhibit junctional conductance induced by Cx50. To examine effects of the D3Y and L11S mutations on hemichannel activity, hemichannel currents were measured in connexin cRNA-injected oocytes. Oocytes expressing D3Y exhibited reduced hemichannel activity as well as alterations in voltage gating and charge selectivity while oocytes expressing L11S showed no hemichannel activity. Moreover, coexpression of mutant with wild-type Cx50 or Cx46 gave rise to hemichannels with distinct electrophysiological properties, suggesting that the mutant connexins were forming heteromeric channels with wild-type connexins. These data suggest D3Y and L11S cause cataracts by similar but not identical mechanisms.

The design and rationale of the Australian Cooperative National Registry of Acute Coronary care, Guideline Adherence and Clinical Events (CONCORDANCE).

BACKGROUND: Cardiovascular observational registries characterise patients and describe the manner and use of therapeutic strategies. They facilitate analyses on the quality of care among participating institutions and document variations in clinical practice which can be benchmarked against best practice recommendations. The Cooperative National Registry of Acute Coronary care, Guideline Adherence and Clinical Events (CONCORDANCE) is an Australian observational registry that describes management and outcomes in patients with acute coronary syndromes (ACS) and feeds back both performance and outcome measures to participating hospitals. METHODS: The CONCORDANCE registry has been designed within a comparative effectiveness research (CER) framework to collect and report data from hospitals located in geographically diverse regions of Australia. Information including patient demographics, presenting characteristics, past medical history, in-hospital management and outcomes at six months and two years are entered into a web-based database using an electronic clinical record form (eCRF). Individual hospital information is returned to the sites in a real time confidential report detailing information on key performance indicator (KPI) process measures and outcomes benchmarked against the aggregated study cohort. Governance rules ensure data security and protect patient and clinician confidentiality. Consistent with a CER framework, additional characteristics of the registry include: (a) the capacity to evaluate associations between the inter and intra hospital systems and the provision of evidence based care and outcomes, (b) ongoing data collection from representative hospitals which allow spatial and temporal analysis of change in practice and the application of treatment modalities in the real world setting and (c) the provision of a data spine for quality improvement strategies and practical clinical trials. CONCLUSION: The CONCORDANCE registry is a clinician-driven initiative describing clinical care for ACS patients admitted to Australian hospitals. The registry generates high quality data which is fed back to clinicians, and key stakeholders in ACS care. Using a CER approach, the registry describes the translation of randomised trial evidence into practice, and provides insights into strategies that could improve care and ultimately patient outcomes.

Takotsubo cardiomyopathy: an Australian single centre experience with medium term follow up.

BACKGROUND: Takotsubo cardiomyopathy (TC) is increasingly recognised in patients presenting with features of acute coronary syndrome. We present a single centre experience of TC with medium term follow up. METHODS: Fifty-two consecutive patients presenting with a diagnosis of TC were included. The clinical presentation, complications, baseline and follow-up echocardiograms and cardiac magnetic resonance imaging were analysed. RESULTS: Fifty-one patients were female. A stressful event preceded presentation in 37 (71%) patients. Chest pain was the most common symptom (83%). Two patients presented with an out-of-hospital cardiac arrest. ST segment elevation (40%) and global T wave inversion (44%) were the most frequent electrocardiogram changes. Left ventricular assessment demonstrated typical apical ballooning in 41 patients and 11 patients demonstrated the mid-wall variant. In-hospital complications occurred in 11 patients (21%) and included acute pulmonary oedema (n = 2), cardiogenic shock (n = 5); two of whom had a significant left ventricular outflow gradient, atrial fibrillation (n = 1), left ventricular thrombus (n = 2) and a cerebrovascular event (n = 2). Left ventricular function at presentation and follow up was compared in 40 patients. The mean ejection fraction in this group at presentation was 47% (20-70%) compared with that at follow up of 63% (44-76%). There were no significant complications or recurrences at follow up. CONCLUSIONS: While TC is a reversible condition with low rates of complications and recurrence at follow up it is, as demonstrated in our cohort, associated with significant in-hospital morbidity in a proportion of patients.

Percutaneous ASD closure in a large Australian series: short- and long-term outcomes.

AIM: To assess the clinical and echocardiographic outcomes in patients referred for device closure of atrial septal defects in a tertiary referral hospital in Australia. METHODS: A prospective follow-up study was performed on all patients who had device closure of a secundum atrial septal defect (ASD) from June 1999 to December 2007. Clinical and echocardiographic data at the time of implantation and follow-up is presented. RESULTS: 176 patients were referred for shunt closure of ASD. All patients had a significant shunt defined as a shunt with right heart dilatation and/or a shunt ratio of at least 1.5:1. The majority were female (67%) and the average age was 36.5 ± 22.7 years; age range 3-84. The average hospital admission time was 2.5 ± 1.7 days. The average follow-up occurred at 3.7 ± 3.6 months for the first follow-up and 26.3 ± 18.2 months (range 3 months-7.8 years) for the long-term follow-up. Baseline echocardiogram findings showed the majority had a normal left ventricular ejection fraction (99%); average LVEF=63.2 ± 7.2% while the right ventricle was dilated in 61% of patients. Procedure information: The average procedure time was 94.8 ± 36.4 min. Procedural imaging was performed using Transoesophageal echocardiography (TOE) in 107 cases (61%); Intracardiac Echocardiography (ICE) in 69 (39%). Device use was as follows: Amplatzer=156 cases, Helex=18, and Starflex=2. Postprocedure shunt assessment by transthoracic echocardiography showed successful closure (no shunt or trivial shunt) in 99% cases. Two patients were referred for inpatient surgery due to a significant residual shunt in one case and an unstable device in another. One patient who had an unstable device had their device repositioned successfully. Atrial arrhythmia was the most common complication occurring in the peri-implantation period in 12 cases (6%) with four further cases at final up. The high prevalence of right ventricular dilatation in 65% patients at baseline had improved significantly at the first and long term follow-up to 2% (p=0.0001). CONCLUSION: Device closure of secundum atrial septal defects in this large Australian cohort demonstrates a high procedural success rate with a low incidence of complications in the short and long term.

The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

Conformational changes in BAK, a pore-forming proapoptotic Bcl-2 family member, upon membrane insertion and direct evidence for the existence of BH3-BH3 contact interface in BAK homo-oligomers.

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.

Computational fluid dynamics simulations of particle deposition in large-scale, multigenerational lung models.

Computational fluid dynamics (CFD) has emerged as a useful tool for the prediction of airflow and particle transport within the human lung airway. Several published studies have demonstrated the use of Eulerian finite-volume CFD simulations coupled with Lagrangian particle tracking methods to determine local and regional particle deposition rates in small subsections of the bronchopulmonary tree. However, the simulation of particle transport and deposition in large-scale models encompassing more than a few generations is less common, due in part to the sheer size and complexity of the human lung airway. Highly resolved, fully coupled flowfield solution and particle tracking in the entire lung, for example, is currently an intractable problem and will remain so for the foreseeable future. This paper adopts a previously reported methodology for simulating large-scale regions of the lung airway (Walters, D. K., and Luke, W. H., 2010, "A Method for Three-Dimensional Navier-Stokes Simulations of Large-Scale Regions of the Human Lung Airway," ASME J. Fluids Eng., 132(5), p. 051101), which was shown to produce results similar to fully resolved geometries using approximate, reduced geometry models. The methodology is extended here to particle transport and deposition simulations. Lagrangian particle tracking simulations are performed in combination with Eulerian simulations of the airflow in an idealized representation of the human lung airway tree. Results using the reduced models are compared with those using the fully resolved models for an eight-generation region of the conducting zone. The agreement between fully resolved and reduced geometry simulations indicates that the new method can provide an accurate alternative for large-scale CFD simulations while potentially reducing the computational cost of these simulations by several orders of magnitude.

Thrombolysis of acute ischaemic stroke in a district general hospital: are we doing enough?

Thrombolysis of acute ischaemic stroke is safe, efficacious and licensed for use in the UK. To date, few studies have looked at the rates of thrombolysis within a district general hospital setting. The aim of the study was to identify whether local thrombolysis protocols for stroke are adhered to; the rate of thrombolysis and strategies for implementation focused at improving the provision of thrombolysis of patients with acute ischaemic stroke. The methodology involved in this was a retrospective study within a district general hospital in the South of England. Data on patients admitted between 15 April 2008 and 14 April 2009 including demographics, use of the local thrombolysis protocol and reasons for non-thrombolysis were collected and analysed. Out of a total of 599 patients with a primary presentation of acute stroke, 18 were considered for thrombolysis. Six patients out of these 18 were thrombolysed without complications. Four out of these six patients had an improved National Institutes of Health Stroke Scale (NIHSS) post-thrombolysis and one eventually died due to the extensiveness of the stroke sustained, despite a slightly improved NIHSS. The rate of thrombolysis of acute ischaemic stroke is low in this hospital (1.001%). Various measures will need to be considered for implementation in order to improve the provision of this service.

Inhibitors of the mitochondrial citrate transport protein: validation of the role of substrate binding residues and discovery of the first purely competitive inhibitor.

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K(ic)) of 0.12 +/- 0.02 mM and an uncompetitive inhibition constant (K(iu)) of 3.04 +/- 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K(ic) values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K(m) for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K(m) approximates the K(d) (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.