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Triggered-release of encapsulated therapeutics from nanoparticles without remote or environmental triggers was demonstrated in this work. Disassembly of the polymer nanoparticles to unimers at precise times allowed the controlled release of oligo DNA. The polymers used in this study consisted of a hydrophilic block for stabilization and second thermoresponsive block for self-assembly and disassembly. At temperatures below the second block's LCST (i.e., below 37 °C for in vitro assays), the diblock copolymer was fully water-soluble, and when heated to 37 °C, the polymer self-assembled into a narrow size distribution of nanoparticles with an average diameter of approximately 25 nm. The thermoresponsive nature of the second block could be manipulated in situ by the self-catalyzed degradation of cationic 2-(dimethylamino)ethyl acrylate (DMAEA) units to negatively charged acrylic acid groups and when the amount of acid groups was sufficiently high to increase the LCST of the second block above 37 °C. The disassembly of the nanoparticles could be controlled from 10 to 70 h. The use of these nanoparticles as a combined therapy, in which one or more agents can be released in a predetermined way, has the potential to improve the personal point of care treatment of patients.

HIV viral load monitoring forms an essential part of the management of patients receiving antiretroviral therapy, but transport of samples without loss of RNA integrity may be problematic in resource limited settings. The use of plasma preparation tubes (PPT) which can be centrifuged to separate cellular components before transport may provide a simple and cost-effective alternative to standard EDTA samples. We investigated whether PPT generated reliable results using the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 test version 2.0 (CAP/CTM HIV-1 v2.0). The mean difference between EDTA and PPT prepared samples (n=261) was acceptable (log 0.04 copies/ml, percentage similarity CV 3.53%). PPT can be used for viral load testing on the CAP/CTM HIV-1 v2.0., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aminoglycoside antibiotics were among the first antibiotics discovered and used clinically. Although they have never completely fallen out of favor, their importance has waned due to the emergence of other broad-spectrum antibiotics with fewer side effects. Today, with the dramatically increasing rate of infections caused by multidrug-resistant bacteria, focus has returned to aminoglycoside antibiotics as one of the few remaining treatment options, particularly for Gram-negative pathogens. Although the mechanisms of resistance are reasonably well understood, our knowledge about the mode of action of aminoglycosides is still far from comprehensive. In the face of emerging bacterial infections that are virtually untreatable, it is time to have a fresh look at this old class to reinvigorate the struggle against multidrug-resistant pathogens.

Purpose: The cells of the mammalian lens must be carefully organized and regulated to maintain clarity. Recent studies have identified the Eph receptor ligand ephrin-A5 as a major contributor to lens development, as mice lacking ephrin-A5 develop abnormal lenses, resulting in cataracts. As a follow-up to our previous study on the cataracts observed in ephrin-A5(-/-) animals, we have further examined the morphological and molecular changes in the ephrin-A5(-/-) lens., Methods: Wild-type and ephrin-A5(-/-) eyes at various ages were fixed, sectioned, and examined using histological techniques. Protein expression and localization were determined using immunohistochemistry and western blot analysis., Results: Lens abnormalities in the ephrin-A5(-/-) animals are observed at postnatal stages, with lens opacity occurring by postnatal day 21. Structural defects in the lens are first observed in the outer lens fiber cell region where cells in the ephrin-A5(-/-) lens are severely disorganized. Ephrin-A5 and the Eph receptor EphA2 are expressed during early ocular development and continue to be expressed into postnatal stages. The cataracts in the ephrin-A5(-/-) mutants occur regardless of the presence of the CP49 mutation., Conclusions: In this follow-up study, we have uncovered additional details explicating the mechanisms underlying ephrin-A5 function in the lens. Furthermore, elucidation of the expression of ephrin-A5 and the Eph receptor EphA2 in the lens supports a fundamental role for this receptor-ligand complex in lens development. These observations, in concert with our previous study, strongly suggest that ephrin-A5 has a critical role in postnatal lens fiber organization to maintain lens transparency.

In vitro passage of Campylobacter jejuni strains results in phenotypic changes and a general loss of virulence, as is the case with the genome-sequenced strain C. jejuni NCTC11168. Re-sequencing of a virulent strain of NCTC11168 identified 41 SNPs or indels involving 20 genes, four intergenic regions and three pseudogenes. The genes include six motility genes, two chemotaxis genes, three hypothetical genes and a capsule biosynthesis gene, which might have a critical role in C. jejuni virulence. Additionally, we found an insertion in both Cj0676 and Cj1470c, pseudogenes in avirulent NCTC11168, but functional proteins in virulent NCTC11168., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Aims: The aim of the study was to compare the effect of carboplatin chemotherapy on the survival of canine patients diagnosed with malignant melanoma after loco-regional control or as a sole therapy., Methods: A retrospective study of 63 dogs with oral, digital or cutaneous malignant melanoma treated with surgery and/or chemotherapy was undertaken. Dogs were grouped based on the anatomical site of melanoma development. For oral melanoma, dogs were subclassified into two groups: loco-regional control and gross disease. All patients in the digital and cutaneous groups had achieved loco-regional control with surgery. Comparisons between survival data for each group at each anatomical site were then made. Within the loco-regional control groups survival time was compared between those treated with and without chemotherapy post surgery. For the oral melanoma patients with gross disease survival was compared between those treated with chemotherapy and palliative therapy. The toxicity of carboplatin chemotherapy was evaluated overall., Results: The overall median survival times for patients with oral, digital and cutaneous melanoma were 389, 1,350 days and not reached (with a median follow-up of 776 days) respectively. Median survival time was defined as "not reached" when less than 50% of the subjects died of the disease at the end of the follow-up period, or at the time they were lost to follow-up. The addition of chemotherapy to surgery did not confer a survival benefit in the loco-regional control setting when assessing survival for each anatomical site. For oral melanoma patients with gross disease there was no difference between survival of patients treated with chemotherapy and palliative intent therapy. There was however an improvement in survival in the three dogs that responded to chemotherapy (978 days; p=0.039) compared to the eight non-responders (147 days). On univariate and multivariate analysis, anatomic location was the only variable that was significantly related to survival (p=0.0002 and p=0.009, respectively)., Conclusions: The addition of chemotherapy to local treatments for canine melanoma at oral, digital and cutaneous sites did not lead to a significant increase in survival times. Carboplatin was well tolerated and appeared to have activity against oral melanoma in a subset of patients with gross disease that responded to treatment., Clinical Relevance: Carboplatin with piroxicam could be considered for patients with gross disease when more traditional therapies, such as surgery or radiation therapy, are declined or are not available. In the loco-regional control setting, prospective randomised blinded studies with matched control groups are required to determine if chemotherapy has a role in the treatment of these types of cancer.

The lacZ gene product, β-galactosidase, has classically been used as a reporter of gene expression. β-Galactosidase activity can be detected using a chromogenic substrate, X-gal, which leaves an intense blue precipitate when cleaved by the enzyme. Insertion of the lacZ coding DNA targeted into a specific gene creates a β-galactosidase-tagged fusion protein that is expressed under the endogenous promoter. Analysis of the hybrid protein takes advantage of the chromogenic detection system, as the distribution and relative abundance of the expressed protein can be efficiently visualized.

Whole genome sequencing was used to characterize the resistome of intensive care unit (ICU) outbreak-associated carbapenem-resistant K. pneumoniae isolates. Importantly, and of particular concern, the carbapenem-hydrolyzing β-lactamase gene bla(OXA-48) and the extended-spectrum β-lactamase gene bla(CTX-M-14), were identified on a single broad host-range conjugative plasmid. This represents the first report of bla(OXA-48) in Australia and highlights the importance of resistance gene surveillance, as such plasmids can silently spread amongst enterobacterial populations and have the potential to drastically limit treatment options. Furthermore, the in vivo evolution of these isolates was also examined after 18 months of intra-abdominal carriage in a patient that transited through the ICU during the outbreak period. Reflecting the clonality of K. pneumoniae, only 11 single nucleotide polymorphisms (SNPs) were accumulated during this time-period and many of these were associated with genes involved in tolerance/resistance to antibiotics, metals or organic solvents, and transcriptional regulation. Collectively, these SNPs are likely to be associated with changes in virulence (at least to some extent) that have refined the in vivo colonization capacity of the original outbreak isolate.

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (∆Hº) and favorable (positive) entropic (∆Sº) contributions to the Gibbs free energy (∆Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Previously developed screening colonoscopy algorithms based on diagnostic and endoscopy procedural variables have not been sufficiently accurate for use in epidemiological and health services research., Objective: To increase understanding of the administrative health database variables that could help to discern screening and nonscreening colonoscopy., Methods: A qualitative study using physician focus groups was conducted in Montreal (Quebec), Calgary (Alberta) and Toronto (Ontario). Specialty-specific focus group sessions were held among family physicians and gastroenterologists - the physicians responsible for referring patients to and performing screening colonoscopy, respectively. Interview guides were developed to better understand physician clinical and billing practices. Discussions were audiotaped, transcribed verbatim and analyzed using the constant comparative approach., Results: Forty family physicians and seven gastroenterologists participated in five focus group sessions. Patient variables included demographics (age) and medical history (colorectal cancer risk factors⁄symptoms, medication for colorectal cancer risk factors⁄symptoms, gastrointestinal disorders, severe disease). Clinical practice variables included timing of the colonoscopy (evenings, weekends, holidays, during hospitalization; same-day endoscopist consultation and colonoscopy), use of services (hospitalization, annual examination, transfer from other facility) and procedure use patterns (large bowel or other medical⁄surgical procedure before and subsequent to colonoscopy). However, wide variability in clinical and billing practices will likely preclude the development of a reasonably accurate screening colonoscopy algorithm. Physicians suggested adding a screening colonoscopy code to the administrative health data., Conclusions: Failure to acknowledge the limitations of the provincial administrative health databases to identify screening colonoscopy may lead to incorrect conclusions and the establishment of inappropriate health care policies.

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Fast methods using balanced steady-state free precession have been developed to reduce the scan time of T₁ and T₂ mapping. However, flip angle (FA) profiles created by the short radiofrequency pulses used in steady-state free precession deviate substantially from the ideal rectangular profile, causing T₁ and T₂ mapping errors. The purpose of this study was to develop a FA profile correction for T₁ and T₂ mapping with Look-Locker 2D inversion recovery steady-state free precession and to validate this method using 2D spin echo as a reference standard. Phantom studies showed consistent improvement in T₁ and T₂ accuracy using profile correction at multiple FAs. Over six human calves, profile correction provided muscle T₁ estimates with mean error ranging from excellent (-0.6%) at repetition time/FA = 18 ms/60° to acceptable (6.8%) at repetition time/FA = 4.9 ms/30°, while muscle T₂ estimates were less accurate with mean errors of 31.2% and 47.9%, respectively., (Copyright © 2012 Wiley Periodicals, Inc.)

Biofilms formed by pathogenic bacteria present a serious threat to human health as the efficacy of standard antibiotic therapeutic regimens is compromised by reduced microbial susceptibility within the biofilm environment. The discovery of improved therapies for biofilm elimination requires an understanding of biofilm formation and dispersal, and the development of assays to specifically analyze these dynamic processes. This review will discuss biofilm screening strategies suitable for drug discovery efforts, especially chemical and biological approaches that specifically target biofilm destruction.

Nonhuman primates show remarkable variation in several aspects of social structure. One way to characterize this variation in the genus Macaca is through the concept of social style, which is based on the observation that several social traits appear to covary with one another in a linear or at least continuous manner. In practice, macaques are more simply characterized as fitting a four-grade scale in which species range from extremely despotic (grade 1) to extremely tolerant (grade 4). Here, we examine the fit of three core measures of social style-two measures of dominance gradients (hierarchical steepness) and another closely related measure (counter-aggression)-to this scale, controlling for phylogenetic relationships. Although raw scores for both steepness and counter-aggression correlated with social scale in predicted directions, the distributions appeared to vary by measure. Counter-aggression appeared to vary dichotomously with scale, with grade 4 species being distinct from all other grades. Steepness measures appeared more continuous. Species in grades 1 and 4 were distinct from one another on all measures, but those in the intermediate grades varied inconsistently. This confirms previous indications that covariation is more readily observable when comparing species at the extreme ends of the scale than those in intermediate positions. When behavioral measures were mapped onto phylogenetic trees, independent contrasts showed no significant consistent directional changes at nodes below which there were evolutionary changes in scale. Further, contrasts were no greater at these nodes than at neutral nodes. This suggests that correlations with the scale can be attributed largely to species' phylogenetic relationships. This could be due in turn to a structural linkage of social traits based on adaptation to similar ecological conditions in the distant past, or simply to unlinked phylogenetic closeness., (© 2012 Wiley Periodicals, Inc.)

Geographic variation has been of interest to both health planners and social epidemiologists. However, while the major focus of interest of planners has been on variation in health care spending, social epidemiologists have focused on health; and while social epidemiologists have observed strong associations between poor health and poverty, planners have concluded that income is not an important determinant of variation in spending. These different conclusions stem, at least in part, from differences in approach. Health planners have generally studied variation among large regions, such as states, counties, or hospital referral regions (HRRs), while epidemiologists have tended to study local areas, such as ZIP codes and census tracts. To better understand the basis for geographic variation in hospital utilization, we drew upon both approaches. Counties and HRRs were disaggregated into their constituent ZIP codes and census tracts and examined the interrelationships between income, disability, and hospital utilization that were examined at both the regional and local levels, using statistical and geomapping tools. Our studies centered on the Milwaukee and Los Angeles HRRs, where per capita health care utilization has been greater than elsewhere in their states. We compared Milwaukee to other HRRs in Wisconsin and Los Angeles to the other populous counties of California and to a region in California of comparable size and diversity, stretching from San Francisco to Sacramento (termed "San-Framento"). When studied at the ZIP code level, we found steep, curvilinear relationships between lower income and both increased hospital utilization and increasing percentages of individuals reporting disabilities. These associations were also evident on geomaps. They were strongest among populations of working-age adults but weaker among seniors, for whom income proved to be a poor proxy for poverty and whose residential locations deviated from the major underlying income patterns. Among working-age adults, virtually all of the excess utilization in Milwaukee was attributable to very high utilization in Milwaukee's segregated "poverty corridor." Similarly, the greater rate of hospital use in Los Angeles than in San-Framento could be explained by proportionately more low-income ZIP codes in Los Angeles and fewer in San-Framento. Indeed, when only high-income ZIP codes were assessed, there was little variation in hospital utilization among California's 18 most populous counties. We estimated that had utilization within each region been at the rate of its high-income ZIP codes, overall utilization would have been 35 % less among working-age adults and 20 % less among seniors. These studies reveal the importance of disaggregating large geographic units into their constituent ZIP codes in order to understand variation in health care utilization among them. They demonstrate the strong association between low ZIP code income and both higher percentages of disability and greater hospital utilization. And they suggest that, given the large contribution of the poorest neighborhoods to aggregate utilization, it will be difficult to curb the growth of health care spending without addressing the underlying social determinants of health.

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

1,3-Benzothiazin-4-ones (BTZs) are a novel class of TB drug candidates with potent activity against M. tuberculosis. An in silico ligand-based model based on structure-activity data from 170 BTZ compounds was used to design a new series. Compounds were tested against a panel of mycobacterial strains and were profiled for cytotoxicity, stability, and antiproliferative effects. Several of the compounds showed improved activity against MDR-TB while retaining low toxicity with higher microsomal, metabolic, and plasma stability.

The vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006-2009. The average number of QCM publications per annum was 124 in the period 2001-2005, 223 in the period 2006-9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces., (Copyright © 2012 John Wiley & Sons, Ltd.)

Here, for the first time, we have characterized binding properties of the polymyxin class of antibiotics for human α-1-acid glycoprotein (AGP) using a combination of biophysical techniques. The binding affinity of colistin, polymyxin B, polymyxin B(3), colistin methansulfonate, and colistin nona-peptide was determined by isothermal titration calorimetry (ITC), surface plasma resonance (SPR) and fluorometric assay methods. All assay techniques indicated colistin, polymyxin B and polymyxin B(3) display a moderate binding affinity for AGP. ITC and SPR showed there was no detectable binding affinity for colistin methansulfonate and colistin nona-peptide, suggesting both the positive charges of the diaminobutyric acid (Dab) side chains and the N-terminal fatty acyl chain of the polymyxin molecule are required to drive binding to AGP. In addition, the ITC and fluorometric data suggested that endogenous lipidic substances bound to AGP provide part of the polymyxin binding surface. A molecular model of the polymyxin B(3)-AGP F1*S complex was presented that illustrates the pivotal role of the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 hydrophobic motif of polymyxin B(3) for binding to the cleft-like ligand binding cavity of AGP F1*S variant. The model conforms with the entropy driven binding interaction characterized by ITC which suggests hydrophobic interactions coupled to desolvation events and conformational changes are the primary driving force for polymyxins binding to AGP. Collectively, the data are consistent with a role of this acute-phase reactant protein in the transport of polymyxins in plasma., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Previous research indicates that serotonin enhances the development of stress-induced changes in behavior, although it is unclear which serotonin receptors mediate this effect. 5-HT2 receptors are potential candidates because activation at these receptors is associated with increased fear and anxiety. In this study, we investigated whether pharmacological treatments targeting 5-HT2 receptors would alter the acquisition and expression of conditioned defeat. Conditioned defeat is a social defeat model in Syrian hamsters in which individuals display increased submissive and defensive behavior and a loss of territorial aggression when tested with a novel intruder 24 hours after an acute social defeat. The nonselective 5-HT2 receptor agonist mCPP (0.0, 0.3, 1.0, or 3.0 mg/kg) was injected either prior to social defeat training or prior to conditioned defeat testing. Also, the 5-HT2A receptor antagonist MDL 11,939 (0.0, 0.5, or 2.0 mg/kg) was injected either prior to social defeat training or prior to conditioned defeat testing. Injection of mCPP prior to testing increased the expression of conditioned defeat, but injection of mCPP prior to training did not alter the acquisition of conditioned defeat. Conversely, injection of MDL 11,939 prior to training reduced the acquisition of conditioned defeat, but injection of MDL 11,939 prior to testing did not alter the expression of conditioned defeat. Our data suggest that mCPP activates 5-HT2C receptors during testing to enhance the display of submissive and defensive behavior, whereas MDL 11,939 blocks 5-HT2A receptors during social defeat to disrupt the development of the conditioned defeat response. In sum, these results suggest that serotonin acts at separate 5-HT2 receptors to facilitate the acquisition and expression of defeat-induced changes in social behavior.

Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

Sensitive and quantitative nucleic acid testing from complex biological samples is now an important component of clinical diagnostics. Whereas nucleic acid amplification represents the gold standard, its utility in resource-limited and point-of-care settings can be problematic due to assay interferants, assay time, engineering constraints, and costs associated with both wetware and hardware. In contrast, amplification-free nucleic acid testing can circumvent these limitations by enabling direct target hybridization within complex sample matrices. In this work, we grew random copolymer brushes from the surface of silica-coated magnetic nanoparticles using azide-modified and hydroxyl oligo ethylene glycol methacrylate (OEGMA) monomers. The azide-functionalized polymer brush was first conjugated, via copper-catalyzed azide/alkyne cycloaddition (CuAAC), with herpes simplex virus (HSV)-specific oligonucleotides and then with alkyne-substituted polyethylene glycol to eliminate all residual azide groups. Our methodology enabled control over brush thickness and probe density and enabled multiple consecutive coupling reactions on the particle grafted brush. Brush- and probe-modified particles were then combined in a 20 min hybridization with fluorescent polystyrene nanoparticles modified with HSV-specific reporter probes. Following magnetic capture and washing, the particles were analyzed with an aggregate fluorescence measurement, which yielded a limit of detection of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Adoption of brush- and probe-modified particles into a particle counting assay will result in the development of diagnostic assays with significant improvements in sensitivity.

Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.

Although exposure to social stress leads to increased depression-like and anxiety-like behavior, some individuals are more vulnerable than others to these stress-induced changes in behavior. Prior social experience is one factor that can modulate how individuals respond to stressful events. In this study, we investigated whether experience-dependent resistance to the behavioral consequences of social defeat was associated with a specific pattern of neural activation. We paired weight-matched male Syrian hamsters in daily aggressive encounters for 2 weeks, during which they formed a stable dominance relationship. We also included control animals that were exposed to an empty cage each day for 2 weeks. Twenty-four hours after the final pairing or empty cage exposure, half of the subjects were socially defeated in 3, 5-min encounters, whereas the others were not socially defeated. Twenty-four hours after social defeat, animals were tested for conditioned defeat in a 5-min social interaction test with a non-aggressive intruder. We collected brains after social defeat and processed the tissue for c-Fos immunoreactivity. We found that dominants were more likely than subordinates to counter-attack the resident aggressor during social defeat, and they showed less submissive and defensive behavior at conditioned defeat testing compared with subordinates. Also, social status was associated with distinct patterns of defeat-induced neural activation in select brain regions, including the amygdala, prefrontal cortex, hypothalamus, and lateral septum. Our results indicate that social status is an important form of prior experience that predicts both initial coping style and the degree of resistance to social defeat. Further, the differences in defeat-induced neural activation suggest possible brain regions that may control resistance to conditioned defeat in dominant individuals., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

These good practice recommendations for outpatient parenteral antimicrobial therapy (OPAT) are an update to a previous consensus statement on OPAT in the UK published in 1998. They are based on previous national and international guidelines, but have been further developed through an extensive consultation process, and are underpinned by evidence from published literature on OPAT. They provide pragmatic guidance on the development and delivery of OPAT services, looking at all aspects of service design, care delivery, outcome monitoring and quality assurance, with the aim of ensuring that OPAT services provide high-quality, low-risk care, whatever the healthcare setting. They will provide a useful resource for teams developing new services, as well as a practical set of quality indicators for existing services.

The rise of antibiotic resistance is of great clinical concern. One approach to reducing the development of resistance is to co-administer two or more antibiotics with different modes of action. However, it can be difficult to control the distribution and pharmacokinetics of two drugs to ensure both concentrations remain within the range of therapeutic efficacy whilst avoiding adverse effects. Hybrid drugs, where two drugs are linked together with a flexible linker, have been explored, but the resultant large, flexible molecules can have poor bioavailability. We have developed a chimeric approach using click chemistry where the pharmacophores of two drugs are overlapped into a single smaller, more drug-like molecule. Design and selection of compounds were assisted by in silico structural docking. We prepared a series of compounds that include candidates showing activity against the targets of both trimethoprim; dihydrofolate reductase, and ciprofloxacin; DNA gyrase and topoisomerase IV. The resultant triazole containing molecules show modest, but broad spectrum activities against drug sensitive and resistant Gram-negative and Gram-positive bacteria, with no observable cytotoxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Free flap monitoring is essential to the early detection of compromise thereby increasing the chance of successful salvage surgery. Many alternatives to classical clinical monitoring have been proposed. This study seeks to investigate a relatively new monitoring technology: near infrared spectroscopy (NIRS). Patients were recruited prospectively to the study from a single center. During the research period, 10 patients underwent reconstruction with a free deep inferior epigastric perforator flap (DIEP). Measurements of flap perfusion were taken using NIRS in the preoperative and intraoperative phases and postoperatively for 72 hours. NIRS showed characteristic changes in all cases which returned to theater for pedicle compromise. In these cases, NIRS identified pedicle compromise prior to clinical identification. There were no false-positives. NIRS accurately identified all compromised flaps in our study. In most cases, there was an evidence of changes in oxygen saturation on NIRS prior to clinical observation. Further research, ideally double blind randomized control trials with large sample groups would be required to definitively establish NIRS as an ideal flap monitoring modality., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The emergence of multi-drug resistant bacteria is one of the most critical medical problems currently facing humankind, which will only get worse if no new antibacterial drugs are launched. This article will first review commonly used screening strategies used to identify potential new antibiotics and then discuss novel screening methods. In addition, new assays, methods, biological targets and compounds with novel modes of action undergoing pre-clinical or clinical development are briefly discussed.

Quantitative assessment of myelination is important for characterizing tissue damage and evaluating response to therapy in white matter diseases such as multiple sclerosis. Conventional multicomponent T(2) relaxometry based on the two-dimensional (2D) multiecho spin echo sequence is a promising method to measure myelin water fraction, but its clinical utility is impeded by the prohibitively long data acquisition and limited brain coverage. The objective of this study was to develop a signal-to-noise ratio efficient 3D T(2) prep spiral gradient echo (3D SPIRAL) sequence for full brain T(2) relaxometry and to validate this sequence using 3D multiecho spin echo as reference standard in healthy brains at 1.5 T. 3D SPIRAL was found to provide similar myelin water fraction in six selected white and gray matter areas using region-of-interest signal averaging analysis (N = 7, P > 0.05). While 3D multiecho spin echo only provided partial brain coverage, 3D SPIRAL enabled whole brain coverage with a fivefold higher acquisition speed per imaging slice and similar signal-to-noise ratio efficiency. Both 3D sequences provided superior signal-to-noise ratio efficiency when compared to the conventional 2D multiecho spin echo approach., (Copyright © 2011 Wiley Periodicals, Inc.)

Introduction: Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free., Areas Covered: This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS., Expert Opinion: The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

Exposure to traumatic events can increase the risk for major depressive disorder (MDD) as well as posttraumatic stress disorder (PTSD), and pharmacological treatments for these disorders often involve the modulation of serotonergic (5-HT) systems. Several behavioral paradigms in rodents produce changes in behavior that resemble symptoms of MDD and these behavioral changes are sensitive to antidepressant treatments. Here we review two animal models in which MDD-like behavioral changes are elicited by exposure to an acute traumatic event during adulthood, learned helplessness (LH) and conditioned defeat. In LH, exposure of rats to inescapable, but not escapable, tailshock produces a constellation of behavioral changes that include deficits in fight/flight responding and enhanced anxiety-like behavior. In conditioned defeat, exposure of Syrian hamsters to a social defeat by a more aggressive animal leads to a loss of territorial aggression and an increase in submissive and defensive behaviors in subsequent encounters with non-aggressive conspecifics. Investigations into the neural substrates that control LH and conditioned defeat revealed that increased 5-HT activity in the dorsal raphe nucleus (DRN) is critical for both models. Other key brain regions that regulate the acquisition and/or expression of behavior in these two paradigms include the basolateral amygdala (BLA), central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST). In this review, we compare and contrast the role of each of these neural structures in mediating LH and conditioned defeat, and discuss the relevance of these data in developing a better understanding of the mechanisms underlying trauma-related depression. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many biologically active compounds are unsuitable for development as drugs due to their poor bioavailability. For hydrophilic compounds, modifications to increase lipophilicity can increase passive diffusion or increase uptake into the lymphatic system. Alternatively, improved bioavailability of hydrophilic drug candidates may be achieved by formulation with absorption promoters such as surfactants, penetration enhancers, or ion pairing agents. This approach to enhancing bioavailability also has the potential to widen the range of compound categories that can be used as chemical probes to study biological systems in cells and in vivo where membrane permeability would otherwise be a significant limitation. Lipidic amino acids, which combine the structural properties of lipids with those of α-amino acids, represent a relatively unexplored class of agents that can improve drug adsorption. This review discusses the potential of absorption promoters possessing lipoamino acids for improving drug bioavailability.

Most drug discovery programs today originate by selection of 'hit' molecules resulting from assays against large compound screening libraries. The chemical space in which these hits reside has implications for its biological activity in vivo and likelihood of progression to a drug candidate. We have created a database of commercially available screening compounds and natural products in order to analyse the drug- and lead-likeness of commercial screening compounds and compare them with i) orally administered drugs, ii) non-orally administered drugs, and iii) compounds with significant biological activity but unspecified or not yet determined route of administration from the public databases DrugBank and ChEMBL. The data set contained 15.5 million entries from 102 vendors, which resulted in just over 8 million unique chemical structures. We review these data for current drug/lead-likeness, then utilise substructure-based filters for promiscuity and unwanted groups, and finally compare chemical properties for structures within the different sub-sets. While the majority of the commercial compounds satisfy various drug-likeness rules, they show a larger molecular weight and higher hydrophobicity compared to orally available drugs, with generally higher aromaticity and lower solubility. This 'right shift' of chemical properties has also been found in the majority of the compounds with significant biological activity in ChEMBL, reflecting a common trend in current drug discovery, towards larger, more hydrophobic compounds and fewer drug-like compounds. In particular, successful drugs were found to possess much lower median logD values than those found for compound collections. In addition, commercial compounds show a quite narrow distribution in molecular weight, with a median absolute deviation of only 78 Da around a median of 387 Da. For high-throughput screening a highly stringent combination of several lead-likeness and substructure filters against unwanted groups could be applied, resulting in 2 million lead-like structures. For fragment based screening approaches the rule of three (Ro3) would select around 400,000 structures.

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

The basolateral nucleus of the amygdala (BLA) is a key brain region regulating behavioral changes following stressful events, including social defeat. Previous research has shown that activation of serotonin (5-HT) 1A receptors in the BLA reduces conditioned fear and anxiety-like behavior. The objective of this study was to test whether 5-HT1A receptors in the BLA contribute to conditioned defeat in male Syrian hamsters (Mesocricetus auratus). We tested whether injection of the selective 5-HT1A receptor agonist flesinoxan (400 ng, 800 ng, or 1200 ng in 200 nl saline) into the BLA prior to social defeat would reduce the acquisition of conditioned defeat, and whether a similar injection prior to testing would reduce the expression of conditioned defeat. We also tested whether injection of the selective 5-HT1A receptor antagonist WAY-100635 (400 ng or 1600 ng in 200 nl saline) into the BLA prior to social defeat would enhance the acquisition of conditioned defeat, and whether a similar injection prior to testing would enhance the expression of conditioned defeat. We found that injection of flesinoxan into the BLA decreased both the acquisition and expression of conditioned defeat. However, injection of WAY-100635 into the BLA did not alter the acquisition or expression of conditioned defeat. These data indicate that pharmacological activation of 5-HT1A receptors in the BLA is sufficient to impair the acquisition and expression of conditioned defeat. Our results suggest that pharmacological treatments that activate 5-HT1A receptors in the BLA are capable of reducing the development of stress-induced changes in behavior., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Molecular dynamics simulations and free energy calculations have been used to examine in detail the mechanism by which a receptor molecule (the glycopeptide antibiotic vancomycin) recognizes and binds to a target molecule (lipid II) embedded within a membrane environment. The simulations show that the direct interaction of vancomycin with lipid II, as opposed to initial binding to the membrane, leads most readily to the formation of a stable complex. The recognition of lipid II by vancomycin occurred via the N-terminal amine group of vancomycin and the C-terminal carboxyl group of lipid II. Despite lying at the membrane-water interface, the interaction of vancomycin with lipid II was found to be essentially identical to that of soluble tripeptide analogs of lipid II (Ac-d-Ala-d-Ala; root mean-square deviation 0.11 nm). Free energy calculations also suggest that the relative binding affinity of vancomycin for native, resistant, and synthetic forms of membrane-bound lipid II was unaffected by the membrane environment. The effect of the dimerization of vancomycin on the binding of lipid II, the position of lipid II within a biological membrane, and the effect of the isoamylene tail of lipid II on membrane fluidity have also been examined., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Helicobacter pylori is a WHO class I carcinogen also associated with nonmalignant gastrointestinal diseases. Effective treatment exists, and all persons infected with H pylori should receive treatment. However, data regarding the rates of treatment prescription in clinical practice are lacking., Objective: To determine the rates of H pylori treatment in usual practice., Methods: Patients with histological evidence of H pylori infection between January 1, 2007, and December 31, 2007, at Sunnybrook Health Sciences Centre (Toronto, Ontario) were identified. Charts were reviewed to determine the rates of H pylori treatment and confirmation of eradication, when indicated. Questionnaires were subsequently sent to endoscopists of patients identified as not having received treatment to determine the reasons for lack of treatment., Results: A total of 102 patients were H pylori positive and were appropriate candidates for treatment, of whom 58 (57%) were male and 78 (76%) were outpatients, with 92 (90%) receiving eradication therapy. When indicated, 15 of 22 (68%) patients received confirmation of eradication, 13 of 18 (72%) patients underwent repeat endoscopy and 86% received complete therapy. Outpatients were more likely to receive eradication therapy (OR 10.3 [95% CI 2.6 to 40.4]; P=0.001) and complete therapy (OR 13.2 [95% CI 3.8 to 45.7]; P=0.0001) compared with inpatients. Having a follow-up appointment resulted in higher treatment rates (OR 12.0 [95% CI 3.0 to 47.5]; P=0.001)., Conclusion: During the time period studied, adequate rates of H pylori treatment were achieved in outpatients and patients who had formal follow-up at Sunnybrook Health Sciences Centre. However, some aspects of care remain suboptimal including treatment of inpatients and care following treatment. Additional studies are required to identify strategies to improve the care of patients infected with H pylori.

Chronic kidney disease (CKD) is a major public health problem. The classification of CKD by KDOQI and KDIGO and the routine eGFR reporting have resulted in increased identification of CKD. It is important to be able to identify those at high risk of CKD progression and its associated cardiovascular disease (CVD). Proteinuria is the most sensitive marker of CKD progression in clinical practice, especially when combined with eGFR, but these have limitations. Hence, early, more sensitive, biomarkers are required. Recently, promising biomarkers have been identified for CKD progression and its associated CVD morbidity and mortality. These may be more sensitive biomarkers of kidney function, the underlying pathophysiological processes, and/or cardiovascular risk. Although there are some common pathways to CKD progression, there are many primary causes, each with its own specific pathophysiological mechanism. Hence, a panel measuring multiple biomarkers including disease-specific biomarkers may be required. Large, longitudinal observational studies are needed to validate candidate biomarkers in a broad range of populations prior to implementation into routine CKD management. Recent renal biomarkers discovered include neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and liver-type fatty acid-binding protein. Although none are ready for use in clinical practice, it is timely to review the role of such biomarkers in predicting CKD progression and/or CVD risk in CKD.

We have developed a sensitive and economical method to directly detect bacteria, based on the interaction between the bacteria and specific antibodies attached to an oscillating surface. By monotonously increasing the amplitude of oscillation of a quartz crystal microbalance (QCM) coated with the antibody, the QCM can be used to sensitively detect the acoustic noise produced when the interactions between the bacteria and the surface were broken. We term this process rupture event scanning (REVS). The method is quantitative over at least 6 orders of magnitude and can detect as few as 10 bacteria. We demonstrate here that this approach allows one to arrange separation of bacteria and follow the process completion on the basis of the acoustic signal. Detection is not significantly affected by non-specific binding of sample contaminants and thus can be achieved both in buffer and in serum., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Since the publication of the original review of piezoelectric acoustic sensors in this series there has been a consistent, gradual expansion in the number of published papers using 'quartz crystal microbalances' (QCM). Between 2001 and 2009, the number of QCM publications per annum has increased from 49 to 273, with a two-fold increase in papers per annum between 2004 and 2008. Within the field, comparing the time covered by the current to the previous review, there are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase) and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterization of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This paper highlights theoretical and practical aspects of the principles that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and membrane interfaces., (Copyright © 2011 John Wiley & Sons, Ltd.)

Past experience can alter how individuals respond to stressful events. The brain serotonin system is a key factor modulating stress-related behavior and may contribute to individual variation in coping styles. In this study we investigated whether dominant and subordinate hamsters respond differently to social defeat and whether their behavioral responses are associated with changes in 5-HT1A and 5-HT2A receptor immunoreactivity in several limbic brain regions. We paired weight-matched hamsters in daily aggressive encounters for two weeks so that they formed a stable dominance relationship. We also included controls that were exposed to an empty cage each day for two weeks. Twenty-four hours after the final pairing or empty cage exposure, subjects were socially defeated in 3, 5-min encounters with a more aggressive hamster. Twenty-four hours after social defeat, animals were tested for conditioned defeat in a 5-min social interaction test with a non-aggressive intruder. We collected brains following conditioned defeat testing and performed immunohistochemistry for 5-HT1A and 5-HT2A receptors. We found that dominants showed less submissive and defensive behavior at conditioned defeat testing compared to both subordinates and controls. Additionally, both dominants and subordinates had an increased number of 5-HT1A immunopositive cells in the basolateral amygdala compared to controls. Subordinates also had more 5-HT1A immunopositive cells in the dorsal medial amygdala than did controls. Finally, dominants had fewer 5-HT1A immunopositive cells in the paraventricular nucleus of the hypothalamus compared to controls. Our results indicate that dominant social status results in a blunted conditioned defeat response and a distinct pattern of 5-HT1A receptor expression, which may contribute to resistance to conditioned defeat., (Copyright © 2011 Elsevier Inc. All rights reserved.)