A method to unmix multiple fluorophores in microscopy images with minimal a priori information.
The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.


Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep.
The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep.


Identifying collagen VI as a target of fibrotic diseases regulated by CREBBP/EP300.
Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren's disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1 Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren's nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.


Frameless image-guided neuronavigation in orbital surgery: practical applications.
Frameless neuronavigation has been increasingly used in intracranial and spinal neurosurgery. However, the application of neuronavigation in orbital surgery has not yet been reported. The purpose of this study was to evaluate whether frameless navigation in the orbit provides sufficient clinical application accuracy and thus a useful tool in orbital surgery. A frameless infrared-based neuronavigation system (VectorVision, BrainLAB, Heimstetten, Germany) was used in the microsurgical removal of 11 orbital tumors (five cavernomas, three lymphomas, two dermoid tumors, and one rhabdomyosarcoma). The transconjunctival approach was performed in seven cases, lateral orbitotomy in three cases, and the extradural pterional approach was performed in one case. The surgery was successful and without any complications in all cases. The registration accuracy of the neuronavigation was between 1.8 and 2.2 mm, with a mean of 1.9 mm. Subtotal tumor debulking was performed in one case with lymphoma. One patient was only biopsied due to suspected systemic lymphoma disease. Total removal of the tumor could be accomplished in the other nine patients. Image guidance during orbital surgery offered excellent three-dimensional guidance on the surface of the intraorbital lesions, allowing a safer, more controlled surgery. The surgical targets in the orbit are fixed structures, thus no shifting occurs and continuous high intraoperative navigation accuracy can be achieved. The use of the navigation clearly reduces the operative risk and increases the effectiveness of microsurgical orbital procedures.


Electroconvulsive shock increases tachykinin NK(1) receptors, but not the encoding mRNA, in rat cortex.
Recent studies have suggested that the substance P (tachykinin NK(1)) receptor may be a pharmacological target for the treatment of mood disorders. Here, the effects of electroconvulsive shock on tachykinin NK(1) receptor gene expression in the rat brain was investigated. Rats received either a single electroconvulsive shock or five shocks on alternate days. Quantitative autoradiography with [(125)I]Bolton Hunter-substance P, and in situ hybridisation histochemistry, were used to measure tachykinin NK(1) receptor-binding site densities and mRNA abundance, respectively. Densities of tachykinin NK(1) receptor-binding sites were significantly increased in the cerebral cortex following repeated electroconvulsive shock compared to sham treated animals. Densities remained unchanged in the hippocampus, striatum and amygdala. Neither single nor repeated electroconvulsive shock altered tachykinin NK(1) receptor mRNA in the brain regions examined. Hence, repeated electroconvulsive shock increases tachykinin NK(1) receptors in the rat brain in a regionally specific way. Upregulation of receptor-binding sites without a change in mRNA indicates that translational or post-translational mechanisms underlie this process.


Group B streptococcus exploits vaginal epithelial exfoliation for ascending infection.
Thirteen percent of pregnancies result in preterm birth or stillbirth, accounting for fifteen million preterm births and three and a half million deaths annually. A significant cause of these adverse pregnancy outcomes is in utero infection by vaginal microorganisms. To establish an in utero infection, vaginal microbes enter the uterus by ascending infection; however, the mechanisms by which this occurs are unknown. Using both in vitro and murine models of vaginal colonization and ascending infection, we demonstrate how a vaginal microbe, group B streptococcus (GBS), which is frequently associated with adverse pregnancy outcomes, uses vaginal exfoliation for ascending infection. GBS induces vaginal epithelial exfoliation by activation of integrin and β-catenin signaling. However, exfoliation did not diminish GBS vaginal colonization as reported for other vaginal microbes. Rather, vaginal exfoliation increased bacterial dissemination and ascending GBS infection, and abrogation of exfoliation reduced ascending infection and improved pregnancy outcomes. Thus, for some vaginal bacteria, exfoliation promotes ascending infection rather than preventing colonization. Our study provides insight into mechanisms of ascending infection by vaginal microbes.


Filtering of distractors during visual search studied by positron emission tomography.
We investigated the sensitivity of brain areas to the presence of filtering operations during overt visual search in crowded displays. Task conditions involved either visual search or predetermined simple eye movements for the detection of target digits. Furthermore, visual displays either contained letter foils that required filtering or contained only target digits. Brain imaging using positron emission tomography showed extensive overlap between areas involved in overt visual search and eye movements. Selective filtering of foils affected visual processing in ventral areas associated with object recognition and in primary visual cortex.


Studies on the role of calcium in the 5-HT-stimulated release of glutamate from C6 glioma cells.
We recently reported that 5-hydroxytryptamine(2A) (5-HT(2A)) receptor activation on cultured glial cells induces glutamate release [J. Neurosci. Res. 67 (2002) 399]. Here we use C6 glioma cells to examine the role of calcium in this response. 5-Hydroxytryptamine (5-HT) increases glutamate release from C6 glioma cells, an effect blocked by low calcium conditions. The calcium ionophores ionomycin and calcimycin also released glutamate from C6 glioma cells in a Ca(2+)-dependent manner. The effect of 5-HT was reduced by the phospholipase C inhibitor U73122 (1-[6[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not its inactive enantomer U73343(1-[6[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione). The protein kinase C inhibitors staurosporine and calphostin C had no effect on the response to 5-HT, whereas the response was blocked by thapsigargin and caffeine. Neither the L-type calcium channel blockers, nifedipine and verapamil, nor the N-type calcium channel blocker omega-conotoxin GVIA inhibited the effect of 5-HT, whereas NiCl(2) and KCl blocked the response to 5-HT. We conclude that the 5-HT-induced efflux of glutamate from C6 glioma cells is Ca(2+)-dependent and involves, at least in part, the mobilisation of Ca(2+) from inositol (1,4,5) tris phosphate (IP(3)) sensitive intracellular stores.


Adequacy of reporting monitoring regimens of risk factors for cardiovascular disease in clinical guidelines: systematic review.
To assess the reporting of monitoring recommendations in guidelines on the prevention and treatment of cardiovascular disease. Medline, Trip database, National Guideline Clearinghouse, and databases containing guidelines published from January 2002 to February 2010. Data selection Three major risk factors for cardiovascular disease: cholesterol level, smoking, and hypertension. The primary outcome was the frequency with which the guidelines dealt with monitoring of risk factors. Secondary outcomes were completeness of monitoring recommendations, defined by the presence of what to monitor, when to monitor, what to do if the targets or variables were not met, and the reported level or strength of the evidence. 117 guidelines were identified, 84 (72%) of which contained a section on lipids. Of those guidelines with a section on lipids, 53% (n = 44) provided no information or specific recommendations on what to monitor, 51% (n = 43) provided no information on when to monitor, and 64% (n = 54) provided no guidance on what to do if the target was out of range. Guidelines for hypertension (n = 79) and smoking (n = 65) were little better, with 63% (n = 50) and 54% (n = 35), respectively, providing no recommendation for what to monitor. The number of guidelines that explicitly referenced the level of evidence for monitoring was low, with most of the recommendations based on weak levels of evidence. Many guidelines for cardiovascular disease do not report clearly what to monitor and what to do if a change is detected. If no evidence is available to support a specific monitoring schedule, this should be explicit in the guideline, with a description of the new research that would fill the gap.


Deformable medical image registration: setting the state of the art with discrete methods.
This review introduces a novel deformable image registration paradigm that exploits Markov random field formulation and powerful discrete optimization algorithms. We express deformable registration as a minimal cost graph problem, where nodes correspond to the deformation grid, a node's connectivity corresponds to regularization constraints, and labels correspond to 3D deformations. To cope with both iconic and geometric (landmark-based) registration, we introduce two graphical models, one for each subproblem. The two graphs share interconnected variables, leading to a modular, powerful, and flexible formulation that can account for arbitrary image-matching criteria, various local deformation models, and regularization constraints. To cope with the corresponding optimization problem, we adopt two optimization strategies: a computationally efficient one and a tight relaxation alternative. Promising results demonstrate the potential of this approach. Discrete methods are an important new trend in medical image registration, as they provide several improvements over the more traditional continuous methods. This is illustrated with several key examples where the presented framework outperforms existing general-purpose registration methods in terms of both performance and computational complexity. Our methods become of particular interest in applications where computation time is a critical issue, as in intraoperative imaging, or where the huge variation in data demands complex and application-specific matching criteria, as in large-scale multimodal population studies. The proposed registration framework, along with a graphical interface and corresponding publications, is available for download for research purposes (for Windows and Linux platforms) from http://www.mrf-registration.net.


Survival in stage II/III colorectal cancer is independently predicted by chromosomal and microsatellite instability, but not by specific driver mutations.
Microsatellite instability (MSI) is an established marker of good prognosis in colorectal cancer (CRC). Chromosomal instability (CIN) is strongly negatively associated with MSI and has been shown to be a marker of poor prognosis in a small number of studies. However, a substantial group of "double-negative" (MSI-/CIN-) CRCs exists. The prognosis of these patients is unclear. Furthermore, MSI and CIN are each associated with specific molecular changes, such as mutations in KRAS and BRAF, that have been associated with prognosis. It is not known which of MSI, CIN, and the specific gene mutations are primary predictors of survival. We evaluated the prognostic value (disease-free survival, DFS) of CIN, MSI, mutations in KRAS, NRAS, BRAF, PIK3CA, FBXW7, and TP53, and chromosome 18q loss-of-heterozygosity (LOH) in 822 patients from the VICTOR trial of stage II/III CRC. We followed up promising associations in an Australian community-based cohort (N=375). In the VICTOR patients, no specific mutation was associated with DFS, but individually MSI and CIN showed significant associations after adjusting for stage, age, gender, tumor location, and therapy. A combined analysis of the VICTOR and community-based cohorts showed that MSI and CIN were independent predictors of DFS (for MSI, hazard ratio (HR)=0.58, 95% confidence interval (CI) 0.36-0.93, and P=0.021; for CIN, HR=1.54, 95% CI 1.14-2.08, and P=0.005), and joint CIN/MSI testing significantly improved the prognostic prediction of MSI alone (P=0.028). Higher levels of CIN were monotonically associated with progressively poorer DFS, and a semi-quantitative measure of CIN was a better predictor of outcome than a simple CIN+/- variable. All measures of CIN predicted DFS better than the recently described Watanabe LOH ratio. MSI and CIN are independent predictors of DFS for stage II/III CRC. Prognostic molecular tests for CRC relapse should currently use MSI and a quantitative measure of CIN rather than specific gene mutations.


Liver transplant and chronic hepatitis B virus infection.
Hepatitis B immune globulin use for preventing hepatitis B virus recurrence after liver transplant has changed our behavior radically, and it now seems that hepatitis B immune globulin has a vital role in preventing recurrence. New nucleotide or nucleoside analogues have promising results in treating chronic hepatitis and in posttransplant hepatitis B virus-infected patients. Hepatitis B immune globulin and other antivirals act on different pathways, so it is logical to combine these drugs to achieve maximum response in suppressing hepatitis B virus (HBV)-replication.


Use of acupuncture in hospices and palliative care services in the UK.
The aim of the present work was to evaluate the availability of acupuncture in UK hospices and specialist palliative care services and to identify any barriers to the use of acupuncture in these settings, to determine the characteristics of available acupuncture services and of practitioners providing acupuncture, and to determine awareness of the evidence base for the use of acupuncture in palliative care. An online questionnaire with an invitation to participate was circulated by email to 263 hospices and specialist palliative care services in the UK. A response rate of 54% was obtained. Acupuncture was provided by 59% of services that responded. In general, small numbers of patients receive acupuncture as part of their palliative care treatment as inpatients, day patients or outpatients. Most practitioners were regulated health professionals who had received a Western-style training in acupuncture and used a Western-style medical acupuncture approach. Where acupuncture was not available the commonest reason given was the lack of a suitable practitioner. Most agreed that if funding and a suitable practitioner were available, acupuncture would be a useful addition to their service. The level of awareness of specific types of evidence supporting the use of acupuncture in palliative care was low, but most respondents were aware that some evidence existed. There is a need to increase training in acupuncture for healthcare professionals working in palliative care. There is also a need to raise awareness of the potential benefits to patients and the evidence base supporting the use of acupuncture in palliative care.


Does science need bioethicists? Ethics and science collaboration in biomedical research.
Biomedical research is an increasingly multidisciplinary activity bringing together a range of different academic fields and forms of expertise to investigate diseases that are increasingly understood to be complex and multifactorial. Recently the discipline of ethics has been starting to find a place in large-scale biomedical collaborations. In this article we draw from our experience of working with the Malaria Genomic Epidemiology Network (MalariaGEN) and other research projects to reflect upon the integration of ethics into biomedical research. We examine the way in which ethics input may be valuable to research, the forms it tends to take, and also the problems and limitations of such collaborations.


The TBC1D15 oncoprotein controls stem cell self-renewal through destabilization of the Numb-p53 complex.
Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.


Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene.
Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.


Human cardiosphere-derived cells from patients with chronic ischaemic heart disease can be routinely expanded from atrial but not epicardial ventricular biopsies.
To investigate the effects of age and disease on endogenous cardiac progenitor cells, we obtained right atrial and left ventricular epicardial biopsies from patients (n = 22) with chronic ischaemic heart disease and measured doubling time and surface marker expression in explant- and cardiosphere-derived cells (EDCs, CDCs). EDCs could be expanded from all atrial biopsy samples, but sufficient cells for cardiosphere culture were obtained from only 8 of 22 ventricular biopsies. EDCs from both atrium and ventricle contained a higher proportion of c-kit+ cells than CDCs, which contained few such cells. There was wide variation in expression of CD90 (atrial CDCs 5-92 % CD90+; ventricular CDCs 11-89 % CD90+), with atrial CDCs cultured from diabetic patients (n = 4) containing 1.6-fold more CD90+ cells than those from non-diabetic patients (n = 18). No effect of age or other co-morbidities was detected. Thus, CDCs from atrial biopsies may vary in their therapeutic potential.


Coffee contains potent opiate receptor binding activity.
Opiate receptor-active peptide fragments (exorphins) have been identified recently in casein and gluten hydrolysates, and morphine has been found in bovine and human milk. To determine whether similar peptides or alkaloids occur in other foodstuffs, we have screened potential sources using a rat brain homogenate assay to detect opiate receptor activity. We report here that instant coffee powders from a variety of manufacturers compete with tritiated naloxone for binding to opiate receptors in the rat brain membrane preparations, with no significant difference between normal and decaffeinated coffee. The receptor binding activity resembles that seen with opiate antagonists, in that there was no change in the half-maximal effective dose (ED50) in the presence of 100 mM Na+; on bioassay, the activity was similarly shown to be antagonistic and specific for opiate-induced inhibition of twitch. Preliminary characterization of the activity reveals that it has a molecular weight (MW) in the range 1,000-3,500, is heat-stable, ether-extractable, not modified by enzymatic digestion with papain, and clearly separable from caffeine and morphine on TLC. As its concentration in an average cup of coffee is five times the ED50, these data suggest that drinking coffee may be followed by effects mediated via opiate receptors, as well as effects of caffeine.


Behavioural characterization of neuregulin 1 type I overexpressing transgenic mice.
Neuregulin 1 (NRG1) is a pleiotropic growth factor involved in diverse aspects of brain development and function. In schizophrenia, expression of the NRG1 type I isoform is selectively increased. However, virtually nothing is known about the roles of this isoform in brain. We have studied transgenic mice overexpressing type I NRG1(NRG1type 1-tg) using a series of behavioural tests. NRG1(type 1-tg) mice have a tremor, are impaired on the accelerating rotarod, and have reduced prepulse inhibition in the context of an increased baseline startle response. There is no overall anxiety or activity phenotype, although female NRG(1type 1-tg) mice show mild increases in anxiety on some measures. The pattern of results shows both similarities and differences to those reported in hypomorphic NRG1 mice, and may be relevant for interpreting the increased NRG1 type I expression observed in schizophrenia.


Use of commercial off-the-shelf digital cameras for scientific data acquisition and scene-specific color calibration.
Commercial off-the-shelf digital cameras are inexpensive and easy-to-use instruments that can be used for quantitative scientific data acquisition if images are captured in raw format and processed so that they maintain a linear relationship with scene radiance. Here we describe the image-processing steps required for consistent data acquisition with color cameras. In addition, we present a method for scene-specific color calibration that increases the accuracy of color capture when a scene contains colors that are not well represented in the gamut of a standard color-calibration target. We demonstrate applications of the proposed methodology in the fields of biomedical engineering, artwork photography, perception science, marine biology, and underwater imaging.


Nitric oxide synthase regulation of cardiac excitation-contraction coupling in health and disease.
Significant advances in our understanding of the ability of nitric oxide synthases (NOS) to modulate cardiac function have provided key insights into the role NOS play in the regulation of excitation-contraction (EC) coupling in health and disease. Through both cGMP-dependent and cGMP-independent (e.g. S-nitrosylation) mechanisms, NOS have the ability to alter intracellular Ca(2+) handling and the myofilament response to Ca(2+), thereby impacting the systolic and diastolic performance of the myocardium. Findings from experiments using nitric oxide (NO) donors and NOS inhibition or gene deletion clearly implicate dysfunctional NOS as a critical contributor to many cardiovascular disease states. However, studies to date have only partially addressed NOS isoform-specific effects and, more importantly, how subcellular localization of NOS influences ion channels involved in myocardial EC coupling and excitability. In this review, we focus on the contribution of each NOS isoform to cardiac dysfunction and on the role of uncoupled NOS activity in common cardiac disease states, including heart failure, diabetic cardiomyopathy, ischemia/reperfusion injury and atrial fibrillation. We also review evidence that clearly indicates the importance of NO in cardioprotection. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".


Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.
Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin → melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.


Altered expression of synaptic protein mRNAs in STOP (MAP6) mutant mice.
Stable tubule-only polypeptide (STOP) proteins are a family of microtubule associated proteins (MAPs) important in microtubule stabilization. Data indicating a role for microtubules in synaptic function has come from studies of the STOP null mouse, which exhibits synaptic deficits, in association with behavioural changes that are alleviated by antipsychotic treatment. These findings suggested that STOP mutant mice may be useful in studies of synaptic function, and could be especially relevant to schizophrenia, postulated to be a disorder of the synapse. Moreover, a genetic association between STOP and schizophrenia has been reported. This study aimed to further characterize synaptic alterations in STOP null and heterozygous mice. Using in situ hybridization histochemistry, the mRNA expression of three pre-synaptic (synaptophysin; growth associated protein-43 (GAP-43); vesicular glutamate transporter-1 (VGlut1)) and two post-synaptic (spinophilin; MAP2) proteins, was quantified in female STOP null (n = 7), heterozygous (n = 5) and wild type (n = 6) mice. For STOP null and heterozygous mice, synaptophysin, VGlut1, GAP-43 and spinophilin mRNAs were decreased in the hippocampus, whilst in addition in the null mice, synaptophysin, VGlut1 and spinophilin mRNAs were decreased in the cerebellum. Alterations in synaptic protein mRNA expression were also detected in the frontal and occipital cortex. MAP2 mRNA expression was unchanged in all brain regions. The profile of mRNA changes is broadly similar to that observed in schizophrenia. Together the data provide supporting evidence for a role for microtubules in synaptic function, and suggest that STOP, or other microtubule proteins, may contribute to the synaptic pathology of schizophrenia.


Lipids in chronic kidney disease.
Patients with chronic kidney disease (CKD) develop premature cardiovascular disease. In the general population (without CKD), there are strong associations between cholesterol fractions and the risk of coronary disease and weaker associations with stroke. Randomised trials in the general population demonstrate that lowering blood cholesterol (chiefly with a statin) reduces the risk of vascular events. Patients with CKD differ significantly from the general population. They have markedly disturbed lipid metabolism manifesting as elevated triglyceride concentrations, reduced HDL cholesterol concentrations and a preponderance of small, dense LDL particles that are potentially more atherogenic; the observed association between lipids and vascular disease is bizarre, and is confounded by co-morbidity; the nature of the vascular disease appears less strongly associated with classical atherosclerosis. Randomised trials are required to determine the relevance of blood lipids to the development of vascular disease in CKD patients, but the results of such studies have been inconclusive to date. CKD patients are at risk of end-stage renal disease. Lipids may be involved in the progression of renal disease. Modifying them may delay the progression of CKD. The current data are based on effects on markers of progression (e.g. proteinuria). The ongoing SHARP (Study of Heart and Renal Protection) trial should provide reliable information about the effects of statins on both vascular and renal risk.


Development of a bacterial propionate-biosensor for anaerobic digestion monitoring.
Monitoring anaerobic digestion (AD) leachate for changes in acetate and propionate concentrations is essential for effective AD operation. In this paper the development of a novel propionate cell-based biosensor is described. A previously designed E. coli mutant (IMD Wldgy) that could selectively determine acetate concentrations in synthetic leachates, based on oxygen uptake measurements, was used as a starting point in the development of a propionate biosensor. However, the propionate-grown IMD Wldgy cells exhibited extremely low propionate:acetate O2 consumption ratios (1:2.4). Screening for alternative propionate-grown E. coli strains naturally possessing a more favourable propionate:acetate O2 consumption ratio identified strain IMD 1, which exhibited a positive ratio (1.6:1). To improve the selectivity of the strain, successive gene knockouts were performed generating the IMD 1 hldgyep mutant. However, propionate-grown IMD 1hdlgyep's O2 consumption ratio was deemed too low to be considered as a propionate detecting bio-element. It was reasoned that the mechanisms by which E. coli activates acetate had to be removed. Deleting acs (acetyl-CoA synthesase) and ackA (acetate kinase) from IMD Wldgyep, resulted in an E. coli IMD Wldgyepak knockout mutant that, when grown on propionate, produced a mean propionate:acetate O2 consumption ratio of approx. 13:1. The resulting IMD Wldgyep and IMD Wldgyepak strains, which formed the acetate- and propionate-biosensor, respectively, were capable of detecting acetate and propionate concentrations ranging from 0.05mM to 4.5mM within two-phase AD synthetic leachates.


Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis.
The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.


Pharmacokinetics and biodistribution of human serum albumin-TIMP-2 fusion protein using near-infrared optical imaging.
TIMP-2 has been studied as an attractive cancer therapeutic candidate, and a TIMP-2 fusion protein (HSA/TIMP-2) displayed effective anticancer activity, despite a lack of information about its pharmacokinetics (PK) and biodistribution. The purpose of this work was to assess the PK and biodistribution of HSA/TIMP-2 as well as to quantify accumulated HSA/TIMP-2 in tumors. Cy5.5 near-infrared (NIR) fluorescence was conjugated to the HSA/TIMP-2 protein (Cy5.5-HSA/TIMP-2) for monitoring spatio-temporal changes in vivo. For PK and biodistribution analysis, 0.2 μg/g body weight of Cy5.5-HSA/TIMP-2 was injected into MAT-LyLu prostate tumor xenografts, which were then imaged using an IVIS-200 optical imaging system. To quantify the accumulated HSA/TIMP-2 in tumors, we introduced a standard curve with depth-corrected fluorescence measurement. In the vascular tube formation assay with human umbilical vein endothelial cells (HUVECs), Cy5.5-HSA/TIMP-2 showed an antiangiogenic effect. In prostate cancer xenografts, Cy5.5-HSA/TIMP-2 exhibited a prolongation of blood half-life to 19.6 h and relatively preferential distribution to the tumor. The amount of tumor-accumulated Cy5.5-HSA/TIMP-2 was calculated to be 4.5 ± 0.5 ng/g body weight at 2 days, representing 2.25 ± 0.25% of the initial dose. We evaluated the pharmacokinetic profile and biodistribution of HSA/TIMP-2 with favorable results, providing new information for more effective approaches to cancer therapeutics using HSA/TIMP-2. Additionally, real-time in vivo fluorescence imaging analysis using a depth-corrected standard curve may serve as a platform to quantify biodistributed drug in anticancer therapeutic studies.


Employee motivation: a powerful new model.
Motivating employees begins with recognizing that to do their best work, people must be in an environment that meets their basic emotional drives to acquire, bond, comprehend, and defend. So say Nohria and Groysberg, of Harvard Business School, and Lee, of the Center for Research on Corporate Performance. Using the results of surveys they conducted with employees at a wide range of Fortune 500 and other companies, they developed a model for how to increase workplace motivation dramatically. The authors identify the organizational levers that companies and frontline managers have at their disposal as they try to meet workers' deep needs. Reward systems that truly value good performance fulfill the drive to acquire. The drive to bond is best met by a culture that promotes collaboration and openness. Jobs that are designed to be meaningful and challenging meet the need to comprehend. Processes for performance management and resource allocation that are fair, trustworthy, and transparent address the drive to defend. Equipped with real-world company examples, the authors articulate how to apply these levers in productive ways. That application should not be selective, they argue, because a holistic approach gets you more than a piecemeal one. By using all four levers simultaneously, and thereby tackling all four drives, organizations can improve motivation levels by leaps and bounds. For example, a company that falls in the 50th percentile on employee motivation improves only to the 56th by boosting performance on one drive, but way up to the 88th percentile by doing better on all four drives. That's a powerful gain in competitive advantage that any business would relish.


Oral corticosteroid use for clinical and cost-effective symptom relief of sore throat: study protocol for a randomized controlled trial.
Management of acute sore throat poses a significant burden on UK general practices, with almost 10% of registered patients attending their GP with sore throat every year. Nearly half of all patients presenting with acute sore throat are treated with antibiotics, despite their limited effect. In a recent systematic review we demonstrated that a single dose of steroids reduced the severity and time to resolution of sore throat. However, all of the trials included looked at the use of steroids alongside antibiotics and only one was in a primary care setting. This trial aims to assess the efficacy and cost-effectiveness of a single oral dose of corticosteroids on symptoms of sore throat in patients receiving either a delayed antibiotic prescription or no antibiotics at all in UK primary care. A double-blind, two arm, randomized, placebo controlled trial in adults (≥ 18 years of age) presenting to primary care with acute sore throat (<seven days). Participants are recruited on the day of presentation to their GP practice. GPs or nurses assess eligibility, record baseline clinical features and obtain a throat swab for bacterial culture. Participants are being randomized to treatment arms at a ratio of 1:1. Treatment arms will be stratified according to whether patients are being given a delayed antibiotic prescription or no antibiotic prescription and by recruiting centre (Oxford, Bristol or Southampton). Outcome data is being collected at 24 and 48 hours via text message or telephone call, from days 0 to 7 using a patient symptom diary and at one month via a GP notes review. This will be the first randomized controlled trial of oral corticosteroids in adults presenting to primary care with sore throat in the UK, and the first to examine the clinical and cost-effectiveness of oral corticosteroids for the treatment of sore throat in the absence of antibiotics. This trial is registered with Current Controlled Trials on 26 March 2013, registration number: ISRCTN17435450.


Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells.
Non-human adenovirus vectors have attractive immunological properties for gene therapy but are frequently restricted by inefficient transduction of human target cells. Using chicken embryo lethal orphan (CELO) virus, we employed a nongenetic mechanism of polymer coating and retargeting with basic fibroblast growth factor (bFGF-pc-CELOluc), a strategy that permits efficient tropism modification of human adenovirus. bFGF-pc-CELOluc showed efficient uptake and transgene expression in chick embryo fibroblasts (CEF), and increased levels of binding and internalization in a variety of human cell lines. Transgene expression was also greater than unmodified CELOluc in PC-3 human prostate cells, although the specific activity (RLU per internalized viral genome) was decreased. In CEF, the specific activity of bFGF-pc-CELOluc was considerably higher than in the human prostate cell line PC-3. Retargeted virus was fully resistant to inhibition by human serum with known adenovirus-neutralizing activity in vitro, while in mice CELOluc was cleared less rapidly from the blood than Adluc following i.v. administration in the presence of adenovirus neutralizing serum. Polymer coating and retargeting with bFGF further reduced rates of clearance for both viruses, suggesting protection against both neutralizing and opsonizing factors. The data indicate that CELO virus may be retargeted to infect human cells via alternative, potentially disease-specific, receptors and resist the effects of pre-existing humoral immunity.


Attentional control constrains visual short-term memory: insights from developmental and individual differences.
The mechanisms by which attentional control biases mnemonic representations have attracted much interest but remain poorly understood. As attention and memory develop gradually over childhood and variably across individuals, assessing how participants of different ages and ability attend to mnemonic contents can elucidate their interplay. In Experiment 1, 7-year-olds, 10-year-olds, and adults were asked to report whether a probe item had been part of a previously presented four-item array. The initial array could either be uncued, be preceded ("precued"), or followed ("retrocued") by a spatial cue orienting attention to one of the potential item locations. Performance across groups was significantly improved by both cue types, and individual differences in children's retrospective attentional control predicted their visual short-term and working memory span, whereas their basic ability to remember in the absence of cues did not. Experiment 2 imposed a variable delay between the array and the subsequent orienting cue. Cueing benefits were greater in adults than in 10-year-olds, but they persisted even when cues followed the array by nearly 3 seconds, suggesting that orienting operated on durable short-term representations for both age groups. The findings indicate that there are substantial developmental and individual differences in the ability to control attention to memory and that in turn these differences constrain visual short-term memory capacity.


Multilayered defense in HLA-B51-associated HIV viral control.
Polymorphism in the HLA region of a chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, -B27, and -B51. Taking advantage of a unique cohort infected with clade B' HIV-1 through contaminated blood, in which many variables such as the length of infection, the infecting viral strain, and host genetic background are controlled, we performed a comprehensive study to understand HLA-B51-associated HIV-1 control. We focused on the T cell responses against three dominant HLA-B51-restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI, and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in position 1 of epitope LI9 was seen in most B51(+) individuals, followed by V289X in position 8 of the TI8, and then, A328S, in position 2 of the NI9 epitope, was also seen in some B51(+) individuals. Good control of viral load and higher CD4(+) counts were significantly associated with at least one detectable T cell response to unmutated epitopes, whereas lower CD4(+) counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defense against the development of immune escape mutations.


Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.


Ultraviolet light provides a major input to non-image-forming light detection in mice.
The change in irradiance at dawn and dusk provides the primary cue for the entrainment of the mammalian circadian pacemaker. Irradiance detection has been ascribed largely to melanopsin-based phototransduction [1-5]. Here we examine the role of ultraviolet-sensitive (UVS) cones in the modulation of circadian behavior, sleep, and suprachiasmatic nucleus (SCN) electrical activity. UV light exposure leads to phase-shifting responses comparable to those of white light. Moreover, UV light exposure induces sleep in wild-type and melanopsin-deficient (Opn4(-/-)) mice with equal efficacy. Electrical recordings from the SCN of wild-type mice show that UV light elicits irradiance-dependent sustained responses that are similar to those induced by white light, with characteristic fast transient components occurring at the light transitions. These responses are retained in Opn4(-/-) mice and preserved under saturating photopic conditions. The sensitivity of phase-shifting responses to UV light is unaffected by the loss of rods but is severely attenuated by the additional loss of cones. Our data show that UVS cones play an important role in circadian and sleep regulation in mice.


Increased temporo-insular engagement in unmedicated bipolar II disorder: an exploratory resting state study using independent component analysis.
Despite numerous structural and functional magnetic resonance imaging studies, the neurobiology of bipolar disorder (BD) is still incompletely understood. Resting-state functional magnetic resonance imaging (rsFMRI) allows for the study of intrinsic functional connections between brain areas, which may reflect the pathophysiology of BD. Most previous rsFMRI studies conducted in BD have included a majority of medicated patients, making simple interpretation with respect to pathophysiology difficult. Participants were 15 antipsychotic agent- and mood-stabilizer-naïve young adults with bipolar II disorder (BD-II), and 20 healthy controls group-matched for gender, age, and cognitive ability. Independent component analysis (ICA) was used to identify eight commonly studied resting-state networks (RSNs). Between-group comparisons were conducted using dual regression and corrected for family-wise error (FWE) across space and the number of components (pFWE < 0.05). In comparison to controls, participants with BD-II had increased coherence across several brain regions, including the bilateral insula and putamen, across a temporo-insular network. No between-group differences in engagement of the default mode network were found. This was the first ICA-based rsFMRI study conducted among unmedicated individuals with BD. Given the young age (mean = 23 years) and antipsychotic agent- and mood-stabilizer-naïve status of our participants with BD-II, temporo-insular functional connectivity is a candidate vulnerability marker for BD. Further work is needed to relate resting-state differences to detailed understanding of pathophysiology.


The Boundary of the Market for Biosecurity Risk.
Imported goods create value in destination countries but also create biosecurity risk. Although widely used in other domains of the economy, risk markets have not been created to manage losses that occur when exotic pests and diseases are introduced with traded goods. In this article we show that not all biosecurity risks are insurable. Losses arising from effort needed to detect and respond to exotic pests and diseases that breach national borders appear to be insurable because entry of these threats and consequent response costs, can be regarded as random events. As pests and diseases establish and spread, however, loss of access to export markets and productivity losses display systematic risk and appear to be uninsurable. Other insurability criteria support this definition of the boundary of biosecurity risk markets. We use the Australian biosecurity system as an example, although the framework described in this study will be applicable to biosecurity systems worldwide. We argue that biosecurity risk insurance could be incorporated into the current biosecurity system but would require legislation mandating importers to purchase insurance. Advantages of actuarial pricing of biosecurity risk are: (i) an increase in economic efficiency to the extent that importers respond to the price of biosecurity risk; (ii) financial sustainability would improve because actuarial pricing creates a structural link between funds available for biosecurity activities and risk exposure; and (iii) equity issues evident in the current biosecurity system could be addressed because risk creators (importers) would fund response activities through the purchase of insurance.


Super-Resolved Traction Force Microscopy (STFM).
Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.


A revised partiality model and post-refinement algorithm for X-ray free-electron laser data.
Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the R(split) value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.


A meta-analysis of four European genome screens (GIFT Consortium) shows evidence for a novel region on chromosome 17p11.2-q22 linked to type 2 diabetes.
Positional cloning is expected to identify novel susceptibility genes underlying complex traits, but replication of genome-wide linkage scan findings has proven erratic. To improve our ability to detect and prioritize chromosomal regions containing type 2 diabetes susceptibility genes, the GIFT consortium has implemented a meta-analysis of four scans conducted in European samples. These included the Botnia I and Botnia II scans, with respectively 58 and 353 pedigrees from Finland and Sweden, the Warren 2 scan performed in 573 multiplex sibships from the UK, and a scan of 143 families from France. The meta-analysis was implemented using the genome-search analysis method (GSMA), an exploratory data analysis technique which is robust across study designs. The analysis provided evidence for linkage of type 2 diabetes to six regions, with the strongest evidence on chromosome 17p11.2-q22 (P=0.0016), followed by 2p22.1-p13.2 (P=0.027), 1p13.1-q22 (P=0.028), 12q21.1-q24.12 (P=0.029), 6q21-q24.1 (P=0.033) and 16p12.3-q11.2 (P=0.033). Linkage analysis of the pooled raw genotype data generated maximum LOD scores in the same regions as identified by GSMA. Altogether, our results have indicated that GSMA is a valuable tool to identify chromosomal regions of interest and that accumulating evidence for linkage from small peaks detected across several samples may be more important than getting a high peak in a single sample. This meta-analysis has led to identification of a novel region on chromosome 17 linked to type 2 diabetes; this region has not been highlighted in any published scan to date but on the basis of these data justifies further exploration.


New tetromycin derivatives with anti-trypanosomal and protease inhibitory activities.
Four new tetromycin derivatives, tetromycins 1-4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001(T) cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV M(pro), and PL(pro). The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with K(i) values in the low micromolar range.


A theory of ethics for forensic psychiatry.
This article offers a justification for a set of principles that constitute the ethical underpinnings of forensic psychiatry. Like professional ethics in general, the principles are based on the particular societal functions performed by forensic psychiatrists and result in the intensification of obligations to promote certain important moral values. For forensic psychiatrists, the primary value of their work is to advance the interests of justice. The two principles on which that effort rests are truth-telling and respect for persons. In the same manner as other physicians who perform functions outside of the usual clinical context (e.g., clinical researchers), forensic psychiatrists cannot simply rely on general medical ethics, embedded as they are in the doctor-patient relationship--which is absent in the forensic setting. Indeed, efforts to retain some residuum of that relationship and its associated ethical principles are likely to create confusion in the minds of both forensic psychiatrists and their evaluees and to heighten the problems of double agency. A virtue of this approach is the clear distinction it offers between clinical and forensic roles.


Gaze fixations predict brain activation during the voluntary regulation of picture-induced negative affect.
Recent studies have identified a distributed network of brain regions thought to support cognitive reappraisal processes underlying emotion regulation in response to affective images, including parieto-temporal regions and lateral/medial regions of prefrontal cortex (PFC). A number of these commonly activated regions are also known to underlie visuospatial attention and oculomotor control, which raises the possibility that people use attentional redeployment rather than, or in addition to, reappraisal as a strategy to regulate emotion. We predicted that a significant portion of the observed variance in brain activation during emotion regulation tasks would be associated with differences in how participants visually scan the images while regulating their emotions. We recorded brain activation using fMRI and quantified patterns of gaze fixation while participants increased or decreased their affective response to a set of affective images. fMRI results replicated previous findings on emotion regulation with regulation differences reflected in regions of PFC and the amygdala. In addition, our gaze fixation data revealed that when regulating, individuals changed their gaze patterns relative to a control condition. Furthermore, this variation in gaze fixation accounted for substantial amounts of variance in brain activation. These data point to the importance of controlling for gaze fixation in studies of emotion regulation that use visual stimuli.


Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers.
Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption. Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs. The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.


Molecular targets for diagnostics and therapeutics of severe acute respiratory syndrome (SARS-CoV).
The large number of deaths in a short period of time due to the spread of severe acute respiratory syndrome (SARS) infection led to the unparalleled collaborative efforts world wide to determine and characterize the new coronavirus (SARS-CoV). The full genome sequence was determined within weeks of the first outbreak by the Canadian group with international collaboration. As per the World Health Organization (WHO), the continual lack of a rapid laboratory test to aid the early diagnosis of suspected cases of SARS makes this area a priority for future research. To prevent deaths in the future, early diagnosis and therapy of this infectious disease is of paramount importance. This review describes the specific molecular targets for diagnostics and therapeutics of viral infection. The three major diagnostic methods available for SARS includes viral RNA detection by reverse transcription polymerase chain reaction (RT-PCR), virus induced antibodies by immunofluorescence assay (IFA) or by enzyme linked immunosorbant assay (ELISA) of nucleocapsid protein (NP). The spike glycoprotein of SARS-CoV is the major inducer of neutralizing antibodies. The receptor binding domain (RBD) in the S1 region of the spike glycoprotein contains multiple conformational epitopes that induces highly potent neutralizing antibodies. The genetically engineered attenuated form of the virus or viral vector vaccine encoding for the SARS-CoV spike glycoprotein has been shown to elicit protective immunity in vaccinated animals. NP is the preferred target for routine detection of SARS-CoV infection by ELISA which is an economical method compared to other methods. The RBD of the spike glycoprotein is both a functional domain for cell receptor binding and also a major neutralizing determinant of SARS-CoV. The progress in evaluating a therapeutic or vaccine would depend on the avail ability of clinically relevant animal model.


[Promoting routine human immunodeficiency virus testing in primary care].
To promote human immunodeficiency virus (HIV) testing in the primary care setting and to describe patients' attitudes toward this practice. A non-randomized intervention was conducted on five physicians of an urban primary care center attending patients aged 18-65 years old, who were scheduled to undergo blood tests for other reasons. The patients were systematically offered HIV blood testing if they reported having had sex without a condom with a person of unknown HIV status. Not being tested required active refusal. The intervention period was from October to December 2008 and the control period was from October to December 2007. The main variable was the difference in the number of HIV tests requested. The proportion of patients accepting the test was also analyzed. Demographic factors were similar in patients in the two periods. The number of HIV tests increased from 3.7% (22/599) to 27.2% (212/780), p <0.001. A total of 209 patients were offered the HIV test. Their mean age was 45.6 years (SD 11.7), 141 were women (68%) and 11 were born outside Spain (5%). One hundred and ninety-five patients (93%) admitted the possibility of having been or being at risk. Of these patients, only three (1.5%), refused the HIV test. Routine HIV testing in the primary care setting is feasible and few patients refuse to be tested.


Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.
A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.


Determinants of response to first HAART regimen in antiretroviral-naïve patients with an estimated time since HIV seroconversion.
To study the determinants of immunological and virological response to highly active antiretroviral therapy (HAART) in naïve patients, adjusting for time since HIV-1 seroconversion. Data from HIV-cohort studies where dates of seroconversion have been reliably estimated. Methods In previously untreated patients, short- and long-term marker responses from HAART initiation (three or more antiretroviral drugs) to the end of follow-up or any treatment modification were considered using mixed effects models accounting for undetectable HIV viral load and informative dropout. In total, 943 patients were treated with a first HAART regimen for a median of 29 months. In adjusted analyses, compared with a reference group of homosexual men without AIDS initiating treatment 4 years after seroconversion, injecting drug users (IDUs) were treated at similar CD4 and HIV RNA levels but had poorer short-term virological response (2.54 vs 2.13 log(10) HIV-1 RNA copies/mL at 1.5 months, P=0.03) and poorer long-term immunological response (522 vs 631 cells/microL at 24 months, P<0.0001). Although individuals with AIDS at HAART initiation had lower CD4 counts (206 vs 382 cells/microL, P<0.0001), their immunological responses were similar to those of individuals without AIDS. Similarly, individuals further from seroconversion started HAART at lower CD4 counts (e.g. 311 vs 382 cells/microL at vs before 9 years from seroconversion, P<0.0001), but had similar CD4 responses. However, they experienced poorer long-term virological response (0.67 log(10) copies/mL/year smaller decline, P<0.0001) compared to those treated before 9 years from seroconversion. Taking into account the time elapsed since seroconversion, this study suggests that careful choices of initial treatment should be made and intensive follow-up carried out in high-risk subgroups such as IDUs who have poorer responses.


Prevalence of neck pain and associated factors with personal characteristics, physical workloads and psychosocial among male rubber workers in FELDA settlement Malaysia.
Rubber tapping processes posed potential risk of various health problems among rubber workers. It ranges from simple musculoskeletal aches to more serious and complicated structural damage to bone, muscles, tendons and nerves of musculoskeletal system. These health problems might be linked directly to the arduous demands of farm labor. A cross-sectional study was conducted to determine the prevalence of neck pain (NP) and musculoskeletal symptoms (MSS) and its association with personal characteristics, physical workloads and psychosocial factors among rubber workers. Stratified random sampling method was adopted and a total of 419 rubber workers in FELDA's scheme Malaysia participated in this study. Data was collected through face to face interview using modified Standardized Nordic Questionnaire (SNQ) and Job Content Questionnaire (JCQ). The results revealed the prevalence of NP was 59.9% and weak correlation with age (?= -0.184, p= 0.001) and a positive weak correlation with working hours per day (?= 0.099, p= 0.043) significantly. All physical workloads (neck flexion or rotation, awkward postures, repetitive motion and static postures) had significant weak to moderate positive correlation with NP (p<0.05). Job insecurity was found to have weak and positive correlation with NP (p<0.05). Binary logistic regression analysis showed risk factors for NP were decreased with age (OR= 3.92, 95% CI 1.61 - 9.58, p=0.003), increase in neck flexion or rotation (OR= 9.52, 95% CI 5.55 - 16.32, p= 0.001), awkward postures (OR=2.23, 95% CI 1.29 - 3.86, p= 0.004) and static postures (OR= 1.86, 95% CI 1.10 - 3.14, p= 0.021). This study showed that high prevalence of NP was associated with neck flexion or rotation, awkward and static postures.


Fibroblast heterogeneity: implications for human disease.
Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining the structural integrity of most tissues. Researchers have long suspected that fibroblasts exhibit functional specialization according to their organ of origin, body site, and spatial location. In recent years, a number of approaches have revealed the existence of fibroblast subtypes in mice. Here, we discuss fibroblast heterogeneity with a focus on the mammalian dermis, which has proven an accessible and tractable system for the dissection of these relationships. We begin by considering differences in fibroblast identity according to anatomical site of origin. Subsequently, we discuss new results relating to the existence of multiple fibroblast subtypes within the mouse dermis. We consider the developmental origin of fibroblasts and how this influences heterogeneity and lineage restriction. We discuss the mechanisms by which fibroblast heterogeneity arises, including intrinsic specification by transcriptional regulatory networks and epigenetic factors in combination with extrinsic effects of the spatial context within tissue. Finally, we discuss how fibroblast heterogeneity may provide insights into pathological states including wound healing, fibrotic diseases, and aging. Our evolving understanding suggests that ex vivo expansion or in vivo inhibition of specific fibroblast subtypes may have important therapeutic applications.


Effects of industrial wind turbine noise on sleep and health.
Industrial wind turbines (IWTs) are a new source of noise in previously quiet rural environments. Environmental noise is a public health concern, of which sleep disruption is a major factor. To compare sleep and general health outcomes between participants living close to IWTs and those living further away from them, participants living between 375 and 1400 m (n = 38) and 3.3 and 6.6 km (n = 41) from IWTs were enrolled in a stratified cross-sectional study involving two rural sites. Validated questionnaires were used to collect information on sleep quality (Pittsburgh Sleep Quality Index - PSQI), daytime sleepiness (Epworth Sleepiness Score - ESS), and general health (SF36v2), together with psychiatric disorders, attitude, and demographics. Descriptive and multivariate analyses were performed to investigate the effect of the main exposure variable of interest (distance to the nearest IWT) on various health outcome measures. Participants living within 1.4 km of an IWT had worse sleep, were sleepier during the day, and had worse SF36 Mental Component Scores compared to those living further than 1.4 km away. Significant dose-response relationships between PSQI, ESS, SF36 Mental Component Score, and log-distance to the nearest IWT were identified after controlling for gender, age, and household clustering. The adverse event reports of sleep disturbance and ill health by those living close to IWTs are supported.


Neonatal administration of thimerosal causes persistent changes in mu opioid receptors in the rat brain.
Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several neurodevelopmental disorders. Our previous study showed that thimerosal administered to suckling rats causes persistent, endogenous opioid-mediated hypoalgesia. Here we examined, using immunohistochemical staining technique, the density of μ-opioid receptors (MORs) in the brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and hippocampus were examined. Thimerosal administration caused dose-dependent statistically significant increase in MOR densities in the periaqueductal gray and caudate putamen, but decrease in the dentate gyrus, where it was accompanied by the presence of degenerating neurons and loss of synaptic vesicle marker (synaptophysin). These data document that exposure to thimerosal during early postnatal life produces lasting alterations in the densities of brain opioid receptors along with other neuropathological changes, which may disturb brain development.


Effect of a combination of genistein, polyunsaturated fatty acids and vitamins D3 and K1 on bone mineral density in postmenopausal women: a randomized, placebo-controlled, double-blind pilot study.
Many postmenopausal women desire non-pharmaceutical alternatives to hormone therapy for protection against osteoporosis. Soybean isoflavones, especially genistein, are being studied for this purpose. This study examined the effects of synthetic genistein in combination with other potential bone-protective dietary molecules on bone mineral density (BMD) in early postmenopausal women. In this 6-month double-blind pilot study, 70 subjects were randomized to receive daily either calcium only or the geniVida™ bone blend (GBB), which consisted of genistein (30 mg/days), vitamin D3 (800 IU/days), vitamin K1 (150 μg/days) and polyunsaturated fatty acids (1 g polyunsaturated fatty acids as ethyl ester: eicosapentaenoic acid/docosahexaenoic acid ratio = ~2/1). Markers of bone resorption and formation and BMD at the femoral neck, lumbar spine, Ward's triangle, trochanter and intertrochanter, total hip and whole body were assessed. Subjects supplemented with the GBB (n = 30) maintained femoral neck BMD, whereas in the placebo group (n = 28), BMD significantly decreased (p = 0.007). There was also a significant difference (p < 0.05) in BMD between the groups at Ward's triangle in favor of the GBB group. Bone-specific alkaline phosphatase and N-telopeptide significantly increased in the GBB group in comparison with those in baseline and in the placebo group. The GBB was well tolerated, and there were no significant differences in adverse events between groups. The GBB may help to prevent osteoporosis and reduce fracture risk, at least at the hip, in postmenopausal women. Larger and longer-term clinical trials are warranted.


Teaching research literacy: a model faculty development program at Oregon College of Oriental Medicine.
Content on integrative healthcare and complementary and alternative medicine is being taught in hundreds of educational programs across the country. Nursing, medical, osteopathic, chiropractic, acupuncture, naturopathic, and other programs are finding creative and innovative ways to include these approaches in new models of education and practice. This column spotlights such innovations in integrative healthcare and CAM education and presents readers with specific educational interventions they can adapt into new or ongoing educational efforts at their institution or programs. We invite readers to submit brief descriptions of efforts in their institutions that reflect the creativity, diversity, and interdisciplinary nature of the field. Please submit to Dr Sierpina at firstname.lastname@example.com or Dr Kreitzer at email@example.com. Submissions should be no more than 500 to 1,500 words. Please include any Web site or other resource that is relevant, as well as contact information.


First steps in using machine learning on fMRI data to predict intrusive memories of traumatic film footage.
After psychological trauma, why do some only some parts of the traumatic event return as intrusive memories while others do not? Intrusive memories are key to cognitive behavioural treatment for post-traumatic stress disorder, and an aetiological understanding is warranted. We present here analyses using multivariate pattern analysis (MVPA) and a machine learning classifier to investigate whether peri-traumatic brain activation was able to predict later intrusive memories (i.e. before they had happened). To provide a methodological basis for understanding the context of the current results, we first show how functional magnetic resonance imaging (fMRI) during an experimental analogue of trauma (a trauma film) via a prospective event-related design was able to capture an individual's later intrusive memories. Results showed widespread increases in brain activation at encoding when viewing a scene in the scanner that would later return as an intrusive memory in the real world. These fMRI results were replicated in a second study. While traditional mass univariate regression analysis highlighted an association between brain processing and symptomatology, this is not the same as prediction. Using MVPA and a machine learning classifier, it was possible to predict later intrusive memories across participants with 68% accuracy, and within a participant with 97% accuracy; i.e. the classifier could identify out of multiple scenes those that would later return as an intrusive memory. We also report here brain networks key in intrusive memory prediction. MVPA opens the possibility of decoding brain activity to reconstruct idiosyncratic cognitive events with relevance to understanding and predicting mental health symptoms.


Witnesses in action: the effect of physical exertion on recall and recognition.
Understanding memory performance under different operational conditions is critical in many occupational settings. To examine the effect of physical exertion on memory for a witnessed event, we placed two groups of law-enforcement officers in a live, occupationally relevant scenario. One group had previously completed a high-intensity physical-assault exercise, and the other had not. Participants who completed the assault exercise showed impaired recall and recognition performance compared with the control group. Specifically, they provided significantly less accurate information concerning critical and incidental target individuals encountered during the scenario, recalled less briefing information, and provided fewer briefing updates than control participants did. Exertion was also associated with reduced accuracy in identifying the critical target from a lineup. These results support arousal-based competition accounts proposing differential allocation of resources under physiological arousal. These novel findings relating to eyewitness memory performance have important implications for victims, ordinary citizens who become witnesses, and witnesses in policing, military, and related operational contexts.


Challenges to the clinical application of pluripotent stem cells: towards genomic and functional stability.
Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and are pluripotent, retaining the ability to differentiate into all cell types in the body. As a renewable source of various types of human cells, hESCs hold great therapeutic potential. Although significant advances have been achieved in defining the conditions needed to differentiate hESCs into various types of biologically active cells, many challenges remain in the clinical development of hESC-based cell therapy, such as the immune rejection of allogeneic hESC-derived cells by recipients. Breakthroughs in the generation of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells with defined factors, raise the hope that autologous cells derived from patient-specific iPSCs can be transplanted without immune rejection. However, recent genomic studies have revealed epigenetic and genetic abnormalities associated with induced pluripotency, a risk of teratomas, and immunogenicity of some iPSC derivatives. These findings have raised safety concerns for iPSC-based therapy. Here, we review recent advances in understanding the genomic and functional stability of human pluripotent stem cells, current challenges to their clinical application and the progress that has been made to overcome these challenges.


Blood pressure-lowering treatment based on cardiovascular risk: a meta-analysis of individual patient data.
We aimed to investigate whether the benefits of blood pressure-lowering drugs are proportional to baseline cardiovascular risk, to establish whether absolute risk could be used to inform treatment decisions for blood pressure-lowering therapy, as is recommended for lipid-lowering therapy. This meta-analysis included individual participant data from trials that randomly assigned patients to either blood pressure-lowering drugs or placebo, or to more intensive or less intensive blood pressure-lowering regimens. The primary outcome was total major cardiovascular events, consisting of stroke, heart attack, heart failure, or cardiovascular death. Participants were separated into four categories of baseline 5-year major cardiovascular risk using a risk prediction equation developed from the placebo groups of the included trials (<11%, 11-15%, 15-21%, >21%). 11 trials and 26 randomised groups met the inclusion criteria, and included 67,475 individuals, of whom 51,917 had available data for the calculation of the risk equations. 4167 (8%) had a cardiovascular event during a median of 4·0 years (IQR 3·4-4·4) of follow-up. The mean estimated baseline levels of 5-year cardiovascular risk for each of the four risk groups were 6·0% (SD 2·0), 12·1% (1·5), 17·7% (1·7), and 26·8% (5·4). In each consecutive higher risk group, blood pressure-lowering treatment reduced the risk of cardiovascular events relatively by 18% (95% CI 7-27), 15% (4-25), 13% (2-22), and 15% (5-24), respectively (p=0·30 for trend). However, in absolute terms, treating 1000 patients in each group with blood pressure-lowering treatment for 5 years would prevent 14 (95% CI 8-21), 20 (8-31), 24 (8-40), and 38 (16-61) cardiovascular events, respectively (p=0·04 for trend). Lowering blood pressure provides similar relative protection at all levels of baseline cardiovascular risk, but progressively greater absolute risk reductions as baseline risk increases. These results support the use of predicted baseline cardiovascular disease risk equations to inform blood pressure-lowering treatment decisions. None.


Research consent from young people in resource-poor settings.
Authoritative international guidelines stipulate that for minors to participate in research, consent must be obtained from their parents or guardians. Significant numbers of mature minors, particularly in low-income settings, are currently being ruled out of research participation because their parents are unavailable or refuse to provide consent despite the possibility that they might wish to do so and that such research has the potential to be of real benefit. These populations are under-represented in all types of clinical research. We propose that, for research with a prospect of direct benefit that has been approved by relevant ethics committees, the default position should be that minors who are able to provide valid consent and meet the following criteria should be able to consent for themselves regardless of age and whether they have reached majority: the minor must be competent and mature relative to the decision; their consent must be voluntary and they must be relatively independent and used to decision making of comparable complexity. In addition, the context must be appropriate, the information related to the research must be provided in a manner accessible to the minor and the consent must be obtained by a trained consent taker in surroundings conducive for decision making by the minor. In this paper, we have argued that consent by mature minors to research participation is acceptable in some situations and should be allowed.


Structural and functional MRI study of the brain, cognition and mood in long-term adequately treated Hashimoto's thyroiditis.
The current study investigated neuropsychological and underlying structural and functional brain alterations in long-term adequately treated patients with Hashimoto's thyroiditis in order to examine much discussed residual complaints in patients in relation to possible long-term neural alterations with a specific interest in the underlying autoimmune process. Eighteen patients with treated hypothyroidism due to Hashimoto's thyroiditis (mean age 32, range 18-54 years; two males; mean treatment duration 4.4 years) and 18 healthy matched control subjects underwent 3-Tesla magnetic resonance imaging (MRI). Voxel-based morphometry was used to investigate grey matter density, resting-state functional MRI to analyse the brain connectivity of areas known to be altered in hypothyroidism and event-related functional MRI to examine brain activity during associative memory encoding. Neuropsychological assessment included memory, working memory, psychomotor speed and attention. We previously reported subclinically reduced mood in this study population and investigated its neural correlates here. Thyroid stimulating hormone, free triiodthyronine, free thyroxine and thyroid peroxidase antibodies were measured in serum. We did not find cognitive deficits or alterations in grey matter density, functional connectivity or associative memory-related brain activity in comparison to the control group and cognition was unrelated to thyroid serum measures in the patient group. Thyroid peroxidase antibodies in the patient group correlated with increased grey matter density in right amygdala and enhanced connectivity between subcallosal and parahippocampal areas. Treatment duration was associated with brain structure in frontal and occipital cortex and connectivity between left amygdala and frontal cortex. Mood correlated with brain areas associated with distinct functional networks, but not with those most prominently affected in depression. In conclusion, no cognitive or neural alterations were detected in this young and otherwise healthy cohort of patients in comparison to a healthy control group and current mood status could not be related to depression-related networks. However, autoimmune activity and treatment duration showed a relationship with depression and hypothyroidism-related brain structure and function. They are thus promising factors to further investigate residual complaints despite biochemically adequate treatment in patients with Hashimoto's thyroiditis. Given the small sample size, all findings require replication.


Randomised controlled trial of topical kanuka honey for the treatment of rosacea.
To investigate the efficacy of topical 90% medical-grade kanuka honey and 10% glycerine (Honevo) as a treatment for rosacea. Randomised controlled trial with blinded assessment of primary outcome variable. Outpatient primary healthcare population from 5 New Zealand sites. 138 adults aged ≥ 16, with a diagnosis of rosacea, and a baseline blinded Investigator Global Assessment of Rosacea Severity Score (IGA-RSS) of ≥ 2. 69 participants were randomised to each treatment arm. 1 participant was excluded from the Honevo group, and 7 and 15 participants withdrew from the Honevo and control groups, respectively. Participants were randomly allocated 1:1 to Honevo or control cream (Cetomacrogol), applied twice daily for 8 weeks. The primary outcome measure was the proportion of participants who had a ≥ 2 improvement in the 7-point IGA-RSS at week 8 compared to baseline. Secondary outcomes included change in IGA-RSS and subject-rated visual analogue score of change in severity (VAS-CS) on a 100 mm scale (0 mm 'much worse', 100 mm 'much improved') at weeks 2 and 8. 24/68 (34.3%) in the Honevo group and 12/69 (17.4%) in the control group had a ≥ 2 improvement in IGA-RSS at week 8 compared to baseline (relative risk 2.03; 95% CI 1.11 to 3.72, p=0.020). The change in IGA-RSS for Honevo compared to control at week 2 minus baseline was -1 (Hodges-Lehman estimate, 95% CI -1 to 0, p=0.03), and at week 8 minus baseline was -1 (Hodges-Lehman estimate, 95% CI -1 to 0, p=0.005). The VAS-CS at week 2 was 9.1 (95% CI 3.5 to 14.7), p=0.002, and at week 8 was 12.3 (95% CI 5.7 to 18.9)¸ p<0.001 for Honevo compared to control. Honevo is an effective treatment for rosacea. This trial was registered in the Australian and New Zealand Clinical Trials Registry ACTRN12614000004662.


Activation of human factor IX (Christmas factor).
Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.


Effects of honey in the management of alveolar osteitis: A study.
Alveolar osteitis (AO) is a complication of tooth extraction which indicates inflammation of alveolar bone of either maxilla or mandible. This study uses Apitherapy where honey catalyses biological reactions to improve immune system, makes local environment unbearable for microorganisms in the affected socket and enhances healing. 50 patients of AO were included in the study. After cleansing of the affected socket, honey dressing was applied. Dressings were changed daily for first 2 days and then altenatively. In biochemical investigations, CRP levels in the body were measured using Nephelometry method. Microbiological examination was done for the identification of microorganism and semi quantitative count of colony forming units. Results were assessed from clinical, microbiological, biochemical and radiological findings at 1st, 2nd, 3rd, 5th, 7th day based on VAS score, erythema, pus discharge, swelling, lymphadenitis, fever, bleeding on probing, exposed bone and necrotic debris. Pre-Treatment CRP was 2.08 ± 1.62 which significantly (P = 0.0001) decreased to 0.82 ± 0.48. Mean change and average percentage change were 1.25 ± 1.51 and 44.1% respectively. Majority of the patients with exposed bone got healed socket with evidance of granulation tissue and healing gingiva in about one week. CRP levels at the completion of treatment of AO with honey dressing showed a significant decrease from the pre-treatment values indicating fast recovery. Microbiological examination showed presence of normal commensal flora at AO sites like Streptococcus, Staphyloccocus and Enterococcus. So, the role of bacteria in the genesis of AO, if any, appears unclear.