Correction: Regulation of dopaminergic function: An [18F]-DOPA PET apomorphine challenge study in humans.

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Correction: Regulation of dopaminergic function: an [18F]-DOPA PET apomorphine challenge study in humans.

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Regulation of dopaminergic function: an [18F]-DOPA PET apomorphine challenge study in humans.

Dopaminergic function has a key role in normal brain function, dopaminergic dysfunction being implicated in numerous neuropsychiatric disorders. Animal studies show that dopaminergic stimulation regulates dopaminergic function, but it is not known whether this exists in humans. In the first study (study 1), we measured dopamine synthesis capacity (indexed as Kicer) to identify the relationship between baseline and change in Kicer under resting conditions for comparison with effects of dopaminergic stimulation. In the second study (study 2), we used a within-subjects design to test effects of dopaminergic stimulation on dopamine synthesis capacity. In study 1, eight volunteers received two 18F-DOPA scans on separate days, both at rest. In study 2, 12 healthy male volunteers received two 18F-DOPA positron emission tomographic (PET) scans after treatment with either the dopamine partial agonist apomorphine (0.03 or 0.005 mg kg-1) or placebo. In study 1, no significant correlation was found between baseline and change in dopamine synthesis capacity between scans (r=-0.57, n=8, P=0.17, two-tailed). In study 2, a significant negative correlation was found between baseline dopamine synthesis capacity and percentage change in dopamine synthesis capacity after apomorphine challenge (r=-0.71, n=12, P=0.01, two-tailed). This correlation was significantly different (P<0.01) from the correlation between baseline and change in dopamine synthesis capacity under unstimulated conditions. One-way repeated-measures analysis of variance showed a significant group (study 1/study 2) × time interaction (F(1,18)=11.5, P=0.003). Our findings suggest that regulation of dopamine synthesis capacity by apomorphine depends on baseline dopamine function, consistent with dopamine stimulation stabilizing dopaminergic function. Loss of this autoregulation may contribute to dopaminergic dysfunction in brain disorders such as schizophrenia, substance dependence, and Parkinson's disease.

Leveraging Industry-Academia Collaborations in Adaptive Biomedical Innovation.

Despite the rapid pace of biomedical innovation, research and development (R&D) productivity in the pharmaceutical industry has not improved broadly. Increasingly, firms need to leverage new approaches to product development and commercial execution, while maintaining adaptability to rapid changes in the marketplace and in biomedical science. Firms are also seeking ways to capture some of the talent, infrastructure, and innovation that depends on federal R&D investment. As a result, a major transition to external innovation is taking place across the industry. One example of these external innovation initiatives is the Sanofi-MIT Partnership, which provided seed funding to MIT investigators to develop novel solutions and approaches in areas of interest to Sanofi. These projects were highly collaborative, with information and materials flowing both ways. The relatively small amount of funding and short time frame of the awards built an adaptable and flexible process to advance translational science.

Chronic exposure to haloperidol and olanzapine leads to common and divergent shape changes in the rat hippocampus in the absence of grey-matter volume loss.

BACKGROUND: One of the most consistently reported brain abnormalities in schizophrenia (SCZ) is decreased volume and shape deformation of the hippocampus. However, the potential contribution of chronic antipsychotic medication exposure to these phenomena remains unclear. METHOD: We examined the effect of chronic exposure (8 weeks) to clinically relevant doses of either haloperidol (HAL) or olanzapine (OLZ) on adult rat hippocampal volume and shape using ex vivo structural MRI with the brain retained inside the cranium to prevent distortions due to dissection, followed by tensor-based morphometry (TBM) and elastic surface-based shape deformation analysis. The volume of the hippocampus was also measured post-mortem from brain tissue sections in each group. RESULTS: Chronic exposure to either HAL or OLZ had no effect on the volume of the hippocampus, even at exploratory thresholds, which was confirmed post-mortem. In contrast, shape deformation analysis revealed that chronic HAL and OLZ exposure lead to both common and divergent shape deformations (q = 0.05, FDR-corrected) in the rat hippocampus. In particular, in the dorsal hippocampus, HAL exposure led to inward shape deformation, whereas OLZ exposure led to outward shape deformation. Interestingly, outward shape deformations that were common to both drugs occurred in the ventral hippocampus. These effects remained significant after controlling for hippocampal volume suggesting true shape changes. CONCLUSIONS: Chronic exposure to either HAL or OLZ leads to both common and divergent effects on rat hippocampal shape in the absence of volume change. The implications of these findings for the clinic are discussed.

Routine measurements of cord arterial blood lactate levels in infants delivering at term and prediction of neonatal outcome.

OBJECTIVE: Our purpose was to evaluate the relationship between umbilical cord arterial blood lactate levels with acid base balance to the mode of delivery and short-term neonatal outcome in a large multiracial population delivering at term in University of Malaya Medical Centre. MATERIALS AND METHODS: Two thousand two hundred and twelve patients of more than 37 weeks of gestation with singleton, liveborn infants with no major anomalies delivering between January 2013 to December 2013 were analysed. Lactate was measured by using portable Lactate analyzer that requires 5 µml of blood and provides the result within 1 minute. The deliveries took place at the Department of Obstetrics and Gynaecology, University Malaya Medical Centre, Malaysia where umbilical cord blood sampling and blood gas analysis is a part of the routine assessment of all newborn. RESULTS: Gestational age ranged from 37 to 43 weeks (mean 39.05 weeks). The highest mean arterial cord lactate values were noted among babies delivered instrumentally (4.87 mmol/L). Infants who had a normal vaginal delivery had the second highest levels (3.36 mmol/L), followed by infants delivered by emergency caesarean section (3.30mmol/L). The lowest lactate values were noted in deliveries by elective caesarean section (3.0mmol/L). Cord arterial lactate levels were significantly higher among infants born with low Apgar scores (7.02 mmol/L vs 4.6mmol/L, P < 0.001). High arterial cord lactate was a significant predictor of admission to Neonatal intensive care unit (NICU) was 6.0mmol/L. Receiver operator curve (ROC) analysis suggests that lactate and pH are virtually equivalent in their correlation with adverse neonatal outcome. CONCLUSION: Cord lactate levels are significantly related to the mode of delivery and is equivalent to cord arterial pH in predicting adverse neonatal outcomes, with similar efficacies; however, its simplicity, less sampling failure and low cost makes lactate analysis an interesting alternative in obstetric care.

Effect of chronic antipsychotic treatment on striatal phosphodiesterase 10A levels: a [¹¹C]MP-10 PET rodent imaging study with ex vivo confirmation.

A number of phosphodiesterase 10A (PDE10) inhibitors are about to undergo clinical evaluation for their efficacy in treating schizophrenia. As phosphodiesterases are in the same signalling pathway as dopamine D2 receptors, it is possible that prior antipsychotic treatment could influence these enzyme systems in patients. Chronic, in contrast to acute, antipsychotic treatment has been reported to increase brain PDE10A levels in rodents. The aim of this study was to confirm these findings in a manner that can be translated to human imaging studies to understand its consequences. Positron emission tomography (PET) scanning was used to evaluate PDE10A enzyme availability, after chronic haloperidol administration, using a specific PDE10A ligand ([(11)C]MP-10). The binding of [(11)C]MP-10 in the striatum and the cerebellum was measured in rodents and a simplified reference tissue model (SRTM) with cerebellum as the reference region was used to determine the binding potential (BPND). In rats treated chronically with haloperidol (2 mg kg(-1) per day), there was no significant difference in PDE10A levels compared with the vehicle-treated group (BPND±s.d.: 3.57 ± 0.64 versus 2.86 ± 0.71). Following PET scans, ex vivo analysis of striatal brain tissue for PDE10A mRNA (Pde10a) and PDE10A enzyme activity showed no significant difference. Similarly, the PDE10A protein content determined by western blot analysis was similar between the two groups, contrary to an earlier finding. The results of the study indicate that prior exposure to antipsychotic medication in rodents does not alter PDE10A levels.

Differential expression of ATP-binding cassette and/or major facilitator superfamily class efflux pumps contributes to voriconazole resistance in Aspergillus flavus.

Invasive aspergillosis remains a life-threatening infection in immunocompromised patients. Although clinical failures are attributed to poor host immunity, antifungal drug resistance may be a contributing factor. Reports of voriconazole (VRC) resistance (VRC-R) in clinical isolates of Aspergillus spp. continue to emerge from various centers around the world, and mechanisms contributing to drug resistance are poorly understood. The aim of this study is to study the role of multidrug resistance efflux pumps (MDR-EPs) in VRC-R in Aspergillus flavus using efflux pump inhibitors and quantitative reverse transcriptase polymerase chain reaction. Relative quantification of various MDR-EPs was performed pre-exposure and postexposure to VRC, which demonstrated an increase in 1 or more efflux pump gene transcripts to varying degrees in VRC-susceptible and VRC-R isolates of A. flavus. Exposure to sub-MIC of VRC causes up-regulation of genes encoding MDR-EPs, contributing to triazole resistance in A. flavus and may not be detected during routine antifungal susceptibility testing in vitro.

Haloperidol and olanzapine mediate metabolic abnormalities through different molecular pathways.

The pathogenesis of antipsychotic-induced disturbances of glucose homeostasis is still unclear. Increased visceral adiposity has been suggested to be a possible mediating mechanism. The aim of this study was to investigate, in an animal model, the differential effects of olanzapine and haloperidol on visceral fat deposition (using magnetic resonance imaging(MRI)) and on critical nodes of the insulin signaling pathway (liver-protein levels of IRS2 (insulin receptor substrate 2), GSK3α (glycogen synthase kinase-3α), GSK3ß, GSK3α-Ser21, GSK3ß-Ser9). To this end, we studied male Sprague-Dawley rats treated with vehicle (n=8), haloperidol (2 mg kg(-1) per day, n=8), or olanzapine (10 mg kg(-1)per day, n=8), using osmotic minipumps, for 8 weeks. The haloperidol group showed a higher percentage of visceral fat than both the olanzapine group and the vehicle group, whereas there was no difference between the olanzapine and the vehicle group. In terms of insulin signaling pathway, the olanzapine group showed significantly reduced IRS2 levels, reduced phosphorylation of GSK3α and increased phosphorylation of GSK3ß, whereas there was no difference between the haloperidol and the vehicle group. Our data suggest that different molecular pathways mediate the disturbances of glucose homeostasis induced by haloperidol and olanzapine with a direct effect of olanzapine on the insulin molecular pathway, possibly partly explaining the stronger propensity of olanzapine for adverse effects on glucose regulation when compared with haloperidol in clinical settings.

In vitro-in vivo correlation of voriconazole resistance due to G448S mutation (cyp51A gene) in Aspergillus fumigatus.

Invasive pulmonary aspergillosis continues to be associated with a high mortality despite timely and appropriate therapy. Although host immunity plays a major role in poor clinical response, antifungal drug resistance cannot be ignored. Our studies were aimed 1) to study the mechanism of drug resistance in voriconazole strains of Aspergillus fumigatus, 2) to establish a causal relationship between cyp51A mutation and voriconazole resistance (VRC-R), and 3) to determine whether VRC-R due to cyp51A mutation correlated with in vivo resistance. A point mutation (G448S) involving cyp51A gene in VRC-R isolate was associated with resistance to VRC but not to posaconazole (POS); POS had superior activity to VRC in reducing lung fungal burden and mortality in mice infected with a VRC-R mutant of A. fumigatus. Our study demonstrated that azole resistance is based on specific site of cyp51A mutation and that in vitro VRC-R correlates with in vivo resistance.

Efficient iodine catalyzed three components domino reaction for the synthesis of 1-((phenylthio)(phenyl)methyl)pyrrolidin-2-one derivatives possessing anticancer activities.

A simple and efficient three components domino reaction of γ-butyrolactam (2-pyrrolidinone), aromatic aldehyde and substituted thiophenol catalyzed by elemental iodine resulted in the formation of 1-((phenylthio)(phenyl)methyl)pyrrolidin-2-one derivatives. The stability of the synthesized analogues was evaluated in stimulated gastric fluid (SGF) and bovine serum albumin (BSA). In vitro anticancer activity was investigated in the low micromolar range and a few analogues were found to possess good activity. This current protocol provides several advantages like shorter reaction time, excellent yield and convenient work-up.

Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus.

AIMS: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. METHODS AND RESULTS: The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis-related mechanism of CPV. CONCLUSIONS: CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic-resistant Staph. aureus.

Terbinafine: a pharmacological and clinical review.

Terbinafine (TRB) is an allylamine antifungal agent that has been available for more than a decade. It is now used for the treatment of dermatophytic infections and onychomycosis. Despite several studies having demonstrated the efficacy of terbinafine against nondermatophytic infections including azole-resistant candidiasis, invasive aspergillosis, disseminated fusariosis and scedosporiosis, the role of TRB in the management of these infections remains greatly underappreciated. A brief review of pharmacodynamic, pharmacokinetic, in vitro and in vivo data and published case reports provides insight into the use of terbinafine as a potential adjunct in combination with azoles, polyenes or echinocandins in the management of severe drug-resistant or refractory mycoses. Despite the lack of intrinsic fungicidal activity against several nondermatophytes, when used in combination, particularly with azoles, TRB has demonstrated good antifungal efficacy that could be exploited in clinical practice. As comprehensive human clinical studies are not feasible with the rare occurence of these mycoses, experiments using animal models are essential to evaluate the in vivo efficacy of drug combinations. In summary, terbinafine has established itself as a drug of choice for dermatophytic infections; it must be considered in combination with other antifungal agents for the management of nondermatophytic refractory or resistant yeast/mold infections as well.

Chromium(III) complexes inhibit transcription factors binding to DNA and associated gene expression.

Role of chromium(III) in biological systems is controversial and no significant conclusions can be arrived with the available data. Chromium(VI) enters in to the cell readily and gets reduced to form stable chromium(III) complexes. Chromium(III) complexes react slowly and were considered non-toxic. Recent studies suggest that chromium(III) indeed produce damage to cellular organelle, DNA and proteins and can be lethal to organisms and their offspring. Several chromium(III) complexes synthesized are shown to interact with DNA and bring changes in the conformation of DNA and proteins. In this work, effects of these interactions in the vital process like transcription have been studied. Three Cr(III) complexes with varied ligand environment, ionic character and charge have been chosen for the study to evaluate their ability to interrupt the binding of two different transcription factors Sp1 and TFIID, with their consensus sequences and interruption of the transcription process. Of the three complexes tested [Cr(salen)(H(2)O)(2)](+) is the most efficient inhibitor of transcription factors-DNA (TF-DNA) complex formation and transcription. Inhibition of TF-DNA complex formation and transcription by this complex is concentration-dependent. [Cr(pic)(3)] does not inhibit both TF-DNA complex formation and transcription. [Cr(ttpy)(2)](+3) inhibits TF-DNA complex formation at higher concentrations but does not inhibit transcription even at very high concentrations compared to that of [Cr(salen)(H(2)O)(2)](+). Results of these studies suggest that chromium(III) complexes, though less reactive can still interfere in the important biological process and this interactions are determined by the ligand environment around the complex.

Compensatory changes in enzymes of arginine metabolism during renal hypertrophy in mice.

The present study investigates enzyme activities of the urea cycle, transamidinase and ornithine-proline inter-conversion in the hypertrophied kidney after unilateral nephrectomy in mice. Surgical removal of the left kidney in mice led to compensatory enlargement of the right kidney after 1 and 14 days. This renal growth was associated with an increase in glomerular volume (but not number) and enlargement of the proximal convoluted tubules. The total renal protein content increased in proportion to the increase in kidney weight, but the protein per gram weight of kidney did not change. The specific activity of only ornithine aminotransferase (OAT), the rate-limiting enzyme in the conversion of ornithine to proline, increased in 2 weeks of hypertrophy. The specific activity of all other enzymes was unchanged. However, the total enzyme activity per kidney of all the enzymes, without exception, was elevated in the hypertrophied kidney. While the increase in total OAT activity was much more than the increase in kidney weight, all other enzymes increased more or less in proportion to the increase in renal mass. The results suggest that compensation in OAT activity to chronic reduction in renal mass was complete, but only partial in the case of other enzymes.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector.

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli.

Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular size between water and urea, through the bacterial membranes changed directly with the rate of respiration (an activity residing in the bacterial plasma membrane) in E. coli and the enhanced permeability and respiratory activity were highly correlated. Hydrogen peroxide permeability and induction of voids (spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these cavities) were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy of the extrinsic fluorescent probes (incorporated by growing bacteria in their presence) decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of osmotic phenomena, as a relevant (thermodynamic) volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth.

A general strategy to enhance the potency of chimeric transcriptional activators.

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.

Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo.

We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome-mediated protein degradation pathway. The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo. Mutations either in the activation domain residues involved in target protein interaction or in the DNA-binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes. Finally, using a rapamycin-regulated gene expression system, we show that recruiting activation domains to DNA-bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators. These observations suggest that in mammalian cells efficient recruitment of activator-target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes. We propose that proteasome-mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.