Antarctic organisms as a source of antimicrobial compounds: a patent review.

Currently, antimicrobial resistance has become a global public health problem, which has made the need for new antimicrobial compounds to deal with resistant infections an emergency. However, environments that once offered so many innovative molecules, now already exhaustively exploited, do not meet this need. In this context, a geographically isolated, under-explored and extreme environment, such as Antarctica, which holds organisms with unique physiological and biochemical characteristics, assumes great importance as a potential source of new compounds with antimicrobial activity. In this patent review, we investigate the state of technological development in the field of antimicrobial compounds obtained from Antarctic organisms, highlighting the main countries and researchers active in the field, the species utilized, the compounds obtained, and their possible therapeutic applications. As results, few patent documents were found, however they encompass a wide diversity of compounds and species, indicating a great antimicrobial potential present in Antarctic biota, including compounds active against the most important human pathogenic microorganisms, such as including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus spp. and multi-resistant Mycobacterium tuberculosis. Furthermore, due to the increasing trend in patent applications, a significant rise in the number of patents in this area is expected in the coming years.

Antarctic environments as a source of bacterial and fungal therapeutic enzymes.

Microbial therapeutic enzymes are the protagonists in the pharmacological treatment of different human diseases. The intrinsic enzymatic characteristics, such as high affinity and specificity to the corresponding substrate, enable effective therapies, with minimal adverse effects and complete remission. However, immunogenicity, short half-life, low enzymatic yield, and low selectivity regarding available enzyme drugs are currently the main obstacles to their development and the broad adherence to therapeutic protocols. By harboring adapted and still unexplored microbial life, environments of extreme conditions, such as Antarctica, become especially important in the prospecting and development of new enzymatic compounds that present higher yields and the possibility of genetic improvement. Antarctic microorganisms have adaptation mechanisms, such as more fluid cell membranes, production of antifreeze proteins and enzymes with more malleable structures, more robust, stable, selective catalytic sites for their respective substrates, and high antioxidant capacity. In this context, this review aims to explore enzymes synthesized by bacteria and fungi from Antarctica as potential drug producers, capable of providing therapeutic efficacy, less adverse effects, and lower production costs with highlight to L-Asparaginase, collagenase, superoxide dismutase and ribonucleases. In addition, this review highlights the unique biotechnological profile of these Antarctic extremophile microorganisms.

Distinguishing Alzheimer's Disease Patients and Biochemical Phenotype Analysis Using a Novel Serum Profiling Platform: Potential Involvement of the VWF/ADAMTS13 Axis.

It is important to develop minimally invasive biomarker platforms to help in the identification and monitoring of patients with Alzheimer's disease (AD). Assisting in the understanding of biochemical mechanisms as well as identifying potential novel biomarkers and therapeutic targets would be an added benefit of such platforms. This study utilizes a simplified and novel serum profiling platform, using mass spectrometry (MS), to help distinguish AD patient groups (mild and moderate) and controls, as well as to aid in understanding of biochemical phenotypes and possible disease development. A comparison of discriminating sera mass peaks between AD patients and control individuals was performed using leave one [serum sample] out cross validation (LOOCV) combined with a novel peak classification valuation (PCV) procedure. LOOCV/PCV was able to distinguish significant sera mass peak differences between a group of mild AD patients and control individuals with a p value of 10-13. This value became non-significant (p = 0.09) when the same sera samples were randomly allocated between the two groups and reanalyzed by LOOCV/PCV. This is indicative of physiological group differences in the original true-pathology binary group comparison. Similarities and differences between AD patients and traumatic brain injury (TBI) patients were also discernable using this novel LOOCV/PCV platform. MS/MS peptide analysis was performed on serum mass peaks comparing mild AD patients with control individuals. Bioinformatics analysis suggested that cell pathways/biochemical phenotypes affected in AD include those involving neuronal cell death, vasculature, neurogenesis, and AD/dementia/amyloidosis. Inflammation, autoimmunity, autophagy, and blood-brain barrier pathways also appear to be relevant to AD. An impaired VWF/ADAMTS13 vasculature axis with connections to F8 (factor VIII) and LRP1 and NOTCH1 was indicated and is proposed to be important in AD development.

Distinguishing and Biochemical Phenotype Analysis of Epilepsy Patients Using a Novel Serum Profiling Platform.

Diagnosis of non-symptomatic epilepsy includes a history of two or more seizures and brain imaging to rule out structural changes like trauma, tumor, infection. Such analysis can be problematic. It is important to develop capabilities to help identify non-symptomatic epilepsy in order to better monitor and understand the condition. This understanding could lead to improved diagnostics and therapeutics. Serum mass peak profiling was performed using electrospray ionization mass spectrometry (ESI-MS). A comparison of sera mass peaks between epilepsy and control groups was performed via leave one [serum sample] out cross-validation (LOOCV). MS/MS peptide analysis was performed on serum mass peaks to compare epilepsy patient and control groups. LOOCV identified significant differences between the epilepsy patient group and control group (p = 10-22). This value became non-significant (p = 0.10) when the samples were randomly allocated between the groups and reanalyzed by LOOCV. LOOCV was thus able to distinguish a non-symptomatic epilepsy patient group from a control group based on physiological differences and underlying phenotype. MS/MS was able to identify potential peptide/protein changes involved in this epilepsy versus control comparison, with 70% of the top 100 proteins indicating overall neurologic function. Specifically, peptide/protein sera changes suggested neuro-inflammatory, seizure, ion-channel, synapse, and autoimmune pathways changing between epilepsy patients and controls.

Implications of Nonmarginal Budgetary Impacts in Health Technology Assessment: A Conceptual Model.

OBJECTIVES: This paper introduces a framework with which to conceptualize the decision-making process in health technology assessment for new interventions with high budgetary impacts. In such circumstances, the use of a single threshold based on the marginal productivity of the healthcare system is inappropriate. The implications of this for potential partial implementation, horizontal equity, and pharmaceutical pricing are illustrated using this framework. RESULTS: Under the condition of perfect divisibility and given an objective of maximizing health, a large budgetary impact of a new treatment may imply that optimal implementation is partial rather than full, even at a given incremental cost-effectiveness ratio that would nevertheless mean the decision to accept the treatment in full would not lead to a net reduction in health. In a one-shot price-setting game, this seems to give rise to potential horizontal equity concerns. When the assumption of fixity of the incremental cost-effectiveness ratio (arising from the assumed exogeneity of the manufacturer's price) is relaxed, it can be shown that the threat of partial implementation may be sufficient to give rise to an incremental cost-effectiveness ratio at which cost the entire potential population is treated, maximizing health at an increased level, and with no contravention of the horizontal equity principle.

Incorporating Affordability Concerns Within Cost-Effectiveness Analysis for Health Technology Assessment.

BACKGROUND: Recent policy developments and journal articles have emphasized a divergence: when interventions are found to be cost-effective but unaffordable. This apparent paradox reflects a conventional practice of cost-effectiveness analysis that does not properly evaluate the opportunity costs of an intervention that imposes non-marginal costs on the healthcare system. OBJECTIVE: Taking the perspective of an exogenously resource constrained decision maker, this paper presents a framework by which concerns for affordability can be appropriately incorporated within cost-effectiveness analysis. METHODS: A net benefit framework is proposed where health opportunity costs are estimated for each simulation iteration within each time period. The framework is applied to a hypothetical case study based on the recent experience of the English NHS with new hepatitis C drugs. RESULTS: Under the proposed framework, but not under conventional cost-effectiveness analysis, estimates of health opportunity costs differ between scenarios involving different profiles of budget impact even when their net present value, or expected value, are the same. CONCLUSIONS: The framework presented here reflects the importance of the scale of budget impacts along with their uncertainty distribution and time profile. In doing so it resolves issues with the conduct of conventional cost-effectiveness analysis where affordability concerns are not explicitly incorporated.

Minimally Invasive Resection of Brain Metastases.

BACKGROUND: Minimally invasive resection of brain metastases aims to maximize resection while minimizing brain trauma. METHODS: Patients with 1 or more metastases that underwent resection following neuro-oncology multidisciplinary meeting discussion from September 2014 to October 2018, with pre- and postoperative magnetic resonance imaging, were included. All patients including posterior fossa metastases or multiple metastases were positioned supine. Hair was not shaved. Volumetric postcontrast T1 magnetic resonance imaging was used for incision planning and neuronavigation. The craniotomy site was tailored to tumor depth according to keyhole principles and ranged between 2 and 5 cm. Intraoperative monitoring and awake mapping were carried out in selected cases. RESULTS: Out of 320 consecutive patients with brain metastases, 44 patients were identified as suitable for minimally invasive resection. Nine patients had no postoperative imaging and were excluded. There were 38 metastases in 35 patients. There were 18 cerebellar metastases, 10 frontal, 3 parietal, 3 occipital, 2 temporal, 1 intraventricular, and 1 basal ganglia. Median length of stay was 3 days (range, 1-24). Average tumor volume was 54.7 cm3 (range, 10-240 cm3). Endoscopic assistance was used in 4 patients. Median performance status improved from 2 to 1 (range, preoperative: 0-4; postoperative: 0-2). Median survival was 14.7 months. CONCLUSIONS: Minimally invasive resection of brain metastasis is safe and effective, and in selected cases confers advantages compared with standard techniques.

Distinguishing and phenotype monitoring of traumatic brain injury and post-concussion syndrome including chronic migraine in serum of Iraq and Afghanistan war veterans.

Traumatic Brain Injury (TBI) and persistent post-concussion syndrome (PCS) including chronic migraine (CM) are major health issues for civilians and the military. It is important to understand underlying biochemical mechanisms of these conditions, and be able to monitor them in an accurate and minimally invasive manner. This study describes the initial use of a novel serum analytical platform to help distinguish TBI patients, including those with post-traumatic headache (PTH), and to help identify phenotypes at play in these disorders. The hypothesis is that physiological responses to disease states like TBI and PTH and related bodily stresses are reflected in biomolecules in the blood in disease-specific manner. Leave one out (serum sample) cross validations (LOOCV) and sample randomizations were utilized to distinguished serum samples from the following TBI patient groups: TBI +PTSD + CM + severe depression (TBI "most affected" group) vs healthy controls, TBI "most affected" vs TBI, TBI vs controls, TBI + CM vs controls, and TBI + CM vs TBI. Inter-group discriminatory p values were ≤ 10-10, and sample group randomizations resulted in p non-significant values. Peptide/protein identifications of discriminatory mass peaks from the TBI "most affected" vs controls and from the TBI plus vs TBI minus CM groups yielded information of the cellular/molecular effects of these disorders (immune responses, amyloidosis/Alzheimer's disease/dementia, neuronal development). More specific biochemical disease effects appear to involve blood brain barrier, depression, migraine headache, autoimmunity, and autophagy pathways. This study demonstrated the ability for the first time of a novel, accurate, biomarker platform to monitor these conditions in serum, and help identify biochemical relationships leading to better understanding of these disorders and to potential therapeutic approaches.

Skin and soft tissue infections and current antimicrobial prescribing practices in Australian aged care residents.

To determine the burden of skin and soft tissue infections (SSTI), the nature of antimicrobial prescribing and factors contributing to inappropriate prescribing for SSTIs in Australian aged care facilities, SSTI and antimicrobial prescribing data were collected via a standardised national survey. The proportion of residents prescribed ⩾1 antimicrobial for presumed SSTI and the proportion whose infections met McGeer et al. surveillance definitions were determined. Antimicrobial choice was compared to national prescribing guidelines and prescription duration analysed using a negative binomial mixed-effects regression model. Of 12 319 surveyed residents, 452 (3.7%) were prescribed an antimicrobial for a SSTI and 29% of these residents had confirmed infection. Topical clotrimazole was most frequently prescribed, often for unspecified indications. Where an indication was documented, antimicrobial choice was generally aligned with recommendations. Duration of prescribing (in days) was associated with use of an agent for prophylaxis (rate ratio (RR) 1.63, 95% confidence interval (CI) 1.08-2.52), PRN orders (RR 2.10, 95% CI 1.42-3.11) and prescription of a topical agent (RR 1.47, 95% CI 1.08-2.02), while documentation of a review or stop date was associated with reduced duration of prescribing (RR 0.33, 95% CI 0.25-0.43). Antimicrobial prescribing for SSTI is frequent in aged care facilities in Australia. Methods to enhance appropriate prescribing, including clinician documentation, are required.

Obesity-induced decreases in muscle performance are not reversed by weight loss.

BACKGROUND/OBJECTIVES: Obesity can affect muscle phenotypes, and may thereby constrain movement and energy expenditure. Weight loss is a common and intuitive intervention for obesity, but it is not known whether the effects of obesity on muscle function are reversible by weight loss. Here we tested whether obesity-induced changes in muscle metabolic and contractile phenotypes are reversible by weight loss. SUBJECTS/METHODS: We used zebrafish (Danio rerio) in a factorial design to compare energy metabolism, locomotor capacity, muscle isometric force and work-loop power output, and myosin heavy chain (MHC) composition between lean fish, diet-induced obese fish, and fish that were obese and then returned to lean body mass following diet restriction. RESULTS: Obesity increased resting metabolic rates (P<0.001) and decreased maximal metabolic rates (P=0.030), but these changes were reversible by weight loss, and were not associated with changes in muscle citrate synthase activity. In contrast, obesity-induced decreases in locomotor performance (P=0.0034), and isolated muscle isometric stress (P=0.01), work-loop power output (P<0.001) and relaxation rates (P=0.012) were not reversed by weight loss. Similarly, obesity-induced decreases in concentrations of fast and slow MHCs, and a shift toward fast MHCs were not reversed by weight loss. CONCLUSION: Obesity-induced changes in locomotor performance and muscle contractile function were not reversible by weight loss. These results show that weight loss alone may not be a sufficient intervention.

Is the Ergogenicity of Caffeine Affected by Increasing Age? The Direct Effect of a Physiological Concentration of Caffeine on the Power Output of Maximally Stimulated EDL and Diaphragm Muscle Isolated from the Mouse.

INTRODUCTION: Caffeine is a well-established performance enhancing nutritional supplement in a young healthy population, however far less is known about how its ergogenicity is affected by increasing age. A recent review has highlighted the value of studies examining the direct effect of caffeine on isolated skeletal muscle contractility, but the present work is the first to assess the direct effect of 70µM caffeine (physiological maximum) on the maximal power output of isolated mammalian muscle from an age range representing developmental to early ageing. METHOD: Female CD1 mice were aged to 3, 10, 30 and 50 weeks (n = 20 in each case) and either whole EDL or a section of the diaphragm was isolated and maximal power output determined using the work loop technique. Once contractile performance was maximised, each muscle preparation was treated with 70µM caffeine and its contractile performance was measured for a further 60 minutes. RESULTS: In both mouse EDL and diaphragm 70µM caffeine treatment resulted in a significant increase in maximal muscle power output that was greatest at 10 or 30 weeks (up to 5% and 6% improvement respectively). This potentiation of maximal muscle power output was significantly lower at the early ageing time point, 50 weeks (up to 3% and 2% improvement respectively), and in mice in the developmental stage, at 3 weeks of age (up to 1% and 2% improvement respectively). CONCLUSION: Uniquely, the present findings indicate a reduced age specific sensitivity to the performance enhancing effect of caffeine in developmental and aged mice which is likely to be attributed to age related muscle growth and degradation, respectively. Importantly, the findings indicate that caffeine may still provide a substantial ergogenic aid in older populations which could prove important for improving functional capacity in tasks of daily living.

What are the similarities and differences in antimicrobial prescribing between Australian public and private hospitals?

BACKGROUND: Identifying themes associated with inappropriate prescribing in Australian public and private hospitals will help target future antimicrobial stewardship initiatives. AIMS: To describe current antimicrobial prescribing practices, identify similarities and differences between hospital sectors and provide target areas for improvement specific to each hospital sector. METHODS: All hospitals included in the study were part of the 2014 national antimicrobial prescribing survey and conducted one of the following: a whole hospital point prevalence survey, serial point prevalence surveys or a sample of randomly selected patients. Data on the types of antibiotics used, their indications for use and the quality of prescription based on compliance with national and local prescribing guidelines were collected. RESULTS: Two hundred and two hospitals (166 public and 36 private) comprising 10 882 patients and 15 967 antimicrobial prescriptions were included. Public hospitals had higher proportions of prescriptions for treatment (81.5% vs 48.4%) and medical prophylaxis (8.8% and 4.6%), whilst private hospitals had significantly higher surgical prophylaxis use (9.6% vs 46.9%) (P < 0.001). In public hospitals, the main reasons for non-compliance of treatment prescriptions were spectrum being too broad (30.5%) while in private it was incorrect dosing. Prolonged duration was the main reason for non-compliance among surgical prophylaxis prescriptions in both types of hospitals. CONCLUSIONS: Australian hospitals need to target specific areas to improve antimicrobial use. Specifically, unnecessary broad-spectrum therapy should be a priority area in public hospitals, whilst emphasis on curtailing antimicrobial overuse in surgical prophylaxis needs to be urgently addressed across in the private hospital sector.

Treatment by specialist surgical neurooncologists improves survival times for patients with malignant glioma.

OBJECT: Surgeries for CNS tumors are frequently performed by general neurosurgeons and by those who specialize in surgical neurooncology. Subspecialization in neurosurgical practice has become common and may improve patient morbidity and mortality rates. However, the potential benefits for patients of having their surgeries performed by surgical neurooncologists remain unclear. Recently, a shift in patient care to those who practice predominantly surgical neurooncology has been promoted. Evidence for this practice is lacking and therefore requires fundamental investigation. METHODS: The authors conducted a case-control study of neurooncology patients who underwent surgery for glioblastoma and anaplastic astrocytoma during 2006-2009. Outcomes were compared for patients whose surgery was performed by general neurosurgeons (generalists) or by specialist neurooncology neurosurgeons (specialists). An electronic record database and a picture archiving and communication system were used to collect data and assess the extent of tumor resection. Mortality rates and survival times were compared. Patient comorbidity and postoperative morbidity were assessed by using the Waterlow, patient handling, and falls risk assessment scores. Effects of case mix were adjusted for by using Cox regression and a hazards model. RESULTS: Outcomes for 135 patients (65 treated by generalists and 70 by specialists) were analyzed. Survival times were longer for patients whose surgery was performed by specialists (p=0.026) and after correction for case mix (p=0.019). Extent of tumor resection was greater when performed by specialists (p=0.005) and correlated with increased survival times (p=0.004). There was a trend toward reduced surgical deaths when surgery was performed by specialists (2.8%) versus generalists (7%) (p=0.102), and inpatient stays were significantly shorter when surgery was performed by specialists (p=0.008). CONCLUSIONS: The prognosis for glioblastoma multiforme remains dire, and improved treatments are urgently needed. This study provides evidence for a survival benefit when surgery is performed by specialist neurooncology neurosurgeons. The benefit might be attributable to increased tumor resection. Furthermore, specialist neurooncology surgical care may reduce the number of surgical patient deaths and length of inpatient stay. These findings support the recommendations for subspecialization within surgical neurooncology and advocate for care of these patients by specialists.

Familiarisation to and reproducibility of cycling at 110% peak power output.

AIM: This study investigated the familiarisation to and test re-test reproducibility of constant load cycling at 110% peak power output (WPEAK). METHODS: Eleven healthy, but not cycle trained, males performed a graded incremental exercise test to ascertain WPEAK followed by three trials (T1, T2 and T3) at 110% WPEAK to exhaustion. Trials were separated by ~7 days. RESULTS: Although there was no difference in time to exhaustion (TLIM) between T1 and T2 (P=0.100) and T2 and T3 (P=0.095) respectively, a difference was observed between T1 and T3 (P=0.046). Correlation coefficients, coefficients of determination, limits of agreement (LoA) and within-subject coefficient of variation (CV) improved across trials demonstrating T2 and T3 had the strongest relationship (T1 vs. T3: r=0.73; r2=0.53; Bias=40 s; CV=14%; T1 vs. T2: r=0.66; r2=0.43; Bias=24 s; CV=10%; T2 vs. T3: r=0.97; r2=0.95; Bias=16 s; CV=7%). There was no difference across trials for HR (P=0.12), BLa (P=0.76), RER (P=0.52), VE, (P=0.32), VO2, (P=0.33), local RPE (RPEL; P=1) and overall RPE (RPEO; P=0.91) at exhaustion or BLa (P=0.76) and pH (P=0.47) 5-minutes post-exercise. CONCLUSION: Constant load cycling at 110% WPEAK is a reliable protocol when assessing supramaximal exercise performance after completion of two familiarisation trials.

Is the whole more than the sum of its parts? Evolutionary trade-offs between burst and sustained locomotion in lacertid lizards.

Trade-offs arise when two functional traits impose conflicting demands on the same design trait. Consequently, excellence in one comes at the cost of performance in the other. One of the most widely studied performance trade-offs is the one between sprint speed and endurance. Although biochemical, physiological and (bio)mechanical correlates of either locomotor trait conflict with each other, results at the whole-organism level are mixed. Here, we test whether burst (speed, acceleration) and sustained locomotion (stamina) trade off at both the isolated muscle and whole-organism level among 17 species of lacertid lizards. In addition, we test for a mechanical link between the organismal and muscular (power output, fatigue resistance) performance traits. We find weak evidence for a trade-off between burst and sustained locomotion at the whole-organism level; however, there is a significant trade-off between muscle power output and fatigue resistance in the isolated muscle level. Variation in whole-animal sprint speed can be convincingly explained by variation in muscular power output. The variation in locomotor stamina at the whole-organism level does not relate to the variation in muscle fatigue resistance, suggesting that whole-organism stamina depends not only on muscle contractile performance but probably also on the performance of the circulatory and respiratory systems.

The effects of elevated levels of sodium bicarbonate (NaHCO3) on the acute power output and time to fatigue of maximally stimulated mouse soleus and EDL muscles.

This study examined the effects of elevated buffer capacity [~32 mM HCO3(-)] through administration of sodium bicarbonate (NaHCO3) on maximally stimulated isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles undergoing cyclical length changes at 37 °C. The elevated buffering capacity was of an equivalent level to that achieved in humans with acute oral supplementation. We evaluated the acute effects of elevated [HCO3(-)] on (1) maximal acute power output (PO) and (2) time to fatigue to 60 % of maximum control PO (TLIM60), the level of decline in muscle PO observed in humans undertaking similar exercise, using the work loop technique. Acute PO was on average 7.0 ± 4.8 % greater for NaHCO3-treated EDL muscles (P < 0.001; ES = 2.0) and 3.6 ± 1.8 % greater for NaHCO3-treated SOL muscles (P < 0.001; ES = 2.3) compared to CON. Increases in PO were likely due to greater force production throughout shortening. The acute effects of NaHCO3 on EDL were significantly greater (P < 0.001; ES = 0.9) than on SOL. Treatment of EDL (P = 0.22; ES = 0.6) and SOL (P = 0.19; ES = 0.9) with NaHCO3 did not alter the pattern of fatigue. Although significant differences were not observed in whole group data, the fatigability of muscle performance was variable, suggesting that there might be inter-individual differences in response to NaHCO3 supplementation. These results present the best indication to date that NaHCO3 has direct peripheral effects on mammalian skeletal muscle resulting in increased acute power output.

Fight versus flight: physiological basis for temperature-dependent behavioral shifts in lizards.

Previous studies have demonstrated that a behavioral shift from flight to aggressive behavior occurs at low temperatures in some lizards. Our data for the agamid lizard Trapelus pallida demonstrate how the effect of temperature on whole organism performance traits such as sprint speed (much lower performance at lower temperature) and bite force (largely independent of temperature) may explain the shift from flight to fight behavior with decreasing temperature. Moreover, our data hint at the physiological basis for this effect as isolated muscle power output, twitch and tetanus time traits, relevant to sprinting, appear to be strongly temperature-dependent muscle properties. Maximal muscle force production, on the other hand, appears largely independent of temperature. Unexpectedly, differences in the physiological properties of jaw versus limb muscle were observed that enhance the ability of the jaw muscle to generate maximal force at all temperatures tested. Thus our data show how behavioral responses may be determined by the limitations set by temperature on physiological processes.

Double-blind, placebo-controlled study of single-dose amphetamine formulations in ADHD.

OBJECTIVE: To compare the efficacy and time course of single morning doses of Adderall, extended-release, and immediate-release dextroamphetamine sulfate. METHOD: Thirty-five children with attention-deficit/hyperactivity disorder, combined type, were given Adderall, immediate-release dextroamphetamine, dextroamphetamine Spansules, and placebo in a randomized, double-blind, crossover study. Behavior ratings, locomotor activity measurements, and academic measures were obtained over a period of 8 weeks. RESULTS: All three drugs exhibited robust efficacy versus placebo on nearly all measures. The effects of dextroamphetamine Spansules were less robust in the morning, particularly compared with Adderall, but they lasted 3 to 6 hours longer, depending on the measure. Although parent behavior ratings and locomotor activity showed improvements up to 12 hours after single doses of all three drugs, the number of math problems attempted and completed correctly 4 hours after dosing were only robustly increased by Spansules. CONCLUSIONS: Both immediate-release amphetamines demonstrated earlier onset of effects, but dextroamphetamine Spansules showed more sustained effects that were present on a wider range of measures.

Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.

PURPOSE: Activation of muscarinic receptors has been implicated in an increased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in other ocular cells. METHODS: Human lens cells were perfused with artificial aqueous humor (35 degrees C) after fura-2 incorporation, and calcium levels were measured using a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by determining the relative effectiveness of pirenzepine and AF-DX 384 in blocking the agonist-induced response. The levels of expression of mRNA for the receptor subtypes M1 through M5 were determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using a sequence detection system (ABI Prism 7700; Perkin-Elmer, Foster City, CA). This was performed using total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS: Acetylcholine induced a similar concentration-dependent increase in peak-amplitude cytosolic calcium in the range 100 nM to 100 microM in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (> 1 microM), a second phase appeared in the HLE-B3 cells that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 microM acetylcholine response by pirenzepine and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that more than 90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origin. mRNA for M3 was also in greatest abundance in other eye tissues, although there was a significant contribution from M1 in iris and sclera. CONCLUSIONS: Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylcholine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subtype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eye tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat glaucoma.

Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction.

Drug-induced changes in expression of cytochrome (CYP) P450 genes are a key cause of drug-drug interactions. Consequently, preclinical prediction of these changes by novel compounds is an integral component of drug development. To date, in vitro models of CYP induction have used mRNA measurement, immunodetection, and substrate metabolism as reporters. Here, we describe the application of quantitative real-time reverse transcriptase polymerase chain reaction to study CYP1A1 and CYP3A4 gene induction in 5-day-old cultures of human hepatocytes by known CYP inducers. After 5 days in culture, CYP1A1 expression was significantly elevated (5.1- to 26-fold; P <.01) in all four livers studied. In direct contrast, CYP3A4 mRNA levels consistently decreased during culture (80- to 300-fold; P <.001). In three independent experiments, a 48-h exposure to 3-methylcholanthrene, omeprazole, and lansoprazole significantly induced CYP1A1 expression in comparison to untreated cultures (P <.05). Rifampicin and solvent were without effect on CYP1A1 expression. Under identical experimental conditions, rifampicin and lansoprazole significantly elevated CYP3A4 mRNA expression (P <.05), whereas 3-methylcholanthrene, omeprazole, and dimethyl sulfoxide were without significant effect. These data demonstrate the applicability of quantitative reverse transcriptase polymerase chain reaction to the determination of gene dynamics in human hepatocytes. This offers a highly specific alternative to quantification of drug effects on CYP expression using immunodetection and substrate metabolism.