Chemogenetic Signaling in Space and Time: Considerations for Designing Neuroscience Experiments Using DREADDs.

The use of designer receptors exclusively activated by designer drugs (DREADDs) has led to significant advances in our understanding of the neural circuits that govern behavior. By allowing selective control over cellular activity and signaling, DREADDs have become an integral tool for defining the pathways and cellular phenotypes that regulate sleep, pain, motor activity, goal-directed behaviors, and a variety of other processes. In this review, we provide a brief overview of DREADDs and discuss notable discoveries in the neurosciences with an emphasis on circuit mechanisms. We then highlight methodological approaches to achieve pathway specific activation of DREADDs. Finally, we discuss spatial and temporal constraints of DREADDs signaling and how these features can be incorporated into experimental designs to precisely dissect circuits of interest.

Chronic modafinil administration to preadolescent rats impairs social play behavior and dopaminergic system.

Some clinical trials are investigating modafinil (Mod) as a treatment for attentional deficit and hyperactivity disorder (ADHD) in children and adolescents. Mod increases dopamine (DA) levels in the reward system by blocking dopamine transporter (DAT). Social interactions are rewarding behaviors and evidence reveals the importance of reward circuitry in social interactions. Chronic psychostimulant treatments alter DA neurotransmission and associated behaviors. The aim of this work was to evaluate the effects of chronic Mod treatment during preadolescence on social play behavior, locomotor activity, and DA in nucleus accumbens (NAc). Preadolescent male Sprague-Dawley rats were injected with Mod (75 mg/kg i.p.) or vehicle for 14 days (PND22 to PND35). After that, we measured social play behavior, content and DA release in NAc by HPLC coupled to electrochemical detection, protein levels of DA type 2 receptor (D2) by Western blot and DA kinetic by fast-scan cyclic voltammetry (FSCV) in NAc. Regarding social play, the total number of pinning events decreased in the Mod group compared with the vehicle. The K+-stimulated DA release in NAc was significantly lower in Mod-treated rats compared with vehicle group. Also, Mod increases locomotor activity at the first injection, but this effect is almost completely lost at day 14 of Mod treatment. Chronic Mod treatment during preadolescence in rats impairs dopaminergic neurotransmission in NAc and decreases the capacity of rats to perceive rewarding effects of social play. Importantly, as Mod is being evaluated to treat ADHD in children and adolescents, potential effects on social behavior should be considered since this kind of behavior in this particular stage is crucial for neurodevelopment.

National osteoarthritis strategy brief report: Advanced care.

BACKGROUND: Osteoarthritis (OA) is one of the most common chronic joint diseases and a leading cause of pain and disability in Australia. A National Osteoarthritis Strategy (the Strategy) was developed to outline a national plan to achieve optimal health outcomes for people at risk of, or with, OA. OBJECTIVE: This article focuses on the theme of advanced care of patients with OA within the Strategy. DISCUSSION: The Strategy was developed in consultation with a leadership group, thematic working groups, an implementation advisory committee, multisectoral stakeholders and the public. This Strategy identified three priorities in advanced care for osteoarthritis. In brief, these include surgical decision making, referral for evidence-informed non-surgical alternatives and surgical services. A set of goals within these priority areas and strategies was also proposed by the working group in consultation with stakeholders nationwide. Peak arthritis bodies and major healthcare professional associations currently endorse the Strategy.

Adult Human Glioblastomas Harbor Radial Glia-like Cells.

Radial glia (RG) cells are the first neural stem cells to appear during embryonic development. Adult human glioblastomas harbor a subpopulation of RG-like cells with typical RG morphology and markers. The cells exhibit the classic and unique mitotic behavior of normal RG in a cell-autonomous manner. Single-cell RNA sequencing analyses of glioblastoma cells reveal transcriptionally dynamic clusters of RG-like cells that share the profiles of normal human fetal radial glia and that reside in quiescent and cycling states. Functional assays show a role for interleukin in triggering exit from dormancy into active cycling, suggesting a role for inflammation in tumor progression. These data are consistent with the possibility of persistence of RG into adulthood and their involvement in tumor initiation or maintenance. They also provide a putative cellular basis for the persistence of normal developmental programs in adult tumors.

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter.

The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.

Biopharmaceutical Industry Approaches to Facility Segregation for Viral Safety: An Effort from the Consortium on Adventitious Agent Contamination in Biomanufacturing.

Appropriate segregation within manufacturing facilities is required by regulators and utilized by manufacturers to ensure that the final product has not been contaminated with (a) adventitious viruses, (b) another pre-/postviral clearance fraction of the same product, or (c) another product processed in the same facility. However, there is no consensus on what constitutes appropriate facility segregation to minimize these risks. In part, this is due to the fact that a wide variety of manufacturing facilities and operational practices exist, including single-product and multiproduct manufacturing, using traditional segregation strategies with separate rooms for specific operations that may use stainless steel or disposable equipment to more modern ballroom-style operations that use mostly disposable equipment (i.e., pre- and postviral clearance manufacturing operations are not physically segregated by walls). Further, consensus is lacking around basic definitions and approaches related to facility segregation. For example, given that several unit operations provide assurance of virus clearance during downstream processing, how does one define pre- and postviral clearance and at which point(s) should a viral segregation barrier be introduced? What is a "functionally closed" system? How can interventions be conducted so that the system remains functionally closed? How can functionally closed systems be used to adequately isolate a product stream and ensure its safety? To address these issues, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a facility segregation project with the following goals: define "pre- and postviral clearance zones" and "pre- and postviral clearance materials"; define "functionally closed" manufacturing systems; and identify an array of facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products. This article reflects the current thinking from this collaborative endeavor.LAY ABSTRACT: Operations in biopharmaceutical manufacturing are segregated to ensure that the final product has not been contaminated with adventitious viruses, another fraction of the same product, or with another product from within the same facility. Yet there is no consensus understanding of what appropriate facility segregation looks like. There are a wide variety of manufacturing facilities and operational practices. There are existing facilities with separate rooms and more modern approaches that use disposable equipment in an open ballroom without walls. There is also no agreement on basic definitions and approaches related to facility segregation approaches. For example, many would like to claim that their manufacturing process is functionally closed, yet exactly how a functionally closed system may be defined is not clear. To address this, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a project with the goal of defining important manufacturing terms relevant to designing an appropriately segregated facility and identifying different facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products.

Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels.

Pressures for cost-effective new therapies and an increased emphasis on emerging markets require technological advancements and a flexible future manufacturing network for the production of biologic medicines. The safety and efficacy of a product is crucial, and consistent product quality is an essential feature of any therapeutic manufacturing process. The active control of product quality in a typical biologic process is challenging because of measurement lags and nonlinearities present in the system. The current study uses nonlinear model predictive control to maintain a critical product quality attribute at a predetermined value during pilot scale manufacturing operations. This approach to product quality control ensures a more consistent product for patients, enables greater manufacturing efficiency, and eliminates the need for extensive process characterization by providing direct measures of critical product quality attributes for real time release of drug product.

Cost and logistics for implementing the American College of Surgeons objective structured clinical examination.

BACKGROUND: The American College of Surgeons has developed a reliable and valid OSCE (objective structured clinical examination) to assess the clinical skills of incoming postgraduate year 1 surgery residents, but the cost and logistics of implementation have not been described. METHODS: Fixed costs included staff time, medical supplies, facility fee, standardized patient (SP) training time, and one OSCE session. Variable costs were incurred for additional OSCE sessions. Costs per resident were calculated and modeled for increasing the number of test takers. RESULTS: American College of Surgeons OSCE materials and examination facilities were free. Fixed costs included training 11 SPs for 4 hours ($1,540), moulage and simulation material ($469), and administrative effort for 44 hours ($2,200). Variable cost for each session was $1,540 (SP time). Total cost for the first session was $6,649 ($664/resident), decreased to $324/resident for 3 sessions, and projected to further decline to $239/resident for 6 sessions. CONCLUSIONS: The cost decreased as the number of residents tested increased. To manage costs, testing more trainees by regional collaboration is recommended.

Cost and logistics of implementing a tissue-based American College of Surgeons/Association of Program Directors in Surgery surgical skills curriculum for general surgery residents of all clinical years.

BACKGROUND: The cost and logistics of deploying the American College of Surgeons (ACS)/Association of Program Directors in Surgery (APDS) National Technical Skills Curriculum across all training years are not known. This information is essential for residency programs choosing to adopt similar curricula. METHODS: A task force evaluated the authors' institution's existing simulation curriculum and enhanced it by implementing the ACS/APDS modules. A 35-module curriculum was administered to 35 general surgery residents across all 5 clinical years. The costs and logistics were noted, and resident satisfaction was assessed. RESULTS: The annual operational cost was $110,300 ($3,150 per resident). Cost per module, per resident was $940 for the cadaveric module compared with $220 and $240 for dry simulation and animal tissue-based modules, respectively. Resident satisfaction improved from 2.45 to 4.78 on a 5-point, Likert-type scale after implementing the ACS/APDS modules. CONCLUSIONS: The ACS/APDS skills curriculum was implemented successfully across all clinical years. Cadaveric modules were the most expensive. Animal and dry simulation modules were equivalent in cost. The addition of tissue-based modules was associated with high satisfaction.

Exploring the nature of interprofessional collaboration and family member involvement in an intensive care context.

Little is known about the nature of interprofessional collaboration on intensive care units (ICUs), despite its recognition as a key component of patient safety and quality improvement initiatives. This comparative ethnographic study addresses this gap in knowledge and explores the different factors that influence collaborative work in the ICU. It aims to develop an empirically grounded team diagnostic tool, and associated interventions to strengthen team-based care and patient family involvement. This iterative study is comprised of three phases: a scoping review, a multi-site ethnographic study in eight ICUs over 2 years; and the development of a diagnostic tool and associated interprofessional intervention-development. This study's multi-site design and the richness and breadth of its data maximize its potential to improve clinical outcomes through an enhanced understanding of interprofessional dynamics and how patient family members in ICU settings are best included in care processes. Our research dissemination strategy, as well as the diagnostic tool and associated educational interventions developed from this study will help transfer the study's findings to other settings.

Vibrio cholerae in an Historically Cholera-Free Country.

We report the autochthonous existence of Vibrio cholerae in coastal waters of Iceland, a geothermally active country where cholera is absent and has never been reported. Seawater, mussel, and macroalgae samples were collected close to and distant from sites where geothermal activity causes a significant increase in water temperature during low tides. V. cholerae was detected only at geothermal-influenced sites during low-tides. None of the V. cholerae isolates encoded cholera toxin (ctxAB) and all were non-O1/non-O139 serogroups. However, all isolates encoded other virulence factors that are associated with cholera as well as extra-intestinal V. cholerae infections. The virulence factors were functional at temperatures of coastal waters of Iceland, suggesting an ecological role. It is noteworthy that V. cholerae was isolated from samples collected at sites distant from anthropogenic influence, supporting the conclusion that V. cholerae is autochthonous to the aquatic environment of Iceland.

Genomic diversity of 2010 Haitian cholera outbreak strains.

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.

Turkish pharmacists' counseling practices and attitudes regarding emergency contraceptive pills.

OBJECTIVE: The aim of this study is to assess Turkish pharmacists' counseling practices and attitudes regarding emergency contraception pills (ECPs). SETTING: This cross-sectional observational study was conducted via a web-based survey in Turkey. METHODS: Pharmacists registered at a professional web site (n = 822) were invited to fill in the study questionnaire; 624 questionnaires were completed and further analyzed. Pharmacists who agreed to participate in the study completed the questionnaire which was structured to elicit their demography, professional experience, counseling practices and attitudes regarding ECP. Attitudes were measured by 18 items under four domains: "reproductive health; information and availability; risk behavior and regulatory restrictions". The answer choices consisted of five items as: "totally agree, agree, neither agree nor disagree, disagree and totally disagree". MAIN OUTCOME MEASURES: The rate of counseling on various aspects of the ECPs and the rate of negative/positive attitudes. RESULTS: The aspects most frequently counseled on by the pharmacists were dosage, timeframes, efficacy and pregnancy testing. Less frequently counseled aspects were mechanism of action, methods of contraception and side-effects. In general, the pharmacists displayed positive attitudes towards all domains of the survey; while they were negative to the items suggesting that ECP should be sold only to women and only on prescription. Another interesting finding is that 58 % of the pharmacists agreed with the item suggesting limiting the ECP sales to those over 18 years of age. CONCLUSIONS: Our results showed that the pharmacists served the clients in need of emergency contraception more frequently than the other health-care providers and in general had favorable attitudes towards ECP. Some aspects of their counseling practices need to be improved. This can be accomplished through continuous education programs that would equip them with the competence to provide counseling on emergency contraception which will in turn help prevent unintended pregnancies and reduce abortion rates.

Can immune parameters be used as predictors to distinguish between pulmonary multidrug-resistant and drug-sensitive tuberculosis?

INTRODUCTION: Despite the development and wide implementation of Directly Observed Therapy Strategies (DOTS), multidrug-resistant tuberculosis (MDR-TB) remains a serious global health threat. In this study, the role of host immune response in patients with MDR-TB is investigated and compared with that of patients with smear-positive drug-sensitive tuberculosis (SP-TB). MATERIAL AND METHODS: 27 patients with SP-TB, 20 patients with MDR-TB, and 20 healthy controls were included in the study. Immune parameters were determined by flow cytometry using monoclonal antibodies in order to compare the percentage values of these markers in the two study groups and the control group. RESULTS: The levels of lymphocyte subgroups in the gate of CD45(+)/CD14(-) lymphocyte: CD45(+), CD3(+), CD4(+), NK, CD3/HLA-DR, CD 95(+) cells were significantly lower; by contrast CD23(+), CD25(+), CD19(+), CD4(+)/CD8(+), HLA-DR cells were found to be lower, but not significantly so in patients with MDR-TB, compared to levels in patients in the SP-TB and control groups. Besides these findings, the levels of NKT cells and (γ)δ TCR(+) cells were significantly higher in the MDR-TB than in the healthy control and SP-TB group. CONCLUSIONS: The lower levels of CD3/ HLA-DR, CD4 (+), Fas (+), and NK, and the higher level of NKT together with (γ)δ T cells in patients with MDR-TB compared to those in SP-TB may indicate a profound immune suppression in MDR-TB patients and thereby may denote an accumulation in the bacterial load. Our findings may shed light on the pathogenesis and prognosis of MDR tuberculosis, and may point towards the use of flow cytometry findings as an aid to early diagnosis in MDR-TB patients.

The relationship between obesity, physical activity, and physical function in older adults.

OBJECTIVE: This study investigated the relationship between overweight and obesity, age, and gender with physical activity and physical function in community-dwelling older men and women. METHOD: Multivariate analysis of covariance was used to analyze differences between normal weight, overweight, and obese adults (n = 821) above the age of 60 years. RESULTS: Obesity but not overweight was associated with lower levels of physical activity and physical function. Within BMI groups, individuals who were physically active were less likely to have abnormal physical function scores compared to those who were sedentary. Compared to men, obese women had lower physical function scores, placing them at higher risk for future disability. Aging was associated with lower levels of physical activity and physical function. DISCUSSION: The study illustrates the importance of avoiding obesity and participating in regular physical activity to prevent or slow down the loss of functioning in older age.

Hematologic characteristics of captive western barred bandicoots (Perameles bougainville) from Western Australia.

BACKGROUND: The western barred bandicoot (Perameles bougainville) is an Australian marsupial species now considered endangered as a consequence of habitat destruction and predation. A recently discovered papillomatosis syndrome is hindering efforts to repopulate this species. Hematology reference intervals have been lacking for P bougainville, preventing optimal interpretation of hematology results from wart-affected and clinically normal animals. OBJECTIVES: The purpose of this study was to establish hematology reference values and describe morphologic characteristics of blood cells of healthy western barred bandicoots. METHODS: Fifty-nine whole blood samples were collected by jugular venipuncture into EDTA from 47 clinically healthy captive western barred bandicoots at 3 locations on the Western Australian mainland. A CBC was performed using an ADVIA-120 analyzer. Data were compared on the basis of geographic location, sex, age, and lactation status, and reference intervals were calculated. Blood cell morphology was evaluated using light microscopy, and transmission and scanning electron microscopy. RESULTS: Significant differences were found based on sex (RBC indices, fibrinogen), age (% polychromatophilic RBCs), and geographic location (RBC, neutrophil, and lymphocyte counts, MCHC, % polychromatophilic RBCs, fibrinogen). Combined reference intervals were calculated for hemoglobin concentration (122-165 g/L), HCT (0.36-0.49 L/L), and total WBC (2.9-14.9 x 10(9)/L), monocyte (0-0.6 x 10(9)/L), eosinophil (0-0.9 x 10(9)/L), and total plasma protein (47-63 g/L) concentrations. Leukocyte, erythrocyte, and platelet morphology were similar to those of other marsupial peramelid species. Nuclei in neutrophils, monocytes, and eosinophils occasionally had an annular configuration. CONCLUSIONS: Reference intervals and blood cell morphology obtained in this study will be useful for the evaluation of laboratory data from ill animals and assist with population health monitoring of western barred bandicoots.

Nonprescription medication purchases and the role of pharmacists as healthcare workers in self-medication in Istanbul.

BACKGROUND: In Turkey, some drugs can be purchased from pharmacists without a prescription and there is no legal classification corresponding to the international term "over-the-counter drugs". The purpose of this study was to identify these non-prescribed drugs and to define the role of pharmacists in their sale and their use in primary healthcare. MATERIAL/METHODS: This cross-sectional study was carried out in Istanbul between December 1, 2003, and August 31, 2004. Seventy-three of the 901 pharmacies in two districts of Istanbul were chosen with systematic sampling, and information concerning drug sales and pharmacists' behavior regarding primary healthcare services was collected by observations as well as by interviews with the pharmacists. RESULTS: The study demonstrated that 41% of the drugs sold were non-prescribed. In pharmacies remote from healthcare institutions, remote from district centers, and in the summer seasons, the rates of non-prescribed drug sales were higher than those of prescribed drugs (Chi(2)=10.5, d.f.=1, p=0.001; Chi(2)=62.8, d.f.=1, p=0.0001; Chi(2)=23.4, d.f.=1, p=0.0001). The pharmacists stated that aside from drug sales, consumers visited them to consult about drugs, to get information on their health status, and to learn about family planning (78.1%, 72.1%, and 72.6% of the pharmacists, respectively). CONCLUSIONS: Extensive sale of non-prescribed drugs as a response to public demand, including those used for serious illnesses, has been observed in private pharmacies in Istanbul. The role of pharmacies and pharmacists in primary healthcare should be discussed more thoroughly and redefined on a legal basis.

Effect of pharmacist-led patient education on adherence to tuberculosis treatment.

PURPOSE: The purpose of this study was to assess the effect of a clinical pharmacist-directed patient education program on the therapy adherence of first-time tuberculosis (TB) patients and to identify the major pharmaceutical care needs and issues of first-time TB and multidrug-resistant (MDR)-TB patients. METHODS: In the first part of the study, first-time TB patients were randomized either to the No EDU group (n = 58) where patients received routine medical and nursing care or to the EDU group (n = 56) where patients were also provided with clinical pharmacist-directed patient education. The patient's adherence to treatment was evaluated by attendance at scheduled visits, medication counting, and urine analysis for the presence of isoniazid metabolites. In the second part of the study, the pharmaceutical care needs and issues were determined for first-time TB patients and for MDR-TB patients (n = 40). RESULTS: The adherence of patients who received pharmacist-directed patient education was greater than that of patients who did not. The attendance at scheduled visits and urine analysis for the presence of isoniazid metabolites yielded better results in respect to adherence for the EDU group (p < 0.05), while medication counting did not differ between the two groups. The major pharmaceutical care needs of first-time TB patients were for pain control, nutrient replacement, appropriate prescribing, respiratory control, and diabetic control. Similar findings were recorded for MDR-TB patients. CONCLUSION: Patients' adherence to TB treatment improved when a pharmacist provided patient education on medication use and addressed patients' pharmaceutical care issues.

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes.

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.