Cellular responsiveness to many neuromodulators is controlled by endocytosis of the transmembrane receptors that transduce their effects. Endocytic membrane trafficking of particular neuromodulator receptors exhibits remarkable diversity and specificity, determined largely by molecular sorting operations that guide receptors at trafficking branch-points after endocytosis. In this review, we discuss recent progress in elucidating mechanisms mediating the molecular sorting of neuromodulator receptors in the endocytic pathway. There is emerging evidence that endocytic trafficking of neuromodulator receptors, in addition to influencing longer-term cellular responsiveness under conditions of prolonged or repeated activation, may also affect the acute response. Physiological and pathological consequences of defined receptor trafficking events are only now being elucidated, but it is already apparent that endocytosis of neuromodulator receptors has a significant impact on the actions of therapeutic drugs. The present data also suggest, conversely, that mechanisms of receptor endocytosis and molecular sorting may themselves represent promising targets for therapeutic manipulation.
In a case-control study at 4 US centers, the effectiveness of influenza vaccine against medically attended polymerase chain reaction–confirmed influenza was 60% (95% confidence interval, 53%–66%). Significant effectiveness was demonstrated in young children, adolescents, and adults, with a lower estimate for those aged ≥65.
Background. Influenza vaccines may be reformulated annually because of antigenic drift in influenza viruses. However, the relationship between antigenic characteristics of circulating viruses and vaccine effectiveness (VE) is not well understood. We conducted an assessment of the effectiveness of US influenza vaccines during the 2010–2011 season.
Methods. We performed a case–control study comparing vaccination histories between subjects with acute respiratory illness with positive real-time reverse transcription polymerase chain reaction for influenza and influenza test-negative controls. Subjects with acute respiratory illness of ≤7 days duration were enrolled in hospitals, emergency departments, or outpatient clinics in communities in 4 states. History of immunization with the 2010–2011 vaccine was ascertained from vaccine registries or medical records. Vaccine effectiveness was estimated in logistic regression models adjusted for study community, age, race, insurance status, enrollment site, and presence of a high-risk medical condition.
Results. A total of 1040 influenza-positive cases and 3717 influenza-negative controls were included from the influenza season, including 373 cases of influenza A(H1N1), 334 cases of influenza A(H3N2), and 333 cases of influenza B. Overall adjusted VE was 60% (95% confidence interval [CI], 53%–66%). Age-specific VE estimates ranged from 69% (95% CI, 56%–77%) in children aged 6 months–8 years to 38% (95% CI, −16% to 67%) in adults aged ≥65 years.
Conclusions. The US 2010–2011 influenza vaccines were moderately effective in preventing medically attended influenza during a season when all 3 vaccine strains were antigenically similar to circulating viruses. Continued monitoring of influenza vaccines in all age groups is important, particularly as new vaccines are introduced.
In April 2009, a pandemic caused by a novel influenza strain, the A(H1N1)pdm09 virus, started. Few age-specific estimates of hospitalizations associated with the first year of circulation of the pandemic virus are available.
To estimate age-specific hospitalization rates associated with laboratory-confirmed A(H1N1)pdm09 virus in Davidson County, TN, from May 2009 to March 2010.
Two separate strategies were applied: capture–recapture and surveillance-sampling methods. For the capture–recapture estimates, we linked data collected via two independent prospective population-based surveillance systems: The Influenza Vaccine Effectiveness Network (Flu-VE) tested consenting county patients hospitalized with respiratory symptoms at selected hospitals using real-time reverse transcriptase polymerase chain reaction (rRT-PCR); the Emerging Infections Program identified county patients with positive influenza tests in all area hospitals. For the surveillance-sampling estimates, we applied the agespecific proportions of influenza-positive patients (from Flu-VE) to the number of acute respiratory illness hospitalizations obtained from the Tennessee Hospital Discharge Data system.
With capture–recapture, we estimated 0.89 (95% CI, 0.72–1.49), 0.62 (0.42–1.11), 1.78 (0.99–3.63), and 0.76 (0.50–1.76) hospitalizations per 1000 residents aged <5, 5–17, 18–49, and ≥50 years, respectively. Surveillance-sampling estimated rates were 0.78 (0.46–1.22), 0.32 (0.14–0.69), 0.99 (0.64–1.52), and 1.43 (0.80–2.48) hospitalizations per 1000 residents aged <5, 5–17, 18–49, and ≥50 years, respectively. In all age-groups combined, we estimated approximately 1 influenza-related hospitalization per 1000 residents.
Two independent methods provided consistent results on the burden of pandemic virus in Davidson County and suggested that the overall incidence of A(H1N1)pdm09-associated hospitalization was 1 per 1000 county residents.
A(H1N1)pdm09 virus; capture–recapture; hospitalization rates; surveillance-sampling
Among infants with prematurity and/or chronic lung disease for whom respiratory syncytial virus immunoprophylaxis is recommended, we examined adherence in infants enrolled during healthcare visits for acute respiratory illness in 3 US counties from 2001 to 2007. Immunoprophylaxis among infants who met national criteria for prophylaxis increased from 33% to 83% over the 6-year period; 17% (11/65) of infants who received immunoprophylaxis did not meet eligibility criteria.
respiratory syncytial virus; adherence; palivizumab
Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.
Optogenetics, the use of light-sensitive ion channels for stimulation of mammalian cells and tissues, offers specificity and superior precision of control compared to traditional chemical or electrical means of stimulation. In particular, Channelrhodospin-2 (ChR2), a light-sensitive ion channel, originally derived from algae, has found wide-spread application in neuroscience for controlled stimulation of different brain regions. More recently, this work was extended to other organs, including the heart, where it opens the possibility for a new generation of optical pacemakers. The development of new optogenetic tools that allow for more efficient optical stimulation can be guided by computational prediction of the response of different cells and tissues to light. In this report, we provide a new computational model of ChR2 that was empirically validated and can be inserted into different cell types – neurons or heart cells – for virtual optical stimulation and prediction of optimal light-delivery arrangements, minimum energy needs etc. Overall, virtual optogenetics can accelerate the development of new optical stimulation tools for better understanding and control of brain and heart function.
Prolonged exposure to high-efficacy agonists results in desensitization of the mu opioid receptor (MOR). Desensitized receptors are thought to be unable to couple to G-proteins, preventing downstream signaling, however the changes to the receptor itself are not well characterized. In the current study, confocal imaging was used to determine whether desensitizing conditions cause a change in agonist-receptor interactions. Using rapid solution exchange, the binding kinetics of fluorescently labeled opioid agonist, dermorphin Alexa594 (derm A594), to MORs was measured in live cells. The affinity of derm A594 binding increased following prolonged treatment of cells with multiple agonists that are known to cause receptor desensitization. In contrast, binding of a fluorescent antagonist, naltrexamine Alexa 594, was unaffected by similar agonist pre-treatment. The increased affinity of derm A594 for the receptor was long-lived and partially reversed after a 45 min wash. Treatment of the cells with pertussis toxin did not alter the increase in affinity of the derm A594 for MOR. Likewise the affinity of derm A594 for MORs expressed in mouse embryonic fibroblasts derived from arrestin 1 and 2 knockout animals increased following treatment of the cells with the desensitization protocol. Thus, opioid receptors were “imprinted” with a memory of prior agonist exposure that was independent of G-protein activation or arrestin binding that altered subsequent agonist-receptor interactions. The increased affinity suggests that acute desensitization results in a long lasting but reversible conformational change in the receptor.
The firing properties of dopamine (DA) neurons in the substantia nigra (SN) pars compacta are strongly influenced by the activity of apamin-sensitive small conductance Ca2+-activated K+ (SK) channels. Of the three SK channel genes expressed in central neurons, only SK3 expression has been identified in DA neurons. The present findings show that SK2 was also expressed in DA neurons. Immuno-electron microscopy (iEM) showed that SK2 was primarily expressed in the distal dendrites, while SK3 was heavily expressed in the soma and, to a lesser extent, throughout the dendritic arbor. Electrophysiological recordings of the effects of the SK channel blocker apamin on DA neurons from wild type and SK−/− mice show that SK2-containing channels contributed to the precision of action potential (AP) timing, while SK3-containing channels influenced AP frequency. The expression of SK2 in DA neurons may endow distinct signaling and subcellular localization to SK2-containing channels. Keywords: Substantia Nigra, Dopamine, SK channels, spontaneous activity, pacemaker
The inpatient and outpatient burden of human metapneumovirus (HMPV) infection among young children has not been well established.
We conducted prospective, population-based surveillance for acute respiratory illness or fever among inpatient and outpatient children less than 5 years of age in three U.S. counties from 2003 through 2009. Clinical and demographic data were obtained from parents and medical records, HMPV was detected by means of a reverse-transcriptase polymerase-chain-reaction assay, and population-based rates of hospitalization and estimated rates of outpatient visits associated with HMPV infection were determined.
HMPV was detected in 200 of 3490 hospitalized children (6%), 222 of 3257 children in outpatient clinics (7%), 224 of 3001 children in the emergency department (7%), and 10 of 770 asymptomatic controls (1%). Overall annual rates of hospitalization associated with HMPV infection were 1 per 1000 children less than 5 years of age, 3 per 1000 infants less than 6 months of age, and 2 per 1000 children 6 to 11 months of age. Children hospitalized with HMPV infection, as compared with those hospitalized without HMPV infection, were older and more likely to receive a diagnosis of pneumonia or asthma, to require supplemental oxygen, and to have a longer stay in the intensive care unit. The estimated annual burden of outpatient visits associated with HMPV infection was 55 clinic visits and 13 emergency department visits per 1000 children. The majority of HMPV-positive inpatient and outpatient children had no underlying medical conditions, although premature birth and asthma were more frequent among hospitalized children with HMPV infection than among those without HMPV infection.
HMPV infection is associated with a substantial burden of hospitalizations and outpatient visits among children throughout the first 5 years of life, especially during the first year. Most children with HMPV infection were previously healthy. (Funded by the Centers for Disease Control and Prevention and the National Institutes of Health.)
Montane forests of western China provide an opportunity to establish baseline studies for climate change. The region is being impacted by climate change, air pollution, and significant human impacts from tourism. We analyzed forest stand structure and climate-growth relationships from Jiuzhaigou National Nature Reserve in northwestern Sichuan province, along the eastern edge of the Tibetan plateau. We conducted a survey to characterize forest stand diversity and structure in plots occurring between 2050 and 3350 m in elevation. We also evaluated seedling and sapling recruitment and tree-ring data from four conifer species to assess: 1) whether the forest appears in transition toward increased hardwood composition; 2) if conifers appear stressed by recent climate change relative to hardwoods; and 3) how growth of four dominant species responds to recent climate. Our study is complicated by clear evidence of 20th century timber extraction. Focusing on regions lacking evidence of logging, we found a diverse suite of conifers (Pinus, Abies, Juniperus, Picea, and Larix) strongly dominate the forest overstory. We found population size structures for most conifer tree species to be consistent with self-replacement and not providing evidence of shifting composition toward hardwoods. Climate-growth analyses indicate increased growth with cool temperatures in summer and fall. Warmer temperatures during the growing season could negatively impact conifer growth, indicating possible seasonal climate water deficit as a constraint on growth. In contrast, however, we found little relationship to seasonal precipitation. Projected warming does not yet have a discernible signal on trends in tree growth rates, but slower growth with warmer growing season climates suggests reduced potential future forest growth.
Endogenous cannabinoid-receptor ligands (endocannabinoids) and over a dozen related metabolites now comprise the “endocannabinoid metabolome”. The diverse (patho)physiological roles of endocannabinoids, the predictive/diagnostic utility of systemic endocannabinoid levels, and the growing interest in endocannabinoid-related pharmacotherapeutics mandate a valid clinical protocol for processing human blood that does not jeopardize profiling of the circulating endocannabinoid metabolome.
We systematically evaluated the potential effect of pre-analytical variables associated with phlebotomy and sample handling/work-up on the human-blood endocannabinoid metabolome as quantified by state-of-the-art liquid chromatography-mass spectrometry.
Neither subject posture during phlebotomy nor moderate activity beforehand influenced the blood levels of the 15 endocannabinoid-system lipids quantified. Storage of fresh blood at 4°C selectively enhanced ethanolamide concentrations artifactually without affecting monoglycerides and nonesterified fatty acids, such as arachidonic acid. In marked contrast, ethanolamides and monoglycerides remained stable through three plasma freeze/thaw cycles, whereas plasma arachidonic acid content increased, probably a reflection of ongoing metabolism.
Class- and compound-selective pre-analytical influences on circulating human endocannabinoid levels necessitate immediate plasma preparation from fresh blood and prompt plasma apportioning and snap-freezing. Repeated plasma thawing and refreezing should be avoided. This protocol ensures sample integrity for evaluating the circulating endocannabinoid metabolome in the clinical setting.
endocannabinoids; human; liquid chromatography-mass spectrometry; plasma; sampling protocol
Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data.
To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting.
Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels.
A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets.
We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity.
Human metapneumovirus; diagnostics; real-time RT-PCR
Healthcare-associated transmission of respiratory viruses is a concerning patient safety issue.
Surveillance for influenza virus among a cohort of healthcare workers (HCWs) was conducted in a tertiary care children’s hospital from November 2009 until April 2010, using biweekly nasal swab collection. If a subject reported respiratory symptoms, an additional specimen was collected. Specimens from ill HCWs and a randomly selected sample from asymptomatic subjects were tested for additional respiratory viruses by multiplex PCR.
From 170 enrolled subjects, 1404 nasal swabs were collected. Influenza circulated at very low levels during the surveillance period and 74.2% of subjects received influenza vaccination. Influenza was not detected in any specimen. Multiplex respiratory virus PCR analysis of all 119 samples from symptomatic subjects and 200 specimens from asymptomatic subjects yielded a total of 42 positive specimens; 7 (16.7%) in asymptomatic subjects. Viral shedding was associated with report of any symptom (OR 13.06, p<0.0001, 95% CI 5.45-31.28) and younger age (OR 0.96, p=0.023, 95% CI 0.92-0.99) when controlled for gender and occupation of physician or nurse. After the surveillance period, 46% of subjects reported working while ill with an influenza-like illness during the previous influenza season.
In this cohort, HCWs working while ill was common, as was viral shedding among those with symptoms. Asymptomatic viral shedding was infrequent, but did occur. HCWs should refrain from patient care duties while ill, and staffing contingencies should accommodate them.
Childhood sexual abuse (CSA), adult sexual abuse (ASA) and intimate partner violence (IVP) are documented risk factors for HIV infection and are often implicated in the presentation of mental health disorders in both males and females, including those who are vulnerable to HIV-infection (African-Americans; trauma survivors). As such, these issues may contribute to health-related challenges among couples, particularly if the individuals are impacted by histories of trauma and HIV. Presented here is a case study of one couple with self-reported histories of CSA and clinically significant symptoms of posttraumatic stress disorder (PTSD) and depression. This couple was selected from a larger National Institute of Mental Health (NIMH)-funded study of 535 African-American HIV-serodiscordant heterosexual couples (see El Bassel, 2010). The study couple completed 8 sessions of an HIV sexual risk reduction intervention program to increase condom use. Although the couple reported an initial increase in condom use at the immediate post intervention assessment, condom use decreased to baseline assessment levels at the 12-month post intervention assessment. The decrease in HIV-transmission protective behaviors over time (i.e., condom use), in part, may be attributable to the clinically significant psychological distress symptoms of PTSD and depression that were maintained from baseline, throughout the trial, and at follow-up assessments. We propose that the success of sexual risk reduction interventions may be attenuated and compromised over time by the presence of sexual trauma histories and the residual mental health issues. We discuss clinical implications for health care professionals in their work with couples, especially those from racially diverse groups.
HIV; sexual trauma; couples therapy
The in vitro activity of five novel Microbiotix bis-indole agents (MBXs) against 30 multidrug-resistant (MDR) A. baumannii (including 18 resistant to carbapenems) was evaluated. Overall, MIC90s ranged from 1-8 μg/ml, whereas those for imipenem were > 64 μg/ml. MBX 1196 was the most potent (MIC90 1 μg/ml). MBXs are compounds that are highly effective against MDR A. baumannii.
bis-indole; multidrug-resistant; MBX; susceptibility; in vitro
There is a paucity of knowledge on the cost of health care services in Ghana. This poses a challenge in the economic evaluation of programmes and inhibits policy makers in making decisions about allocation of resources to improve health care. This study analysed the overall cost of providing health services in selected primary health centres and how much of the cost is attributed to the provision of antenatal and delivery services.
The study has a cross-sectional design and quantitative data was collected between July and December 2010. Twelve government run primary health centres in the Kassena-Nankana and Builsa districts of Ghana were randomly selected for the study. All health-care related costs for the year 2010 were collected from a public service provider’s perspective. The step-down allocation approach recommended by World Health Organization was used for the analysis.
The average annual cost of operating a health centre was $136,014 US. The mean costs attributable to ANC and delivery services were $23,063 US and $11,543 US respectively. Personnel accounted for the largest proportion of cost (45%). Overall, ANC (17%) and delivery (8%) were responsible for less than a quarter of the total cost of operating the health centres. By disaggregating the costs, the average recurrent cost was estimated at $127,475 US, representing 93.7% of the total cost. Even though maternal health services are free, utilization of these services at the health centres were low, particularly for delivery (49%), leading to high unit costs. The mean unit costs were $18 US for an ANC visit and $63 US for spontaneous delivery.
The high unit costs reflect underutilization of the existing capacities of health centres and indicate the need to encourage patients to use health centres .The study provides useful information that could be used for cost effectiveness analyses of maternal and neonatal care interventions, as well as for policy makers to make appropriate decisions regarding the allocation and sustainability of health care resources.
Cost; Step-down allocation approach; Antenatal care; Delivery; Maternal health service; Ghana
To define the utility of using routine diagnostic methods to detect influenza in older, hospitalized adults.
One academic hospital and 1 community hospital during the 2006–2007 and 2007–2008 influenza seasons.
Hospitalized adults 50 years of age or older.
Adults who were 50 years of age or older and hospitalized with symptoms of respiratory illness were enrolled and tested for influenza by use of reverse-transcriptase polymerase chain reaction (RT-PCR). Using RT-PCR as the gold standard, we assessed the performances of rapid antigen tests and conventional influenza culture and the diagnostic use of the clinical definition of influenza-like illness.
Influenza was detected by use of RT-PCR in 26 (11%) of 228 patients enrolled in our study. The sensitivity of the rapid antigen test performed at bedside by research staff members was 19.2% (95% confidence interval, 8.51%–37.9%); the sensitivity of conventional influenza culture was 34.6% (95% confidence interval, 19.4%–53.8%). The ability to detect influenza with both the rapid antigen test and culture was associated with patients with a higher viral load (P=.002 and P=.001, respectively). The ability to diagnose influenza by use of the clinical definition of influenza-like illness had a higher sensitivity (80.8%) but lacked specificity (40.6%).
Because rapid antigen testing and viral culture have poor sensitivity (19.2% and 34.6%, respectively), neither testing method is sufficient to use to determine what type of isolation procedures to implement in a hospital setting.
Background. We performed a prospective study to determine the disease burden of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) in older adults in comparison with influenza virus.
Methods. During 3 consecutive winters, we enrolled Davidson County (Nashville, TN) residents aged ≥50 years admitted to 1 of 4 hospitals with acute respiratory illness (ARI). Nasal/throat swabs were tested for influenza, RSV, and HMPV with reverse-transcriptase polymerase chain reaction. Hospitalization rates were calculated.
Results. Of 1042 eligible patients, 508 consented to testing. Respiratory syncytial virus was detected in 31 participants (6.1%); HMPV was detected in 23 (4.5%) patients; and influenza was detected in 33 (6.5%) patients. Of those subjects aged ≥65 years, 78% received influenza vaccination. Compared with patients with confirmed influenza, patients with RSV were older and more immunocompromised; patients with HMPV were older, had more cardiovascular disease, were more likely to have received the influenza vaccination, and were less likely to report fever than those with influenza. Over 3 years, average annual rates of hospitalization were 15.01, 9.82, and 11.81 per 10 000 county residents due to RSV, HMPV, and influenza, respectively.
Conclusions. In adults aged ≥50 years, hospitalization rates for RSV and HMPV were similar to those associated with influenza.
The human cannabinoid 1 receptor (hCB1), a ubiquitous G protein-coupled receptor (GPCR), transmits cannabinergic signals that participate in diverse (patho)physiological processes. Pharmacotherapeutic hCB1 targeting is considered a tractable approach for treating such prevalent diseases as obesity, mood disorders, and drug addiction. The hydrophobic nature of the transmembrane helices of hCB1 presents a formidable difficulty to its direct structural analysis. Comprehensive experimental characterization of functional hCB1 by mass spectrometry (MS) is essential to the targeting of affinity probes that can be used to define directly hCB1 binding domains using a ligand-assisted experimental approach. Such information would greatly facilitate the rational design of hCB1-selective agonists/antagonists with therapeutic potential. We report the first high-coverage MS analysis of the primary sequence of the functional hCB1 receptor, one of the few such comprehensive MS-based analyses of any GPCR. Recombinant C-terminal hexa-histidine-tagged hCB1 (His6-hCB1) was expressed in cultured insect (Spodoptera frugiperda) cells, solubilized by a procedure devised to enhance receptor purity following metal-affinity chromatography, desalted by buffer exchange, and digested in solution with (chymo)-trypsin. “Bottom-up” nanoLC-MS/MS of the (chymo)tryptic digests afforded a degree of overall hCB1 coverage (>94%) thus far reported for only two other GPCRs. This MS-compatible procedure devised for His6-hCB1 sample preparation, incorporating in-solution (chymo)trypsin digestion in the presence of a low concentration of CYMAL-5 detergent, may be applicable to the MS-based proteomic characterization of other GPCRs. This work should help enable future ligand-assisted structural characterization of hCB1 binding motifs at the amino-acid level using rationally designed and targeted covalent cannabinergic probes.
transmembrane protein; G protein-coupled receptor; protein expression; affinity purification; endocannabinoid signaling system; proteomic analysis; signal transduction; ligand-assisted protein analysis; ligand targeting; drug design; binding motifs; nanoLC-MS/MS
The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (≈30 mg/L). With 2-AG as substrate, hMGL displayed an apparent Vmax of 25 μmol/(μg min) and Km of 19.7 μM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (Km = 33.6 μM). hMGL also demonstrated a comparable affinity (Km ≈8–9 μM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34 126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzyme’s N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.
Monoacylglycerol lipase; MGL; 2-Arachidonoyl glycerol; 2-AG; Endocannabinoids; Fatty acid amide hydrolase; FAAH; Cannabinoid receptors; Protein expression; E. coli; Mass spectrometry; Fluorogenic substrate; Post-translational processing; Signal transduction; Proteomic analysis; MALDI-TOF MS
A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.
Outpatient burden of laboratory-confirmed influenza among children in Knox and Davidson Counties was compared in 2006–2007 when only Knox County had a school-based influenza vaccine campaign. Of 1016 eligible children seeking outpatient care, 87% were enrolled and 20% were influenza-positive. Estimated influenza vaccination coverage was similar in Knox and Davidson for preschool-age children (36% versus 33%) but higher for school-age children (44% versus 12%). Influenza detection was higher among Knox than Davidson County preschool-age children (18% versus 10%) but similar among school-age children (28% versus 27%). These data are consistent with a direct effect of the campaign in school-age children.
influenza vaccine; epidemiology; school-based vaccination
RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.
Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intra-retinal vascular architecture. In genetic mosaics, wild type endothelial cells (ECs) instruct neighboring Fz4−/− ECs to produce an architecturally normal mosaic vasculature, a cell non-autonomous effect. However, over the ensuing weeks, Fz4−/− ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier (BRB) and blood brain barrier (BBB) function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.
Because evidence-based psychotherapies of 12 to 20 sessions can be perceived as too lengthy and time intensive for the treatment of depression in primary care, a number of studies have examined abbreviated psychotherapy protocols. The purpose of this study was to conduct a systematic review and meta-analysis to determine the efficacy of brief psychotherapy (i.e., ≤ 8 sessions) for depression.
We used combined literature searches in PubMed, EMBASE, PsycINFO, and an Internet-accessible database of clinical trials of psychotherapy to conduct two systematic searches: one for existing systematic reviews and another for randomized controlled trials (RCTs). Included studies examined evidence-based psychotherapy(s) of 8 or fewer sessions, focused on adults with depression, contained an acceptable control condition, were published in English, and used validated measures of depressive symptoms.
We retained 2 systematic reviews and 15 RCTs evaluating cognitive behavioral therapy, problem-solving therapy, and mindfulness-based cognitive therapy. The systematic reviews found brief psychotherapies to be more efficacious than control, with effect sizes ranging from −0.33 to −0.25. Our meta-analysis found six to eight sessions of cognitive behavioral therapy to be more efficacious than control (ES −0.42, 95% CI −0.74 to −0.10, I2=56%). A sensitivity analysis controlled for statistical heterogeneity but showed smaller treatment effects (ES −0.24, 95% CI −0.42 to −0.06, I2=0%).
Depression can be efficaciously treated with six to eight sessions of psychotherapy, particularly cognitive behavioral therapy and problem-solving therapy. Access to non-pharmacologic treatments for depression could be improved by training health care providers to deliver brief psychotherapies.
depression; psychotherapy; primary health care; meta-analysis; review; systematic; psychotherapy; brief
During the 1990s, researchers at the Navrongo Health Research Centre in northern Ghana developed a highly successful community health program. The keystone of the Navrongo approach was the deployment of nurses termed community health officers to village locations. A trial showed that, compared to areas relying on existing services alone, the approach reduced child mortality by half, maternal mortality by 40%, and fertility by nearly a birth — from a total fertility rate of 5.5 in only five years. In 2000, the government of Ghana launched a national program called Community-based Health Planning and Services (CHPS) to scale up the Navrongo model. However, CHPS scale-up has been slow in districts located outside of the Upper East Region, where the “Navrongo Experiment” was first carried out. This paper describes the Ghana Essential Health Intervention Project (GEHIP), a plausibility trial of strategies for strengthening CHPS, especially in the areas of maternal and newborn health, and generating the political will to scale up the program with strategies that are faithful to the original design.
Description of the intervention
GEHIP improves the CHPS model by 1) extending the range and quality of services for newborns; 2) training community volunteers to conduct the World Health Organization service regimen known as integrated management of childhood illness (IMCI); 3) simplifying the collection of health management information and ensuring its use for decision making; 4) enabling community health nurses to manage emergencies, particularly obstetric complications and refer cases without delay; 5) adding $0.85 per capita annually to district budgets and marshalling grassroots political commitment to financing CHPS implementation; and 6) strengthening CHPS leadership at all levels of the system.
GEHIP impact is assessed by conducting baseline and endline survey research and computing the Heckman “difference in difference” test for under-5 mortality in three intervention districts relative to four comparison districts for core indicators of health status and survival rates. To elucidate results, hierarchical child survival hazard models will be estimated that incorporate measures of health system strength as survival determinants, adjusting for the potentially confounding effects of parental and household characteristics. Qualitative systems appraisal procedures will be used to monitor and explain GEHIP implementation innovations, constraints, and progress.
By demonstrating practical means of strengthening a real-world health system while monitoring costs and assessing maternal and child survival impact, GEHIP is expected to contribute to national health policy, planning, and resource allocation that will be needed to accelerate progress with the Millennium Development Goals.