The subthalamic nucleus contributes causally to perceptual decision-making in monkeys.

The subthalamic nucleus (STN) plays critical roles in the motor and cognitive function of the basal ganglia (BG), but the exact nature of these roles is not fully understood, especially in the context of decision-making based on uncertain evidence. Guided by theoretical predictions of specific STN contributions, we used single-unit recording and electrical microstimulation in the STN of healthy monkeys to assess its causal, computational roles in visual-saccadic decisions based on noisy evidence. The recordings identified subpopulations of STN neurons with distinct task-related activity patterns that related to different theoretically predicted functions. Microstimulation caused changes in behavioral choices and response times that reflected multiple contributions to an "accumulate-to-bound"-like decision process, including modulation of decision bounds and evidence accumulation, and to non-perceptual processes. These results provide new insights into the multiple ways that the STN can support higher brain function.

Does non-implanted electrical stimulation reduce post-stroke urinary or fecal incontinence? A systematic review with meta-analysis.

BACKGROUND: Urinary and fecal incontinence are disabling impairments after stroke that can be clinically managed with electrical stimulation. AIM: The purpose of this systematic review was to determine the effectiveness of non-implanted electrical stimulation to reduce the severity of post-stroke incontinence. SUMMARY OF REVIEW: Clinical trials of non-implanted electrical stimulation applied for the purposes of treating post-stroke incontinence were searched in MEDLINE, EMBASE, CINAHL, PEDro, and CENTRAL. From a total of 5043 manuscripts, 10 trials met the eligibility criteria (n = 894 subjects). Nine trials reported urinary incontinence severity outcomes enabling meta-analysis of transcutaneous electrical nerve stimulation (TENS; five trials) and electroacupuncture (four trials). Studies provide good-to-fair quality evidence that TENS commenced <3 months post-stroke has a large effect on urinary continence (SMD = -3.40, 95% CI -4.46 to -2.34) and a medium effect when commenced >3 months after stroke (SMD = -0.67, 95% CI -1.09 to -0.26). Electroacupuncture has a large effect when administered >5 times a week (SMD = -2.32, 95% CI -2.96 to -1.68) and a small effect when administered five times a week (SMD = -0.44, 95% CI -0.69 to -0.18). Only one trial reported the effect of non-implanted electrical stimulation on post-stroke fecal incontinence. CONCLUSIONS: Published trials evaluating the effect of non-implanted electrical stimulation on post-stroke incontinence are few and heterogenous. Synthesized trials suggest that early and frequent treatment using electrical stimulation is probably more effective than sham or no treatment. Further trials measuring incontinence in an objective manner are required.

Epidemiology, sexual risk behavior, and HIV prevention practices of men who have sex with men using GRINDR in Los Angeles, California.

Young men who have sex with men (YMSM) are at alarming risk for HIV acquisition, demonstrating the highest rates of incident infection of any age-risk group. GRINDR is a global positioning service-based social networking application popular with YMSM for sexual partnering. To assess the characteristics of YMSM who use GRINDR, we conducted a computer-assisted self-interview-based survey of 375 YMSM using GRINDR in metropolitan Los Angeles, recruited using the GRINDR platform. The median age was 25 (interquartile range, 22-27) years old, 42.4 % caucasian, 6.4 % African American, 33.6 % Latino, and 14.1 % Asian/Pacific Islander. Participants reported high rates of sexual partnering and unprotected anal intercourse (UAI). The majority (70 %) of those reporting unprotected anal intercourse reported low perception of HIV-acquisition risk. Of the participants, 83.1 % reported HIV testing within the past 12 months; 4.3 % had never been HIV tested. Of the participants, 4.5 % reported HIV-positive serostatus; 51.7 % indicated that they would be interested in participating in a future HIV prevention trial. Latinos were more likely than either caucasians or African Americans to endorse trial participation interest (odds ratio, 1.9; 95 % confidence interval [1.1-3.3]). HIV-positive test results were associated with increased number of anal sex partners in the past 3 months (adjusted odds ratio (AOR), 1.53 [0.97-2.40]), inconsistent inquiry about partners' serostatus (AOR, 3.63 [1.37-9.64]), reporting the purpose for GRINDR use including "friendship" (AOR, 0.17 [0.03-1.06), and meeting a sexual partner in a bookstore in the past 3 months (AOR, 33.84 [0.99-1152]). Men recruited via GRINDR were high risk for HIV acquisition or transmission and interested in clinical trial participation, suggesting potential for this method to be used for recruitment of YMSM to HIV prevention trials.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

BACKGROUND: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aß42 and Aß40. Many drug discovery efforts have focused on decreasing the production of Aß42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aß production to favor shorter, less amyloidogenic peptides than Aß42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer's disease. RESULTS: Here we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aß42 in H4 cells (IC50 = 67 nM) and increased the shorter Aß38 by 1.7 fold at the IC50 for lowering of Aß42. AßTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aß42 and did not alter AßTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aß deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aß aggregates, amyloid plaques, inflammatory markers, and cognitive deficits. CONCLUSIONS: EVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aß42, attenuated memory deficits, and reduced Aß plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer's disease.

The impact of decreased bead count to determine concentrations of amyloid beta1-42, total-tau, and phosphorylated-tau181 in human cerebrospinal fluid using xMAP technology.

Alzheimer's disease is the leading cause of human dementia. The lack of diagnostic tests and limited therapeutic options has driven the search for endogenous biomarkers. The INNO-BIA AlzBio3 assay is a multiplex flow-based immunoassay measuring Aß42, tau, and p-tau in cerebrospinal fluid (CSF). This study assesses assays performance under varying bead count (BC) parameters. Original method validation parameters at 100 BC were acceptable. Reanalyses performed at 3, 10, 25, and 50 BCs were compared to 100 BC data by ANOVA, Bland-Altman analysis, evaluation of concordance correlation coefficients, and frequency distribution of coefficient of variation (CV) ranges. Method validation characteristics were acceptable with 100 BCs. Equivalency for 25 and 50 versus 100 BCs was demonstrated, but not for 3 and 10 BCs. A general trend of decreasing agreement between decreasing BCs and the 100 BC reference resulted in decreases in concordance coefficients ρ(c) . The frequency of CV values greater than 20% increased with decreasing BCs, and CV values of 5% or less decreased with decreased BCs. Statistical analyses demonstrate that BCs of 3 and 10 are not equivalent with the reference and should not be used as a basis for determination of Aß42, tau, and p-tau concentration in human CSF.

Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs.

Regulatory recommendations for providing bioanalytical support for biological therapeutics have co-evolved with the increasing success of these unique pharmaceuticals. Immunoassays have been used to quantify biological macromolecules for more than 50 years. These assays rely on the use of antigen-specific antibodies. More recently, LC-MS methods have being adapted to quantitate biologics. LC-MS has attributes that complement the limitations encountered by immunoassays. Whether employing immunoassay or LC-MS methods, compared with traditional chemical-based therapeutics, biological therapeutics present unique analytical challenges to analysts. In this article, we review bioanalytical strategies for supporting biologics and discuss the regulatory and analytical challenges that must be met.

Read-across to rank skin sensitization potential: subcategories for the Michael acceptor domain.

BACKGROUND: Eliminating animal testing for skin sensitization is a significant challenge in consumer safety risk assessment. To be able to perform resilient risk assessments in the future, one will need alternative approaches to fill the data gaps. OBJECTIVES: To this end, we propose a subcategory-based read-across approach to estimate and rank skin sensitization potential of chemicals. The example described here is for the mechanism of Michael-type nucleophilic addition with subcategories being limited to carbonyl-containing compounds. PATIENTS/METHODS: In this approach, in silico tools based on structural alerts were used to determine both the mechanism of protein binding and the relative subcategories within that mechanism. RESULTS: Fifty compounds previously evaluated in the in vivo mouse local lymph node assay (LLNA) were placed in 10 subcategories defined by their polarized alpha,beta-unsaturated substructure. To offset the limitations and skewness of the published in vivo data, in chemico glutathione (GSH) depletion data also were included. CONCLUSIONS: It was shown that the read-across approach can be successfully used to rank qualitatively skin sensitization potential of an untested carbonyl-containing Michael acceptor chemical by using subcategories. Moreover, the use of the more resilient in chemico GSH depletion data added further support to the read-across result.

Phase II study of risk-adapted therapy of newly diagnosed, aggressive non-Hodgkin lymphoma based on midtreatment FDG-PET scanning.

In newly diagnosed aggressive non-Hodgkin lymphoma (NHL), a positive midtreatment fluorine-18 fluorodeoxyglucose positron emission tomography (PET) scan often carries a poor prognosis, with reported 2-year event-free survival (EFS) rates of 0% to 30% after standard therapy. To determine the outcome of early treatment intensification for midtreatment PET-positive disease, a phase II trial of risk-adapted therapy was conducted. Fifty-nine newly diagnosed patients, 98% with B cell lymphoma, had PET/CT performed after 2 or 3 cycles of first-line chemotherapy. Those with negative PET on semiquantitative visual interpretation completed standard therapy. Those with positive PET received platinum-based salvage chemotherapy, high-dose therapy, and autologous stem cell transplantation (ASCT). Midtreatment PET was positive in 33 (56%); 28 received ASCT with an actuarial 2-year EFS of 75% (95% confidence interval, 60%-93%). On intention-to-treat analysis, 2-year EFS was 67% (53%-86%) in all PET-positive patients and 89% (77%-100%) in PET-negative patients. No association was found between the International Prognostic Index category and the midtreatment PET result. The favorable outcome achieved here in historically poor-risk patients warrants further, more definitive investigation of treatment modification based on early PET scanning.

Management of side effects of novel therapies for multiple myeloma: consensus statements developed by the International Myeloma Foundation's Nurse Leadership Board.

Nurses play an essential role in managing the care of patients with multiple myeloma, who require education and support to receive and adhere to optimal therapy. The International Myeloma Foundation created a Nurse Leadership Board comprised of oncology nurses from leading cancer centers and community practices. An assessment survey identified the need for specific recommendations for managing key side effects of novel antimyeloma agents. Myelosuppression, thromboembolic events, peripheral neuropathy, steroid toxicities, and gastrointestinal side effects were selected for the first consensus statements. The board developed recommendations for healthcare providers in any medical setting, including grading of side-effect toxicity and strategies for managing the side effects in general, with specific recommendations pertaining to the novel agents.

Steroid-associated side effects in patients with multiple myeloma: consensus statement of the IMF Nurse Leadership Board.

Steroids have been the foundation of multiple myeloma therapy for more than 30 years and continue to be prescribed as single agents and in combination with other antimyeloma drugs, including novel therapies. Steroids cause a wide range of side effects that affect almost every system of the body. Identification and prompt management of the toxicities contribute to the success of steroid-containing antimyeloma regimens. By following patients carefully and educating them and their caregivers, nurses can promote adherence to therapy and improve quality of life. The International Myeloma Foundation's Nurse Leadership Board developed this consensus statement for the management of steroid-associated side effects to be used by healthcare providers in any medical setting.

Identification of critical amino acid residues for human iNOS functional activity.

Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.

Biochemical investigation is no substitute for clinical examination!

CONTEXT: Toxic shock syndrome has been shown previously to be associated with hyperamylasaemia. However, serum amylase levels do not usually exceed three times upper limit of normal in these cases. CASE REPORT: We report a case of a young girl of 17 years who presented with upper abdominal pain, severe shock and raised serum amylase level of 3,898 U/L, giving an impression of severe acute pancreatitis. It was only after finding a tampon in her vagina, and subsequently growing Staphylococcus aureus in her blood cultures, did the diagnosis of toxic shock syndrome become apparent. She recovered fully with supportive treatment and appropriate antibiotics. CONCLUSIONS: Toxic shock syndrome with such a high level of serum amylase has not been previously reported. This case exemplifies the importance of repeated clinical evaluation of patients in this era of multiple investigations, and not simply relying on biochemical values for diagnosis.

Tramadol and several anticonvulsants synergize in attenuating nerve injury-induced allodynia.

Neuropathic pain results from injury or dysfunction of the central or peripheral nervous system. The treatment of neuropathic pain is challenging, in part because of its multiple etiologies. The present study explores combinations of the analgesic tramadol and each of four anticonvulsants in the treatment of surgically induced (ligation of the L5 spinal nerve) allodynia in rats. Each of the five drugs studied exhibited a dose-dependent antiallodynic effect. When studied in combination, tramadol and each of two of the anticonvulsants (topiramate and RWJ-333369) interacted synergistically at all three ratios studied, whereas tramadol and each of the other two anticonvulsants (gabapentin and lamotrigine) exhibited a synergistic antiallodynic effect at only one of three ratios investigated. In addition, tramadol and topiramate were found to interact synergistically in a nociceptive pain model, the mouse hot-plate test. These studies suggest the benefit of using combinations of analgesics and anticonvulsants in the relief of neuropathic pain.

Direct inhibition of Ih by analgesic loperamide in rat DRG neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.

Use of a murine cell line for identification of human nitric oxide synthase inhibitors.

INTRODUCTION: Nitric oxide (NO) has been implicated in a wide range of physiological and pathological processes. Low concentrations of this mediator play homeostatic roles, whereas many acute and chronic responses are associated with excessive production of NO. This upregulation is due in part to the induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines in several different cell types, including macrophages and their CNS derivative, microglia. METHODS: The crystal structures of the oxygenase domains of mouse and human iNOS were superimposed using the "align by homology" feature in Sybyl (SYBYL 7.0, Tripos Inc.). NOS isoform expression was assessed by TaqMan, Western blotting, and activity assays. RESULTS: We demonstrate that there is a high degree of three-dimensional overlap between the mouse and human iNOS active centers and propose that the murine isoform can serve as a suitable substitute for the human in assays. We also demonstrate that LPS stimulation of the mouse macrophage cell line RAW 264.7 induces the expression of iNOS, but not nNOS or eNOS, at the levels of mRNA transcription and protein expression. Furthermore, the pharmacology and calcium dependency of the NO formation support the finding that it is due to iNOS alone. Also reported is the demonstration of LPS-induced RAW 264.7 macrophages in simple cell-based and cell-free screening assays for iNOS inhibitors. Both assays were reproducible, as demonstrated by Z' factors of 0.69 and 0.71, and had high signal to noise ratios of 11- and 6-fold for the cell-based and cell-free assay, respectively. DISCUSSION: Our computational analyses indicate that there is a high degree of three-dimensional overlap between the oxygenase domains of human and murine iNOS. This observation together with the selective induction of murine iNOS in RAW 264.7 macrophages demonstrates the potential utility of the mouse iNOS assay to identify inhibitors of the human enzyme.

Differential and synergistic effects of selective norepinephrine and serotonin reuptake inhibitors in rodent models of pain.

There is increasing recognition that norepinephrine (NE) and serotonin (5-HT) reuptake inhibitors (NRIs and SRIs) are efficacious in treating some types of pain. To date, studies have not systematically evaluated the relative activity at the NE and/or 5-HT transporter required for maximal efficacy in rodent pain models. Known selective NE and 5-HT reuptake inhibitors reboxetine, desipramine, fluoxetine, and paroxetine were evaluated in both in vitro and in vivo assays. Using the spinal nerve ligation model of neuropathic pain, the compounds differentially reversed tactile allodynia. Evaluation of a broader spectrum of reuptake inhibitors in the para-phenylquinone (PPQ)-induced abdominal constriction model, a model of acute visceral pain, demonstrated that both the SRIs and the NRIs significantly blocked abdominal constrictions. However, the magnitude of this effect was greater following treatment with compounds having greater affinity for NRI compared with SRI affinity. In addition, isobolographic analyses indicated significant synergistic effects for all combinations of desipramine and fluoxetine in the PPQ model of visceral pain. Collectively, the present results support the hypothesis that compounds with greater NRI activity should be more effective for the treatment of pain than compounds having only SRI activity, and this hypothesis is also supported by clinical data. These studies also suggest that the potency and effectiveness of NRIs might be enhanced by the presence of 5-HT activity.

An estrogen receptor-beta agonist is active in models of inflammatory and chemical-induced pain.

ERB-041 (2-(3-Fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol) is a selective estrogen receptor-beta agonist with activity in rodent models of rheumatoid arthritis and endometriosis. Clinical trials for these diseases are underway: however, the role of estrogen receptor-beta in modulating pain associated with inflammation remains unknown. These studies demonstrate that acutely administered ERB-041 is anti-hyperalgesic in preclinical models of chemical-induced and acute inflammatory pain, thus suggesting that ERB-041 may be useful for modulating pain associated with some types of inflammation.

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay.

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening.

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.