SARS-CoV-2 vaccine-related neurological complications.

OBJECTIVE: To describe three cases with neurological symptoms after SARS-CoV-2 vaccination. METHODS: A case series followed by a review of the literature, describing hypotheses on how neurological symptoms might develop after vaccination. RESULTS: The different temporal relationship between the onset or worsening of different neurological symptoms suggests different pathophysiological mechanisms. Progression of post-infectious myoclonus, caused by a previous SARS-CoV-2-infection, shortly after vaccination suggests a renewed auto-immune mediated crossreaction of antibodies to both viral epitopes and central nervous system components. Thunderclap headache after vaccination suggests a similar pathophysiological mechanism to the headache and other flu-like symptoms described after vaccination against other viruses. This might be ascribed to the activation of immunoinflammatory mediators or accompanying fever. Although headache accompanied by encephalopathy and focal neurological deficit might occur as part of a cytokine release syndrome, this is clinically less likely. CONCLUSIONS: A variety of symptoms, including thunderclap headache, focal deficits and movement disorders, can occur after SARS-CoV-2 vaccination, and an activation or reactivation of the immune system is suggested as most likely cause. However, one should be careful about claiming a direct correlation. It remains important to exclude other causes, such as structural lesions, infections or subarachnoid hemorrhage, and future research is required to understand possible pathophysiological mechanisms and associations with the SARS-CoV-2 vaccine.

Experimental testing of hypotheses for temperature- and pH-based niche specialization of ammonia oxidizing archaea and bacteria.

Investigation of niche specialization in microbial communities is important in assessing consequences of environmental change for ecosystem processes. Ammonia oxidizing bacteria (AOB) and archaea (AOA) present a convenient model for studying niche specialization. They coexist in most soils and effects of soil characteristics on their relative abundances have been studied extensively. This study integrated published information on the influence of temperature and pH on AOB and AOA into several hypotheses, generating predictions that were tested in soil microcosms. The influence of perturbations in temperature was determined in pH 4.5, 6 and 7.5 soils and perturbations in pH were investigated at 15°C, 25°C and 35°C. AO activities were determined by analysing changes in amoA gene and transcript abundances, stable isotope probing and nitrate production. Experimental data supported major predictions of the effects of temperature and pH, but with several significant discrepancies, some of which may have resulted from experimental limitations. The study also provided evidence for unpredicted activity of AOB in pH 4.5 soil. Other discrepancies highlighted important deficiencies in current knowledge, particularly lack of consideration of niche overlap and the need to consider combinations of factors when assessing the influence of environmental change on microbial communities and their activities.

Blame It on the Metabolite: 3,5-Dichloroaniline Rather than the Parent Compound Is Responsible for the Decreasing Diversity and Function of Soil Microorganisms.

Pesticides are key stressors of soil microorganisms with reciprocal effects on ecosystem functioning. These effects have been mainly attributed to the parent compounds, while the impact of their transformation products (TPs) has been largely overlooked. We assessed in a meadow soil (soil A) the transformation of iprodione and its toxicity in relation to (i) the abundance of functional microbial groups, (ii) the activity of key microbial enzymes, and (iii) the diversity of bacteria, fungi, and ammonia-oxidizing microorganisms (AOM) using amplicon sequencing. 3,5-Dichloroaniline (3,5-DCA), the main iprodione TP, was identified as a key explanatory factor for the persistent reduction in enzymatic activities and potential nitrification (PN) and for the observed structural changes in the bacterial and fungal communities. The abundances of certain bacterial (Actinobacteria, Hyphomicrobiaceae, Ilumatobacter, and Solirubrobacter) and fungal (Pichiaceae) groups were negatively correlated with 3,5-DCA. A subsequent study in a fallow agricultural soil (soil B) showed limited formation of 3,5-DCA, which concurred with the lack of effects on nitrification. Direct 3,5-DCA application in soil B induced a dose-dependent reduction of PN and NO3--N, which recovered with time. In vitro assays with terrestrial AOM verified the greater toxicity of 3,5-DCA over iprodione. "Candidatus Nitrosotalea sinensis" Nd2 was the most sensitive AOM to both compounds. Our findings build on previous evidence on the sensitivity of AOM to pesticides, reinforcing their potential utilization as indicators of the soil microbial toxicity of pesticides in pesticide environmental risk analysis and stressing the need to consider the contribution of TPs in the toxicity of pesticides on the soil microbial community.IMPORTANCE Pesticide toxicity on soil microorganisms is an emerging issue in pesticide risk assessment, dictated by the pivotal role of soil microorganisms in ecosystem services. However, the focus has traditionally been on parent compounds, while transformation products (TPs) are largely overlooked. We tested the hypothesis that TPs can be major contributors to the soil microbial toxicity of pesticides using iprodione and its main TP, 3,5-dichloroaniline, as model compounds. We demonstrated, by measuring functional and structural endpoints, that 3,5-dichloroaniline and not iprodione was associated with adverse effects on soil microorganisms, with nitrification being mostly affected. Pioneering in vitro assays with relevant ammonia-oxidizing bacteria and archaea verified the greater toxicity of 3,5-dichloroaniline. Our findings are expected to advance environmental risk assessment, highlighting the potential of ammonia-oxidizing microorganisms as indicators of the soil microbial toxicity of pesticides and stressing the need to consider the contribution of TPs to pesticide soil microbial toxicity.

Outcome analysis of cubital tunnel decompression.

BACKGROUND: Cubital tunnel decompression is a commonly undertaken upper limb procedure. Most studies compare the different techniques of decompression; however, only a few have specifically investigated the outcome of ulnar nerve decompression. AIM: The aim of this study was to investigate the outcome of ulnar nerve decompression following cubital tunnel syndrome. METHODS AND RESULTS: A total of 174 ulnar nerve decompression cases were identified from the upper limb surgery database with complete data available for 136 cases. Simple decompression was performed in 110 (80.88%) cases, and in 26 (19.12%), anterior subcutaneous transposition was also supplemented. These operations were performed at three different hospitals by surgeons of different levels of experience. The most common cause of cubital tunnel syndrome was idiopathic. The outcome was satisfactory in 86% of cases. No obvious association was demonstrated between the outcome of surgery and duration of symptoms, presence of co-morbidities or the type of surgery performed. CONCLUSION: This is the largest outcome analysis of the results of ulnar nerve decompression at the elbow. Good results following nerve decompression were attained in 86% of cases without any significant effect of duration of symptoms or co-morbidities on the outcome of surgery. It is hoped that the findings of the current study will help general practitioners, junior doctors and surgeons in their management and pre-operative consultation with patients having cubital tunnel syndrome.

Structured medication reviews in Parkinson's disease: pharmacists' views, experiences and needs - a qualitative study.

Background: Executing structured medication reviews (SMRs) in primary care to optimize drug treatment is considered standard care of community pharmacists in the Netherlands. Patients with Parkinson's disease (PD) often face complex drug regimens for their symptomatic treatment and might, therefore, benefit from an SMR. However, previously, no effect of an SMR on quality of life in PD was found. In trying to improve the case management of PD, it is interesting to understand if and to what extent SMRs in PD patients are of added value in the pharmacist's opinion and what are assumed facilitating and hindering factors. Objectives: To analyse the process of executing SMRs in PD patients from a community pharmacist's point of view. Design: A cross-sectional, qualitative study was performed, consisting of face-to-face semi-structured in-depth interviews. Methods: The interviews were conducted with community pharmacists who executed at least one SMR in PD, till data saturation was reached. Interviews were transcribed verbatim, coded and analysed thematically using an iterative approach. Results: Thirteen pharmacists were interviewed. SMRs in PD were considered of added value, especially regarding patient contact and bonding, individualized care and its possible effect in the future, although PD treatment is found already well monitored in secondary care. Major constraints were time, logistics and collaboration with medical specialists. Conclusion: Although community pharmacist-led SMRs are time-consuming and sometimes logistically challenging, they are of added value in primary care in general, and also in PD, of which treatment occurs mainly in secondary care. It emphasizes the pharmacist's role in PD treatment and might tackle future drug-related issues. Improvements concern multidisciplinary collaboration for optimized SMR execution and results.

Structured medication reviews in Parkinson's disease: pharmacists' views, experiences and needs Why is this research done? In Parkinson's disease (PD), drug therapy is still the most important treatment strategy. Due to disease progression, patients often face complicated medication regimens, polypharmacy, and potential drug-related problems. The execution of structured medication reviews (SMR) in primary care is considered standard care of community pharmacists in the Netherlands, aiming to optimise drug treatment. Although it might also affect clinical outcomes, we found no effect of an SMR in PD on quality of life in our previous study. In trying to improve case management of PD, we need to understand if and to what extent SMRs in PD patients are of added value in the pharmacist's opinion, and what are assumed facilitating and hindering factors. What did the researchers do? We conducted semi-structured interviews with 13 community pharmacists who recently executed ⩾1 SMR in PD. What did the researchers find? We found that SMRs in PD are considered of added value with regard to patient bonding and individualised care. By being known by the pharmacist, and vice versa, by knowing the patient's situation, future drug problems might be tackled earlier. However, executing SMRs comes with barriers, of which lack of time, logistic constraints and difficulties in cooperation with the medical specialist are the most important. What do these findings mean? Taking into account both the pharmacist's effort and additional costs when performing an SMR in the current setting, the valuable time of a pharmacist could potentially better be spent on more (cost-)effective interventions, or a structural solution should be sought for the experienced hindering factors. Since we do not doubt the importance of periodic medication optimization in complex diseases or high-risk patients, we have to focus on either improving the current setting of SMRs in PD, or searching for other strategies in which this can be achieved.

Expression of sphingosine 1-phosphate receptors in the rat dorsal root ganglia and defined single isolated sensory neurons.

Previously, we demonstrated that sphingosine 1-phosphate (S1P) increased the excitability of small-diameter sensory neurons, in part, through activation of S1P receptor 1 (S1PR(1)), suggesting that other S1PRs can modulate neuronal excitability. Therefore, studies were undertaken to establish the expression profiles of S1PRs in the intact dorsal root ganglion (DRG) and in defined single isolated sensory neurons. To determine mRNA expression of S1PRs in the DRG, SYBR green quantitative PCR (qPCR) was used. To determine the expression of S1PR mRNAs in single neurons of defined diameters, a preamplification protocol utilizing Taqman primer and probes was used to enhance the sensitivity of detection. The preamplification protocol also permitted detection of mRNA for two hallmark neuronal receptor/ion channels, TRPV1 and P(2)X(3). Expression profiles of S1PR mRNA isolated from lung and brain were used as positive control tissues. In the intact DRG, the order of expression of S1PRs was S1PR(3)>>R(1)≈R(2)>R(5)≈R(4). In the single neurons, the expression of S1PRs was quite variable with some neurons expressing all five subtypes, whereas some expressing only one subtype. In contrast to the DRG, S1PR(1) was the highest expressing subtype in 10 of the 18 small-, medium-, and large-diameter sensory neurons. S1PR(1) was the second highest expressor in -50% of those remaining neurons. Overall, in the single neurons, the order of expression was S1PR(1)>>R(3)≈R(5)>R(4)>R(2). The results obtained from the single defined neurons are consistent with our previous findings wherein S1PR(1) plays a prominent but not exclusive role in the enhancement of neuronal excitability.

Sphingosine 1-phosphate receptor 2 antagonist JTE-013 increases the excitability of sensory neurons independently of the receptor.

Previously we demonstrated that sphingosine 1-phosphate receptor 1 (S1PR(1)) played a prominent, but not exclusive, role in enhancing the excitability of small-diameter sensory neurons, suggesting that other S1PRs can modulate neuronal excitability. To examine the potential role of S1PR(2) in regulating neuronal excitability we used the established selective antagonist of S1PR(2), JTE-013. Here we report that exposure to JTE-013 alone produced a significant increase in excitability in a time- and concentration-dependent manner in 70-80% of recorded neurons. Internal perfusion of sensory neurons with guanosine 5'-O-(2-thiodiphosphate) (GDP-ß-S) via the recording pipette inhibited the sensitization produced by JTE-013 as well as prostaglandin E(2). Pretreatment with pertussis toxin or the selective S1PR(1) antagonist W146 blocked the sensitization produced by JTE-013. These results indicate that JTE-013 might act as an agonist at other G protein-coupled receptors. In neurons that were sensitized by JTE-013, single-cell RT-PCR studies demonstrated that these neurons did not express the mRNA for S1PR(2). In behavioral studies, injection of JTE-013 into the rat's hindpaw produced a significant increase in the mechanical sensitivity in the ipsilateral, but not contralateral, paw. Injection of JTE-013 did not affect the withdrawal latency to thermal stimulation. Thus JTE-013 augments neuronal excitability independently of S1PR(2) by unknown mechanisms that may involve activation of other G protein-coupled receptors such as S1PR(1). Clearly, further studies are warranted to establish the causal nature of this increased sensitivity, and future studies of neuronal function using JTE-013 should be interpreted with caution.

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ.

Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

The Effect of a Structured Medication Review on Quality of Life in Parkinson's Disease.

BACKGROUND: Drug therapy is important for controlling symptoms in Parkinson's disease (PD). However, it often results in complex medication regimens and could easily lead to drug related problems (DRP), suboptimal adherence and reduced treatment efficacy. A structured medication review (SMR) could address these issues and optimize therapy, although little is known about clinical effects in PD patients. OBJECTIVE: To analyze whether an SMR improves quality of life (QoL) in PD. METHODS: In this multicenter randomized controlled trial, half of the 202 PD patients with polypharmacy received a community pharmacist-led SMR. The control group received usual care. Assessments at baseline, and after three and six months comprised six validated questionnaires. Primary outcome was PD specific QoL [(PDQ-39; range 0 (best QoL) - 100 (worst QoL)]. Secondary outcomes were disability score, non-motor symptoms, general health status, and personal care giver's QoL. Furthermore, DRPs, proposed interventions, and implemented modifications in medication schedules were analyzed. RESULTS: No improvement in QoL was seen six months after an SMR, with a non-significant treatment effect difference of 2.09 (-0.63;4.80) in favor of the control group. No differences were found in secondary outcomes. In total, 260 potential DRPs were identified (2.6 (±1.8) per patient), of which 62% led to drug therapy optimization. CONCLUSION: In the current setting, a community pharmacist-led SMR did not improve QoL in PD patients, nor improved other pre-specified outcomes.

The Impact of Non-dopaminergic Medication on Quality of Life in Parkinson's Disease.

BACKGROUND AND OBJECTIVES: Quality of life (QoL) in Parkinson's disease (PD) depends on multiple factors. Due to PD treatment and accompanying, age-related or independent comorbidities, pill burden is often high. The relation of QoL and pharmacotherapy for comorbidities in PD has not been widely studied. This study investigated if and to what extent non-dopaminergic drugs are related to QoL in PD. Second, the impact of demographics and non-motor symptoms were evaluated. A better understanding of the impact of different non-dopaminergic drugs and polypharmacy on QoL will have added value in selecting appropriate (medication) interventions. METHODS: In a cross-sectional analysis, medication prescription data of 209 PD patients were analyzed and grouped according to the Rx-Risk comorbidity index. QoL was measured using the PDQ-39 questionnaire. Non-motor symptoms were analyzed with the Non-Motor Symptoms questionnaire. Independent factors associated with a reduced QoL were identified with a multivariate linear regression analysis. RESULTS: Non-dopaminergic drugs, subdivided into Rx-Risk comorbidity categories, were not associated with reduced QoL, except for the use of anti-epileptic drugs. However, using more daily non-dopaminergic drugs was also negatively associated with QoL, as well as female sex, increased PD severity, and more non-motor symptoms. Contraindicated non-dopaminergic medication was barely prescribed (0.4%). CONCLUSION: Non-dopaminergic drugs are frequently prescribed, and higher numbers are associated with impaired QoL in PD. However, when divided in drug types, only anti-epileptic drugs were negatively associated with QoL. In these patients, physicians might improve QoL by further optimizing the condition it was prescribed for (e.g., pain or anxiety), or managing of side effects. TRIAL REGISTRATION: Netherlands Trial Register; NL4360.

The sphingosine 1-phosphate receptor, S1PR1, plays a prominent but not exclusive role in enhancing the excitability of sensory neurons.

Sphingosine 1-phosphate (S1P) through its interaction with a family of G protein-coupled receptors (S1PR) is proving to have a significant impact on the activation of a variety of cell types, most notably those cells mediating the inflammatory response. Previously, we showed that S1P enhanced the excitability of small diameter sensory neurons, and mRNA for S1PR(1-4) was expressed in sensory neurons. These initial findings did not determine which S1PR subtype(s) mediated the increased excitability. Here, we report that exposure to the selective S1PR(1) agonist, SEW2871, produced a significant increase in excitability of some, but not all, sensory neurons. To further examine the role of S1PR(1), neurons were treated with siRNA targeted to S1PR(1). siRNA reduced S1PR(1) protein expression by 75% and blocked the sensitization produced by SEW2871, although some neurons remained responsive to subsequent exposure to S1P. Treatment with scramble siRNA did not alter S1PR(1) expression. Recordings from siRNA- and scramble-treated neurons suggested three distinct populations based on their sensitivities to SEW2871 and S1P. Approximately 50% of the neurons exhibited a significant increase in excitability after exposure to SEW2871 and subsequent S1P produced no additional increase; ∼25% were not affected by SEW2871 but S1P significantly increased excitability; and ∼25% of the neurons were not sensitized by either SEW2871 or S1P. RT-PCR measurements obtained from single neurons showed that 50% of the small diameter neurons expressed the mRNA for S1PR(1). These results indicate that S1PR(1) plays a prominent, although not exclusive, role in mediating the enhancement of excitability produced by S1P.

Augmented sodium currents contribute to the enhanced excitability of small diameter capsaicin-sensitive sensory neurons isolated from Nf1+/⁻ mice.

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Quantitative assessment of ammonia-oxidizing bacterial communities in the epiphyton of submerged macrophytes in shallow lakes.

In addition to the benthic and pelagic habitats, the epiphytic compartment of submerged macrophytes in shallow freshwater lakes offers a niche to bacterial ammonia-oxidizing communities. However, the diversity, numbers, and activity of epiphytic ammonia-oxidizing bacteria have long been overlooked. In the present study, we analyzed quantitatively the epiphytic communities of three shallow lakes by a potential nitrification assay and by quantitative PCR of 16S rRNA genes. On the basis of the m(2) of the lake surface, the gene copy numbers of epiphytic ammonia oxidizers were not significantly different from those in the benthic and pelagic compartments. The potential ammonia-oxidizing activities measured in the epiphytic compartment were also not significantly different from the activities determined in the benthic compartment. No potential ammonia-oxidizing activities were observed in the pelagic compartment. No activity was detected in the epiphyton of Chara aspera, the dominant submerged macrophyte in Lake Nuldernauw in The Netherlands. The presence of ammonia-oxidizing bacterial cells in the epiphyton of Potamogeton pectinatus was also demonstrated by fluorescent in situ hybridization microscopy images. By comparing the community composition as assessed by the 16S rRNA gene PCR-denaturing gradient gel electrophoresis approach, it was concluded that the epiphytic ammonia-oxidizing communities consisted of cells that were also present in the benthic and pelagic compartments. Of the environmental parameters examined, only the water retention time, the Kjeldahl nitrogen content, and the total phosphorus content correlated with potential ammonia-oxidizing activities. None of these parameters correlated with the numbers of gene copies related to ammonia-oxidizing betaproteobacteria.

Getting to precision psychopharmacology: Combining clinical and genetic information to predict fat gain from aripiprazole.

INTRODUCTION: All atypical antipsychotics are associated with some degree of weight gain. We applied a novel statistical approach to identify moderators of aripiprazole-induced fat gain using clinical and genetic data from a randomized clinical trial (RCT) of treatment resistant depression in older adults. MATERIALS AND METHODS: Adults aged ≥60 years with non-response to a prospective trial of venlafaxine were randomized to 12 weeks of aripiprazole augmentation (n = 91) or placebo (n = 90). Dual energy x-ray absorptiometry (DEXA) measured adiposity at baseline and 12 weeks. Independent moderators of total body fat gain were used to generate two combined multiple moderators, one including clinical data alone and one including both clinical and genetic data to characterize individuals who gained fat during aripiprazole augmentation. RESULTS: The value of the combined genetic + clinical multiple moderator (Mcg) was 0.57 [95% CI 0.46, 0.68] (effect size: 0.57), compared to the combined clinical moderator (Mc) value of 0.49 [0.34, 0.63] (effect size: 0.49). Individuals who gained adiposity in this study were more likely to be female and younger in age, have lower weight, fasting glucose and lipids at baseline and positive for the HTR2C polymorphism. DISCUSSION: These results demonstrate a combined multiple moderator approach, including both clinical and genetic moderators, can be applied to existing clinical trial data to understand adverse treatment effects. This method allowed for more specific characterization of individuals at risk for the outcome of interest. Further work is needed to identify additional genetic moderators and to validate the approach.

Brain-derived neurotrophic factor enhances the excitability of rat sensory neurons through activation of the p75 neurotrophin receptor and the sphingomyelin pathway.

Neurotrophin-mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75(NTR)) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75(NTR) to liberate sphingosine 1-phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75(NTR), then brain-derived neurotrophic factor (BDNF) should produce the same sensitizing action as did NGF. In this report, we show that focally applied BDNF increases the number of action potentials (APs) evoked by a ramp of depolarizing current by reducing the rheobase without altering the firing threshold. This increased excitability results, in part, from the capacity of BDNF to enhance a tetrodotoxin-resistant sodium current (TTX-R I(Na)) and to suppress a delayed rectifier-like potassium current (I(K)). The idea that BDNF acts via p75(NTR) is supported by the following observations. The sensitizing action of BDNF is prevented by pretreatment with a blocking antibody to p75(NTR) or an inhibitor of sphingosine kinase (dimethylsphingosine), but not by inhibitors of tyrosine kinase receptors (K252a or AG879). Furthermore, using single-cell RT-PCR, neurons that were sensitized by BDNF expressed the mRNA for p75(NTR) but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin-sensitive sensory neurons through the activation of p75(NTR) and its downstream sphingomyelin signalling cascade. Neurotrophins released upon activation of a variety of immuno-competent cells may be important mediators that give rise to the enhanced neuronal sensitivity associated with the inflammatory response.

Interactions of peripheral endothelin-1 and nerve growth factor as contributors to persistent cutaneous pain.

Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.

ATP-sensitive potassium currents reduce the PGE2-mediated enhancement of excitability in adult rat sensory neurons.

Behavioral studies have shown that the hyperalgesia arising from inflammatory agents, such as prostaglandin E(2) (PGE(2)), can be antagonized by activators of the ATP-sensitive potassium current (K(ATP)). This observation raises questions as to whether this suppression results from a direct action on sensory neurons and what are the cellular mechanisms giving rise to this inhibition. We found that small to medium diameter sensory neurons isolated from the L4-6 DRGs expressed the mRNAs for Kir6.1, Kir6.2, and SUR1. In perforated-patch clamp recordings from acutely dissociated sensory neurons from the young adult rat, exposure to 300 microM diazoxide, a K(ATP) channel agonist, significantly hyperpolarized the resting membrane potential, reduced the number of action potentials evoked by a ramp of depolarizing current, and increased the amplitude of inward K(ATP) currents evoked by the voltage ramp. Similar results were obtained with the protonophore FCCP, which is known to reduce the levels of intracellular ATP and lead to the activation of K(ATP). Only a subpopulation of sensory neurons was sensitive to diazoxide whereas other neurons were unaffected. Treatment with 1 microM PGE(2) significantly enhanced the excitability of these small to medium diameter capsaicin-sensitive sensory neurons; this enhancement was reversed by subsequent exposure to diazoxide in a subpopulation of neurons. Similar to diazoxide, exposure to 8-Br-cyclic GMP antagonized the PGE(2)-induced increase in excitability. The effects of 8-Br-cyclic GMP could be reversed by exposure to glibenclamide, an antagonist of K(ATP) channels. As with diazoxide, only a subpopulation of sensory neurons were affected by 8-Br-cyclic GMP. These results demonstrate that activation of K(ATP) can reverse the sensitization produced by PGE(2) and may be an important means to modulate the enhanced excitability that results from inflammatory or injury conditions.

Measurement of neural respiratory drive via parasternal intercostal electromyography in healthy adult subjects.

Neural respiratory drive, quantified by the parasternal intercostal muscle electromyogram (EMGpara), provides a sensitive measure of respiratory system load-capacity balance. Reference values for EMGpara-based measures are lacking and the influence of individual anthropometric characteristics is not known. EMGpara is conventionally expressed as a percentage of that obtained during a maximal inspiratory effort (EMGpara%max), leading to difficulty in applying the technique in subjects unable to reliably perform such manoeuvres. To measure EMGpara in a large, unselected cohort of healthy adult subjects in order to evaluate relevant technical and anthropometric factors. Surface second intercostal space EMGpara was measured during resting breathing and maximal inspiratory efforts in 63 healthy adult subjects, median (IQR) age 31.0 (25.0-47.0) years, 28 males. Detailed anthropometry, spirometry and respiratory muscle strength were also recorded. Median (IQR EMGpara was 4.95 (3.35-6.93) µV, EMGpara%max 4.95 (3.39-8.65)% and neural respiratory drive index (NRDI, the product of EMGpara%max and respiratory rate) was 73.62 (46.41-143.92) %.breath/min. EMGpara increased significantly to 6.28 (4.26-9.93) µV (p < 0.001) with a mouthpiece, noseclip and pneumotachograph in situ. Median (IQR) EMGpara was higher in female subjects (5.79 (4.42-7.98) µV versus 3.56 (2.81-5.35) µV, p = 0.003); after controlling for sex neither EMGpara, EMGpara%max or NRDI were significantly related to anthropometrics, age or respiratory muscle strength. In subjects undergoing repeat measurements within the same testing session (n = 48) or on a separate occasion (n = 19) similar repeatability was observed for both EMGpara and EMGpara%max. EMGpara is higher in female subjects than males, without influence of other anthropometric characteristics. Reference values are provided for EMGpara-derived measures. Expressing EMGpara as a percentage of maximum confers no advantage with respect to measurement repeatability, expanding the potential application of the technique. Raw EMGpara is a useful marker of respiratory system load-capacity balance.

Calcium regulates some, but not all, aspects of light adaptation in rod photoreceptors.

The role of calcium as a regulator of light adaptation in rod photoreceptors was examined by manipulation of the intracellular Ca2+ concentration through the use of the calcium ionophore A23187 and external Ca2+ buffers. These studies utilized suspensions of isolated and purified frog rod outer segments that retain their mitochondria-rich inner segments (OS-IS). Three criteria of the dark- and light-adapted flash response were characterized as a function of the Ca2+ concentration: (a) the time to peak, (b) the rate of recovery, and (c) the response amplitude or sensitivity. For all Ca2+ concentrations examined, the time to peak of the flash response was accelerated in the presence of background illumination, suggesting that mechanisms controlling this aspect of adaptation are independent of the Ca2+ concentration. The recovery kinetics of the flash response appeared to depend on the Ca2+ concentration. In 1 mM Ca2+-Ringer's and 300 nM Ca2+-Ringer's + A23187, background illumination enhanced the recovery rate of the response; however, in 10 and 100 nM Ca2+-Ringer's + A23187, the recovery rates were the same for dark- and light-adapted responses. This result implies that a critical level of Ca2+ may be necessary for background illumination to accelerate the recovery of the flash response. The sensitivity of the flash response in darkness (SDF) was dependent on the Ca2+ concentration. In 1 mM Ca2+-Ringer's SDF was 0.481 pA per bleached rhodopsin (Rh*); a background of four Rh*/s decreased SDF by half (Io). At 300 nM Ca2+ + A23187, SDF was reduced to 0.0307 pA/Rh* and Io increased to 60 Rh*/s. At 100 nM Ca2+ + A23187, SDF was reduced further to 0.0025 pA/Rh* and Io increased to 220 Rh*/s. In 10 nM Ca2+ + A23187, SDF was lowered to 0.00045 pA/Rh* and Io raised to 760 RhI/s. Using these values of SDF and Io for each respective Ca2+ concentration, the dependence of the flash sensitivity on background intensity could be described by the Weber-Fechner relation. Under low Ca2+ conditions + A23187, bright background illumination could desensitize the flash response. These results are consistent with the idea that the concentration of Ca2+ may set the absolute magnitude of response sensitivity in darkness, and that there exist mechanisms capable of adapting the photoresponse in the absence of significant changes in cytoplasmic Ca2+ concentration.