Assessment of liver graft quality during hypothermic oxygenated perfusion: the first international validation study.

BACKGROUND AND AIM: While it is currently assumed that liver assessment is only possible during normothermic machine perfusion (NMP), there is uncertainty regarding a reliable and quick prediction of graft injury during ex situ hypothermic oxygenated perfusion (HOPE). We therefore intended to test, in an international liver transplant cohort, recently described mitochondrial injury biomarkers measured during HOPE before liver transplantation. STUDY DESIGN: Perfusate samples of human livers from 10 centers in 7 countries with HOPE-experience were analyzed for released mitochondrial compounds, i.e. flavin mononucleotide (FMN), NADH, purine derivates and inflammatory markers. Perfusate FMN was correlated with graft loss due to primary non-function or symptomatic non-anastomotic biliary strictures (NAS), and kidney failure, as well as liver injury after transplantation. Livers deemed unsuitable for transplantation served as negative control. RESULTS: We collected 473 perfusate samples of human DCD (n=315) and DBD livers (n=158). Fluorometric assessment of FMN in perfusate was validated by mass spectrometry (R=0.7011,p<0.0001). Graft loss due to primary non-function or cholangiopathy was predicted by perfusate FMN values (c-statistic mass spectrometry 0.8418 (95%CI 0.7466-0.9370,p<0.0001), c-statistic fluorometry 0.7733 (95%CI 0.7006-0.8461,p<0.0001). Perfusate FMN values were also significantly correlated with symptomatic NAS and kidney failure, and superior in prediction of graft loss when compared to conventional scores derived from donor and recipient parameters, such as the donor risk index and the balance of risk score. Mitochondrial FMN values in liver tissues of non-utilized livers were low, and inversely correlated to high perfusate FMN values and purine metabolite release. CONCLUSIONS: This first international study validates the predictive value of the mitochondrial co-factor FMN, released from complex I during HOPE, and may therefore contribute to a better risk stratification of injured livers before implantation. IMPACT AND IMPLICATIONS: Analysis of 473 perfusates, collected from 10 international centers during hypothermic oxygenated perfusion (HOPE), revealed that mitochondria derived flavin mononucleotide (FMN) values in perfusate is predictive for graft loss, cholangiopathy, and kidney failure after liver transplantation. This result is of high clinical relevance, as recognition of graft quality is urgently needed to improve the safe utilization of marginal livers. Ex-situ machine perfusion approaches, such as HOPE, are therefore likely to increase the number of useable liver grafts.

Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation.

Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we "digitalized" XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.

ABCB1 polymorphisms are associated with cyclosporine-induced nephrotoxicity and gingival hyperplasia in renal transplant recipients.

PURPOSE: There is a great deal of controversy regarding the clinical impact of genetic variants in patients receiving cyclosporine (CsA) as immunosuppressant therapy. We have investigated the effect of polymorphisms in the CYP3A and ABCB1 genes on CsA pharmacokinetics, acute rejection incidence and drug-related side effects in renal transplant recipients METHODS: The presence of CYP3A5*3, CYP3A4*1B and ABCB1 C1236T, G2677T/A and C3435T polymorphisms was assessed in 68 patients and retrospectively associated with pharmacokinetic and clinical parameters at 1 week and 1, 5 and 12 months after transplantation. RESULTS: Only minor associations were found between the tested polymorphisms and CsA pharmacokinetics. Most notably, CYP3A5 expressers showed lower blood trough levels than non-expressers in the first week after grafting (32.5 ± 14.7 vs. 55.1 ± 3.8 ng/ml per mg/day per kilogram). In terms of CsA-induced adverse effects, the incidence of nephrotoxicity was higher in carriers of the ABCB1 3435TT genotype and in those patients carrying four to six variants in the three ABCB1 loci [odds ratio (OR) 4.2, 95 % confidence interval (CI) 1.3-13.9, p = 0.02 and OR 3.6, 95 % CI 1.1-11.8, p = 0.05, respectively]. These subjects with four to six ABCB1 variants were also at higher risk for gingival hyperplasia (OR 3.29, 95 % CI 1.1-10.3, p = 0.04). Renal function and the incidence of neurotoxicity and of acute rejection did not vary across the different genotypes. CONCLUSIONS: ABCB1 polymorphisms may be helpful in predicting certain CsA-related side effects in renal transplant recipients. Our results also suggest that the mechanisms underlying these genetic associations are most likely independent of the drug's trough blood concentrations.

Single-Cell mRNA-Seq of In Vitro-Derived Human Neurons Using Smart-Seq2.

Single-cell mRNA sequencing can dissect heterogeneous cell populations as it can identify cell types and cellular states based on their unique transcriptional signatures. We use fluorescence-activated cell sorting (FACS) to isolate individual cultured neurons derived from human-induced pluripotent stem cells (hiPSCs) followed by polyA-based Smart-Seq2 RNA sequencing to analyze the single-cell transcriptional profiles. We provide protocols and guidelines on dissociation, cell selection, and library preparation that can be readily adapted to other cell types or tissue samples.

Preventable risk factors for type 2 diabetes can be detected using noninvasive spontaneous electroretinogram signals.

Given the ever-increasing prevalence of type 2 diabetes and obesity, the pressure on global healthcare is expected to be colossal, especially in terms of blindness. Electroretinogram (ERG) has long been perceived as a first-use technique for diagnosing eye diseases, and some studies suggested its use for preventable risk factors of type 2 diabetes and thereby diabetic retinopathy (DR). Here, we show that in a non-evoked mode, ERG signals contain spontaneous oscillations that predict disease cases in rodent models of obesity and in people with overweight, obesity, and metabolic syndrome but not yet diabetes, using one single random forest-based model. Classification performance was both internally and externally validated, and correlation analysis showed that the spontaneous oscillations of the non-evoked ERG are altered before oscillatory potentials, which are the current gold-standard for early DR. Principal component and discriminant analysis suggested that the slow frequency (0.4-0.7 Hz) components are the main discriminators for our predictive model. In addition, we established that the optimal conditions to record these informative signals, are 5-minute duration recordings under daylight conditions, using any ERG sensors, including ones working with portative, non-mydriatic devices. Our study provides an early warning system with promising applications for prevention, monitoring and even the development of new therapies against type 2 diabetes.

Impact of genetic polymorphisms on tacrolimus pharmacokinetics and the clinical outcome of renal transplantation.

We retrospectively examined the association of polymorphisms in the CYP3A, CYP2J2, CYP2C8, and ABCB1 genes with pharmacokinetic (PKs) and pharmacodynamic (PDs) parameters of tacrolimus in 103 renal transplant recipients for a period of 1 year. CYP3A5 expressers had lower predose concentrations (C(0) )/dose and higher dose requirements than nonexpressers throughout the study. Among CYP3A5*1 carriers, those also carrying the CYP3A4*1B allele showed the lowest C(0) /dose, as compared with CYP3A4*1/CYP3A5*3 carriers (54.28±26.45, 59.12±24.00, 62.43±41.12, and 57.01±17.34 vs. 112.37± 76.60, 123.21±59.57, 163.34±76.23, and 183.07±107.82 at 1 week, 1 month, 5 months, and 1 year after transplantation). In addition, CYP3A4*1B/CYP3A5*1 carriers showed significantly lower dose-corrected exposure than CYP3A4*1/CYP3A5*1 carriers 1 year after transplantation (57.01±17.34 vs. 100.09±24.78; P=0.016). Only the ABCB1 TGC (3435-2677-1236) haplotype showed a consistent association with PDs (nephrotoxicity; OR=4.73; CI: 1.3-16.7; P=0.02). Our findings indicate that the CYP3A4*1B-CYP3A5*1 haplotype may have a more profound impact in tacrolimus PKs than the CYP3A5*1 allele. This study does not support a critical role of the CYP450 or ABCB1 single nucleotide polymorphisms in the occurrence of toxicity or acute rejection in renal transplant recipients treated with tacrolimus.

A risk evaluation framework for the best maintenance strategy: The case of a marine salt manufacture firm.

This paper intends to contribute with a multi-criteria decision-making (MCDM) framework to support risk evaluation for maintenance activities carried out on critical systems in industry. We propose to first select the best maintenance strategy tailored to companies' requirements and systems' features, and second to perform a risk prioritisation aimed at highlighting priorities of intervention. The Analytic Network Process (ANP) is suggested to select the maintenance policy representing the best trade-off considering the complex and varied interdependencies amongst a diversity of clustered elements characterising the system. Then, the main risks related to the interventions associated to the selected maintenance policy are ranked using the ELimination Et Choix Traduisant la REalité III (ELECTRE III) method, using the same criteria weighted by the previous ANP application. This hybrid MCDM framework is applied to a core subsystem of a real-world marine salt manufacture firm.

CYP1A1 gene polymorphisms increase lung cancer risk in a high-incidence region of Spain: a case control study.

BACKGROUND: A rural region in south-west Spain has one of the highest lung cancer incidence rates of the country, as revealed by a previous epidemiological 10-year follow-up study. The present work was undertaken to ascertain the role of CYP1A1 gene polymorphisms and their interaction with tobacco smoking in the development of the disease in this location. METHODS: One-hundred-and-three cases of lung cancer and 265 controls participated in the study. The participants were screened for the presence of four CYP1A1 polymorphisms, namely MspI, Ile462Val, T3205C, and Thr461Asn. Lung cancer risk was estimated as odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression models adjusting for age, sex, and smoking. RESULTS: The distribution of the variant CYP1A1 alleles was different from that described for other Caucasian populations, with CYP1A1*2A showing an uncommonly high frequency (p < 0.01). The CYP1A1*2B allele (carrying MspI and Ile462Val mutations) was strongly associated with high lung cancer risk (OR = 4.59, CI:1.4-12.6, p <0.01). The Ile462Val polymorphism was also shown to increase the risk for the disease (OR = 4.51, CI:1.8-11.9; p <0.01) and particularly for squamous-cell (OR = 5.01; CI: 1.6-14.3, p < 0.01) and small-cell lung carcinoma (SCLC) (OR = 6.97, CI: 1.2-81.3; p = 0.04). Moreover, the Thr461Asn polymorphism was found to be associated with SCLC in a Caucasian population for the first time to our knowledge (OR = 8.33, CI: 1.3-15.2, p = 0.04). CONCLUSION: The results suggest that CYP1A1 polymorphisms contribute to increase lung cancer susceptibility in an area with an uncommon high incidence rate.

GST polymorphisms interact with dietary factors to modulate lung cancer risk: study in a high-incidence area.

The aim of this study was to explore possible correlations between glutathione S-transferases (GST) polymorphisms, smoking, diet, and lung cancer susceptibility in a rural Spanish region with one of the highest incidence rates of the country. All lung cancer patients living in the area (103) and 247 matched controls were genotyped for the GST mu 1 (GSTM1) null, GST theta 1 (GSTT1) null, and GST pi 1 (GSTP1) Isoleucine (Ile) 105 valine (Val) polymorphisms and interviewed to gather information on smoking and dietary habits. Neither the presence of GST polymorphisms nor their interaction with smoking was independently associated to lung cancer risk. The intake of carotenoid-rich red and yellow vegetables was inversely associated with lung cancer (P < 0.05). Interestingly, this was observed only in carriers of the GSTM1 (P = 0.04), GSTT1 (P = 0.03), or GSTM1/T1 (P = 0.04) positive genotypes. Similarly, the consumption of citrus fruits was more frequent among cancer-free subjects who carried functional GSTM1 (P = 0.04) or both GSTM1 and GSTT1 enzymes (P = 0.04). The results show that the inverse association observed between the intake of dietary carotenoid-rich vegetables and lung cancer risk is dependent on the GST genotype. These results warrant further investigations to confirm the observed associations.

Pharmacogenetic testing and therapeutic drug monitoring are complementary tools for optimal individualization of drug therapy.

Genetic factors contribute to the phenotype of drug response, but the translation of pharmacogenetic outcomes into drug discovery, drug development or clinical practice has proved to be surprisingly disappointing. Despite significant progress in pharmacogenetic research, only a few drugs, such as cetuximab, dasatinib, maraviroc and trastuzumab, require a pharmacogenetic test before being prescribed. There are several gaps that limit the application of pharmacogenetics based upon the complex nature of the drug response itself. First, pharmacogenetic tests could be more clinically applicable if they included a comprehensive survey of variation in the human genome and took into account the multigenic nature of many phenotypes of drug disposition and response. Unfortunately, much of the existing research in this area has been hampered by limitations in study designs and the nonoptimal selection of gene variants. Secondly, although responses to drugs can be influenced by the environment, only fragmentary information is currently available on how the interplay between genetics and environment affects drug response. Third, the use of a pharmacogenetic test as a standard of care for drug therapy has to overcome significant scientific, economic, commercial, political and educational barriers, among others, in order for clinically useful information to be effectively communicated to practitioners and patients. Meanwhile, the lack of efficacy is in this process is quite as costly as drug toxicity, especially for very expensive drugs, and there is a widespread need for clinically and commercially robust pharmacogenetic testing to be applied. In this complex scenario, therapeutic drug monitoring of parent drugs and/or metabolites, alone or combined with available pharmacogenetic tests, may be an alternative or complementary approach when attempts are made to individualize dosing regimen, maximize drug efficacy and enhance drug safety with certain drugs and populations (e.g. antidepressants in older people).

Modeling Motor Neuron Resilience in ALS Using Stem Cells.

Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Toward this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of PHOX2A in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro-generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro that display a resilience similar to that seen in vivo.

LCM-Seq: A Method for Spatial Transcriptomic Profiling Using Laser Capture Microdissection Coupled with PolyA-Based RNA Sequencing.

LCM-seq couples laser capture microdissection of cells from frozen tissues with polyA-based RNA sequencing and is applicable to single neurons. The method utilizes off-the-shelf reagents and direct lysis of the cells without RNA purification, making it a simple and relatively cheap method with high reproducibility and sensitivity compared to previous methods. The advantage with LCM-seq is also that tissue sections are kept intact and thus the positional information of each cell is preserved.

CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells.

CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to ∼50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.

Engineering Human Epidermal Growth Receptor 2-Targeting Hepatitis B Virus Core Nanoparticles for siRNA Delivery in Vitro and in Vivo.

Hepatitis B virus core (HBc) particles acquire the capacity to disassemble and reassemble in a controlled manner, allowing entrapment and delivery of drugs and macromolecules to cells. HBc particles are made of 180-240 copies of 21 kDa protein monomers, assembled into 30-34 nm diameter icosahedral particles. In this study, we aimed at formulating HBc particles for the delivery of siRNA for gene silencing in vitro and in vivo. We have previously reported recombinant HBc particles expressing ZHER2 affibodies, specifically targeting human epidermal growth receptor 2 (HER2)-expressing cancer cells (ZHER2-ΔHBc). siRNA was encapsulated within the ZHER2-ΔHBc particles following disassembly and reassembly. The ZHER2-ΔHBc-siRNA hybrids were able to secure the encapsulated siRNA from serum and nucleases in vitro. Enhanced siRNA uptake in HER2-expressing cancer cells treated with ZHER2-ΔHBc-siRNA hybrids was observed compared to the nontargeted HBc-siRNA hybrids in a time- and dose-dependent manner. A successful in vitro polo-like kinase 1 (PLK1) gene knockdown was demonstrated in cancer cells treated with ZHER2-ΔHBc-siPLK1 hybrids, to levels comparable to commercial transfecting reagents. Interestingly, ZHER2-ΔHBc particles exhibit intrinsic capability of reducing the solid tumor mass, independent of siPLK1 therapy, in an intraperitoneal tumor model following intraperitoneal injection.

Retinal toxic reactions following photopheresis.

BACKGROUND: Extracorporeal photochemotherapy (ECP), also known as photopheresis, is a generally well-tolerated therapeutic, immunomodulatory approach successfully used in cutaneous T-cell lymphoma and other diseases produced by T-lymphocytes such as graft vs host disease. OBSERVATIONS: On 2 separate occasions, a 54-year-old white man with Sézary syndrome developed cutaneous phototoxic reactions and chorioretinitis after being treated with ECP. A pharmacokinetic study showed therapeutic blood levels of 8-methoxypsoralen as long as 18 weeks after therapy had been terminated. However, the analysis of mutations in genes involved in the drug's disposition could not explain these abnormal levels. CONCLUSIONS: To our knowledge, there has been no previous description of ECP-related retinal toxic effects. This adverse effect was probably linked to impaired drug elimination. Further studies would be needed to determine the underlying mechanism.

Revealing allele-specific gene expression by single-cell transcriptomics.

Single-cell sequencing has emerged as a revolutionary method that reveals biological processes with unprecedented resolution and scale, and has already greatly impacted biology and medicine. To investigate processes such as alternative splicing, novel exon detection and allele-specific expression (ASE), full-length based single-cell RNA-seq methods are required for broad sequence coverage and single nucleotide polymorphism (SNP) identification. In this review, we revisit recent achievements from studies that used single-cell RNA-seq to advance our understanding of ASE in the context of both autosomal and X-chromosome genes. We also recapitulate useful bioinformatic tools developed to identify haplotype phase.

Direct Reprogramming of Resident NG2 Glia into Neurons with Properties of Fast-Spiking Parvalbumin-Containing Interneurons.

Converting resident glia into functional and subtype-specific neurons in vivo by delivering reprogramming genes directly to the brain provides a step forward toward the possibility of treating brain injuries or diseases. To date, it has been possible to obtain GABAergic and glutamatergic neurons via in vivo conversion, but the precise phenotype of these cells has not yet been analyzed in detail. Here, we show that neurons reprogrammed using Ascl1, Lmx1a, and Nurr1 functionally mature and integrate into existing brain circuitry and that the majority of the reprogrammed neurons have properties of fast-spiking, parvalbumin-containing interneurons. When testing different combinations of genes for neural conversion with a focus on pro-neural genes and dopamine fate determinants, we found that functional neurons can be generated using different gene combinations and in different brain regions and that most of the reprogrammed neurons become interneurons, independently of the combination of reprogramming factors used.

Evaluation of cellular uptake, cytotoxicity and cellular ultrastructural effects of heteroleptic oxidovanadium(IV) complexes of salicylaldimines and polypyridyl ligands.

Searching for prospective vanadium-based drugs for cancer treatment, a new series of structurally related [VIVO(L-2H)(NN)] compounds (1-8) was developed. They include a double deprotonated salicylaldimine Schiff base ligand (L-2H) and different NN-polypyridyl co-ligands having DNA intercalating capacity. Compounds were characterized in solid state and in solution. EPR spectroscopy suggests that the NN ligands act as bidentate and bind through both nitrogen donor atoms in an axial-equatorial mode. The cytotoxicity was evaluated in human tumoral cells (ovarian A2780, breast MCF7, prostate PC3). The cytotoxic activity was dependent on type of cell and incubation time. At 24h PC3 cells presented low sensitivity, but at 72h all complexes showed high cytotoxic activity in all cells. Human kidney HEK293 and ovarian cisplatin resistant A2780cisR cells were also included to evaluate selectivity towards cancer cells and potency to overcome cisplatin resistance, respectively. Most complexes showed no detectable interaction with plasmid DNA, except 2 and 7 which depicted low ability to induce single strand breaks in supercoiled DNA. Based on the overall cytotoxic profile, complexes with 2,2´-bipyridine and 1,10-phenanthroline ligands (1 and 2) were selected for further studies, which consisted on cellular distribution and ultrastructural analyses. In the A2780 cells both depicted different distribution profiles; the former accumulates mostly at the membrane and the latter in the cytoskeleton. Morphology of treated cells showed nuclear atypia and membrane alterations, more severe for 1. Complexes induce different cell death pathways, predominantly necrosis for 1 and apoptosis for 2. Complexes alternative mode of cell death motivates the possibility for further developments.

Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3ß phosphorylation and ß-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling.

Laser capture microscopy (LCM) coupled with global transcriptome profiling could enable precise analyses of cell populations without the need for tissue dissociation, but has so far required relatively large numbers of cells. Here we report a robust and highly efficient strategy for LCM coupled with full-length mRNA-sequencing (LCM-seq) developed for single-cell transcriptomics. Fixed cells are subjected to direct lysis without RNA extraction, which both simplifies the experimental procedures as well as lowers technical noise. We apply LCM-seq on neurons isolated from mouse tissues, human post-mortem tissues, and illustrate its utility down to single captured cells. Importantly, we demonstrate that LCM-seq can provide biological insight on highly similar neuronal populations, including motor neurons isolated from different levels of the mouse spinal cord, as well as human midbrain dopamine neurons of the substantia nigra compacta and the ventral tegmental area.