Harnessing Escherichia coli for Bio-Based Production of Formate under Pressurized H2 and CO2 Gases.

Escherichia coli is a Gram-negative bacterium that is a workhorse for biotechnology. The organism naturally performs a mixed-acid fermentation under anaerobic conditions where it synthesizes formate hydrogenlyase (FHL-1). The physiological role of the enzyme is the disproportionation of formate into H2 and CO2. However, the enzyme has been observed to catalyze hydrogenation of CO2 given the correct conditions, and so it has possibilities in bio-based carbon capture and storage if it can be harnessed as a hydrogen-dependent CO2 reductase (HDCR). In this study, an E. coli host strain was engineered for the continuous production of formic acid from H2 and CO2 during bacterial growth in a pressurized batch bioreactor. Incorporation of tungsten, in place of molybdenum, in FHL-1 helped to impose a degree of catalytic bias on the enzyme. This work demonstrates that it is possible to couple cell growth to simultaneous, unidirectional formate production from carbon dioxide and develops a process for growth under pressurized gases. IMPORTANCE Greenhouse gas emissions, including waste carbon dioxide, are contributing to global climate change. A basket of solutions is needed to steadily reduce emissions, and one approach is bio-based carbon capture and storage. Here, we present our latest work on harnessing a novel biological solution for carbon capture. The Escherichia coli formate hydrogenlyase (FHL-1) was engineered to be constitutively expressed. Anaerobic growth under pressurized H2 and CO2 gases was established, and aqueous formic acid was produced as a result. Incorporation of tungsten into the enzyme in place of molybdenum proved useful in poising FHL-1 as a hydrogen-dependent CO2 reductase (HDCR).

Visualizing cortical response to optogenetic stimulation and sensory inputs using multispectral handheld optoacoustic imaging.

To date, the vast majority of intra-vital neuroimaging systems applied in clinic and diagnostics is stationary with a rigid scanning element, requires specialized facilities and costly infrastructure. Here, we describe a simple yet radical approach for optoacoustic (photoacoustic) brain imaging in vivo using a light-weight handheld probe. It enables multispectral video-rate visualization of hemoglobin gradient changes in the cortex of adult rats induced by whisker and forelimb sensory inputs, as well as by optogenetic stimulation of intra-cortical connections. With superb penetration and molecular specificity, described here in method holds major promises for future applications in research, routine ambulatory neuroimaging, and diagnostics.

A comparison of stress levels, coping styles and psychological morbidity between graduate-entry and traditional undergraduate medical students during the first 2 years at a UK medical school.

BACKGROUND: Stress levels and psychological morbidity are high among undergraduate medical students (UGs), but there is a lack of research into the psychological health of UK graduate-entry medical students (GEs). GEs are likely to experience different (perhaps more severe) stressors and to cope with stress differently. We compared stress levels, psychological morbidity and coping styles in GE versus UG medical students studying at the same UK medical school in the same academic year. A cross-sectional self-rated questionnaire study of all first- and second-year GE and UG medical students was conducted. Perceived stress, psychological morbidity, recent adverse life events, stress-related personality traits and coping styles were assessed using standard questionnaires. RESULTS: 75% GEs and 46% UGs responded to the questionnaire. Both groups reported equally high levels, and similar profiles of, perceived stress and psychological morbidity. Levels of recent adverse life events and stress-related personality traits were similar in both groups. Compared to UGs, GEs were more likely to use active coping (p = 0.02) and positive reframing (p = 0.03), but were also more likely to use substances (alcohol and other drugs; p < 0.001) to help them cope. Unlike UGs, second-year GEs showed less perceived stress (p = 0.007) and psychological morbidity (p = 0.006) than first-year GEs although levels of both were still high. CONCLUSION: Our results show that both GE students and their younger UG counterparts on a traditional medical course have similar profiles of stress symptoms. They do, however, cope with stress differently. GEs are more likely to use active problem-focused coping strategies, and they are also more likely to cope by using substances (alcohol or other drugs). GE students need interventions to prevent maladaptive coping styles and encourage adaptive coping that are tailored to their needs. Such interventions should be targeted at first-year students. It is vital that these students develop positive coping skills to benefit them during training and in a future career that is inherently stressful.

Screening of Living Kidney Donors for Genetic Diseases Using a Comprehensive Genetic Testing Strategy.

Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs.

An Unexpected Surge in Plasma BKPyV Viral Load Heralds the Development of BKPyV-Associated Metastatic Bladder Cancer in a Lung Transplant Recipient With BKPyV Nephropathy.

We report a lung transplant recipient who developed BK polyoma virus (BKPyV) DNAemia and BKPyV nephropathy. With careful management of his immunosuppression he achieved significant reduction in BKPyV DNAemia and stabilization of his kidney function. He later developed a high-grade bladder cancer and shortly thereafter he experienced a major upsurge in the level of BKPyV DNAemia that coincided with the discovery of hepatic metastasis. Retrospectively, the bladder cancer and the hepatic secondary tumor stained uniformly for SV40 large T antigen, and the BKPyV DNA sequences identified in plasma corresponded to BKPyV DNA within hepatic tissue, indicating that the spike in BKPyV load was likely derived from the circulating tumor cells or cell-free tumor DNA following metastases of a BKV-associated cancer. To the best of our knowledge, this is the first description of a surge in BKPyV load in a patient with controlled BKPyVN that heralded the appearance of a metastatic urothelial malignancy. This report discusses the literature on BKPyV-associated malignancies and the possibility that unexplained increases in BKPyV DNAemia may be a biomarker for metastatic BKPyV-related urothelial cancer.

Light Chain Deposition Disease After Kidney Transplantation With Long Graft Survival: Case Report.

Light Chain Deposition Disease (LCDD) is a monoclonal immunoglobulin deposition disease that commonly affects kidneys among other organs. It leads to end-stage renal disease and has a high disease recurrence rate after kidney transplantation. This has led some authors to advise against transplantation in view of the poor long-term graft and patient outcomes. Recent literature has shown improvement/stabilization of native kidney disease following the use of bortezomib. We present 2 cases of LCDD after transplantation with graft dysfunction. They were both treated with different therapeutic agents to induce remission. Because sustained remission was not achieved they received bortezomib following which they have experienced a prolonged period of stable renal function with no clinically detectable disease. These unique cases highlight the possibility to achieve long-term stable graft function and disease remission after renal transplantation for LCDD.

Structural and Functional Analysis of Intact Hair Follicles and Pilosebaceous Units by Volumetric Multispectral Optoacoustic Tomography.

Visualizing anatomical and functional features of hair follicle development in their unperturbed environment is key in understanding complex mechanisms of hair pathophysiology and in discovery of novel therapies. Of particular interest is in vivo visualization of the intact pilosebaceous unit, vascularization of the hair bulb, and evaluation of the hair cycle, particularly in humans. Furthermore, noninvasive visualization of the sebaceous glands could offer crucial insight into the pathophysiology of follicle-related diseases and dry or seborrheic skin, in particular by combining in vivo imaging with other phenotyping, genotyping, and microbial analyses. The available imaging techniques are limited in their ability for deep tissue in vivo imaging of hair follicles and lipid-rich sebaceous glands in their entirety without biopsy. We developed a noninvasive, painless, and risk-free volumetric multispectral optoacoustic tomography method for deep tissue three-dimensional visualization of whole hair follicles and surrounding structures with high spatial resolution below 80 µm. Herein we demonstrate on-the-fly assessment of key morphometric parameters of follicles and lipid content as well as functional oxygenation parameters of the associated capillary bed. The ease of handheld operation and versatility of the newly developed approach poise it as an indispensable tool for early diagnosis of disorders of the pilosebaceous unit and surrounding structures, and for monitoring the efficacy of cosmetic and therapeutic interventions.

Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging.

Sentinel lymph node (SLN) excision is included in various cancer guidelines to identify microscopic metastatic disease. Although effective, SLN excision is an invasive procedure requiring radioactive tracing. Novel imaging approaches assessing SLN metastatic status could improve or replace conventional lymph node excision protocols. In our first-in-human study, we used noninvasive multispectral optoacoustic tomography (MSOT) to image SLNs ex vivo and in vivo in patients with melanoma, to determine metastatic status. MSOT significantly improved the tumor metastasis detection rate in excised SLN (506 SLNs from 214 melanoma patients) compared with the conventional EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group protocol (22.9% versus 14.2%). MSOT combined with the near-infrared fluorophore indocyanine green reliably visualized SLNs in vivo in 20 patients, up to 5-cm penetration and with 100% concordance with (99m)Tc-marked SLN lymphoscintigraphy. MSOT identified cancer-free SLNs in vivo and ex vivo without a single false negative (189 total lymph nodes), with 100% sensitivity and 48 to 62% specificity. Our findings indicate that a noninvasive, nonradioactive MSOT-based approach can identify and determine SLN status and confidently rule out the presence of metastasis. The study further demonstrates that optoacoustic imaging strategies can improve the identification of SLN metastasis as an alternative to current invasive SLN excision protocols.

Forecasting carbapenem resistance from antimicrobial consumption surveillance: Lessons learnt from an OXA-48-producing Klebsiella pneumoniae outbreak in a West London renal unit.

This study aimed to forecast the incidence rate of carbapenem resistance and to assess the impact of an antimicrobial stewardship intervention using routine antimicrobial consumption surveillance data. Following an outbreak of OXA-48-producing Klebsiella pneumoniae (January 2008-April 2010) in a renal cohort in London, a forecasting ARIMA model was derived using meropenem consumption data [defined daily dose per 100 occupied bed-days (DDD/100OBD)] from 2005-2014 as a predictor of the incidence rate of OXA-48-producing organisms (number of new cases/year/100,000OBD). Interrupted times series assessed the impact of meropenem consumption restriction as part of the outbreak control. Meropenem consumption at lag -1 year (the preceding year), highly correlated with the incidence of OXA-48-producing organisms (r=0.71; P=0.005), was included as a predictor within the forecasting model. The number of cases/100,000OBD for 2014-2015 was estimated to be 4.96 (95% CI 2.53-7.39). Analysis of meropenem consumption pre- and post-intervention demonstrated an increase of 7.12 DDD/100OBD/year (95% CI 2.97-11.27; P<0.001) in the 4 years preceding the intervention, but a decrease thereafter. The change in slope was -9.11 DDD/100OBD/year (95% CI -13.82 to -4.39). Analysis of alternative antimicrobials showed a significant increase in amikacin consumption post-intervention from 0.54 to 3.41 DDD/100OBD/year (slope +0.72, 95% CI 0.29-1.15; P=0.01). Total antimicrobials significantly decreased from 176.21 to 126.24 DDD/100OBD/year (P=0.05). Surveillance of routinely collected antimicrobial consumption data may provide a key warning indicator to anticipate increased incidence of carbapenem-resistant organisms. Further validation using real-time data is needed.

Aspirin inhibits expression of sFLT1 from human cytotrophoblasts induced by hypoxia, via cyclo-oxygenase 1.

INTRODUCTION: Elevated circulating soluble FLT1 (sFLT1) levels seen in preeclampsia may play a role in its development. Aspirin is recommended for prevention of preeclampsia. We hypothesized that aspirin may inhibit the production of sFlt1. METHODS: Placentas from women with and without preeclampsia were collected. Primary cytotrophoblasts (CTBs) were cultured from normal placentas and treated with aspirin, sc-560, a COX1 inhibitor or celecoxib, a COX2 inhibitor. The expression of sFLT1, FLT1, COX1 and COX2 was studied. The effect of aspirin on sFlt1 expression was also studied in HEK293 cells and in HTR-8/SVNeo cells. RESULTS: The expression of sFLT1 was increased in preeclamptic placentas compared to control placentas and the expression and release of sFLT1 increased in CTBs exposed to 2% O2 compared to controls. Aspirin at 3 and 12 mM concentration reduced the expression and release of sFLT1 in CTBs. Aspirin also inhibited sFlt1 expression from HTR-8/SVNeo and HEK293 cells. Sc-560, but not celecoxib, reduced sFLT1 expression and release from CTBs. Aspirin and sc-560 also reduced hypoxia-induced FLT1 mRNA expression and inhibited COX1 mRNA in CTBs. DISCUSSION: This study confirms that sFLT1 expression is increased in preeclamptic placentas and in CTBs exposed to hypoxia. Aspirin inhibits the production sFLT1 in CTBs and in HTR-8/SVNeo. Sc-560 recapitulated the effects of aspirin on sFLT1 expression and release in CTBs suggesting that the aspirin effect may be mediated via inhibition of COX1. The study increases our understanding of the mechanisms regulating sFlt1 expression and provides a plausible explanation for the effect of aspirin to prevent preeclampsia.

Conversion to a sirolimus-based regimen is associated with lower incidence of BK viremia in low-risk kidney transplant recipients.

BACKGROUND: BK viral nephropathy is an increasingly recognized cause of early allograft loss in kidney transplantation. This study aimed to determine whether a sirolimus (Sir)-based calcineurin inhibitor-sparing regimen is associated with a lower incidence of BK viremia. METHODS: This was a single-center retrospective study. Patients were either on tacrolimus (Tac)-based or on Sir-based immunosuppression. Conversion from Tac to Sir occurred at or after 3 months if patients were <62 years of age, had calculated panel reactive antibodies of <20%, and did not have acute early rejection. RESULTS: Incidence of clinically significant BK viremia was 17.9% in the Tac group and 4.3% in the Sir group. Cox regression multivariate analysis showed that male gender (hazard ratio [HR] = 2.87) and switch to Sir (HR = 0.333) impacted the incidence of BK viremia. Kaplan-Meier analysis showed a higher BK-free survival in the Sir group. A trend was seen toward shorter time to resolution of BK viremia and lower peak viremia in the Sir group. Patients on Sir had a higher estimated glomerular filtration rate at each time point; 34% of patients discontinued Sir because of side effects. CONCLUSION: Conversion to Sir-based maintenance immunosuppression at or about 3 months after kidney transplantation correlates with a lower incidence of BK viremia.

Late-onset BK viral nephropathy in a kidney transplant recipient.

BK polyoma viral infection occurs as an asymptomatic infection in a high proportion of normal hosts without obvious sequelae. In the kidney transplant population, the virus is reactivated because of reduced immunity and, if not appropriately managed, can lead to BK viral nephropathy, which has emerged as a common cause of acute kidney injury and progressive chronic kidney disease in renal transplant recipients. BK viremia almost always occurs during the first 2 years after transplantation, when immunosuppressive therapy is high, or at other periods when immunosuppression is intensified. BK viremia is now detected by routine screening of transplant patients for the first few years, and BK viral nephropathy is considered to be high in the differential diagnosis of acute kidney injury in recently transplanted patients. We report a case of BK viral nephropathy developing 10 years after transplantation and present the challenges of managing advanced disease.

Functional differences between neurochemically defined populations of inhibitory interneurons in the rat spinal dorsal horn.

In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I-III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 non-overlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrate that these form distinct functional populations that differ in terms of sst(2A) receptor expression and in their responses to painful stimulation. The sst(2A) receptor was expressed by nearly all of the nNOS- and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin- and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection. Parvalbumin cells did not express either activity-dependent marker following any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations.

Tailored eculizumab therapy in the management of complement factor H-mediated atypical hemolytic uremic syndrome in an adult kidney transplant recipient: a case report.

Atypical hemolytic uremic syndrome (aHUS) is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury (AKI) which frequently progresses to end-stage renal disease (ESRD). In 50% of affected patients, mutations in complement regulatory proteins cause inappropriate complement activation with endothelial injury. Complement factor H (CFH) mutations cause 25% of aHUS cases; these patients have an 80% recurrence risk after kidney transplantation. Eculizumab, an anti-C5 antibody, is effective in limiting hemolysis episodes in patients with aHUS, but less is known about preventing recurrence after kidney transplantation. Herein we report the use of prophylactic eculizumab in an adult with aHUS who underwent kidney transplantation. A 31-year-old female presented with aHUS and progressive AKI associated with low complement 3 level leading to ESRD despite plasmapheresis and corticosteroids. She had a heterozygous nonsense mutation in CFH and reduced plasma CFH levels. She was given preoperative plasmapheresis and eculizumab and underwent living unrelated renal transplantation. Postoperatively, eculizumab was dosed to achieve low functional complement 5 levels and low soluble membrane attack complex levels and she has maintained excellent graft function without aHUS recurrence. We propose that eculizumab with titrated dosing should be used in CFH-mediated aHUS patients who are at a high risk of recurrence.

Case report: Eculizumab rescue of severe accelerated antibody-mediated rejection after ABO-incompatible kidney transplant.

ABO-incompatible (ABOI) living donor kidney transplantation has become a well-accepted practice with standard protocols using perioperative antibody-depleting therapies to lower blood group titers to an acceptable threshold for transplantation. However, a subset of patients will experience accelerated antibody-mediated rejection (AMR) after ABOI kidney transplantation and require aggressive intervention to prevent allograft loss. Here in we report the successful use of terminal complement inhibition with eculizumab to rescue an ABOI kidney allograft with accelerated AMR refractory to salvage splenectomy and daily plasmapheresis. This case emphasizes the fact that, despite close postoperative surveillance and aggressive intervention, graft loss from accelerated AMR after ABOI kidney transplantation remains a very real risk. Eculizumab may offer a graft-saving therapeutic option for isolated cases of severe AMR after ABOI kidney transplantation refractory to standard treatment.

Labelling of olfactory ensheathing cells with micron-sized particles of iron oxide and detection by MRI.

A crucial issue in transplant-mediated repair of the damaged central nervous system (CNS) is serial non-invasive imaging of the transplanted cells, which has led to interest in the application of magnetic resonance imaging (MRI) combined with designated intracellular magnetic labels for cell tracking. Micron-sized particles of iron oxide (MPIO) have been successfully used to track cells by MRI, yet there is relatively little known about either their suitability for efficient labelling of specific cell types, or their effects on cell viability. The purpose of this study was to develop a suitable MPIO labelling protocol for olfactory ensheathing cells (OECs), a type of glia used to promote the regeneration of CNS axons after transplantation into the injured CNS. Here, we demonstrate an OEC labelling efficiency of >90% with an MPIO incubation time as short as 6 h, enabling intracellular particle uptake for single-cell detection by MRI without affecting cell proliferation, migration and viability. Moreover, MPIO are resolvable in OECs transplanted into the vitreous body of adult rat eyes, providing the first detailed protocol for efficient and safe MPIO labelling of OECs for non-invasive MRI tracking of transplanted OECs in real time for use in studies of CNS repair and axon regeneration.

In vivo MRI of olfactory ensheathing cell grafts and regenerating axons in transplant mediated repair of the adult rat optic nerve.

The purpose of the present study was to use magnetic resonance imaging (MRI) as a tool for monitoring transplant-mediated repair of the adult rat visual pathway. We labelled rat olfactory ensheathing cells (OECs) using micron-sized particles of iron oxide (MPIO) and transplanted them by: i) intravitreal injection (ivit) and ii) intra-optic nerve (ON) injection (iON) in adult rats with ON crush (ONC) injury. We applied T(2)-weighted MRI and manganese-enhanced MRI (MEMRI) to visualise transplanted cells and ON axons at specific times after injury and cell engraftment. Our findings demonstrate that ivit MPIO-labelled OECs are unequivocally detected by T(2)-weighted MRI in vivo and that the T(1)-weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn(2+-) enhanced regenerating retinal ganglion cell (RGC) axons and MPIO-labelled OEC grafts. Furthermore, analysis of MRI data and ultrastructural findings supports the hypothesis that iON OEC transplants mediate regeneration and remyelination of RGC axons post injury.

Dynorphin is expressed primarily by GABAergic neurons that contain galanin in the rat dorsal horn.

BACKGROUND: The opioid peptide dynorphin is expressed by certain neurons in the superficial dorsal horn of the spinal cord, but little is known about the types of cell that contain dynorphin. In this study, we have used an antibody against the dynorphin precursor preprodynorphin (PPD), to reveal the cell bodies and axons of dynorphin-expressing neurons in the rat spinal cord. The main aims were to estimate the proportion of neurons in each of laminae I-III that express dynorphin and to determine whether they are excitatory or inhibitory neurons. RESULTS: PPD-immunoreactive cells were concentrated in lamina I and the outer part of lamina II (IIo), where they constituted 17% and 8%, respectively, of all neurons. Around half of those in lamina I and 80% of those in lamina II were GABA-immunoreactive. We have previously identified four non-overlapping neurochemical populations of inhibitory interneurons in this region, defined by the presence of neuropeptide Y, galanin, parvalbumin and neuronal nitric oxide synthase. PPD co-localised extensively with galanin in both cell bodies and axons, but rarely or not at all with the other three markers. PPD was present in around 4% of GABAergic boutons (identified by the presence of the vesicular GABA transporter) in laminae I-II. CONCLUSIONS: These results show that most dynorphin-expressing cells in the superficial dorsal horn are inhibitory interneurons, and that they largely correspond to the population that is defined by the presence of galanin. We estimate that dynorphin is present in ~32% of inhibitory interneurons in lamina I and 11% of those in lamina II. Since the proportion of GABAergic boutons that contain PPD in these laminae was considerably lower than this, our findings suggest that these neurons may generate relatively small axonal arborisations.

Quantitative study of NPY-expressing GABAergic neurons and axons in rat spinal dorsal horn.

Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cγ (PKCγ)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron.