RESUMEN
Schistosomes require both molluscan and mammalian hosts for development. The larval cercaria exits the snail host and swims to identify and invade the mammalian host. The cercaria has two macrostructures, the head and the tail. The head invades the host, where it matures into an adult worm. The tail is lost after host invasion. Translation in the cercaria differs in each macrostructure, with higher levels of translation in the cercarial tail and little to no translational activity in the cercarial head. We compared the transcriptome and proteome of the cercarial head and tail and observed stark differences between the two macrostructures. We identified unique and differentially expressed transcripts and proteins, including ribosomal components expressed in higher levels in tails than in heads, which may explain the differences in translation levels between heads and tails. We also characterized the weak correlation between transcription and translation in infectious cercarial heads and tails.
Asunto(s)
Proteínas del Helminto/metabolismo , Proteoma/metabolismo , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Transcriptoma/genética , Animales , Cercarias/genética , Cercarias/crecimiento & desarrollo , Cercarias/metabolismo , Cromatografía Liquida/métodos , Perfilación de la Expresión Génica/métodos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteómica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Schistosoma mansoni/crecimiento & desarrollo , Espectrometría de Masas en Tándem/métodosRESUMEN
Parasites remainunderstudied members of most ecosystems, especially free-living infectious stages, such as the aquatic cercariae of trematodes (flatworms). Recent studies are shedding more light on their roles, particularly as prey for a diverse array of aquatic predators, but the possible fates of cercariae remain unclear. While this is critical to elucidate because cercariae represent a large potential source of energy and nutrients, determining the fate of cercariae-derived organic matter involves many logistical challenges. Previous studies utilized elemental and stable isotope analysis when examining host-parasite interactions, but none has used such approaches to track the movement of cercariae biomass within food webs. Here we report that Plagiorchis sp. cercariae were effectively labelled with 13C by introducing this compound in the food of their snail host. We then added 13C-labelled cercariae as a potential food source to experimental mesocosms containing a simplified model freshwater food web represented by diving beetles (Dytiscidae sp.), dragonfly larvae (Leucorrhinia intacta), oligochaete worms (Lumbriculus variegatus), and a zooplankton community dominated by Daphnia pulex. The oligochaetes had the highest ratio of 13C to 12C, suggesting benthic detritivores are substantial, but previously unrecognized, consumers of cercariae biomass. In an experiment where L. variegatus were fed mass equivalents of dead D. pulex or cercariae, growth was greater with the latter diet, supporting the importance of cercariae as food source for benthic organisms. Given the substantial cercariae biomass possible in natural settings, understanding their contributions to energy flow and nutrient cycling is important, along with developing methods to do so.
Asunto(s)
Ciclo del Carbono , Cercarias/fisiología , Agua Dulce , Trematodos/fisiología , Animales , Organismos Acuáticos/clasificación , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/metabolismo , Biomasa , Isótopos de Carbono/análisis , Isótopos de Carbono/metabolismo , Cercarias/crecimiento & desarrollo , Cercarias/metabolismo , Ecosistema , Cadena Alimentaria , Agua Dulce/química , Agua Dulce/parasitología , Trematodos/crecimiento & desarrollo , Trematodos/metabolismoRESUMEN
Schistosomiasis is one of the most important helminthic parasitic infections in the world, with over 700 million people at risk of infection. Species of Schistosoma have a complex life cycle involving the infection of freshwater snails before infecting their mammalian definitive host. Taking about 130,000 lives per annum, S. mansoni is the major cause of intestinal schistosomiasis worldwide. Within Biomphalaria glabrata snails, asexual replication of the parasite gives rise to cercariae larvae. Cercariae actively penetrate the host's skin to complete their life cycle and eventually transform into adult worms. If left untreated, intestinal schistosomiasis can lead to peripheral destruction of the portal vein system, gastric hemorrhage from esophageal varices, as well as hepatic failure. Mass spectrometry (MS) is the method of choice for proteomics analysis. The bottom-up proteomics approach-also known as "shotgun proteomics"-typically includes a protein extraction and solubilization step followed by proteolytic digestion and tandem MS (MS/MS) analysis. Proteins are later identified by peptide de novo sequencing upon MS and MS/MS spectra of digest peptides. In this chapter, we introduce an analytical workflow for proteome profiling of S. mansoni cercariae using bottom-up proteomics. The cercariae were isolated and lysed. Proteins were then extracted, enzymatically digested, and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins were identified using MaxQuant software. Cercariae are the first life stage of the parasite S. mansoni which humans encounter, and conducting proteomic analysis on this life cycle stage can shed light on possible drug or vaccine candidates to help disable the parasite's ability to infect or arm the immune system for parasite clearance.
Asunto(s)
Cercarias/metabolismo , Proteínas del Helminto/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Schistosoma mansoni/metabolismo , Animales , Biomphalaria/parasitología , Cromatografía Liquida , Proteínas del Helminto/aislamiento & purificación , Parásitos/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used. PRINCIPAL FINDINGS: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity. CONCLUSIONS/SIGNIFICANCE: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated.
Asunto(s)
Biomphalaria/parasitología , Proteínas del Helminto/genética , Schistosoma mansoni/genética , Animales , Biomphalaria/clasificación , Cercarias/genética , Cercarias/crecimiento & desarrollo , Cercarias/metabolismo , Vectores de Enfermedades , Perfilación de la Expresión Génica , Proteínas del Helminto/metabolismo , Schistosoma mansoni/crecimiento & desarrollo , Schistosoma mansoni/metabolismo , TranscriptomaRESUMEN
In schistosomiasis, egg deposition in the liver contributes to the formation of hepatic granuloma and fibrosis, which are the most serious clinical pathological features. It has been proposed that activation of the nuclear factor kappa B (NF-κB) signaling pathways is closely associated with the development of hepatic granuloma and fibrosis. Genistein has been shown to inhibit the activity of NF-κB signaling pathways, which might be a potential agent to protect against Schistosoma japonicum egg-induced liver granuloma and fibrosis. In this study, liver granuloma and fibrosis were induced by infecting BALB/c mice with 18 ± 3 cercariae of S. japonicum. At the beginning of egg granuloma formation (early phase genistein treatment from 4 to 6 weeks after infection) or after the formation of liver fibrosis (late phase genistein treatment from 6 to 10 weeks after infection), the infected mice were injected with genistein (25, 50 mg/kg). The results revealed that genistein treatment significantly decreased the extent of hepatic granuloma and fibrosis in infected mice. The activity of NF-κB signaling declined sharply after the treatment with genistein, as evidenced by the inhibition of NF-κB-p65, phospho-NF-κB-p65, and phospo-IκB-α expressions, as well as the expression of IκB-α and the messenger RNA (mRNA) expression of inflammatory cytokines (MCP1, TNFα, IL1ß, IL4, IL10) mediated by NF-κB signaling pathways in the early phase of the infection. Moreover, western blot and immunohistochemistry assays demonstrated that the contents of α-smooth muscle actin (α-SMA) and transforming growth factor-ß were dramatically reduced in liver tissue under the treatment of genistein in the late phase of the infection. At the same time, the mRNA expression of MCP1, TNFα, and IL10 was inhibited markedly. These results provided evidence that genistein reduces S. japonicum egg-induced liver granuloma and fibrosis, at least partly due to decreased NF-κB signaling, and subsequently decreased MCP1, TNFα, and IL10 expressions. This implies that genistein can be a potential natural agent against schistosomiasis.
Asunto(s)
Antiprotozoarios/uso terapéutico , Genisteína/uso terapéutico , Granuloma/tratamiento farmacológico , Quinasa I-kappa B/antagonistas & inhibidores , Cirrosis Hepática/tratamiento farmacológico , Schistosoma japonicum/efectos de los fármacos , Esquistosomiasis Japónica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Animales , Cercarias/metabolismo , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Activación Enzimática , Granuloma/parasitología , Granuloma/patología , Interleucina-10/biosíntesis , Interleucina-10/genética , Hígado/parasitología , Hígado/patología , Cirrosis Hepática/parasitología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Schistosoma japonicum/genética , Esquistosomiasis Japónica/parasitología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The parasitic flatworm Schistosoma mansoni, causative agent of human intestinal schistosomiasis in South America, relies importantly on the freshwater snail Biomphalaria glabrata as intermediate host to achieve development of cercariae that infect humans. The recommendation from the World Health Organization (WHO) to integrate snail control in efforts to counter schistosomiasis transmission provides impetus for in depth study of B. glabrata biology. Our analysis indicates that two distinct hemocyanin-like genes (hcl-1 and hcl-2) are present in B. glabrata, a snail that uses hemoglobin for oxygen transport. Characterization of BAC clones yielded the full length hcl-1 gene, which is comprised of three functional unit (FU) domains at the amino acid level. Database searches and in silico analyses identified the second hcl gene (hcl-2), composed of six FU domains. Both genes are unusual for lacking canonical residues and having fewer FU domains than typical molluscan hemocyanins that contain 7-8 FUs. Reverse transcription PCR demonstrated that Hcl-1 is expressed in a manner that correlates with reproductive maturity in the albumen gland (AG), an immune- and reproduction-relevant organ. Immune cross-reactivity with anti-keyhole limpet hemocyanin (α-KLH) antiserum and tandem-mass spectrometry validated the presence of Hcl-1 protein in the AG and egg mass fluid (EMF). The evolutionary conservation of hemocyanin-like sequences in B. glabrata in the presence of the oxygen carrier hemoglobin, combined with our results, suggest that the Hcl-1protein has a functional role in general and/or reproductive biology. Further investigations are needed to explore Hcl-1 as a potential target for snail control.
Asunto(s)
Biomphalaria/metabolismo , Glándulas Exocrinas/metabolismo , Hemocianinas/metabolismo , Caracoles/metabolismo , Animales , Cercarias/metabolismo , Humanos , Schistosoma mansoni/metabolismo , Esquistosomiasis mansoni/parasitología , América del SurRESUMEN
Schistosoma mansoni cercariae display specific behavioral responses to abiotic/biotic stimuli enabling them to locate and infect the definitive human host. Here we report the effect of such stimulants on signaling pathways of cercariae in relation to host finding and invasion. Cercariae exposed to various light/temperature regimens displayed modulated protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activities, with distinct responses at 37 °C and intense light/dark, when compared to 24 °C under normal light. Kinase activities were localized to regions including the oral sensory papillae, acetabular ducts, tegument, acetabular glands, and nervous system. Furthermore, linoleic acid modulated PKC and ERK activities concurrent with the temporal release of acetabular gland components. Attenuation of PKC, ERK, and p38 MAPK activities significantly reduced gland component release, particularly in response to linoleic acid, demonstrating the importance of these signaling pathways to host penetration mechanisms.
Asunto(s)
Cercarias , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas/metabolismo , Schistosoma mansoni , Animales , Cercarias/efectos de los fármacos , Cercarias/enzimología , Cercarias/metabolismo , Cercarias/efectos de la radiación , Humanos , Ácido Linoleico/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , Schistosoma mansoni/metabolismo , Schistosoma mansoni/efectos de la radiaciónRESUMEN
Schistosomiasis is a parasitic zoonosis posing great threat to human health. The infection is acquired by larval cercariae penetrating host skin and transforming into juveniles, schistosomula. Proteolytic enzymes secreted from the cercarial acetabular glands are known to aid to the skin penetration, but molecular mechanisms remain largely unclear. To profile the protein composition and identify potential invasive proteases, we developed a new method for simulating cercarial transformation and collecting schistosomula, and for the first time, we compared the proteomes of Schistosoma japonicum cercariae and schistosomula by using in-gel shotgun proteomic analysis. Totally, 1972 proteins were identified in association with ten main biological processes based on Gene Ontology analysis; 46 proteases were detected in cercariae, and among them, 25 proteases disappeared after penetrated. Notably, leishmanolysins and serine and cysteine proteases were found abundant but differentially expressed. Recombinant serine protease SjCE2b and cysteine protease SjCB2 were produced and used for validation of native proteins. Immunofluorescence and Western blotting assays detected SjCE2b and SjCB2 in cercariae but not in schistosomula, suggesting the two enzymes might be consumed upon skin migration. Our data comprehensively chart the proteomic changes during cercarial invasion, revealing the potential proteases involved, providing a platform for the development of molecular anti-infection strategy.
Asunto(s)
Cercarias/metabolismo , Proteasas de Cisteína/química , Proteínas del Helminto/química , Fragmentos de Péptidos/aislamiento & purificación , Proteoma/química , Schistosoma japonicum/metabolismo , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Animales , Cercarias/genética , Cercarias/crecimiento & desarrollo , Cromatografía Liquida , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Estadios del Ciclo de Vida/genética , Ratones , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/genética , Schistosoma japonicum/crecimiento & desarrollo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Piel/parasitología , Caracoles/parasitología , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Chromatin structure can control gene expression and can define specific transcription states. For example, bivalent methylation of histone H3K4 and H3K27 is linked to poised transcription in vertebrate embryonic stem cells (ESC). It allows them to rapidly engage specific developmental pathways. We reasoned that non-vertebrate metazoans that encounter a similar developmental constraint (i.e. to quickly start development into a new phenotype) might use a similar system. Schistosomes are parasitic platyhelminthes that are characterized by passage through two hosts: a mollusk as intermediate host and humans or rodents as definitive host. During its development, the parasite undergoes drastic changes, most notable immediately after infection of the definitive host, i.e. during the transition from the free-swimming cercariae into adult worms. METHODOLOGY/PRINCIPAL FINDINGS: We used Chromatin Immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) to analyze genome-wide chromatin structure of S. mansoni on the level of histone modifications (H3K4me3, H3K27me3, H3K9me3, and H3K9ac) in cercariae, schistosomula and adults (available at http://genome.univ-perp.fr). We saw striking differences in chromatin structure between the developmental stages, but most importantly we found that cercariae possess a specific combination of marks at the transcription start sites (TSS) that has similarities to a structure found in ESC. We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells. CONCLUSIONS/SIGNIFICANCE: We describe here a broad view on the epigenome of a metazoan parasite. Most notably, we find bivalent histone H3 methylation in cercariae. Methylation of H3K27 is removed during transformation into schistosomula (and stays absent in adults) and transcription is activated. In addition, shifts of H3K9 methylation and acetylation occur towards upstream and downstream of the transcriptional start site (TSS). We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism, i.e. not restricted to stem cells, to poise transcription. Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.
Asunto(s)
Cercarias/genética , Schistosoma mansoni/genética , Transcripción Genética , Acetilación , Animales , Cercarias/crecimiento & desarrollo , Cercarias/metabolismo , Inmunoprecipitación de Cromatina , Femenino , Gastrópodos , Genoma de los Helmintos , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Histonas/genética , Humanos , Masculino , Metilación , Ratones , Schistosoma mansoni/crecimiento & desarrollo , Schistosoma mansoni/metabolismo , Caracoles/parasitologíaRESUMEN
Total IgG ELISA was assessed comparing soluble adult worm (SWA), cercarial (SCA), and egg antigens (SEA) using sera of chronic schistosomiasis patients in two different concentrations (1/50 and 1/100). This response showed reactivity against all antigens. Concerning 1/100 serum concentration the SWA gave the best sensitivity (100%) followed by SCA (86.6%) and SEA (80%). The best specificity was obtained from SCA (94.11%) with an equivalent result from the SWA and SEA (76.47%). Concerning the 1/50 serum concentration, the SWA gave the best sensitivity (100%) with an equivalent result from the SCA and SEA (80%). The best specificity was obtained from SEA (82.35%) followed by SCA (76.47%) then SWA (64.7%). The best AUC value (0.992) was that for SWA indicating its high ability to predict the disease in comparison to (0.914) for SCA and 0.871 for SEA in serum dilution 1/100. The highest AUC value (0.955) was that for SWA in comparison to (0.914) for SEA and (0.88) for SCA in serum dilution 1/50. Comparison between the AUCs from the three mentioned antigens revealed highly significant differences when the data were analyzed by ROC. Each antigen showed a highly statistically significant correlation within its optical density values at both concentrations; 1/100 and 1/50. Comparing the two concentrations in each antigen revealed a non significant correlation obtained from the SWA which signifies a near outcome from both concentrations while the SCA and SEA showed a highly significant difference between the two concentrations. In conclusion, for the diagnosis of chronic schistosomiasis mansoni, total IgG reactivity revealed the best sensitivity by SWA. There was a difference in the type of antigen showing best specificity results between SCA and SEA according to serum concentration used.
Asunto(s)
Antígenos Helmínticos/inmunología , Cercarias/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Óvulo/metabolismo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/diagnóstico , Adulto , Anciano , Animales , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
Schistosome cercariae must penetrate skin as an initial step to successfully infect the final host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. Nowadays, the researches of molecular mechanism of schistosome infection mainly focus on the cercarial secretions including serine protease and cysteine protease. Previous researches already showed that Schistosoma mansoni penetrates the skin mainly depend on cercarial elastease secreted by cercariae while Schistosoma japonicum penetrates the skin chiefly by cathepsin B2. The illustration of molecular mechanism of schistosome cecariae infection will accelerate the identification of novel vaccines and drug targets.
Asunto(s)
Cercarias , Esquistosomiasis/etiología , Animales , Cercarias/metabolismo , Proteasas de Cisteína/fisiología , Humanos , Schistosoma/enzimología , Serina Proteasas/fisiologíaRESUMEN
Miracidia are short-lived, non-feeding (lecithotrophic) free-living stages of trematodes, whose survival is potentially influenced by temperature. Climate change may result in elevated temperatures affecting trematode transmission. Therefore understanding their thermobiology forms an important step in determining the future dynamics of parasite populations. An empirical relationship exists between the mean expected life span of lecithotrophic larvae and the half life of their population (t(0·5)) and therefore t(0·5) is a good indicator of glycogen utilization. In this study experimental data on the effects of temperature on miracidial survival were compiled from the scientific literature and evaluated in terms of metabolism using Q 10 and Arrhenius activation energy (E* or µ). Temperature poorly influenced survival/metabolism with all miracidia having distinct zone(s) of thermostability. Overall there were few differences in Q(10) and E* values between most species temperature ranges whilst there were only limited strain-specific variations in thermal responses of laboratory-maintained Schistosoma mansoni. Miracidia demonstrated a trend of greater thermal resistance than cercariae. In particular, comparative studies on 4 strains of the same species of miracidia and cercariae showed little correlation in thermal biology between the 2 life-history stages. The importance of these results for trematode transmission under global climate change is discussed.
Asunto(s)
Schistosoma mansoni/fisiología , Temperatura , Animales , Cercarias/metabolismo , Cercarias/fisiología , Glucógeno/metabolismo , Estadios del Ciclo de Vida/fisiología , Schistosoma mansoni/metabolismo , Análisis de Supervivencia , TermodinámicaRESUMEN
A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.