RESUMEN

Lipid-rich deposits called drusen accumulate under the retinal pigment epithelium (RPE) in the eyes of patients with age-related macular degeneration (AMD) and Sorsby's fundus dystrophy (SFD). Drusen may contribute to photoreceptor and RPE degeneration in these blinding diseases. We hypothesize that stimulating ß-oxidation of fatty acids could decrease the availability of lipid with which RPE cells can generate drusen. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate ß-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, can diminish lipid deposition by RPE cells. We probed metabolism and cellular function in mouse RPE-choroid tissue and in cultured human RPE cells. We used 13C6-glucose, 13C16-palmitate, and gas chromatography-linked mass spectrometry to monitor effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. We quantified lipid abundance, apolipoprotein E (ApoE) and vascular endothelial growth factor (VEGF) release using liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assays and localized ApoE deposits by immunostaining. RPE barrier function was assessed by trans-epithelial electrical resistance (TEER). Firsocostat-mediated ACC inhibition increases ß-oxidation, decreases intracellular lipid levels, diminishes lipoprotein release, and increases TEER. When human serum or outer segments are used to stimulate lipoprotein release, fewer lipoproteins are released in the presence of either lipid source and Firsocostat. In a culture model of SFD, Firsocostat stimulates fatty acid oxidation, increases TEER, and decreases ApoE release. We conclude that Firsocostat remodels RPE metabolism and can limit lipid deposition. This suggests that ACC inhibition could be an effective strategy for diminishing pathologic drusen in the eyes of patients with AMD or SFD.

RESUMEN

The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.

Asunto(s)

Metanol , Methylobacterium , Methylobacterium/metabolismo , Methylobacterium/genética , Methylobacterium/enzimología , Methylobacterium/crecimiento & desarrollo , Metanol/metabolismo , Simbiosis , Mutación , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hojas de la Planta/microbiología , Hojas de la Planta/crecimiento & desarrollo , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Methylobacterium extorquens/crecimiento & desarrollo , Methylobacterium extorquens/enzimología , Desarrollo de la Planta , Microbiota/genética , Biomasa

RESUMEN

Purpose: In age-related macular degeneration (AMD) and Sorsby's fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating ß-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate ß-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. Methods: We probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13 C6-glucose and 13 C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13 C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). Results: ACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsby's fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. Conclusions: Firsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

RESUMEN

Newborn screening (NBS) for the full set of mucopolysaccharidoses (MPSs) is now possible by either measuring all of the relevant enzymatic activities in dried blood spots (DBS) using tandem mass spectrometry followed by measurement of accumulated glycosaminoglycans (GAGs) or the vice-versa approach. In this study we considered multiple factors in detail including reagent costs, time per analysis, false positive rates, instrumentation requirements, and multiplexing capability. Both NBS approaches are found to provide acceptable solutions for comprehensive MPS NBS, but the enzyme-first approach allows for better multiplexing to include numerous additional diseases that are appropriate for NBS expansion. By using a two-tier NBS approach, the false positive and false negatives rates are expected to acceptably low and close to zero.

Asunto(s)

Glicosaminoglicanos , Mucopolisacaridosis , Recién Nacido , Humanos , Tamizaje Neonatal/métodos , Espectrometría de Masas en Tándem/métodos , Pruebas de Enzimas

RESUMEN

Metabolism is adapted to meet energetic needs. Based on the amount of ATP required to maintain plasma membrane potential, photoreceptor energy demands must be high. The available evidence suggests that photoreceptors primarily generate metabolic energy through aerobic glycolysis, though this evidence is based primarily on protein expression and not measurement of metabolic flux. Aerobic glycolysis can be validated by measuring flux of glucose to lactate. Aerobic glycolysis is also inefficient and thus an unexpected adaptation for photoreceptors to make. We measured metabolic rates to determine the energy-generating pathways that support photoreceptor metabolism. We found that photoreceptors indeed perform aerobic glycolysis and this is associated with mitochondrial uncoupling.

Asunto(s)

Glucólisis , Células Fotorreceptoras , Células Fotorreceptoras/metabolismo , Mitocondrias/metabolismo , Ácido Láctico/metabolismo , Metabolismo Energético , Glucosa/metabolismo

RESUMEN

Fumarate can be a surrogate for O2 as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O2 to H2O. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous 13C4-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O2 consumption by uncoupling adjacent RPE mitochondria.

Asunto(s)

Epitelio Pigmentado de la Retina , Ácido Succínico , Adenosina Trifosfato/metabolismo , Fumaratos/metabolismo , Respiración , Epitelio Pigmentado de la Retina/metabolismo , Succinatos/metabolismo , Ácido Succínico/metabolismo

RESUMEN

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Asunto(s)

Histonas/metabolismo , ARN Polimerasa II/genética , Sumoilación , Transcripción Genética , Extractos Celulares , Humanos , ARN Polimerasa II/metabolismo

RESUMEN

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

Asunto(s)

Envejecimiento/fisiología , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Visión de Colores/fisiología , Electrorretinografía , Fusión de Flicker/fisiología , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Glutamina/metabolismo , Luz , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Visión Nocturna/fisiología , Consumo de Oxígeno/fisiología , Estimulación Luminosa

RESUMEN

PURPOSE: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ARSA), which results in the accumulation of sulfatides. Newborn screening for MLD may be considered in the future as innovative treatments are advancing. We carried out a research study to assess the feasibility of screening MLD using dried blood spots (DBS) from de-identified newborns. METHODS: To minimize the false-positive rate, a two-tier screening algorithm was designed. The primary test was to quantify C16:0-sulfatide in DBS by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The screening cutoff was established based on the results from 15 MLD newborns to achieve 100% sensitivity. The secondary test was to measure the ARSA activity in DBS from newborns with abnormal C16:0-sulfatide levels. Only newborns that displayed both abnormal C16:0-sulfatide abundance and ARSA activity were considered screen positives. RESULTS: A total of 27,335 newborns were screened using this two-tier algorithm, and 2 high-risk cases were identified. ARSA gene sequencing identified these two high-risk subjects to be a MLD-affected patient and a heterozygote. CONCLUSION: Our study demonstrates that newborn screening for MLD is highly feasible in a real-world scenario with near 100% assay specificity.

Asunto(s)

Leucodistrofia Metacromática , Cerebrósido Sulfatasa/genética , Cromatografía Liquida , Humanos , Recién Nacido , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Tamizaje Neonatal , Espectrometría de Masas en Tándem

RESUMEN

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5ß-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). METHODS: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. CONCLUSION: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.

Asunto(s)

Xantomatosis Cerebrotendinosa , Biomarcadores , Cromatografía Liquida , Humanos , Recién Nacido , Tamizaje Neonatal , Espectrometría de Masas en Tándem , Xantomatosis Cerebrotendinosa/diagnóstico , Xantomatosis Cerebrotendinosa/genética

RESUMEN

When O2 is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives in a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by reversing the succinate dehydrogenase reaction to use fumarate to accept electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by hypoxia-induced downregulation of cytochrome oxidase. Retinas can export the succinate they produce to the neighboring O2-rich retinal pigment epithelium-choroid complex. There, succinate enhances O2 consumption by severalfold. Malate made from succinate in the pigment epithelium can then be imported into the retina, where it is converted to fumarate to again accept electrons in the reverse succinate dehydrogenase reaction. This malate-succinate shuttle can sustain these two tissues by transferring reducing power from an O2-poor tissue (retina) to an O2-rich one (retinal pigment epithelium-choroid).

Asunto(s)

Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Succinatos/farmacología , Animales , Complejo IV de Transporte de Electrones/efectos de los fármacos , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/fisiología , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Succinatos/metabolismo

RESUMEN

PURPOSE: To develop a multiplexed assay for the newborn screening of lysosomal storage disorders and additional inborn errors in a flexible, comprehensive, and affordable manner to keep up with the expansion of the newborn screening panel. METHODS: Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was chosen as the detection platform for its superiority compared to traditional flow-injection MS/MS. RESULTS: A high-throughput, 18-plex UPLC-MS/MS assay was developed for screening purposes with a sample turnaround time of 2.7 minutes. The assay was consolidated such that only four dried blood spot punches were required, and it displayed good precision and reproducibility. CONCLUSION: We report a highly multiplexed UPLC-MS/MS assay that is appropriate for the newborn screening of 15 lysosomal storage diseases and 3 additional inborn errors. It can be further expanded to include additional conditions for which presymptomatic diagnosis may facilitate optimum treatment outcome.

Asunto(s)

Enfermedades por Almacenamiento Lisosomal , Tamizaje Neonatal , Cromatografía Liquida , Pruebas con Sangre Seca , Humanos , Recién Nacido , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Enfermedades por Almacenamiento Lisosomal/genética , Lisosomas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem

RESUMEN

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

Asunto(s)

Cerebrósido Sulfatasa/análisis , Pruebas con Sangre Seca , Leucocitos/enzimología , Leucodistrofia Metacromática/sangre , Enfermedad por Deficiencia de Múltiples Sulfatasas/sangre , Cerebrósido Sulfatasa/metabolismo , Cromatografía Liquida , Humanos , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/enzimología , Estructura Molecular , Enfermedad por Deficiencia de Múltiples Sulfatasas/diagnóstico , Enfermedad por Deficiencia de Múltiples Sulfatasas/enzimología , Sulfoglicoesfingolípidos/química , Espectrometría de Masas en Tándem

RESUMEN

Studies were conducted to determine the distribution and elimination of imidacloprid (IMI) in rainbow trout. Animals were injected with a low (47.6 µg/kg), medium (117.5 µg/kg) or high (232.7 µg/kg) dose directly into the bloodstream and allowed to depurate. The fish were then sampled to characterize the loss of IMI from plasma and its appearance in expired water (all dose groups) and urine (medium dose only). In vitro biotransformation of IMI was evaluated using trout liver S9 fractions. Mean total clearance (CLT) values determined by non-compartmental analysis of plasma time-course data were 21.8, 27.0 and 19.5 mL/h/kg for the low, medium and high dose groups, respectively. Estimated half-lives for the same groups were 67.0, 68.4 and 68.1 h, while fitted values for the steady-state volume of distribution (VSS) were 1.72, 2.23 and 1.81 L/kg. Branchial elimination rates were much lower than expected, suggesting that IMI is highly bound in blood. Renal clearance rates were greater than measured rates of branchial clearance (60% of CLT in the medium dose group), possibly indicating a role for renal membrane transporters. There was no evidence for hepatic biotransformation of IMI. Collectively, these findings suggest that IMI would accumulate in trout in continuous waterborne exposures.

Asunto(s)

Colinérgicos/toxicidad , Insecticidas/toxicidad , Neonicotinoides/toxicidad , Nitrocompuestos/toxicidad , Oncorhynchus mykiss/metabolismo , Animales , Acuicultura , Bilis/metabolismo , Biotransformación , Colinérgicos/administración & dosificación , Colinérgicos/sangre , Colinérgicos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Eliminación Hepatobiliar , Inyecciones Intravenosas , Insecticidas/administración & dosificación , Insecticidas/sangre , Insecticidas/metabolismo , Masculino , Tasa de Depuración Metabólica , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Neonicotinoides/administración & dosificación , Neonicotinoides/sangre , Neonicotinoides/metabolismo , Nitrocompuestos/administración & dosificación , Nitrocompuestos/sangre , Nitrocompuestos/metabolismo , Oncorhynchus mykiss/sangre , Oncorhynchus mykiss/orina , Eliminación Pulmonar , Eliminación Renal , Factores Sexuales , Distribución Tisular , Toxicocinética , Contaminantes Químicos del Agua/administración & dosificación , Contaminantes Químicos del Agua/sangre , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/toxicidad

RESUMEN

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.

Asunto(s)

Adaptación Ocular , Metabolismo Energético , Células Ependimogliales/fisiología , Células Fotorreceptoras/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Células Ependimogliales/metabolismo , Glucosa/metabolismo , Humanos , Lactatos/metabolismo , Ratones , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pez Cebra

RESUMEN

Sperm selection by females is an important process influencing fertilization and, particularly in broadcast-spawning organisms, often occurs before sperm reach the egg. Waterborne sperm chemoattractants are one mechanism by which eggs selectively influence conspecific sperm behavior, but it remains an open question whether the eggs from different females produce different amounts of sperm chemoattractant, and how that might influence sperm behavior. Here, we quantify the differences in attractant production between females of the sea urchin species Lytechinus pictus and use computational models and microfluidic sperm chemotaxis assays to determine how differences in chemoattractant production between females affects their ability to attract sperm. Our study demonstrates that there is significant individual female variation in egg chemoattractant production, and that this variation changes the scope and strength of sperm attraction. These results provide evidence for the importance of individual female variability in differential sperm attraction and fertilization success.

Asunto(s)

Factores Quimiotácticos/biosíntesis , Quimiotaxis/fisiología , Fertilización/fisiología , Lytechinus/fisiología , Óvulo/metabolismo , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/citología , Animales , Cromatografía Líquida de Alta Presión , Simulación por Computador , Femenino , Masculino , Espectrometría de Masas , Microfluídica , Óvulo/citología

RESUMEN

The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current work sets an important basis for further investigations including novel metabolites discovery and biosynthetic capacity improvement.

RESUMEN

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD+ precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.

Asunto(s)

Carbono/química , Ojo/embriología , Ácidos Cetoglutáricos/química , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/metabolismo , Anciano de 80 o más Años , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Ácidos Grasos/química , Femenino , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Degeneración Macular/patología , Ratones , NAD/química , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción , Estrés Oxidativo , Oxígeno/química , Poli(ADP-Ribosa) Polimerasas/metabolismo

RESUMEN

Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-ß-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.

Asunto(s)

Ganoderma/metabolismo , Glicósidos/metabolismo , Trametes/metabolismo , Secuencia de Aminoácidos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Secuencia Conservada , Evaluación Preclínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ganoderma/genética , Glicósidos/química , Glicósidos/aislamiento & purificación , Glicósidos/farmacología , Humanos , Metabolómica , Interacciones Microbianas , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Filogenia , Trametes/genética , UDP Xilosa Proteína Xilosiltransferasa