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BACKGROUND: Contrast-enhanced computed tomography angiography (CTA) and magnetic resonance angiography (MRA) are the primary modalities to assess donors' vessels before transplant surgery. Radiation and contrast medium are potentially harmful to donors. PURPOSE: To compare the image quality and visualization scores of hepatic arteries on CTA and balanced steady-state free-precession (bSSFP) non-contrast-enhanced MRA (NC-MRA), and to evaluate if bSSFP NC-MRA can potentially be a substitute for CTA. STUDY TYPE: Prospective. POPULATION: Fifty-six consecutive potential living-related liver donors (30.9 ± 8.4 years; 31 men). FIELD STRENGTH/SEQUENCE: 1.5T; four bSSFP NC-MRA sequences: respiratory-triggered (Inhance inflow inversion recovery [IFIR]) and three breath-hold (BH); and CTA. ASSESSMENT: The artery-to-liver contrast (Ca-l) was quantified. Three radiologists independently assigned visualization scores using a four-point scale to potential origins, segments, and branches of the hepatic arteries, determined the anatomical variants based on Hiatt's classification, and assessed the image quality of NC-MRA sequences. STATISTICAL TESTS: Fleiss' kappa to evaluate the readers' agreement. Repeat measured ANOVA or Friedman test to compare Ca-l of each NC-MRA. Friedman test to compare overall image quality and visualization scores; post hoc analysis using Wilcoxon signed-rank test. P-value <0.05 was considered statistically significant. RESULTS: Inhance IFIR Ca-l was significantly higher than all BH bSSFP Ca-l (0.56 [0.45-0.64] vs. 0.37 [0.29-0.47] to 0.41 [0.23-0.51]). Overall image quality score of BH bSSFP TI1200 was significantly higher than other NC-MRA (4 [4-4] vs. 4 [3 to 4-4]). The median visualization scores of almost all arteries on CTA were significantly higher than on NC-MRA (4 [3 to 4-4] vs. 1 [1-2] to 4 [4-4]). The median visualization scores were all 4 [4-4 ] on Inhance IFIR with >92.3% observed scores ≥3, except the segment 4 branch (3 [1-4], 53.6%). The identification rates of arterial variants were 92.9%-97% on Inhance IFIR. DATA CONCLUSIONS: Although CTA is superior to the NC-MRA, all NC-MRA depict the donor arterial anatomy well. Inhance IFIR can potentially be an alternative image modality for CTA to evaluate the arterial variants of living donors. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.
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Medios de Contraste , Donadores Vivos , Masculino , Humanos , Estudios Prospectivos , Hígado/diagnóstico por imagen , Hígado/irrigación sanguínea , Angiografía por Resonancia Magnética/métodos , Tomografía Computarizada por Rayos X , Reproducibilidad de los ResultadosRESUMEN
Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.
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Ligasas de Carbono-Nitrógeno , Histidina , Animales , Citidina Trifosfato , Histidina/genética , QueratinasRESUMEN
Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors, little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). Here, we identify and characterize a bilateral pair of gustatory second-order neurons (G2Ns) in Drosophila. Previous studies identified GSNs that relay taste information to distinct subregions of the primary gustatory center (PGC) in the gnathal ganglia (GNG). To identify candidate G2Ns, we screened â¼5,000 GAL4 driver strains for lines that label neural fibers innervating the PGC. We then combined GRASP (GFP reconstitution across synaptic partners) with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate G2Ns and the axonal terminals of Gr5a-expressing GSNs, which are known to respond to sucrose. Results of the GRASP analysis, followed by a single-cell analysis by FLP-out recombination, revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, we expressed fluorescence-tagged acetylcholine receptor subunit (Dα7) and active-zone marker (Brp) in the G2Ns. We found that G2N input sites overlaid GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. GRASP analysis and further tests with the Syb-GRASP method suggested that the identified G2Ns receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the GNG, and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). Our findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.
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Encéfalo/citología , Proteínas de Drosophila/metabolismo , Receptores de Superficie Celular/metabolismo , Células Receptoras Sensoriales/citología , Gusto/genética , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Drosophila , Proteínas de Drosophila/genética , Receptores de Superficie Celular/genética , Células Receptoras Sensoriales/metabolismo , SacarosaRESUMEN
The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.
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Visión de Colores/fisiología , Bulbo Raquídeo/fisiología , Neuronas/fisiología , Vías Visuales/fisiología , Animales , Discriminación en Psicología/fisiología , Drosophila/fisiología , Estimulación Luminosa , Células Fotorreceptoras de Invertebrados/fisiología , Sinapsis/fisiologíaRESUMEN
BACKGROUND: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. RESULTS: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. CONCLUSIONS: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.
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Bases de Datos Genéticas , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Genoma de los Insectos , Algoritmos , Animales , Secuencia de Bases , Biología Computacional/métodos , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , TransgenesRESUMEN
Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.
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Axones/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Morfogénesis/fisiología , Neurogénesis/fisiología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Sinapsis/fisiología , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Axones/ultraestructura , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neurotransmisores/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-abl/genética , Sinapsis/ultraestructura , Transmisión Sináptica/fisiologíaRESUMEN
Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.
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Proteínas de Drosophila , Células Madre Oogoniales , Animales , Femenino , Drosophila , Microscopía , MitocondriasRESUMEN
Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1-R6 through Tm3 and Mi1 is sufficient to confer Dm8's UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1-R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8's spatial opponency and animals' phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.
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Drosophila , Neuronas , Animales , Percepción de Color/fisiología , Neuronas/fisiología , RetinaRESUMEN
Stereotypic dendrite arborizations are key morphological features of neuronal identity, as the size, shape and location of dendritic trees determine the synaptic input fields and how information is integrated within developed neural circuits. In this review, we focus on the actions of extrinsic intercellular communication factors and their effects on intrinsic developmental processes that lead to dendrite patterning. Surrounding neurons or supporting cells express adhesion receptors and secreted proteins that respectively, act via direct contact or over short distances to shape, size, and localize dendrites during specific developmental stages. The different ligand-receptor interactions and downstream signaling events appear to direct dendrite morphogenesis by converging on two categorical mechanisms: local cytoskeletal and adhesion modulation and global transcriptional regulation of key dendritic growth components, such as lipid synthesis enzymes. Recent work has begun to uncover how the coordinated signaling of multiple extrinsic factors promotes complexity in dendritic trees and ensures robust dendritic patterning.
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Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.
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Activinas/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Activinas/farmacología , Animales , Drosophila/fisiología , Proteínas de Drosophila/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Modelos Biológicos , Mutación , Neuronas/efectos de los fármacos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and their breakdown during aging, remain unclear. Here, we identify a mitochondrial protein trafficking pathway in Drosophila melanogaster involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic protein within mitochondria. The rate of vesicle formation increases with age. Vesicles originate from the outer mitochondrial membrane as observed by tracking Tom20 localization, and the process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of ubiquitinated protein aggregation, which are themselves associated with age-related diseases. The mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future age-related mitochondrial disease therapies.
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Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Factores de Edad , Animales , Animales Modificados Genéticamente , Proteínas del Citoesqueleto/metabolismo , Drosophila melanogaster/fisiología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Longevidad , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Dominios Proteicos , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMEN
Glioblastoma multiforme (GBM), the most prevalent brain tumor in adults, has extremely poor prognosis. Frequent genetic alterations that activate epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling, as well as metabolic remodeling, have been associated with gliomagenesis. To establish a whole-animal approach that can be used to readily identify individual pathometabolic signaling factors, we induced glioma formation in the adult Drosophila brain by activating the EGFR-PI3K pathway. Glioma-induced animals showed significantly enlarged brain volume, early locomotor abnormalities, memory deficits, and a shorter lifespan. Combining bioinformatics analysis and glial-specific gene knockdown in the adult fly glioma model, we identified four evolutionarily conserved metabolic genes, including ALDOA, ACAT1, ELOVL6, and LOX, that were involved in gliomagenesis. Silencing of ACAT1, which controls cholesterol homeostasis, reduced brain enlargement and increased the lifespan of the glioma-bearing flies. In GBM patients, ACAT1 is overexpressed and correlates with poor survival outcomes. Moreover, pharmacological inhibition of ACAT1 in human glioma cell lines revealed that it is essential for tumor proliferation. Collectively, these results imply that ACAT1 is a potential therapeutic target, and cholesterol homeostasis is strongly related to glioma formation. This in vivo model provides several rapid and robust phenotypic readouts, allowing determination of the pathometabolic pathways involved in gliomagenesis, as well as providing valuable information for novel therapeutic strategies.
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Envejecimiento/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Carcinogénesis/metabolismo , Drosophila melanogaster/metabolismo , Glioma/metabolismo , Glioma/patología , Redes y Vías Metabólicas , Animales , Conducta Animal , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/fisiopatología , Humanos , Longevidad , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/fisiopatología , Actividad Motora , Esterol O-Aciltransferasa/metabolismo , Análisis de SupervivenciaRESUMEN
In this study, we aim to rapidly fabricate an aptachip with a dual colorimetric and fluorometric sensing strategy for easy dopamine (DA) detection with high sensitivity and selectivity. To construct an aptachip with high DA capture efficiency, molecular dynamics (MD) simulations were utilitized to predict the most stable configuration of the DA-binding aptamer (DBA) for DA recognition. The DA in the specimen would be specifically captured on the DBA-aptachip, then released from the DBA in alkaline solution to form DA-quinone (DAQ), thus leading to a color change (from colorless to brown) and inducing a dramatic decrease in the fluorescence intensity as a result of the photoinduced electron transfer (PET) for bovine serum albumin (BSA)-stabilized Au nanoclusters (BSA-Au NCs). The detection limit of DA is as low as 0.1 µg mL-1 for the colorimetric system and 0.5 ng mL-1 for the fluorometric system. In addition, this biosensing of DA is easy to implement for visual detection owing to the DA oxidation and fluorescence quenching by BSA-AuNCs in the presence of the alkaline solution. Both the colorimetric and fluorometric systems showed excellent selectivity toward DA over interfering substances. Furthermore, we demonstrated the application of the present approach to analysis of artificial cerebrospinal fluid (ACSF) and serum samples, suggesting that this system holds promise for diagnostics.
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Kawasaki disease (KD) is a type of acute febrile vasculitis syndrome and is the most frequent cause of cardiac illness in children under the age of five years old. Although the etiology of KD remains largely unknown, some recent genome-wide studies have indicated that epigenetic factors may be important in its pathogenesis. We enrolled 24 KD patients and 24 non-KD controls in this study to access their DNA methylation status using HumanMethylation450 BeadChips. Another 34 KD patients and 62 control subjects were enrolled for expression validation. Of the 3193 CpG methylation regions with a methylation difference ≥ 20% between KD and controls, 3096 CpG loci revealed hypomehtylation, with only 3% being hypermethylated. Pathway buildup identified 11 networked genes among the hypermethylated regions, including four transcription factors: nuclear factor of activated T-cells 1, v-ets avian erythroblastosis virus E26 oncogene homolog 1, runt related transcription factor 3, and retinoic acid receptor gamma, as well as the activator ß-catenin. Ten of these network-selected genes demonstrated a significant decrease in mRNA in KD patients, whereas only CTNNB1 significantly decreased in correlation with coronary artery lesions in KD patients. Furthermore, CTNNB1-silenced THP-1 monocytic cells drastically increased the expression of CD40 and significantly increased the expression of both CD40 and CD40L in cocultured human coronary artery endothelial cells. This study is the first to identify network-based susceptible genes of hypermethylated CpG loci, their expression levels, and the functional impact of ß-catenin, which may be involved in both the cause and the development of KD.
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CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.
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Ligasas de Carbono-Nitrógeno/metabolismo , Histidina/metabolismo , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Ácido Fólico/metabolismo , Homocisteína/metabolismo , Humanos , Metilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Early diagnosis of prostate cancer (PCa) is critical for the prevention of metastasis and for early treatment; therefore, a simple and accurate device must be developed for this purpose. In this study, we reported a novel fabrication method for producing a dual-modality biosensor that can simultaneously detect vascular endothelial growth factor (VEGF) and prostate-specific antigen (PSA) in human serum for early diagnosis of PCa. This biosensor was constructed by coating graphene oxide/ssDNA (GO-ssDNA) on an Au-electrode for VEGF detection, and incorporated with poly-L-lactide nanoparticles (PLLA NPs) for signal amplification and PSA detection. The results showed that this biosensor has wide liner detection ranges (0.05-100ng/mL for VEGF and 1-100ng/mL for PSA), as well as high levels of sensitivity and selectivity (i.e., resisting interference from external factors, such as glucose, ascorbic acid human serum protein, immunoglobulin G, and immunoglobulin M), and demonstrated a high correlation with an enzyme-linked immunosorbent assay for sample detection in patients. Therefore, this biosensor could be utilized for early clinical diagnosis of PCa in the future.
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ADN de Cadena Simple/química , Grafito/química , Nanopartículas/química , Poliésteres/química , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Anticuerpos Inmovilizados/química , Técnicas Biosensibles/métodos , Detección Precoz del Cáncer , Técnicas Electroquímicas/métodos , Electrodos , Humanos , Masculino , Nanopartículas/ultraestructura , Óxidos/química , Neoplasias de la Próstata/diagnósticoRESUMEN
In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a "two-tag" double-labeling method to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals.
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Mapeo Encefálico , Color , Neuronas , Neurópilo/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Vías Visuales/citología , Animales , Animales Modificados Genéticamente , Drosophila/anatomía & histología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Bulbo Raquídeo/citología , Microscopía Confocal , Neuronas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vías Visuales/metabolismoRESUMEN
Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.
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Drosophila/fisiología , Neuronas/química , Coloración y Etiquetado/métodos , Sinapsis/química , Animales , Drosophila/química , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Neuronas/fisiología , Coloración y Etiquetado/instrumentación , Sinapsis/fisiologíaRESUMEN
BACKGROUND: In the fly's visual motion pathways, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF channel from L1's partner, L2, to T5 are so far not known, however. RESULTS: Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3's combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9's input synapses are spatially segregated on T5's dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). CONCLUSIONS: We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.
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Drosophila melanogaster/fisiología , Percepción de Movimiento , Animales , Microscopía Electrónica , Neuronas/fisiología , Vías VisualesRESUMEN
How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. Here, we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive â¼16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.