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BACKGROUND: Iodine deficiency is a significant public health problem for many populations worldwide, including India, particularly during the "first 1000 days" of life. Though Universal Salt iodization (USI) is mandatory in India, prior to 2018-19, there was no state-wide survey with estimates of iodine concentrations in salt using iodometric titration. Taking cognizance of this fact, Nutrition International commissioned the first-of-its-kind national-level survey in India, titled the India Iodine Survey 2018-19. OBJECTIVES: The study was conducted across the country to provide national and subnational estimates of iodine concentrations in household salt using iodometric titration and iodine nutrition status among women of reproductive age (15-49 y). METHODS: The survey adopted a multi-stage randomcluster probability proportional to size sampling design, covering 21,406 households in all the states and union territories (UTs) of India. RESULTS: At the national level, the household coverage of edible salt with adequate iodine (content ≥15 parts/million) was 76.3%. At the sub-national level, the coverage varied, with 10 states and 3 UTs achieving USI and 11 states and 2 UTs falling below the national average, with the highest among all the states and UTs, being Jammu and Kashmir and the lowest being Tamil Nadu. At the national level, the median urinary iodine concentration for pregnant women was 173.4 µg/L, for lactating women was 172.8 µg/L, and for non-pregnant, non-lactating women, it was 178.0 µg/L, which is within the adequate iodine nutrition range according to the WHO guidelines. CONCLUSIONS: The survey results can be widely used by various stakeholders, including government, academia, and industry, to understand the iodine nutrition status of the population, enable the scale-up of sustained efforts toward consolidating gains and achieving USI, leading to the reduction and elimination of Iodine Deficiency Disorders.
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Yodo , Estado Nutricional , Humanos , Femenino , Embarazo , India/epidemiología , Estudios Transversales , Cloruro de Sodio Dietético , Yodo/orinaRESUMEN
Exposure of rodents to <20 cGy Space Radiation (SR) impairs performance in several hippocampus-dependent cognitive tasks, including spatial memory. However, there is considerable inter-individual susceptibility to develop SR-induced spatial memory impairment. In this study, a robust label-free mass spectrometry (MS)-based unbiased proteomic profiling approach was used to characterize the composition of the hippocampal proteome in adult male Wistar rats exposed to 15 cGy of 1 GeV/n 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.
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Radiación Cósmica , Hipocampo/efectos de la radiación , Proteoma/metabolismo , Ubiquitina/metabolismo , Animales , Cognición/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Medio Ambiente Extraterrestre , Hipocampo/metabolismo , Masculino , Proteómica/métodos , Ratas , Ratas Wistar , Memoria Espacial/efectos de la radiaciónRESUMEN
The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.
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Antígenos de Carbohidratos Asociados a Tumores/análisis , Biomarcadores de Tumor/análisis , Carcinoma/diagnóstico , Glicoproteínas/análisis , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Carcinoma/genética , Carcinoma/inmunología , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Lactante , Masculino , Ratones , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).
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MicroARNs/genética , Enfermedades por Prión/metabolismo , Sinapsis/metabolismo , Animales , Dendritas/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Priones/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label-free LC-MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO-P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) were identified to be key mediators of pro- and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO-P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.
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Bleomicina , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/metabolismo , Mapas de Interacción de Proteínas , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Uniones Adherentes/metabolismo , Animales , Línea Celular , Factores de Crecimiento Endotelial/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/metabolismo , Péptidos Cíclicos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.
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Acinetobacter/metabolismo , Proteínas Bacterianas/metabolismo , Polisacáridos/metabolismo , Cromatografía Liquida , Glicopéptidos/metabolismo , Glicosilación , Espectrometría de Masas en TándemRESUMEN
Vesicular stomatitis virus (VSV) is an oncolytic virus that induces cancer cell death through activation of the apoptotic pathway. Intrinsic resistance to oncolysis is found in some cell lines and many primary tumors as a consequence of residual innate immunity to VSV. In resistant-tumor models, VSV oncolytic potential can be reversibly stimulated by combination with epigenetic modulators, such as the histone deacetylase inhibitor vorinostat. Based on this reversible effect of vorinostat, we reasoned that critical host genes involved in oncolysis may likewise be reversibly regulated by vorinostat. A transcriptome analysis in prostate cancer PC3 cells identified a subset of NF-κB target genes reversibly regulated by vorinostat, as well as a group of interferon (IFN)-stimulated genes (ISGs). Consistent with the induction of NF-κB target genes, vorinostat-mediated enhancement of VSV oncolysis increased hyperacetylation of NF-κB RELA/p65. Additional bioinformatics analysis revealed that NF-κB signaling also increased the expression of several autophagy-related genes. Kinetically, autophagy preceded apoptosis, and apoptosis was observed only when cells were treated with both VSV and vorinostat. VSV replication and cell killing were suppressed when NF-κB signaling was inhibited using pharmacological or genetic approaches. Inhibition of autophagy by 3-methyladenine (3-MA) enhanced expression of ISGs, and either 3-MA treatment or genetic ablation of the autophagic marker Atg5 decreased VSV replication and oncolysis. Together, these data demonstrate that vorinostat stimulates NF-κB activity in a reversible manner via modulation of RELA/p65 signaling, leading to induction of autophagy, suppression of the IFN-mediated response, and subsequent enhancement of VSV replication and apoptosis.
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Autofagia , Inhibidores de Histona Desacetilasas/farmacología , FN-kappa B/metabolismo , Virus Oncolíticos/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Acetilación , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Cromatina/metabolismo , Análisis por Conglomerados , Técnicas de Silenciamiento del Gen , Humanos , Ácidos Hidroxámicos/farmacología , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , Viroterapia Oncolítica , Virus Oncolíticos/genética , Neoplasias de la Próstata/terapia , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Transcriptoma , Virus de la Estomatitis Vesicular Indiana/genética , Replicación Viral , VorinostatRESUMEN
SAMHD1 is a host protein responsible, at least in part, for the inefficient infection of dendritic, myeloid, and resting T cells by HIV-1. Interestingly, HIV-2 and SIVsm viruses are able to counteract SAMHD1 by targeting it for proteasomal degradation using their Vpx proteins. It has been proposed that SAMHD1 is a dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) that restricts HIV-1 by reducing cellular dNTP levels to below that required for reverse transcription. However, nothing is known about SAMHD1 posttranslational modifications and their potential role in regulating SAMHD1 function. We used (32)P labeling and immunoblotting with phospho-specific antibodies to identify SAMHD1 as a phosphoprotein. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry. Mutation of these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation had no effect on the nuclear localization of SAMHD1 or its sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions had a significant effect on SAMHD1 dNTPase activity in an in vitro assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive phosphorylation at a main phosphorylation site, severely affected the ability of SAMHD1 to restrict HIV-1 in a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is supported by our finding that SAMHD1 is hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions.
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VIH-1/inmunología , Proteínas de Unión al GTP Monoméricas/inmunología , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleósido-Trifosfatasa/inmunología , Nucleósido-Trifosfatasa/metabolismo , Procesamiento Proteico-Postraduccional , Línea Celular , Análisis Mutacional de ADN , Humanos , Immunoblotting , Marcaje Isotópico , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Radioisótopos de Fósforo/metabolismo , Fosforilación , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive spectroscopic technique that generates signal-enhanced fingerprint vibrational spectra of small molecules. However, without rigorous control of SERS substrate active sites, geometry, surface area, or surface functionality, SERS is notoriously irreproducible, complicating the consistent quantitative analysis of small molecules. While evaporatively prepared samples yield significant SERS enhancement resulting in lower detection limits, the distribution of these enhancements along the SERS surface is inherently stochastic. Acquiring spatially resolved SERS spectra of these dried surfaces, we have shown that this enhancement is governed by a power law as a function of analyte concentration. Consequently, by definition, there is no true mean of SERS enhancement, requiring an alternative approach to achieve reproducible quantitative results. In this study, we introduce a new method of analysis of SERS data using a cumulative distribution function (CDF). The antiviral drug tenofovir (TFV) in an aqueous matrix was quantified down to a clinically relevant concentration of 25 ng/mL using hydroxylamine-reduced silver colloids evaporated to dryness. The data presented in this study provide a rationale for the benefits of combining a novel statistical approach using CDFs with simple and inexpensive experimental techniques to increase the precision, accuracy, and analytical sensitivity of aqueous TFV quantification by SERS. TFV calibration curves generated using CDF analysis showed higher analytical sensitivity (in the form of a normalized calibration curve average slope increase of 0.25) compared to traditional SERS intensity calculations. A second aliquot of nanoparticles and analyte dried on the SERS surface followed by CDF analysis showed further analytical sensitivity with a normalized calibration curve slope increase of 0.23 and decreased variation among replicates represented by an average standard deviation decrease of 0.02 with a second aliquot. The quantitative analysis of SERS data using CDFs presented here shows promise to be a reproducible method for quantitative analysis of SERS data, a significant step toward implementing SERS as an analytical method in clinical and industrial settings.
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In drug discovery and development, in vitro absorption and metabolism assays along with in vivo pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic (TK) studies are used to evaluate a potential drug candidate. More recently, imaging mass spectrometry approaches have been successfully reported to aid in the preclinical assessment of drug candidates, resulting in the rapid and noteworthy acceptance of the technique in pharmaceutical research. Traditionally, drug distribution studies via mass spectrometric imaging (MSI) are performed as targeted MS/MS analyses, where the analytes of interest, drug and/or metabolite, are known before the imaging experiment is performed. The study presented here describes a whole-body mass spectrometric imaging (WB-MSI) approach using a hybrid MALDI-LTQ-Orbitrap-MS to detect the distribution of reserpine at 2 h post a 20 mg/kg oral dose. This study effectively demonstrates the utility of obtaining accurate mass measurements across a wide mass range combined with postprocessing tools to efficiently identify drug and metabolite distributions without the need for any a priori knowledge.
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Imagen Molecular/métodos , Reserpina/metabolismo , Imagen de Cuerpo Entero/métodos , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Reserpina/farmacocinética , Factores de TiempoRESUMEN
NASA's planned mission to Mars will result in astronauts being exposed to â¼350 mSv/yr of Galactic Cosmic Radiation (GCR). A growing body of data from ground-based experiments indicates that exposure to space radiation doses (approximating those that astronauts will be exposed to on a mission to Mars) impairs a variety of cognitive processes, including cognitive flexibility tasks. Some studies report that 33% of individuals may experience severe cognitive impairment. Translating the results from ground-based rodent studies into tangible risk estimates for astronauts is an enormous challenge, but it would be germane for NASA to use the vast body of data from the rodent studies to start developing appropriate countermeasures, in the expectation that some level of space radiation (SR) -induced cognitive impairment could occur in astronauts. While some targeted studies have reported radiation-induced changes in the neurotransmission properties and/or increased neuroinflammation within space radiation exposed brains, there remains little information that can be used to start the development of a mechanism-based countermeasure strategy. In this study, we have employed a robust label-free mass spectrometry (MS) -based untargeted quantitative proteomic profiling approach to characterize the composition of the medial prefrontal cortex (mPFC) proteome in rats that have been exposed to 15 cGy of 600 MeV/n28Si ions. A variety of analytical techniques were used to mine the generated expression data, which in such studies is typically hampered by low and variable sample size. We have identified several pathways and proteins whose expression alters as a result of space radiation exposure, including decreased mitochondrial function, and a further subset of proteins differs in rats that have a high level of cognitive performance after SR exposure in comparison with those that have low performance levels. While this study has provided further insight into how SR impacts upon neurophysiology, and what adaptive responses can be invoked to prevent the emergence of SR-induced cognitive impairment, the main objective of this paper is to outline strategies that can be used by others to analyze sub-optimal data sets and to identify new information.
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Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.
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Glicopéptidos/química , Análisis por Micromatrices/métodos , Anticuerpos/inmunología , Cromatografía Líquida de Alta Presión , Polarización de Fluorescencia , Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Biblioteca de Péptidos , Polisacáridos/inmunología , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Electrostatic interactions play an important role in the formation of noncovalent complexes. Our previous work has highlighted the role of certain amino acid residues, such as arginine, glutamate, aspartate, and phosphorylated/sulfated residues, in the formation of salt bridges resulting in noncovalent complexes between peptides. Tandem mass spectrometry (MS) studies of these complexes using collision-induced dissociation (CID) have provided information on their relative stability. However, product-ion spectra produced by CID have been unable to assign specifically the site of interaction for the complex. In this work, tandem MS experiments were conducted on noncovalent complexes using both electron capture dissociation (ECD) and electron-transfer dissociation (ETD). The resulting spectra were dominated by intramolecular fragments of the complex with the electrostatic interaction site intact. Based upon these data, we were able to assign the binding site for the peptides forming the noncovalent complex.
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Aminoácidos/química , Péptidos/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Arginina/química , Fragmentos de Péptidos/química , Espectrometría de Masa por Ionización de Electrospray , Electricidad EstáticaRESUMEN
Influenza A viruses can bind sialic acid-terminating glycan receptors, and species specificity is often correlated with sialic acid linkage with avian strains recognizing α2,3-linked sialylated glycans and mammalian strains preferring α2,6-linked sialylated glycans. These paradigms derive primarily from studies involving erythrocyte agglutination, binding to synthetic receptor analogs or binding to undefined surface markers on cells or tissues. Here, we present the first examination of the N-glycome of the human lung for identifying natural receptors for a range of avian and mammalian influenza viruses. We found that the human lung contains many α2,3- and α2,6-linked sialylated glycan determinants bound by virus, but all viruses also bound to phosphorylated, nonsialylated glycans.
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Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Pulmón/metabolismo , Pulmón/virología , Polisacáridos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Fosforilación , Polisacáridos/química , Proteómica/métodos , Proteínas ViralesRESUMEN
NASA is planning future missions to Mars, which will result in astronauts being exposed to â¼13 cGy/year of galactic cosmic radiation (GCR). Previous ground-based experiments have demonstrated that low (15 cGy) doses of 1 GeV/n 56Fe ions impair hippocampus-dependent spatial memory in rats. However, some irradiated rats maintain a spatial memory performance comparable to that seen in the sham-irradiated rats, suggesting that some of these animals are able to ameliorate the deleterious effects of the GCR, while others are not. This rat model provides a unique opportunity to increase our understanding of how GCR affects neurophysiology, what adaptive responses can be invoked to prevent the emergence of GCR-induced spatial memory impairment, as well as the pathways that are altered when spatial memory impairment occurs. A label-free, unbiased proteomic profiling approach involving quantitative protein/peptide profiling followed by Cytoscape analysis has established the composition of the hippocampal proteome in male Wistar rats after exposure to 15 cGy of 1 GeV/n 56Fe, and identified proteins whose expression is altered with respect to: 1. radiation exposure and 2. impaired spatial memory performance. We identified 30 proteins that were classified as "GCR exposure marker" (GEM) proteins (expressed solely or at higher levels in the irradiated rats but not related to spatial memory performance), most notably CD98, Cadps and GMFB. Conversely, there were 252 proteins that were detected only in the sham-irradiated samples, i.e., they were not detected in either of the irradiated cohorts; of these 10% have well-documented roles in neurotransmission. The second aspect of our data mining was to identify proteins whose expression was associated with either impaired or functional spatial memory. While there are multiple changes in the hippocampal proteome in the irradiated rats that have impaired spatial memory performance, with 203 proteins being detected (or upregulated) only in these rats, it would appear that spatial memory impairment may also arise from an inability of these rats to express "good spatial memory" (GSM) proteins, many of which play an important role in neuronal homeostasis and function, axonogenesis, presynaptic membrane organization and G-protein coupled receptor (GCPR) signaling. It may be possible to use this knowledge to develop two alternative countermeasure strategies, one that preserves critical pathways prophylactically and one that invokes restorative pathways after GCR exposure.
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Radiación Cósmica/efectos adversos , Hipocampo/fisiología , Hipocampo/efectos de la radiación , Proteómica , Memoria Espacial/efectos de la radiación , Animales , Masculino , Ratas , Ratas WistarRESUMEN
We have discovered an unexpected connection between a critical lung development and cancer gene termed thyroid transcription factor 1 (TTF-1 also known as NKX2-1) and cholesterol metabolism. Our published work implicates that TTF-1 positively regulates miR-33a which is known to repress ATP-binding cassette transporter 1 (ABCA1) and thus its cholesterol efflux activity. We set out to demonstrate that a higher TTF-1 expression would presumably inhibit cholesterol efflux and consequently raise intracellular cholesterol level. Surprisingly, raising TTF-1 expression actually lowers intracellular cholesterol level, which, we believe, is attributed to a direct transactivation of ABCA1 by TTF-1. Subsequently, we show that lung cancer cells primed with a TTF-1-driven decrease of cholesterol were more vulnerable to simvastatin, a frequently prescribed cholesterol biosynthesis inhibitor. In view of the fact that pathologists routinely interrogate human lung cancers for TTF-1 immunopositivity to guide diagnosis and the prevalent use of statins, TTF-1 should be further investigated as a putative biomarker of lung cancer vulnerability to statins.
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Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Factor Nuclear Tiroideo 1/metabolismo , Factores de Transcripción/metabolismo , Células A549 , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , MicroARNs/metabolismo , Simvastatina/farmacología , Factor Nuclear Tiroideo 1/genética , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Poor molecular characterization of idiopathic pulmonary fibrosis (IPF) has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies and poor prognosis. Particularly, the role of lipid imbalance due to impaired lipid metabolism in the pathogenesis of IPF has been poorly studied. EXPERIMENTAL DESIGN: The authors have used shotgun lipidomics in a bleomycin (BLM) mouse model of pulmonary fibrosis with vascular endothelial growth factor (VEGF)-inhibitor CBO-P11 as a therapeutic measure, to identify a comprehensive set of lipids that contribute to the pathogenesis of pulmonary fibrosis. RESULTS: The authors report that attenuation of BLM-induced fibrotic response with CBO-P11 cotreatment is accompanied by a decrease in total lipid content and specific downregulation of lipids, which are upregulated in response to BLM treatment. CONCLUSION AND CLINICAL RELEVANCE: Dysregulated lipids identified in this study hold the potential of being future biomarkers for IPF.
Asunto(s)
Bleomicina/efectos adversos , Biología Computacional , Factores de Crecimiento Endotelial/farmacología , Metabolismo de los Lípidos , Péptidos Cíclicos/farmacología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Factores de Crecimiento Endotelial/uso terapéutico , Ácidos Grasos/biosíntesis , Metabolismo de los Lípidos/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos Cíclicos/uso terapéutico , Fosfolípidos/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Regulación hacia Arriba/efectos de los fármacosRESUMEN
INTRODUCTION: Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS: Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION: Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE: This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.
Asunto(s)
Matriz Extracelular/química , Regeneración Tisular Dirigida/métodos , Hidrogeles/química , Isquemia Miocárdica/terapia , Miocitos Cardíacos/fisiología , Placenta/química , Células Madre/fisiología , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocitos Cardíacos/citología , Embarazo , Ratas , Ratas Sprague-Dawley , Células Madre/citologíaRESUMEN
Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding.