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Idiopathic Pulmonary Fibrosis (IPF) is a progressively fatal and incurable disease characterized by the loss of alveolar structures, increased epithelial-mesenchymal transition (EMT), and aberrant tissue repair. In this study, we investigated the role of Nuclear Factor I-B (NFIB), a transcription factor critical for lung development and maturation, in IPF. Using both human lung tissue samples from patients with IPF, and a mouse model of lung fibrosis induced by bleomycin, we showed that there was a significant reduction of NFIB both in the lungs of patients and mice with IPF. Furthermore, our in vitro experiments using cultured human lung cells demonstrated that the loss of NFIB was associated with the induction of EMT by transforming growth factor beta (TGF-ß). Knockdown of NFIB promoted EMT, while overexpression of NFIB suppressed EMT and attenuated the severity of bleomycin-induced lung fibrosis in mice. Mechanistically, we identified post-translational regulation of NFIB by miR-326, a miRNA with anti-fibrotic effects that is diminished in IPF. Specifically, we showed that miR-326 stabilized and increased the expression of NFIB through its 3'UTR target sites for Human antigen R (HuR). Moreover, treatment of mice with either NFIB plasmid or miR-326 reversed airway collagen deposition and fibrosis. In conclusion, our study emphasizes the critical role of NFIB in lung development and maturation, and its reduction in IPF leading to EMT and loss of alveolar structures. Our study highlights the potential of miR-326 as a therapeutic intervention for IPF. The schema shows the role of NFIB in maintaining the normal epithelial cell characteristics in the lungs and how its reduction leads to a shift towards mesenchymal cell-like features and pulmonary fibrosis. A In normal lungs, NFIB is expressed abundantly in the epithelial cells, which helps in maintaining their shape, cell polarity and adhesion molecules. However, when the lungs are exposed to factors that induce pulmonary fibrosis, such as bleomycin, or TGF-ß, the epithelial cells undergo epithelial to mesenchymal transition (EMT), which leads to a decrease in NFIB. B The mesenchymal cells that arise from EMT appear as spindle-shaped with loss of cell junctions, increased cell migration, loss of polarity and expression of markers associated with mesenchymal cells/fibroblasts. C We designed a therapeutic approach that involves exogenous administration of NFIB in the form of overexpression plasmid or microRNA-326. This therapeutic approach decreases the mesenchymal cell phenotype and restores the epithelial cell phenotype, thus preventing the development or progression of pulmonary fibrosis.
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Fibrosis Pulmonar Idiopática , MicroARNs , Humanos , Ratones , Animales , Transición Epitelial-Mesenquimal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factores de Transcripción NFI/metabolismo , Factores de Transcripción NFI/farmacología , Pulmón/metabolismo , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , MicroARNs/metabolismo , Células Epiteliales/metabolismo , Bleomicina/toxicidadRESUMEN
Fungal infections are the inevitable limiting factor for productivity of tea. Transcriptome reprogramming recruits multiple regulatory pathways during pathogen infection. A comprehensive meta-analysis was performed utilizing previously reported, well-replicated transcriptomic datasets from seven fungal diseases of tea. The study identified a cumulative set of 18,517 differentially expressed genes (DEGs) in tea, implicated in several functional clusters, including the MAPK signaling pathway, transcriptional regulation, and the biosynthesis of phenylpropanoids. Gene set enrichment analyses under each pathogen stress elucidated that DEGs were involved in ethylene metabolism, secondary metabolism, receptor kinase activity, and various reactive oxygen species detoxification enzyme activities. Expressional fold change of combined datasets highlighting 2258 meta-DEGs shared a common transcriptomic response upon fungal stress in tea. Pervasive duplication events caused biotic stress-responsive core DEGs to appear in multiple copies throughout the tea genome. The co-expression network of meta-DEGs in multiple modules demonstrated the coordination of appropriate pathways, most of which involved cell wall organization. The functional coordination was controlled by a number of hub genes and miRNAs, leading to pathogenic resistance or susceptibility. This first-of-its-kind meta-analysis of host-pathogen interaction generated consensus candidate loci as molecular signatures, which can be associated with future resistance breeding programs in tea.
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Perfilación de la Expresión Génica , MicroARNs , Transcriptoma , Regulación Fúngica de la Expresión Génica , Té/genética , Redes Reguladoras de GenesRESUMEN
Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.
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COVID-19 , Proteínas de la Nucleocápside de Coronavirus/metabolismo , ADN Mitocondrial , Proteínas no Estructurales Virales/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Humanos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosfoproteínas/metabolismo , SARS-CoV-2RESUMEN
Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.
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Axones , Neurregulinas , Receptor ErbB-4 , Transcitosis , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Axones/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Neurregulinas/genética , Neurregulinas/metabolismo , Ratas , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismoRESUMEN
The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: ⢠Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera ⢠Green-tissue-specific expression of Cry1Ac conferred partial pest resistance ⢠Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.
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Cajanus , Mariposas Nocturnas , Agrobacterium/genética , Animales , Cajanus/genética , Cajanus/metabolismo , Mariposas Nocturnas/genética , Plantas Modificadas Genéticamente/genética , TecnologíaRESUMEN
Vascular wilt caused by Fusarium udum Butler is the most important disease of pigeonpea throughout the world. F. udum isolate MTCC 2204 (M1) inoculated pigeonpea plants of susceptible (ICP 2376) and resistant (ICP 8863) cultivars were taken at invasion stage of pathogenesis process for transcriptomic profiling to understand defense signaling reactions that interplay at early stage of this plant-pathogen encounter. Differential transcriptomic profiles were generated through cDNA-AFLP from M1 inoculated resistant and susceptible pigeonpea root tissues. Twenty five percent of transcript derived fragments (TDFs) were found to be pathogen induced. Among them 73 TDFs were re-amplified and sequenced. Homology search of the TDFs in available databases and thorough study of scientific literature identified several pathways, which could play crucial role in defense responses of the F. udum inoculated resistant plants. Some of the defense responsive pathways identified to be active during this interaction are, jasmonic acid and salicylic acid mediated defense responses, cell wall remodeling, vascular development and pattering, abscisic acid mediated responses, effector triggered immunity, and reactive oxygen species mediated signaling. This study identified important wilt responsive regulatory pathways in pigeonpea which will be helpful for further exploration of these resistant components for pigeonpea improvement.
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Cajanus , Resistencia a la Enfermedad/genética , Fusarium/crecimiento & desarrollo , Perfilación de la Expresión Génica , Enfermedades de las Plantas , Cajanus/genética , Cajanus/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
The most consumed and economically important beverage plant, tea (Camellia sinensis), and its pests have coevolved so as to maintain the plant-insect interaction. In this review, findings of different research groups on pest responsive tolerance mechanisms that exist in tea manifested through the production of secondary metabolites and their inducers are presented. The phytochemicals of C. sinensis have been categorized into volatiles, nonvolatiles, enzymes, and phytohormones for convenience. Two types of pests, namely the piercing-sucking pests and chewing pests, are associated with tea. Both the insect groups can trigger the production of those metabolites and inducers through several primary and secondary biosynthetic pathways. These induced biomolecules can act as insect repellents and most of them are associated with lowering the nutrient quality of plant tissue and increasing the indigestibility in the pest's gut. Moreover, some of them also act as predator attractants of particular pests. The herbivore-induced plant volatiles secreted from tea plants during pest infestation were (E)-nerolidol, α-farnesene, (Z)-3-hexenol, (E)-4,8-dimethyl-1,3,7-nonatriene, indole, benzyl nitrile (BN), linalool, and ocimenes. The nonvolatiles like theaflavin and L-theanine were increased in response to the herbivore attack. Simultaneously, S-adenosyl-L-methionine synthase, caffeine synthase activities were affected, whereas flavonoid synthesis and wax formation were elevated. Defense responsive enzymes like peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, ascorbate peroxidase, and catalase are involved in pest prevention mechanisms. Phytohormones like jasmonic acid, salicylic acid, abscisic acid, and ethylene act as the modulator of the defense system. The objective of this review is to discuss the defensive roles of these metabolites and their inducers against pest infestation in tea with an aim to develop environmentally sustainable pesticides in the future.Key points⢠Herbivore-induced volatile signals and their effects on neighboring tea plant protection⢠Stereochemical conversion of volatiles, effects of nonvolatiles, expression of defense-responsive enzymes, and phytohormones due to pest attack⢠Improved understanding of metabolites for bio-sustainable pesticide development.
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Camellia sinensis , Animales , Insectos , TéRESUMEN
Aim: To differentiate mesenchymal stem cells into functional dopaminergic neurons using an electrospun polycaprolactone (PCL) and graphene (G) nanocomposite. Methods: A one-step approach was used to electrospin the PCL nanocomposite, with varying G concentrations, followed by evaluating their biocompatibility and neuronal differentiation. Results: PCL with exiguous graphene demonstrated an ideal nanotopography with an unprecedented combination of guidance stimuli and substrate cues, aiding the enhanced differentiation of mesenchymal stem cells into dopaminergic neurons. These newly differentiated neurons were seen to exhibit unique neuronal arborization, enhanced intracellular Ca2+ influx and dopamine secretion. Conclusion: Having cost-effective fabrication and room-temperature storage, the PCL-G nanocomposites could pave the way for enhanced neuronal differentiation, thereby opening a new horizon for an array of applications in neural regenerative medicine.
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Grafito , Células Madre Mesenquimatosas , Nanocompuestos , Nanofibras , Diferenciación Celular , Humanos , Poliésteres , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Calcium (Ca2+) plays fundamental and diverse roles in brain cells as a second messenger of many signaling pathways. Given the high energy demand in the brain and the generally non-regenerative state of neurons, the role of brain mitochondrial calcium [Ca2+]m in particular, in regulating ATP generation and determination of cell fate by initiation or inhibition of programmed cell death (PCD) becomes critical. Since [Ca2+]m signaling has a central role in brain physiology, it represents an ideal target for viruses to hijack the Ca2+ machinery to favor their own persistence, replication and/or dissemination by modulating cell death. This review discusses the ways by which neurotropic viruses are known to exploit the [Ca2+]m signaling of their host cells to regulate cell death in the brain, particularly in neurons. We hope our review will highlight the importance of [Ca2+]m handling in the virus-infected brain and stimulate further studies towards exploring novel [Ca2+]m related therapeutic strategies for viral effects on the brain.
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Encéfalo/virología , Señalización del Calcio , Mitocondrias/metabolismo , Virosis/metabolismo , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Regulación de la Expresión Génica , HumanosRESUMEN
Hyperactivation of the host immune system during infection by SARS-CoV-2 is the leading cause of death in COVID-19 patients. It is also evident that patients who develop mild/moderate symptoms and successfully recover display functional and well-regulated immune response. Whereas a delayed initial interferon response is associated with severe disease outcome and can be the tipping point towards immunopathological deterioration, often preceding death in COVID-19 patients. Further, adaptive immune response during COVID-19 is heterogeneous and poorly understood. At the same time, some studies suggest activated T and B cell response in severe and critically ill patients and the presence of SARS-CoV2-specific antibodies. Thus, understanding this problem and the underlying molecular pathways implicated in host immune function/dysfunction is imperative to devise effective therapeutic interventions. In this comprehensive review, we discuss the emerging immunopathological determinants and the mechanism of virus evasion by the host cell immune system. Using the knowledge gained from previous respiratory viruses and the emerging clinical and molecular findings on SARS-CoV-2, we have tried to provide a holistic understanding of the host innate and adaptive immune response that may determine disease outcome. Considering the critical role of the adaptive immune system during the viral clearance, we have presented the molecular insights of the plausible mechanisms involved in impaired T cell function/dysfunction during various stages of COVID-19.
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BACKGROUND: In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation. METHODS: Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed. RESULTS: HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat. CONCLUSION: Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.
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Gliosis/metabolismo , VIH-1/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas Mitocondriales/biosíntesis , Neuronas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Feto , Gliosis/genética , Gliosis/patología , VIH-1/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas Mitocondriales/genética , Neuronas/patología , Células Madre/metabolismo , Células Madre/patología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
A reliable pigeon pea transformation system can assist the rapid improvement of this important grain legume through transgenic development. Here we describe two methods of Agrobacterium tumefaciens-mediated pigeon pea transformation. In the tissue culture based embryonic explant transformation method, microshoot grafting was included to obtain rapid root induction, while the other method was culture independent and designated as plumular meristem transformation. Both methods drastically enhanced the transformation frequency and have the potential to provide reasonable solutions for maximum transgenic recovery in biotechnological breeding programs.
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Agrobacterium tumefaciens/genética , Cajanus/microbiología , Meristema/citología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Agrobacterium tumefaciens/fisiología , Cajanus/genética , Cajanus/crecimiento & desarrollo , Meristema/crecimiento & desarrollo , Fitomejoramiento , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/microbiología , Técnicas de Cultivo de Tejidos , Transformación GenéticaRESUMEN
The original article [1] contains errors in Fig. 1. The authors noticed a potentially misleading aspect of the original article Fig. 1 where representative flow cytometry data for different panels were from different data sets and thus the gates were not in the same line. This may cause confusion to the readers who attempt to compare panels and, thus the amended Fig. 1 shown ahead represents data from a single data set that is suitable for between panel comparisons.
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The asthma candidate gene inositol polyphosphate 4-phosphatase type I A (INPP4A) is a lipid phosphatase that negatively regulates the PI3K/Akt pathway. Destabilizing genetic variants of INPP4A increase the risk of asthma, and lung-specific INPP4A knockdown induces asthma-like features. INPP4A is known to localize intracellularly, and its extracellular presence has not been reported yet. Here we show for the first time that INPP4A is secreted by airway epithelial cells and that extracellular INPP4A critically inhibits airway inflammation and remodeling. INPP4A was present in blood and BAL fluid, and this extracellular INPP4A was reduced in patients with asthma and mice with allergic airway inflammation. In both naive mice and mice with allergic airway inflammation, antibody-mediated neutralization of extracellular INPP4A potentiated PI3K/Akt signaling and induced airway hyperresponsiveness, with prominent airway remodeling, subepithelial fibroblast proliferation, and collagen deposition. The link between extracellular INPP4A and fibroblasts was investigated in vitro. Cultured airway epithelial cells secreted enzymatically active INPP4A in extracellular vesicles and in a free form. Extracellular vesicle-mediated transfer of labeled INPP4A, from epithelial cells to fibroblasts, was observed. Inhibition of such transfer by anti-INPP4A antibody increased fibroblast proliferation. We propose that secretory INPP4A is a novel "paracrine" layer of the intricate regulation of lung homeostasis, by which airway epithelium dampens PI3K/Akt signaling in inflammatory cells or local fibroblasts, thereby limiting inflammation and remodeling.
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Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Asma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Hipersensibilidad Respiratoria/patología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Animales , Asma/sangre , Asma/genética , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Monoéster Fosfórico Hidrolasas/sangre , Monoéster Fosfórico Hidrolasas/genética , Hipersensibilidad Respiratoria/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Recent studies have demonstrated mesenchymal stem cells (MSCs) as effective mitochondrial donors with therapeutic success in multiple experimental models of human disease. MSCs obtained from different tissue sources such as bone marrow (BM), adipose (AD), dental pulp (DP), and Wharton's jelly (WJ) are routinely used in clinical trials with no known study of their mitochondrial donor capacity. Here, we show for the first time that MSCs derived from different tissue sources have different mitochondrial donor properties and that this is correlated with their intrinsic respiratory states. METHODS: MitoTracker®-labeled MSCs were co-cultured with Cell Trace-labeled U87-MG cells or rat cardiomyocytes. Mitochondrial transfer abilities of MSCs were assessed by using flow cytometry analysis and fluorescence imaging. Mitochondrial reactive oxygen species (mtROS) levels were analyzed by using MitoSOX red-based staining, and mitochondrial respiration parameters were analyzed by using a Seahorse XF Analyzer. RESULTS: AD-MSCs and BM-MSCs displayed higher mitochondrial transfer than DP-MSCs and WJ-MSCs. Counterintuitively, DP-MSCs and WJ-MSCs were more effective in suppressing mtROS levels in stressed recipient cells than AD-MSCs or BM-MSCs. Interestingly, the oxygen consumption rates and intrinsic mitochondrial respiration parameters like ATP levels, basal and maximal respiration, and mitochondrial DNA copy number in donor MSCs showed a highly significant inverse correlation with their mitochondrial donation. CONCLUSIONS: We find that there are intrinsic differences in the mitochondrial respiration, donation capacity, and therapeutic efficacy among MSCs of different tissue origin. MSCs with high mitochondrial respiration capacities are associated with lower mitochondrial transfer but more effective suppression of mtROS in stressed recipient cells. This is most compatible with a model where recipient cells optimally regulate mitochondrial transfer such that they take more mitochondria from MSCs with lower mitochondrial function. Furthermore, it appears to be advantageous to use MSCs such as DP-MSCs or WJ-MSCs with higher mitochondrial respiratory abilities that achieved better therapeutic effect with lower mitochondrial transfer in our study. This opens up a new direction in stem cell therapeutics.
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Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Especificidad de Órganos , Regeneración , Adenosina Trifosfato/metabolismo , Tejido Adiposo/citología , Animales , Línea Celular Tumoral , Respiración de la Célula , Pulpa Dental/citología , Metabolismo Energético , Humanos , Miocitos Cardíacos/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Gelatina de Wharton/citologíaRESUMEN
Inositol polyphosphate 4 phosphatase type I enzyme (INPP4A) has a well-documented function in the cytoplasm where it terminates the phosphatidylinositol 3-kinase (PI 3-K) pathway by acting as a negative regulator. In this study, we demonstrate for the first time that INPP4A shuttles between the cytoplasm and the nucleus. Nuclear INPP4A is enzymatically active and in dynamic equilibrium between the nucleus and cytoplasm depending on the cell cycle stage, with highest amounts detected in the nucleus during the G0/G1 phase. Moreover, nuclear INPP4A is found to have direct proliferation suppressive activity. Cells constitutively overexpressing nuclear INPP4A exhibit massive apoptosis. In human tissues as well as cell lines, lower nuclear localization of INPP4A correlate with cancerous growth. Together, our findings suggest that nuclear compartmentalization of INPP4A may be a mechanism to regulate cell cycle progression, proliferation and apoptosis. Our results imply a role for nuclear-localized INPP4A in tumor suppression in humans.
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The past decade has witnessed an upsurge in studies demonstrating mitochondrial transfer as one of the emerging mechanisms through which mesenchymal stem cells (MSCs) can regenerate and repair damaged cells or tissues. It has been found to play a critical role in healing several diseases related to brain injury, cardiac myopathies, muscle sepsis, lung disorders and acute respiratory disorders. Several studies have shown that various mechanisms are involved in mitochondrial transfer that includes tunnel tube formation, micro vesicle formation, gap junctions, cell fusion and others modes of transfer. Few studies have investigated the mechanisms that contribute to mitochondrial transfer, primarily comprising of signaling pathways involved in tunnel tube formation that facilitates tunnel tube formation for movement of mitochondria from one cell to another. Various stress signals such as release of damaged mitochondria, mtDNA and mitochondrial products along with elevated reactive oxygen species levels trigger the transfer of mitochondria from MSCs to recipient cells. However, extensive cell signaling pathways that lead to mitochondrial transfer from healthy cells are still under investigation and the changes that contribute to restoration of mitochondrial bioenergetics in recipient cells remain largely elusive. In this review, we have discussed the phenomenon of mitochondrial transfer from MSCs to neighboring stressed cells, and how this aids in cellular repair and regeneration of different organs such as lung, heart, eye, brain and kidney. The potential scope of mitochondrial transfer in providing novel therapeutic strategies for treatment of various pathophysiological conditions has also been discussed.
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Trasplante de Células Madre Mesenquimatosas/estadística & datos numéricos , Células Madre Mesenquimatosas/fisiología , Mitocondrias/trasplante , RegeneraciónRESUMEN
KEY MESSAGE: Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.