RESUMEN
BACKGROUND: Weibel-Palade bodies (WPBs) are endothelial cell-specific cigar-shaped secretory organelles containing various biologically active molecules. WPBs play crucial roles in thrombosis, hemostasis, angiogenesis, and inflammation. The main content of WPBs is the procoagulant protein vWF (von Willebrand factor). Physical contacts and functional cross talk between mitochondria and other organelles have been demonstrated. Whether an interorganellar connection exists between mitochondria and WPBs is unknown. METHODS: We observed physical contacts between mitochondria and WPBs in human umbilical vein endothelial cells by electron microscopy and living cell confocal microscopy. We developed an artificial intelligence-assisted method to quantify the duration and length of organelle contact sites in live cells. RESULTS: We found there existed physical contacts between mitochondria and WPBs. Disruption of mitochondrial function affected the morphology of WPBs. Furthermore, we found that Rab3b, a small GTPase on the WPBs, was enriched at the mitochondrion-WPB contact sites. Rab3b deficiency reduced interaction between the two organelles and impaired the maturation of WPBs and vWF multimer secretion. CONCLUSIONS: Our results reveal that Rab3b plays a crucial role in mediating the mitochondrion-WPB contacts, and that mitochondrion-WPB coupling is critical for the maturation of WPBs in vascular endothelial cells.
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Cuerpos de Weibel-Palade , Factor de von Willebrand , Humanos , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/metabolismo , Inteligencia Artificial , Exocitosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mitocondrias/metabolismo , Células CultivadasRESUMEN
Cytoskeletal-based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)-labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3-6-nanometer accuracy and 3.4-ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin-II and OSM-3-kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin-II, a torque-generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM-3-kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia-dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.
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Animales Modificados Genéticamente/metabolismo , Axonema/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Cinesinas/metabolismo , Animales , Animales Modificados Genéticamente/genética , Axonema/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cilios/genética , Cinesinas/genéticaRESUMEN
Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Cilios/genética , Mutación , Espectrina/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Cilios/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Microscopía Confocal , Microscopía Electrónica de Transmisión , Análisis de Secuencia de ARN , Espectrina/metabolismoRESUMEN
BACKGROUND: Early prediction of acute kidney injury (AKI) after liver transplantation (LT) facilitates timely recognition and intervention. We aimed to build a risk predictor of post-LT AKI via supervised machine learning and visualize the mechanism driving within to assist clinical decision-making. METHODS: Data of 894 cases that underwent liver transplantation from January 2015 to September 2019 were collected, covering demographics, donor characteristics, etiology, peri-operative laboratory results, co-morbidities and medications. The primary outcome was new-onset AKI after LT according to Kidney Disease Improving Global Outcomes guidelines. Predicting performance of five classifiers including logistic regression, support vector machine, random forest, gradient boosting machine (GBM) and adaptive boosting were respectively evaluated by the area under the receiver-operating characteristic curve (AUC), accuracy, F1-score, sensitivity and specificity. Model with the best performance was validated in an independent dataset involving 195 adult LT cases from October 2019 to March 2021. SHapley Additive exPlanations (SHAP) method was applied to evaluate feature importance and explain the predictions made by ML algorithms. RESULTS: 430 AKI cases (55.1%) were diagnosed out of 780 included cases. The GBM model achieved the highest AUC (0.76, CI 0.70 to 0.82), F1-score (0.73, CI 0.66 to 0.79) and sensitivity (0.74, CI 0.66 to 0.8) in the internal validation set, and a comparable AUC (0.75, CI 0.67 to 0.81) in the external validation set. High preoperative indirect bilirubin, low intraoperative urine output, long anesthesia time, low preoperative platelets, and graft steatosis graded NASH CRN 1 and above were revealed by SHAP method the top 5 important variables contributing to the diagnosis of post-LT AKI made by GBM model. CONCLUSIONS: Our GBM-based predictor of post-LT AKI provides a highly interoperable tool across institutions to assist decision-making after LT.
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Lesión Renal Aguda , Trasplante de Hígado , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/etiología , Adulto , Humanos , Trasplante de Hígado/efectos adversos , Donadores Vivos , Aprendizaje Automático , Medición de Riesgo , Aprendizaje Automático SupervisadoRESUMEN
Nonmuscle myosin II has been implicated in regulation of von Willebrand factor (VWF) release from endothelial Weibel-Palade bodies (WPBs), but the specific role of myosin IIa isoform is poorly defined. Here, we report that myosin IIa is expressed both in primary human endothelial cells and intact mouse vessels, essential for cyclic adenosine monophosphate (cAMP)-mediated endothelial VWF secretion. Downregulation of myosin IIa by shRNAs significantly suppressed both forskolin- and epinephrine-induced VWF secretion. Endothelium-specific myosin IIa knockout mice exhibited impaired epinephrine-stimulated VWF release, prolonged bleeding time, and thrombosis. Further study showed that in resting cells, myosin IIa deficiency disrupted the peripheral localization of Rab27-positive WPBs along stress fibers; on stimulation by cAMP agonists, myosin IIa in synergy with zyxin promotes the formation of a functional actin framework, which is derived from preexisting cortical actin filaments, around WPBs, facilitating fusion and subsequent exocytosis. In summary, our findings not only identify new functions of myosin IIa in regulation of WPB positioning and the interaction between preexisting cortical actin filaments and exocytosing vesicles before fusion but also reveal myosin IIa as a physiological regulator of endothelial VWF secretion in stress-induced hemostasis and thrombosis.
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AMP Cíclico/farmacología , Células Endoteliales/efectos de los fármacos , Miosina Tipo IIA no Muscular/fisiología , Factor de von Willebrand/metabolismo , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miosina Tipo IIA no Muscular/genética , Vías Secretoras/efectos de los fármacos , Vías Secretoras/genéticaRESUMEN
Deep learning-based reconstruction has emerged as an effective tool in fluorescence microscopy, with the potential to resolve diffraction-limited structures. However, most deep-learning reconstruction methods employed an end-to-end strategy, which ignored physical laws in the imaging process and made the preparation of training data highly challenging as well. In this study, we have proposed a novel deconvolution algorithm based on an imaging model, deep-learning priors and the alternating direction method of multipliers. This scheme decouples the reconstruction into two separate sub-problems, one for deblurring and one for restraining noise and artifacts. As a result of the decoupling, we are able to introduce deep-learning image priors and a variance stabilizing transform against targeted image degeneration due to the low photon budget. Deep-learning priors are learned from the general image dataset, in which biological images do not have to be involved, and are more powerful than hand-designed ones. Moreover, the use of the imaging model ensures high fidelity and generalization. Experiments on various kinds of measurement data show that the proposed algorithm outperforms existing state-of-the-art deconvolution algorithms in resolution enhancement and generalization.
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The power factories in cells, mitochondria, play important roles in all physiological processes. It is reported that progressive mitochondrial swelling and outer mitochondrial membrane rupture could be induced by a wide variety of apoptotic and necrotic stimuli. Regrettably, although a variety of mitochondrial probes have been developed, most of them are based on the detection of active species in mitochondria. Probes that can monitor the status and distribution of mitochondria for a long time are still urgently needed. In this study, a fluorescent sensor with excellent properties, EtNBEn, is described. Outstanding performance allows it to be observed not only in cells but also in living Daphnia and zebrafish under confocal microscopy for a long time. Moreover, the swelling process of mitochondria under light stimulation is also visualized under super-resolution (SR) microscopy. All these results suggest that EtNBEn could be employed for tagging mitochondria in various physiological processes, which makes a great contribution to the cure of diseases.
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Colorantes Fluorescentes/química , Microscopía Confocal/métodos , Mitocondrias/química , Animales , Daphnia/química , Daphnia/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Células MCF-7 , Mitocondrias/metabolismo , Imagen Óptica , Fotólisis , Pez Cebra/metabolismoRESUMEN
Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.
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Células/ultraestructura , Microscopía Fluorescente/métodos , Animales , Luz , FotonesRESUMEN
Although research regarding antibiotic resistance genes (ARGs) in aquaculture environments has gained increasing scientific interest, further studies are required to understand the abundances and removal mechanisms of ARGs during the entire rearing period of shrimp aquaculture. Thus, in this study, abundances, distributions and removal rates of ARGs in different environmental compartments of intensive shrimp farms in South China were investigated during the entire rearing period. The results indicated that sul1 and cmlA were the predominant ARGs in the water and sediment samples. Additionally, the total abundance of ARGs was higher in shrimp pond water than in the source water and farm effluent. Moreover, sediment samples indicated significantly higher ARG abundances than water samples from the shrimp ponds (P < 0.05). Environmental factors were found to significantly affect the distribution of ARGs in shrimp rearing environments. Furthermore, stable ponds aided the removal of ARGs from shrimp pond water. This study accounted for temporal variations in ARG abundances as well as removal of ARGs in different environmental compartments during the entire shrimp rearing period. However, additional research is required to optimize the water treatment process for removal of ARGs from the aquaculture.
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Acuicultura/métodos , Farmacorresistencia Microbiana/genética , Sedimentos Geológicos/microbiología , Estanques/microbiología , Animales , China , Crustáceos , Genes Bacterianos , Microbiología del AguaRESUMEN
We demonstrate a robust high power 930 nm femtosecond Nd:fiber laser system with hollow-core photonic bandgap fiber (HC-PBGF) as the output delivery, which can be easily integrated into compact two-photon microscopy system for bio-imaging. The whole laser system can deliver up to 17.4 nJ, 220-fs pulses at 930 nm with repetition rate of 46 MHz. In this paper, this laser was demonstrated as the light source for volumetric imaging of zebrafish blood vessel.
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Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (â¼10-10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG.
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Andrógenos/metabolismo , Braquiuros/metabolismo , Proteínas Portadoras/metabolismo , Insulina/metabolismo , Ovario/embriología , Ovario/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , ADN Complementario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Insulina/química , Insulina/genética , Péptidos y Proteínas de Señalización Intracelular , Funciones de Verosimilitud , Filogenia , Estaciones del Año , Análisis de Secuencia de ADNRESUMEN
Vitellogenin (vtg) synthesis, known as vitellogenesis, is one of most important processes in the ovarian development of oviparous animals. Recently, multiple insulin-like peptides (ILPs) have been reported in crustacean species due to the application of transcriptome sequencing. In this context, the present study reports that the addition of an exogenous ILP, bovine insulin, stimulates vtg (termed Sp-vtg) expression in hepatopancreatic explants from the mud crab, Scylla paramamosain, by in vitro experiments. Homologous genes of key factors in ILP signaling, Sp-PI3K, Sp-Akt, Sp-Rheb and Sp-TOR, have been isolated in S. paramamosain based on a transcriptome database. Further experiments reveal that the RNAi-mediated Sp-Akt gene knockdown and the inhibitors of Sp-PI3K and Sp-TOR block the stimulation of Sp-vtg expression by insulin. The combined results implicate the endogenous ILP and its corresponding signaling in the regulation of Sp-vtg synthesis in S. paramamosain.
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Braquiuros/metabolismo , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Vitelogeninas/metabolismo , Animales , Braquiuros/efectos de los fármacos , Bovinos , ADN Complementario/genética , Femenino , Técnicas de Silenciamiento del Gen , Hepatopáncreas/efectos de los fármacos , Hepatopáncreas/metabolismo , Péptidos/metabolismoRESUMEN
Insulin-like peptides (ILPs) have regulatory roles in reproduction, development and metabolism in invertebrates. The mode of ILP actions has not been well studied in invertebrates in regard to the role of binding partners, i.e., ILP binding protein (ILPBP). In this study, the full-length cDNA of Callinectes sapidus ILPBP (Cas-ILPBP, 960 bp) has been isolated using RACE cloning, having short 5' and 3' UTRs of 30 and 162 bp, respectively. The predicted precursor of Cas-ILPBP (255 aa) contains, in order a signal peptide (23 aa), an insulin-like growth factor (IGF) binding (IB) domain (79 aa), a kazal-type serine protease inhibitor (KI) domain (36 aa) and an immunoglobulin (Ig) domain (101 aa). Phylogenetic analysis shows that Cas-ILPBP is grouped with the ILPBPs of other crustacean species, and it shares the closest relationship with the ILPBP from another crab species, Scylla paramamosain. Transcripts of Cas-ILPBP are found in all examined tissues, with the highest levels in the nervous tissues (eyestalk ganglia, brain and thoracic ganglia complex) and followed by midgut, the pericardial organ, abdominal muscle and the heart. As Cas-ILPBP contains a putative Ig domain, it is hypothesized that this protein may be involved in immunity, particularly in the adult females infected with a reo-like virus (CsRV1). The expression levels of Cas-ILPBP are examined in several tissues (hemocytes, midgut, eyestalk ganglia) from the animals carrying varying levels of CsRV1 at 17 and 23 °C water temperatures. Cas-ILPBP levels in the midgut are most significantly affected by high levels of CsRV1 infection. Reduction in Cas-ILPBP levels in the midguts is noted from the animals infected with high levels of CsRV1 that show reduced or stop feeding activity, indicating that it may play an important role in midgut functions such as digestion and nutrient absorption.
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Proteínas de Artrópodos/genética , Braquiuros/genética , Braquiuros/virología , Proteínas Portadoras/genética , Expresión Génica , Inmunidad Innata , Reoviridae/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/metabolismo , Braquiuros/inmunología , Braquiuros/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intracelular , Especificidad de Órganos , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , TemperaturaRESUMEN
Insulin-like growth factor (IGF) signaling system holds a central position in regulating growth and metabolism in vertebrates. As critical components of this system, the IGF-binding proteins (IGFBPs) play important roles in regulating the biological activities of IGFs. Recently, the single IGF-binding domain protein (SIBD) was identified in invertebrates and its sequence was highly homologous with the N-terminal domain of IGFBP. In view of the possible role as counterparts of vertebrate IGFBPs, SIBDs have attracted the ever-increasing attention. This study reports the identification of a 1284bp SIBD gene (Sp-SIBD) from a member of commercially important family of Portunidae. The tissue distribution analysis showed that Sp-SIBD was mainly expressed in the nervous tissues and hepatopancreas. RNA in situ hybridization analysis showed that the positive signals were predominantly distributed in the secretory cells of the hepatopancreas. Subsequently, we examined the effects of various stresses, including hyperosmotic stress, hyperthermia, activated stress and fasting, on glucose levels in the hemolymph and Sp-SIBD expressions in the hepatopancreas. Interestingly, we found that Sp-SIBD expression was strongly up-regulated in response to these catabolic circumstances. Given the previous findings of insulin-like peptides (ILPs) in invertebrates, we speculate that invertebrate ILPs and SIBDs promise to serve as a pair of counterparts of IGFs and IGFBPs from vertebrate species respectively. In this context, the combined results suggested, by analogy with IGFBP 1 from vertebrates, for the first time that SIBD might play a key physiological role by sequestering ILPs to inhibit energy-expensive growth until conditions are more favorable.
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Proteínas de Artrópodos/metabolismo , Braquiuros/metabolismo , Hemolinfa/metabolismo , Hepatopáncreas/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Estrés Fisiológico , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Secuencia de Bases , Braquiuros/genética , Braquiuros/crecimiento & desarrollo , Clonación Molecular , Metabolismo Energético , Ayuno/fisiología , Fiebre , Glucosa/metabolismo , Hibridación in Situ , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Datos de Secuencia Molecular , Presión Osmótica , Fosforilación , Homología de Secuencia de Aminoácido , Transducción de Señal , Somatomedinas/genética , Somatomedinas/metabolismoRESUMEN
Insulin-like androgenic gland hormone (IAG) produced by androgenic gland (AG) in male crustaceans is regarded as a key regulator of sex differentiation. As a member of the insulin/insulin-like growth factor family, IAG is also likely involved in regulating somatic growth. In this study, a full-length cDNA of IAG (termed Sp-IAG) was isolated from the mud crab, Scylla paramamosain. Genomic DNA of Sp-IAG was also cloned, analysis of which reveals that Sp-IAG gene is organized in a 4 exon/3 intron manner. RNA in situ hybridization analysis detected positive signals in both type I and type II AG cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Sp-IAG was expressed not only in AG, but also in many other tissues. Sp-IAG expression levels in ovaries were examined at different stages of ovarian development (stages I to V); it was found that the expression was maintained at low levels during undeveloped stage (stage I) to late vitellogenic stage (stage IV) and then increased significantly at mature stage (stage V), suggesting that Sp-IAG may participate in inhibiting oocyte growth and vitellogenesis. The expression pattern of Sp-IAG during the molting cycle of the first stage crabs (C1) was also determined. Sp-IAG expression level continuously decreased from 0 h C1 (postmolt) crabs to 96 h C1 (premolt) crabs, and then increased significantly in the newly molted second stage crabs (C2, postmolt). The combined results suggested for the first time that IAG is involved in regulating ovarian development and somatic growth in crustaceans.
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Andrógenos/metabolismo , Braquiuros/genética , Insulina/genética , Hormonas de Invertebrados/genética , Ovario/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Glándulas Exocrinas/metabolismo , Femenino , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Oogénesis/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Diferenciación Sexual/genética , Vitelogénesis/fisiologíaRESUMEN
Investigating the fine structure of mitochondria and their dynamic interactions with other organelles is crucial for unraveling the mechanisms underlying mitochondrial-related diseases. The development of super-resolution techniques has provided powerful visualization tools for mitochondrial research, which is significant for investigating mitochondrial cristae structure, the localization of mitochondrial-related protein complex, and the interactions between mitochondria and other organelles. In this perspective, we introduce several advanced super-resolution techniques and their applications in mitochondrial research, and discuss the potential roles these techniques may play in future studies of mitochondria.
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Hsp60 play a crucial role in the process of pathogenic and protective immune responses and is implicated in autoimmune disease. In order to understand the environmental response and immune response of this gene, we cloned a Hsp60 (SpHsp60) gene from the mud crab Scylla paramamosain, localized SpHsp60 in hemocytes by in situ hybridization, and detected the expression of SpHsp60 after stresses in relation to three housekeeping genes (ß-actin, 18S rRNA and GAPDH). The full-length of the SpHsp60 cDNA was found to be 2424 bp. The predicted ORF encoded a protein of 576 amino acids with a predicted molecular mass of 61.19 kDa and a theoretical isoelectric point (pI) of 5.46. It shared high scoring identity 95% with the swimming crab Portunus trituberculatus. In situ hybridization assay showed that a higher expression occurred in the granular and semigranular cells when compared to the hyaline hemocytes. It suggested that SpHsp60 was mainly contributed from the granular and semigranular cells in hemolymph. The expression level of SpHsp60 in hemocytes showed a clear time-dependent pattern during the 96 h after stimulated by Vibrio alginolyticus. During this experiment the gene was induced and the highest expression level was observed at 3 h. The significantly up-regulated expression and rapid response of SpHsp60 indicated that the crabs were sensitive to bacterial challenge. After osmotic stress, the expression of SpHsp60 in hemocytes showed that this gene was induced by the high salinity (30) and the crabs had its adaptive responses to high salinity, when compared to the normal salinity (15). SpHsp60 mRNA expression in hemocytes was analyzed after thermal stress at 6 h, the highest and the lowest expression levels of SpHsp60 were observed at 36 and 32 °C, respectively. This study demonstrated that SpHsp60 was easily induced at the higher temperatures. Based on our research, SpHsp60 participate in innate immune and environmental response of S. paramamosain. It could be used as a biomarker to test the stress caused by the local aquaculture environment.
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Proteínas de Artrópodos/genética , Braquiuros/genética , Braquiuros/inmunología , Chaperonina 60/genética , Hemocitos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Braquiuros/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Datos de Secuencia Molecular , Presión Osmótica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Salinidad , Homología de Secuencia de Aminoácido , Estrés Fisiológico , Vibrio alginolyticus/fisiologíaRESUMEN
Mitochondrial export into the extracellular space is emerging as a fundamental cellular process implicated in diverse physiological activities. Although a few studies have shed light on the process of discarding damaged mitochondria, how mitochondria are exported and the functions of mitochondrial release remain largely unclear. Here we describe mitopherogenesis, a formerly unknown process that specifically secretes mitochondria through a unique extracellular vesicle termed a 'mitopher'. We observed that during sperm development in male Caenorhabditis elegans, healthy mitochondria are exported out of the spermatids through mitopherogenesis and each of the generated mitophers harbours only one mitochondrion. In mitopherogenesis, the plasma membrane first forms mitochondrion-embedding outward buds, which then promptly bud off and thereby result in the generation of mitophers. Mechanistically, extracellular protease signalling in the testis triggers mitopher formation from spermatids, which is partially mediated by the tyrosine kinase SPE-8. Moreover, mitopherogenesis requires normal microfilament dynamics, whereas myosin VI antagonizes mitopher generation. Strikingly, our three-dimensional electron microscopy analyses indicate that mitochondrial quantity requires precise modulation during sperm development, which is critically mediated by mitopherogenesis. Inhibition of mitopherogenesis causes accumulation of mitochondria in sperm, which may lead to sperm motility and fertility defects. Our findings identify mitopherogenesis as a previously undescribed process for mitochondria-specific ectocytosis, which may represent a fundamental branch of mechanisms underlying mitochondrial quantity control to regulate cell functions during development.
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Semen , Motilidad Espermática , Animales , Masculino , Semen/metabolismo , Espermatozoides/metabolismo , Fertilidad , Caenorhabditis elegans/genética , Mitocondrias/metabolismoRESUMEN
In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) method to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution-scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Overall, we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.