RESUMEN

N6-methyladenosine (m6A) is the most abundant post-transcriptional dynamic RNA modification process in eukaryotes, extensively implicated in cellular growth, embryonic development and immune homeostasis. One of the most profound biological functions of m6A is to regulate RNA metabolism, thereby determining the fate of RNA. Notably, the regulation of m6A-mediated organized RNA metabolism critically relies on the assembly of membraneless organelles (MLOs) in both the nucleus and cytoplasm, such as nuclear speckles, stress granules and processing bodies. In addition, m6A-associated MLOs exert a pivotal role in governing diverse RNA metabolic processes encompassing transcription, splicing, transport, decay and translation. However, emerging evidence suggests that dysregulated m6A levels contribute to the formation of pathological condensates in a range of human diseases, including tumorigenesis, reproductive diseases, neurological diseases and respiratory diseases. To date, the molecular mechanism by which m6A regulates the aggregation of biomolecular condensates associated with RNA metabolism is unclear. In this review, we comprehensively summarize the updated biochemical processes of m6A-associated MLOs, particularly focusing on their impact on RNA metabolism and their pivotal role in disease development and related biological mechanisms. Furthermore, we propose that m6A-associated MLOs could serve as predictive markers for disease progression and potential drug targets in the future.

Asunto(s)

Adenosina , ARN , Humanos , Adenosina/metabolismo , Adenosina/análogos & derivados , ARN/metabolismo , Orgánulos/metabolismo , Animales , Procesamiento Postranscripcional del ARN , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Núcleo Celular/metabolismo , Citoplasma/metabolismo

RESUMEN

How intracellular bacteria subvert the major histocompatibility complex (MHC) class I pathway is poorly understood. Here, we show that the obligate intracellular bacterium Orientia tsutsugamushi uses its effector protein, Ank5, to inhibit nuclear translocation of the MHC class I gene transactivator, NLRC5, and orchestrate its proteasomal degradation. Ank5 uses a tyrosine in its fourth ankyrin repeat to bind the NLRC5 N-terminus while its F-box directs host SCF complex ubiquitination of NLRC5 in the leucine-rich repeat region that dictates susceptibility to Orientia- and Ank5-mediated degradation. The ability of O. tsutsugamushi strains to degrade NLRC5 correlates with ank5 genomic carriage. Ectopically expressed Ank5 that can bind but not degrade NLRC5 protects the transactivator during Orientia infection. Thus, Ank5 is an immunoevasin that uses its bipartite architecture to rid host cells of NLRC5 and reduce surface MHC class I molecules. This study offers insight into how intracellular pathogens can impair MHC class I expression.

Asunto(s)

Antígenos de Histocompatibilidad Clase I , Péptidos y Proteínas de Señalización Intracelular , Orientia tsutsugamushi , Orientia tsutsugamushi/metabolismo , Orientia tsutsugamushi/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Citoplasma/metabolismo , Células HEK293 , Proteolisis , Tifus por Ácaros/inmunología , Tifus por Ácaros/microbiología , Tifus por Ácaros/metabolismo , Ratones , Ubiquitinación , Interacciones Huésped-Patógeno/inmunología

RESUMEN

Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.

Asunto(s)

Membrana Celular , Péptidos de Penetración Celular , Citoplasma , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Células HeLa , Animales

RESUMEN

The coordinated action of transcriptional and post-transcriptional machineries shapes gene expression programs at steady state and determines their concerted response to perturbations. We have developed Nanodynamo, an experimental and computational workflow for quantifying the kinetic rates of nuclear and cytoplasmic steps of the RNA life cycle. Nanodynamo is based on mathematical modelling following sequencing of native RNA from cellular fractions and polysomes. We have applied this workflow to triple-negative breast cancer cells, revealing widespread post-transcriptional RNA processing that is mutually exclusive with its co-transcriptional counterpart. We used Nanodynamo to unravel the coupling between transcription, processing, export, decay and translation machineries. We have identified a number of coupling interactions within and between the nucleus and cytoplasm that largely contribute to coordinating how cells respond to perturbations that affect gene expression programs. Nanodynamo will be instrumental in unravelling the determinants and regulatory processes involved in the coordination of gene expression responses.

Asunto(s)

Núcleo Celular , Humanos , Núcleo Celular/metabolismo , Línea Celular Tumoral , ARN/metabolismo , ARN/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Procesamiento Postranscripcional del ARN , Citoplasma/metabolismo , Cinética , Polirribosomas/metabolismo , Transcripción Genética , ARN Mensajero/metabolismo , ARN Mensajero/genética

RESUMEN

Cytoplasmic male sterility (CMS) is important for commercial hybrid seed production. However, it is still not used in eggplant (Solanum melongena L.), and corresponding regulatory genes and mechanisms of action have not been reported. We report CMS line 327A, which was derived from the hybridization between cultivated and wild eggplants. By looking at different stages of anther development under a microscope, we saw that the 327A anther's tapetum layer vacuolized during meiosis, which caused abortion. To investigate the 327A CMS regulatory genes, the mitochondrial genomes of 327A and its maintainer line 327B were assembled de novo. It was found that 15 unique ORFs (Open Reading Frame) were identified in 327A. RT-PCR and RT-QPCAR tests confirmed that orf312a and orf172a, 327A-specific ORFs with a transmembrane domain, were strongly expressed in sterile anthers of 327A. In addition, orf312a has a chimeric structure with the ribosomal protein subunit rpl16. Therefore, orf312a and orf172a can be considered strong candidate genes for CMS. Concurrently, we analyzed the characteristics of CMS to develop a functional molecular marker, CMS312, targeting a future theoretical basis for eggplant CMS three-line molecular breeding.

Asunto(s)

Genoma Mitocondrial , Infertilidad Vegetal , Solanum melongena , Solanum melongena/genética , Infertilidad Vegetal/genética , Sistemas de Lectura Abierta/genética , Regulación de la Expresión Génica de las Plantas , Citoplasma/genética , Citoplasma/metabolismo , Genes de Plantas

RESUMEN

CircRNAs are an important class of RNAs with diverse cellular functions in human physiology and disease. A thorough knowledge of circRNAs including their biogenesis and subcellular distribution is important to understand their roles in a wide variety of processes. However, the analysis of circRNAs from total RNA sequencing data remains challenging. Therefore, we developed Calcifer, a versatile workflow for circRNA annotation. Using Calcifer, we analysed APEX-Seq data to compare circRNA occurrence between whole cells, nucleus and subnuclear compartments. We generally find that circRNAs show higher abundance in whole cells compared to nuclear samples, consistent with their accumulation in the cytoplasm. The notable exception is the single-exon circRNA circCANX(9), which is unexpectedly enriched in the nucleus. In addition, we observe that circFIRRE prevails over the linear lncRNA FIRRE in both the cytoplasm and the nucleus. Zooming in on the subnuclear compartments, we show that circRNAs are strongly depleted from nuclear speckles, indicating that excess splicing factors in this compartment counteract back-splicing. Our results thereby provide valuable insights into the subnuclear distribution of circRNAs. Regarding circRNA function, we surprisingly find that the majority of all detected circRNAs possess complete open reading frames with potential for cap-independent translation. Overall, we show that Calcifer is an easy-to-use, versatile and sustainable workflow for the annotation of circRNAs which expands the repertoire of circRNA tools and allows to gain new insights into circRNA distribution and function.

Asunto(s)

Núcleo Celular , ARN Circular , ARN Circular/genética , ARN Circular/metabolismo , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Citoplasma/metabolismo , Citoplasma/genética , Sistemas de Lectura Abierta , Anotación de Secuencia Molecular , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Empalme del ARN , Biología Computacional/métodos , Análisis de Secuencia de ARN

RESUMEN

Two-pore domain K+ (K2P) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that directly target the SF gate (i.e., pHe., Rb+ permeation, membrane depolarization) also opened the cytoplasmic gate. Reciprocally, opening of the lower gate reduced the electric work to open the SF via voltage driven ion binding. Therefore, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate and, vice versa, opening of the lower gate concurrently promote SF gate opening. This concept might extent to other K+ channels that contain two gates (e.g., voltage-gated K+ channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated.

Asunto(s)

Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem , Animales , Humanos , Citoplasma/metabolismo , Células HEK293 , Iones/metabolismo , Mutación , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/genética , Xenopus laevis

RESUMEN

Cytoplasmic male sterility (CMS) arises from the incompatibility between the nucleus and cytoplasm as typical representatives of the chimeric structures in the mitochondrial genome (mitogenome), which has been extensively applied for hybrid seed production in various crops. The frequent occurrence of chimeric mitochondrial genes leading to CMS is consistent with the mitochondrial DNA (mtDNA) evolution. The sequence conservation resulting from faithfully maternal inheritance and the chimeric structure caused by frequent sequence recombination have been defined as two major features of the mitogenome. However, when and how these chimeric mitochondrial genes appear in the context of the highly conserved reproduction of mitochondria is an enigma. This review, therefore, presents the critical view of the research on CMS in plants to elucidate the mechanisms of this phenomenon. Generally, distant hybridization is the main mechanism to generate an original CMS source in natural populations and in breeding. Mitochondria and mitogenomes show pleomorphic and dynamic changes at key stages of the life cycle. The promitochondria in dry seeds develop into fully functioning mitochondria during seed imbibition, followed by massive mitochondria or mitogenome fusion and fission in the germination stage along with changes in the mtDNA structure and quantity. The mitogenome stability is controlled by nuclear loci, such as the nuclear gene Msh1. Its suppression leads to the rearrangement of mtDNA and the production of heritable CMS genes. An abundant recombination of mtDNA is also often found in distant hybrids and somatic/cybrid hybrids. Since mtDNA recombination is ubiquitous in distant hybridization, we put forward a hypothesis that the original CMS genes originated from mtDNA recombination during the germination of the hybrid seeds produced from distant hybridizations to solve the nucleo-cytoplasmic incompatibility resulting from the allogenic nuclear genome during seed germination.

Asunto(s)

Productos Agrícolas , ADN Mitocondrial , Genoma Mitocondrial , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , ADN Mitocondrial/genética , Infertilidad Vegetal/genética , Citoplasma/genética , Citoplasma/metabolismo , Fitomejoramiento/métodos , Mitocondrias/genética , Mitocondrias/metabolismo , Genes Mitocondriales

RESUMEN

Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, belonging to the family Spinareoviridae. Members of the Spinareoviridae family are known to replicate and assemble in cytoplasmic inclusion bodies termed viroplasms; however, the detailed mechanism underlying GCRV viroplasm formation and its specific roles in virus infection remains largely unknown. Here, we demonstrate that GCRV viroplasms form through liquid-liquid phase separation (LLPS) of the nonstructural protein NS80 and elucidate the specific role of LLPS during reovirus infection and immune evasion. We observe that viroplasms coalesce within the cytoplasm of GCRV-infected cells. Immunofluorescence and transmission electron microscopy indicate that GCRV viroplasms are membraneless structures. Live-cell imaging and fluorescence recovery after photobleaching assay reveal that GCRV viroplasms exhibit liquid-like properties and are highly dynamic structures undergoing fusion and fission. Furthermore, by using a reagent to inhibit the LLPS process and constructing an NS80 mutant defective in LLPS, we confirm that the liquid-like properties of viroplasms are essential for recruiting viral dsRNA, viral RdRp, and viral proteins to participate in viral genome replication and virion assembly, as well as for sequestering host antiviral factors for immune evasion. Collectively, our findings provide detailed insights into reovirus viroplasm formation and reveal the specific functions of LLPS during virus infection and immune evasion, identifying potential targets for the prevention and control of this virus. IMPORTANCE: Grass carp reovirus (GCRV) poses a significant threat to the aquaculture industry, particularly in China, where grass carp is a vital commercial fish species. However, detailed information regarding how GCRV viroplasms form and their specific roles in GCRV infection remains largely unknown. We discovered that GCRV viroplasms exhibit liquid-like properties and are formed through a physico-chemical biological phenomenon known as liquid-liquid phase separation (LLPS), primarily driven by the nonstructural protein NS80. Furthermore, we confirmed that the liquid-like properties of viroplasms are essential for virus replication, assembly, and immune evasion. Our study not only contributes to a deeper understanding of GCRV infection but also sheds light on broader aspects of viroplasm biology. Given that viroplasms are a universal feature of reovirus infection, inhibiting LLPS and then blocking viroplasms formation may serve as a potential pan-reovirus inhibition strategy.

Asunto(s)

Carpas , Evasión Inmune , Infecciones por Reoviridae , Reoviridae , Proteínas no Estructurales Virales , Replicación Viral , Reoviridae/genética , Reoviridae/fisiología , Animales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Carpas/virología , Infecciones por Reoviridae/virología , Cuerpos de Inclusión Viral/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/inmunología , Citoplasma/virología , Citoplasma/metabolismo , Genoma Viral , Línea Celular , ARN Viral/genética , Separación de Fases

RESUMEN

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.

Asunto(s)

Actomiosina , ARN Mensajero , Proteínas de Unión al ARN , Transducción de Señal , Proteína de Unión al GTP rhoA , Humanos , Actomiosina/metabolismo , Citoplasma/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Proteína de Unión al GTP rhoA/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo

RESUMEN

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Asunto(s)

Proteínas no Estructurales Virales , Virus del Nilo Occidental , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/fisiología , Humanos , Animales , Replicación Viral , ARN Helicasas/metabolismo , ARN Helicasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Células Vero , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fiebre del Nilo Occidental/virología , Línea Celular , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-box

RESUMEN

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.

Asunto(s)

Células Artificiales , Núcleo Celular , Biología Sintética , Biología Sintética/métodos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Células Artificiales/metabolismo , Transducción de Señal , Citoplasma/metabolismo , Comunicación Celular

RESUMEN

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Asunto(s)

Fagocitosis , Vacuolas , Fagocitosis/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Animales , Humanos , Ratones , Citoplasma/metabolismo , Citoplasma/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos

RESUMEN

The multifunctional tegument protein pUL21 of HSV-2 is phosphorylated in infected cells. We have identified two residues in the unstructured linker region of pUL21, serine 251 and serine 253, as phosphorylation sites. Both phosphorylation sites are absent in HSV-1 pUL21, which likely explains why phosphorylated pUL21 was not detected in cells infected with HSV-1. Cells infected with HSV-2 strain 186 viruses deficient in pUL21 phosphorylation exhibited reductions in both cell-cell spread of virus infection and virus replication. Defects in secondary envelopment of cytoplasmic nucleocapsids were also observed in cells infected with viruses deficient in pUL21 phosphorylation as well as in cells infected with multiple strains of HSV-2 and HSV-1 deleted for pUL21. These results confirm a role for HSV pUL21 in the secondary envelopment of cytoplasmic nucleocapsids and indicate that phosphorylation of HSV-2 pUL21 is required for this activity. Phosphorylation of pUL21 was substantially reduced in cells infected with HSV-2 strain 186 mutants lacking the viral serine/threonine kinase pUL13, indicating a requirement for pUL13 in pUL21 phosphorylation. IMPORTANCE: It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.

Asunto(s)

Herpesvirus Humano 2 , Nucleocápside , Replicación Viral , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiología , Fosforilación , Animales , Chlorocebus aethiops , Humanos , Células Vero , Nucleocápside/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Citoplasma/metabolismo , Citoplasma/virología , Línea Celular , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/genética , Ensamble de Virus , Herpes Simple/virología , Herpes Simple/metabolismo

RESUMEN

In a Comment to the Editor, Skóra raises a concern that the modeling framework implemented in Garner et al. (Biophysical Journal, 2023) neglects a potentially important term in the Brownian dynamics simulation of diffusion. Omission of this diffusivity gradient term may lead to an underestimation of the mean and overestimation of the variance of the cytoplasmic viscosity. In this response, we directly address this concern by incorporating this term into our model and showing that for this data set, its effect is negligible and does not alter the conclusions of this work.

Asunto(s)

Citoplasma , Difusión , Citoplasma/metabolismo , Viscosidad , Modelos Biológicos , Reología , Nanotecnología

RESUMEN

Objective: To analyze the sensitivity of cytoplasmic light-chain immunofluorescence with fluorescence in situ hybridization in bone marrow smears (new FISH) for detecting cytogenetic abnormalities in multiple myeloma (MM) . Methods: 42 MM patients admitted to the First Affiliated Hospital of Nanjing Medical University from April 2022 to October 2023 were enrolled. The patients with MM were detected by new FISH and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) or cytoplasmic immunoglobulin FISH (cIg-FISH) to analyze cytogenetic detection results using combination probes which included 1q21/1p32, p53, IgH, IgH/FGFR3 [t (4;14) ], and IgH/MAF [t (14;16) ]. Results: In 23 patients with MM, the abnormality detection rates of cIg-FISH and new FISH were 95.7% and 100.0%, respectively (P>0.05). The detection rates of 1q21+, 1p32-, p53 deletion, and IgH abnormalities by cIg-FISH and new FISH were consistent, which were 52.2%, 8.7%, 17.4%, and 65.2%, respectively. The results of the two methods further performed with t (4;14) and t (14;16) in patients with IgH abnormalities were identical. The positive rate of t (4;14) was 26.7%, whereas t (14;16) was not detected. In 19 patients with MM, the abnormality detection rates of MACS-FISH and new FISH were 73.7% and 63.2%, respectively (P>0.05). The positivity rate of 1q21+, 1p32- and IgH abnormalities detected by MACS-FISH were slightly higher than those detected by new FISH; however, the differences were not statistically significant (all P values >0.05) . Conclusion: The new FISH method has a higher detection rate of cytogenetic abnormalities in patients with MM and has good consistency with MACS-FISH and cIg-FISH.

Asunto(s)

Médula Ósea , Aberraciones Cromosómicas , Hibridación Fluorescente in Situ , Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/diagnóstico , Hibridación Fluorescente in Situ/métodos , Cadenas Ligeras de Inmunoglobulina/genética , Masculino , Citoplasma/metabolismo , Persona de Mediana Edad , Femenino

RESUMEN

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is the most common primary liver tumor and the second leading cause of cancer-related deaths worldwide. The current study aimed to investigate the clinical relevance of the epidermal growth factor-like domain multiple 6 (EGFL6) expression in HCC and to evaluate whether the expression of EGFL6 in HCC has diagnostic and prognostic significance. PATIENTS AND METHODS: This study aimed to investigate EGFL6 protein expression levels in 260 HCC tissue specimens using immunohistochemical analyses. The immunohistochemical study demonstrated strong EGFL6 expression in the cytoplasm of non-tumor or normal hepatocytes. RESULTS: The findings revealed that 98 patients exhibited low EGFL6 expression, while 162 patients displayed high EGFL6 expression. We explored the associations between cytoplasmic EGFL6 expression and the clinicopathological features of HCC. Decreased cytoplasmic EGFL6 expression exhibited significant correlations with worse cellular differentiation, higher T classification, vascular invasion, higher stage, and tumor recurrence. Survival analyses, using Kaplan-Meier survival curves for HCC patients, revealed that those with reduced cytoplasmic EGFL6 expression experienced significantly worse disease-free survival (DFS) and disease-specific survival (DSS). Univariate and multivariate analyses identified EGFL6 as an independent predictor for decreased expression, differentiation grade, vascular invasion, stage, or recurrence in cases of DFS or DSS in HCC. CONCLUSION: This study represents, to the best of our knowledge, the first investigation into the expression of EGFL6 protein in HCC. Taken together, our findings strongly suggest that EGFL6 likely plays a crucial role in the pathogenesis of HCC and indicates that targeting EGFL6 could be a promising therapeutic strategy.

Asunto(s)

Biomarcadores de Tumor , Proteínas de Unión al Calcio , Carcinoma Hepatocelular , Citoplasma , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Masculino , Femenino , Persona de Mediana Edad , Pronóstico , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Biomarcadores de Tumor/metabolismo , Citoplasma/metabolismo , Anciano , Adulto , Estimación de Kaplan-Meier , Inmunohistoquímica , Estadificación de Neoplasias , Moléculas de Adhesión Celular

Asunto(s)

Mitocondrias , Membranas Mitocondriales , Animales , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Citoplasma/metabolismo

RESUMEN

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are core-shell-type membrane-less organelles typically found in the nucleus of mammalian somatic cells but are absent in mouse oocytes. Here, we deliberately induced the assembly of PML-NBs by injecting mRNA encoding human PML protein (hPML VI -sfGFP) into oocytes and investigated their impact on fertilization in which oocyte/embryos undergo multiple types of stresses. Following nuclear membrane breakdown, preassembled hPML VI -sfGFP mRNA-derived PML-NBs (hmdPML-NBs) persisted in the cytoplasm of oocytes, forming less-soluble debris, particularly under stress. Parthenogenetic embryos that successfully formed pronuclei were capable of removing preassembled hmdPML-NBs from the cytoplasm while forming new hmdPML-NBs in the pronucleus. These observations highlight the beneficial aspect of the PML-NB-free nucleoplasmic environment and suggest that the ability to eliminate unnecessary materials in the cytoplasm of metaphase oocytes serves as a potential indicator of the oocyte quality.

Asunto(s)

Oocitos , Proteína de la Leucemia Promielocítica , Oocitos/metabolismo , Animales , Ratones , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Femenino , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , División Celular Asimétrica , Citoplasma/metabolismo , Núcleo Celular/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Leucemia Promielocítica Aguda/genética

RESUMEN

Nucleoporins, essential proteins building the nuclear pore, are pivotal for ensuring nucleocytoplasmic transport. While traditionally confined to the nuclear envelope, emerging evidence indicates their presence in various cytoplasmic structures, suggesting potential non-transport-related roles. This review consolidates findings on cytoplasmic nucleoporin assemblies across different states, including normal physiological conditions, stress, and pathology, exploring their structural organization, formation dynamics, and functional implications. We summarize the current knowledge and the latest concepts on the regulation of nucleoporin homeostasis, aiming to enhance our understanding of their unexpected roles in physiological and pathological processes.

Asunto(s)

Citoplasma , Proteínas de Complejo Poro Nuclear , Proteínas de Complejo Poro Nuclear/metabolismo , Humanos , Citoplasma/metabolismo , Animales , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular