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The opportunistic pathogen Pseudomonas aeruginosa PAO1 is infected by the filamentous bacteriophage Pf4. Pf4 virions promote biofilm formation, protect bacteria from antibiotics, and modulate animal immune responses in ways that promote infection. Furthermore, strains cured of their Pf4 infection (ΔPf4) are less virulent in animal models of infection. Consistently, we find that strain ΔPf4 is less virulent in a Caenorhabditis elegans nematode infection model. However, our data indicate that PQS quorum sensing is activated and production of the pigment pyocyanin, a potent virulence factor, is enhanced in strain ΔPf4. The reduced virulence of ΔPf4 despite high levels of pyocyanin production may be explained by our finding that C. elegans mutants unable to sense bacterial pigments through the aryl hydrocarbon receptor are more susceptible to ΔPf4 infection compared to wild-type C. elegans. Collectively, our data support a model where suppression of quorum-regulated virulence factors by Pf4 allows P. aeruginosa to evade detection by innate host immune responses.
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Inovirus , Fagos Pseudomonas , Animales , Pseudomonas aeruginosa , Caenorhabditis elegans/microbiología , Piocianina , Percepción de Quorum , Factores de Virulencia , Biopelículas , Antibacterianos/farmacología , Proteínas BacterianasRESUMEN
Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.
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Células Madre Pluripotentes Inducidas , Humanos , Afidicolina/farmacología , Cafeína , Cromosomas Humanos Par 12 , Hibridación Fluorescente in Situ , Péptidos y Proteínas de Señalización IntracelularRESUMEN
We present a formalism of a neural network encoding bonded interactions in molecules. This intramolecular encoding is consistent with the models of intermolecular interactions previously designed by this group. Variants of the encoding fed into a corresponding neural network may be used to economically improve the representation of torsional degrees of freedom in any force field. We test the accuracy of the reproduction of the ab initio potential energy surface on a set of conformations of two dipeptides, methyl-capped ALA and ASP, in several scenarios. The encoding, either alone or in conjunction with an analytical potential, improves agreement with ab initio energies that are on par with those of other neural network-based potentials. Using the encoding and neural nets in tandem with an analytical model places the agreements firmly within "chemical accuracy" of ±0.5 kcal/mol.
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Dipéptidos , Redes Neurales de la Computación , Conformación MolecularRESUMEN
A key goal of molecular modeling is the accurate reproduction of the true quantum mechanical potential energy of arbitrary molecular ensembles with a tractable classical approximation. The challenges are that analytical expressions found in general purpose force fields struggle to faithfully represent the intermolecular quantum potential energy surface at close distances and in strong interaction regimes; that the more accurate neural network approximations do not capture crucial physics concepts, e.g., nonadditive inductive contributions and application of electric fields; and that the ultra-accurate narrowly targeted models have difficulty generalizing to the entire chemical space. We therefore designed a hybrid wide-coverage intermolecular interaction model consisting of an analytically polarizable force field combined with a short-range neural network correction for the total intermolecular interaction energy. Here, we describe the methodology and apply the model to accurately determine the properties of water, the free energy of solvation of neutral and charged molecules, and the binding free energy of ligands to proteins. The correction is subtyped for distinct chemical species to match the underlying force field, to segment and reduce the amount of quantum training data, and to increase accuracy and computational speed. For the systems considered, the hybrid ab initio parametrized Hamiltonian reproduces the two-body dimer quantum mechanics (QM) energies to within 0.03 kcal/mol and the nonadditive many-molecule contributions to within 2%. Simulations of molecular systems using this interaction model run at speeds of several nanoseconds per day.
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Implantation and subsequent behaviour of heavy noble gases (Ar, Kr, and Xe) in few-layer graphene sheets and in nanodiamonds are studied both using computational methods and experimentally using X-ray absorption spectroscopy. X-ray absorption spectroscopy provides substantial support for Xe-vacancy (Xe-V) defects as main sites for Xe in nanodiamonds. It is shown that noble gases in thin graphene stacks distort the layers, forming bulges. The energy of an ion placed in between flat graphene sheets is notably lower than that in domains with high curvature. However, if the ion is trapped in the curved domain, considerable additional energy is required to displace it. This phenomenon is likely responsible for strong binding of noble gases implanted into disordered carbonaceous phase in meteorites (the Q-component).
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PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Dineínas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión/genética , Proteínas de Caenorhabditis elegans/genética , Dineínas/genética , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Células Madre/citologíaRESUMEN
In the C. elegans germline, maintenance of undifferentiated stem cells depends on the PUF family RNA-binding proteins FBF-1 and FBF-2. FBF-1 and FBF-2 are 89% identical and are required redundantly to silence the expression of mRNAs that promote meiosis. Here we show that, despite their extensive sequence similarity, FBF-1 and FBF-2 have different effects on target mRNAs. FBF-1 promotes the degradation and/or transport of meiotic mRNAs out of the stem cell region, whereas FBF-2 prevents translation. FBF-2 activity depends on the P granule component PGL-1. PGL-1 is required to localize FBF-2 to perinuclear P granules and for efficient binding of FBF-2 to its mRNA targets. We conclude that multiple regulatory mechanisms converge on meiotic RNAs to ensure silencing in germline stem cells. Our findings also support the view that P granules facilitate mRNA silencing by providing an environment in which translational repressors can encounter their mRNA targets immediately upon exit from the nucleus.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Células Madre Embrionarias/metabolismo , Células Germinativas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Meiosis/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN/genéticaRESUMEN
RNA-binding proteins FBF-1 and FBF-2 (FBFs) are required for germline stem cell maintenance and the sperm/oocyte switch in Caenorhabditis elegans, although the mechanisms controlling FBF protein levels remain unknown. We identified an interaction between both FBFs and CSN-5), a component of the constitutive photomorphogenesis 9 (COP9) signalosome best known for its role in regulating protein degradation. Here, we find that the Mpr1/Pad1 N-terminal metalloprotease domain of CSN-5 interacts with the Pumilio and FBF RNA-binding domain of FBFs and the interaction is conserved for human homologs CSN5 and PUM1. The interaction between FBF-2 and CSN-5 can be detected in vivo by proximity ligation. csn-5 mutation results in the destabilization of FBF proteins, which may explain previously observed decrease in the numbers of germline stem and progenitor cells, and disruption of oogenesis. The loss of csn-5 does not decrease the levels of a related PUF protein PUF-3, and csn-5(lf) phenotype is not enhanced by fbf-1/2 knockdown, suggesting that the effect is specific to FBFs. The effect of csn-5 on oogenesis is largely independent of the COP9 signalosome and is cell autonomous. Surprisingly, the regulation of FBF protein levels involves a combination of COP9-dependent and COP9-independent mechanisms differentially affecting FBF-1 and FBF-2. This work supports a previously unappreciated role for CSN-5 in the stabilization of germline stem cell regulatory proteins FBF-1 and FBF-2.
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Complejo del Señalosoma COP9 , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Complejo del Señalosoma COP9/metabolismo , Complejo del Señalosoma COP9/genética , Células Germinativas/metabolismo , Oogénesis/genética , Estabilidad Proteica , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células Madre/metabolismo , Células Madre/citologíaRESUMEN
We model the autoionization of water by determining the free energy of hydration of the major intermediate species of water ions. We represent the smallest ionsâthe hydroxide ion OH-, the hydronium ion H3O+, and the Zundel ion H5O2+âby bonded models and the more extended ionic structures by strong nonbonded interactions (e.g., the Eigen H9O4+ = H3O+ + 3(H2O) and the Stoyanov H13O6+ = H5O2+ + 4(H2O)). Our models are faithful to the precise QM energies and their components to within 1% or less. Using the calculated free energies and atomization energies, we compute the pKa of pure water from first principles as a consistency check and arrive at a value within 1.3 log units of the experimental one. From these calculations, we conclude that the hydronium ion, and its hydrated state, the Eigen cation, are the dominant species in the water autoionization process.
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We incorporate nuclear quantum effects (NQE) in condensed matter simulations by introducing short-range neural network (NN) corrections to the ab initio fitted molecular force field ARROW. Force field NN corrections are fitted to average interaction energies and forces of molecular dimers, which are simulated using the Path Integral Molecular Dynamics (PIMD) technique with restrained centroid positions. The NN-corrected force field allows reproduction of the NQE for computed liquid water and methane properties such as density, radial distribution function (RDF), heat of evaporation (HVAP), and solvation free energy. Accounting for NQE through molecular force field corrections circumvents the need for explicit computationally expensive PIMD simulations in accurate calculations of the properties of chemical and biological systems. The accuracy and locality of pairwise NN NQE corrections indicate that this approach could be applicable to complex heterogeneous systems, such as proteins.
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Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.
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Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.
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Caenorhabditis elegans/embriología , Poro Nuclear/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulación hacia Abajo , Embrión no Mamífero , Mitosis , Poro Nuclear/genética , Poro Nuclear/ultraestructura , Interferencia de ARN , Ribonucleoproteínas Nucleares Pequeñas/antagonistas & inhibidores , Ribonucleoproteínas Nucleares Pequeñas/genética , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
C. elegans P granules are conserved cytoplasmic ribonucleoprotein complexes that are unique to the germline and essential for fertility. During most of germline development, P granules are perinuclear and associate with clusters of nuclear pores. In an RNAi screen against nucleoporins, we have identified a specific nucleoporin essential for P granule integrity and function. The C. elegans homolog of vertebrate Nup98 (CeNup98) is enriched in P granules and associates with the translationally repressed, P granule-enriched mRNA nos-2 (nanos homolog). Loss of CeNup98 causes P granules to disperse in the cytoplasm and to release nos-2 mRNA. Embryos depleted for CeNup98 express a nos-2 3'UTR reporter prematurely. In the mouse, Nup98 immunoprecipitates with the germ granule component MVH. Our findings suggest that, in germ cells, the function of Nup98 extends beyond transport at the nuclear pore to include mRNA regulation in the cytoplasm.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Células Germinativas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Embrión no Mamífero/metabolismo , Inmunoprecipitación , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Proteínas de Complejo Poro Nuclear/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
P-granules are conserved cytoplasmic organelles, similar to nuage, that are present in Caenorhabditis elegans germ cells. Based on the prevailing sterility phenotype of the component mutants, P-granules have been seen as regulators of germ cell development and function. Yet, specific germline defects resulting from P-granule failure vary, depending on which component(s) are inactivated, at which stage of development, as well as on the presence of stress factors during animal culture. This review discusses the unifying themes in many P-granule functions, with the main focus on their role as organizing centers nucleating RNA regulation in the germ cell cytoplasm.
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Gránulos Citoplasmáticos/metabolismo , Células Germinativas/citología , Procesos de Determinación del Sexo/genética , Transporte Activo de Núcleo Celular , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Elementos Transponibles de ADN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Skin fibroblasts obtained from a 5-year-old girl with genetically proven (two heterozygous mutations in ARSB gene) and clinically manifested mucopolysaccharidosis type VI were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors namely SOX2, OCT3/4, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, had a normal karyotype and the potential to differentiate into three germ layers in spontaneous differentiation assay. The line may be used for cell differentiation and pharmacological investigations, and also may provide a model for development of a personalized treatment including drug screening and genome editing.
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Células Madre Pluripotentes Inducidas , Mucopolisacaridosis VI , Femenino , Humanos , Preescolar , Células Madre Pluripotentes Inducidas/metabolismo , Mucopolisacaridosis VI/metabolismo , Factor 4 Similar a Kruppel , Diferenciación Celular/genética , Fibroblastos/metabolismo , Reprogramación CelularRESUMEN
Urine cells obtained from a 14-year-old man with genetically proven (ACVR1: c.6176G > A) and clinically manifested fibrodysplasia ossificans progressiva were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors such as OCT3/4, SOX2, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay and had a normal karyotype. The iPSC line may provide a model for development of a personalized treatment including genome editing and drug screening, may be used for disease modelling, cell differentiation and pharmacological investigations. .
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Células Madre Pluripotentes Inducidas , Miositis Osificante , Masculino , Humanos , Adolescente , Células Madre Pluripotentes Inducidas/metabolismo , Miositis Osificante/metabolismo , Factor 4 Similar a Kruppel , Diferenciación Celular/genética , Virus Sendai/genética , Reprogramación CelularRESUMEN
Dynein light chain (DLC-1) is a light chain component of the dynein motor complex, it functions as an allosteric regulator of multi-subunit protein complexes and promotes P granule integrity in the C. elegans embryo. P granules are RNA-protein complexes located in the C. elegans germline that are important for RNA regulation and fertility. To further study the role of DLC-1 during C. elegans embryogenesis we performed quantitative tandem mass tag mass spectrometry on embryos after dlc-1 knock down. The amount of core P granule components and nucleoporin proteins did not change after dlc-1(RNAi). These results show that DLC-1 does not help regulate P granule protein levels and support the model that DLC-1 facilitates phase separation of P granule components in vivo .
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Germ granules are cytoplasmic assemblies of RNA-binding proteins (RBPs) required for germ cell development and fertility. During the first four cell divisions of the Caenorhabditis elegans zygote, regulated assembly of germ (P) granules leads to their selective segregation to the future germ cell. Here we investigate the role of DLC-1, a hub protein implicated in stabilization and function of diverse protein complexes, in maintaining P granule integrity. We find that DLC-1 directly interacts with several core P granule proteins, predominantly during embryogenesis. The loss of dlc-1 disrupts assembly of P granule components into phase-separated organelles in the embryos, regardless of whether or not DLC-1 directly interacts with these proteins. Finally, we infer that P granule dispersal in the absence of dlc-1 is likely independent of DLC-1's function as a subunit of the dynein motor and does not result from a loss of cell polarity.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Dineínas/metabolismo , Gránulos de Ribonucleoproteína de Células Germinales , Células Germinativas/metabolismoRESUMEN
Skin fibroblasts obtained from a 20-year-old woman with clinically manifested and genetically proven (F508del/CFTRdele2.3) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using Sendai virus-based reprogramming vectors including the four Yamanaka factors, OCT3/4, SOX2, KLF4, and c-MYC. The iPSCs showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. This iPSC line may be used for development of a personalized treatment including genome editing, disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.
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Fibrosis Quística , Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular/genética , Reprogramación Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Adulto JovenRESUMEN
Induced pluripotent stem cells (iPSCs) was successfully generated from skin fibroblast obtained from patient with cystic fibrosis by using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit, which contain three vectors preparation: polycistronic Klf4-Oct3/4-Sox2, cMyc, and Klf4. Created iPSC lines showed a normal karyotype, expressed pluripotency markers and demonstrated the potential to differentiate into three germ layers in spontaneous differentiation assay.