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[This corrects the article DOI: 10.1371/journal.pgen.1004749.].
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Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.
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Enzimas Reparadoras del ADN , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Humanos , Ratones , Acetilación , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Mamíferos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Polinucleótido 5'-Hidroxil-Quinasa/genéticaRESUMEN
SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.
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Receptores Quiméricos de Antígenos , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Proteínas Ligadas a GPI/metabolismo , Mesotelina , Linfocitos TRESUMEN
A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.
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Roturas del ADN de Doble Cadena , Escherichia coli , Animales , Humanos , Escherichia coli/genética , Reparación del ADN , Reparación del ADN por Unión de Extremidades , ADN , ARN/genética , ADN Ligasa (ATP) , Mamíferos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Enzimas Reparadoras del ADN/genéticaRESUMEN
Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.
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Enzimas Reparadoras del ADN , Leucocitos Mononucleares , Fosfotransferasas (Aceptor de Grupo Alcohol) , Ataxias Espinocerebelosas , Humanos , ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ataxias Espinocerebelosas/genéticaRESUMEN
As part of the antiviral response, cells activate the expressions of type I interferons (IFNs) and proinflammatory mediators to control viral spreading. Viral infections can impact DNA integrity; however, how DNA damage repair coordinates antiviral response remains elusive. Here we report Nei-like DNA glycosylase 2 (NEIL2), a transcription-coupled DNA repair protein, actively recognizes the oxidative DNA substrates induced by respiratory syncytial virus (RSV) infection to set the threshold of IFN-ß expression. Our results show that NEIL2 antagonizes nuclear factor κB (NF-κB) acting on the IFN-ß promoter early after infection, thus limiting gene expression amplified by type I IFNs. Mice lacking Neil2 are far more susceptible to RSV-induced illness with an exuberant expression of proinflammatory genes and tissue damage, and the administration of NEIL2 protein into the airway corrected these defects. These results suggest a safeguarding function of NEIL2 in controlling IFN-ß levels against RSV infection. Due to the short- and long-term side effects of type I IFNs applied in antiviral therapy, NEIL2 may provide an alternative not only for ensuring genome fidelity but also for controlling immune responses.
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ADN Glicosilasas , Interferón beta , Infecciones por Virus Sincitial Respiratorio , Virus Sincitiales Respiratorios , Animales , Ratones , ADN , ADN Glicosilasas/genética , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Interferón beta/genética , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/inmunologíaRESUMEN
Development of protein-enriched chickpea varieties necessitates an understanding of specific genes and key regulatory circuits that govern the synthesis of seed storage proteins (SSPs). Here, we demonstrated the novel involvement of Ca-miR164e-CaNAC100 in regulating SSP synthesis in chickpea. Ca-miRNA164e was significantly decreased during seed maturation, especially in high-protein accessions. The miRNA was found to directly target the transactivation conferring C-terminal region of a nuclear-localized transcription factor, CaNAC100 as revealed using RNA ligase-mediated-rapid amplification of cDNA ends and target mimic assays. The functional role of CaNAC100 was demonstrated through seed-specific overexpression (NACOE) resulting in significantly augmented seed protein content (SPC) consequential to increased SSP transcription. Further, NACOE lines displayed conspicuously enhanced seed weight but reduced numbers and yield. Conversely, a downregulation of CaNAC100 and SSP transcripts was evident in seed-specific overexpression lines of Ca-miR164e that culminated in significantly lowered SPC. CaNAC100 was additionally demonstrated to transactivate the SSP-encoding genes by directly binding to their promoters as demonstrated using electrophoretic mobility shift and dual-luciferase reporter assays. Taken together, our study for the first time established a distinct role of CaNAC100 in positively influencing SSP synthesis and its critical regulation by CamiR164e, thereby serving as an understanding that can be utilized for developing SPC-rich chickpea varieties.
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Cicer , Regulación de la Expresión Génica de las Plantas , MicroARNs , Proteínas de Almacenamiento de Semillas , Factores de Transcripción , Secuencia de Bases , Cicer/genética , Cicer/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Proteínas de Almacenamiento de Semillas/metabolismo , Proteínas de Almacenamiento de Semillas/genética , Semillas/metabolismo , Semillas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Activación Transcripcional/genéticaRESUMEN
DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
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Enzimas Reparadoras del ADN/genética , Reparación del ADN , ADN/genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Inhibidoras de STAT Activados/genética , Procesamiento Proteico-Postraduccional , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Animales , Diferenciación Celular , ADN/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Cultivo Primario de Células , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Transcripción GenéticaRESUMEN
Oral Squamous cell carcinoma (OSCC) is the 14th most frequent cancer with 300,000 new cases and 100,000 deaths reported annually. Even with advanced therapy, the treatment outcomes are poor at advanced stages of the disease. The diagnosis of early OSCC is of paramount clinical value given the high mortality rate associated with the late stages of the disease. Recently, the role of microbiome in the disease manifestation, including oral cancer, has garnered considerable attention. But, to establish the role of bacteria in oral cancer, it is important to determine the differences in the colonization pattern in non-tumour and tumour tissues. In this study, 16S rRNA based metagenomic analyses of 13 tumorous and contralateral anatomically matched normal tissue biopsies, obtained from patients with advanced stage of OSCC were evaluated to understand the correlation between OSCC and oral microbiome. In this study we identified Fusobacterium, Prevotella, Capnocytophaga, Leptotrichia, Peptostreptococcus, Parvimonas and Bacteroidetes as the most significantly enriched taxa in OSCC lesions compared to the non-cancerous tissues. Further, PICRUSt2 analysis unveiled enhanced expression of metabolic pathways associated with L-lysine fermentation, pyruvate fermentation, and isoleucine biosynthesis in those microbes associated with OSCC tissues. These findings provide valuable insights into the distinctive microbial signatures associated with OSCC, offering potential biomarkers and metabolic pathways underlying OSCC pathogenesis. While our focus has primarily centred on microbial signatures, it is essential to recognize the pivotal role of host factors such as immune responses, genetic predisposition, and the oral microenvironment in shaping OSCC development and microbiome composition.
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Green-synthesis of biodegradable polymeric curcumin-nanoparticles using affordable biodegradable polymers to enhance curcumin's solubility and anti-oxidative potential. The curcumin-nanoparticle was prepared based on the ionic-interaction method without using any chemical surfactants, and the particle-size, zeta-potential, surface-morphology, entrapmentefficiency, and in-vitro drug release study were used to optimise the formulation. The antioxidant activity was investigated using H2DCFDA staining in the zebrafish (Danio rerio) model. The mean-diameter of blank nanoparticles was 178.2 nm (±4.69), and that of curcuminnanoparticles was about 227.7 nm (±10.4), with a PDI value of 0.312 (±0.023) and 0.360 (±0.02). The encapsulation-efficacy was found to be 34% (±1.8), with significantly reduced oxidative-stress and toxicity (â¼5 times) in the zebrafish model compared to standard curcumin. The results suggested that the current way of encapsulating curcumin using affordable, biodegradable, natural polymers could be a better approach to enhancing curcumin's water solubility and bioactivity, which could further be translated into potential therapeutics.
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Antioxidantes , Quitosano , Curcumina , Tecnología Química Verde , Goma Arábiga , Nanopartículas , Pez Cebra , Animales , Curcumina/farmacología , Curcumina/química , Curcumina/administración & dosificación , Curcumina/farmacocinética , Nanopartículas/química , Antioxidantes/farmacología , Antioxidantes/química , Antioxidantes/administración & dosificación , Quitosano/química , Goma Arábiga/química , Portadores de Fármacos/química , Liberación de Fármacos , Solubilidad , Estrés Oxidativo/efectos de los fármacos , Tamaño de la PartículaRESUMEN
Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.
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Aeromonas hydrophila , Proteínas de la Membrana Bacteriana Externa , Vacunas Bacterianas , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Nanopartículas , Proteínas Recombinantes , Pez Cebra , Animales , Pez Cebra/inmunología , Aeromonas hydrophila/inmunología , Aeromonas hydrophila/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Administración Oral , Infecciones por Bacterias Gramnegativas/prevención & control , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Vacunación , NanovacunasRESUMEN
Utilizing a combinatorial approach of quantitative trait locus (QTL)-Seq and candidate gene-based association mapping, the QTLs and genes responsible for seed protein content (SPC), a major quality trait in chickpea, were identified. Whole genome re-sequencing based QTL-Seq analysis of bulked recombinant inbred lines from a mapping population contrasting for SPC led to the identification of two QTLs [0.94 Mb on Linkage Group (LG)5 and 1.16 Mb on LG6] encompassing three SNPs, displaying the highest ΔSNP index. These highly significant SNPs and their associated genes were validated in 211 chickpea mini-core accessions varying in SPC, revealing a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) and explaining a phenotypic variation of 23%. This SNP was subsequently converted into a cost effective allele-specific PCR-based marker that could be utilized for rapid screening of SPC during marker assisted breeding. Furthermore, in planta functional validation via knockdown of CaREN1 transcripts led to significant reduction in SPC of chickpea. This decrease in seed protein is likely due to disruption in the formation of CaREN1 protein complexes comprising chaperones, phosphopeptide-binding proteins, and GTPases that mediate folding, transport and accumulation of seed storage proteins, as indicated through affinity purification-mass spectrometry. Taken together, our data will expedite tailoring of chickpea cultivars with augmented SPC.
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Cicer , Cicer/genética , Genoma de Planta/genética , Fitomejoramiento , Polimorfismo de Nucleótido Simple , Genómica/métodos , Semillas/genéticaRESUMEN
Eco-friendly alternatives such as probiotics are needed to prevent economically relevant infectious diseases for a successful disease-free harvest in aquaculture. The use of antibiotics has been the favored practice, but its empirical and indiscriminate use has led to antibiotic resistance in the aquatic environment and residues in the food fish. With this rationale, a probiotic was isolated from tilapia, a commercially important cultured fish worldwide. The characteristics of the probiotic were checked against common bacterial pathogens affecting aquaculture. In vitro tests demonstrated the inhibitory effects of the isolated probiotic on the growth of Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and V. alginolyticus. The candidate probiotic, referred to as TLDK301120C24, was identified as Bacillus subtilis by a battery of biochemical tests and genotypic confirmation by 16S rDNA sequencing. The in vitro results revealed the ability of the probiotic to withstand the gut conditions that included pH range of 3-9, salt concentration of 0.5-6%, and bile salt concentration of up to 6%. The isolate could hydrolyze starch (12-14 mm clearance zone), protein (20-22 mm clearance zone), and cellulose (22-24 mm clearance zone). Further, the inhibitory ability of the probiotic against aquatic pathogens was determined in vivo using gnotobiotic zebrafish by employing a novel approach that involved tagging the probiotic with a red fluorescent protein and the pathogens with a green fluorescent protein, respectively. The colonizing ability of probiotics and its inhibitory effects against the pathogens were evaluated by fluorescence microscopy, PCR, and estimation of viable counts in LBA + Amp plates. Finally, the competitive inhibition and exclusion of fish pathogens A. hydrophila and E. tarda by B. subtilis was confirmed semi-quantitatively, through challenge experiments. This study shows the potential of B. subtilis as a probiotic and its excellent ability to inhibit major fish pathogens in vivo and in vitro. It also shows promise as a potent substitute for antibiotics.
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Enfermedades de los Peces , Probióticos , Tilapia , Animales , Bacillus subtilis/genética , Pez Cebra , Probióticos/farmacología , Antibacterianos/farmacología , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/microbiologíaRESUMEN
Iron deficiency is a major nutritional stress that severely impacts crop productivity worldwide. However, molecular intricacies and subsequent physiological and metabolic changes in response to Fe starvation, especially in leguminous crops like chickpea, remain elusive. In the present study, we investigated physiological, transcriptional, and metabolic reprogramming in two chickpea genotypes (H6013 and L4958) with contrasting seed iron concentrations upon Fe deficiency. Our findings revealed that iron starvation affected growth and physiological parameters of both chickpea genotypes. Comparative transcriptome analysis led to the identification of differentially expressed genes between the genotypes related to strategy I uptake, metal ions transporters, reactive oxygen species-associated genes, transcription factors, and protein kinases that could mitigate Fe deficiency. Our gene correlation network discovered several putative candidate genes like CIPK25, CKX3, WRKY50, NAC29, MYB4, and PAP18, which could facilitate the investigation of the molecular rationale underlying Fe tolerance in chickpea. Furthermore, the metabolite analysis also illustrated the differential accumulation of organic acids, amino acids and other metabolites associated with Fe mobilization in chickpea genotypes. Overall, our study demonstrated the comparative transcriptional dynamics upon Fe starvation. The outcomes of the current endeavor will enable the development of Fe deficiency tolerant chickpea cultivars.
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Cicer , Transcriptoma , Cicer/genética , Perfilación de la Expresión Génica , Genotipo , Hierro/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
RNA helicases couple nucleotide-driven conformational changes to the unwinding of RNA duplexes. Interaction partners can regulate helicase activity by altering the rate constants of these conformational changes. Single-molecule FRET experiments on donor/acceptor-labeled, immobilized molecules are ideally suited to monitor conformational changes in real time and to extract rate constants for these processes. This article provides guidance on how to design, perform, and analyze single-molecule FRET experiments by TIRF microscopy. It covers the theoretical background of FRET and single-molecule TIRF microscopy, the considerations to prepare proteins of interest for donor/acceptor labeling and surface immobilization, and the principles and procedures of data analysis, including image analysis and the determination of FRET time traces, the extraction of rate constants from FRET time traces, and the general conclusions that can be drawn from these data. A case study, using the DEAD-box protein eIF4A as an example, highlights how single-molecule FRET studies have been instrumental in understanding the role of conformational changes for duplex unwinding and for the regulation of helicase activities. Selected examples illustrate which conclusions can be drawn from the kinetic data obtained, highlight possible pitfalls in data analysis and interpretation, and outline how kinetic models can be related to functionally relevant states.
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Transferencia Resonante de Energía de Fluorescencia , Microscopía , ARN Helicasas DEAD-box/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , ARN/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Marine cold seeps transmit fluids between the subseafloor and seafloor biospheres through upward migration of hydrocarbons that originate in deep sediment layers. It remains unclear how geofluids influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. Here we analyzed 172 marine surficial sediments from the deep-water Eastern Gulf of Mexico to assess whether hydrocarbon fluid migration is a mechanism for upward microbial dispersal. While 132 of these sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well-known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the uncultivated bacterial phyla Atribacteria and Aminicenantes and the archaeal order Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum, in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of nonrespiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. These results point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.
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Sedimentos Geológicos/microbiología , Hidrocarburos/metabolismo , Microbiota/fisiología , Agua de Mar/microbiología , Alcanos/metabolismo , Archaea/clasificación , Archaea/metabolismo , Bacterias/clasificación , Bacterias/metabolismo , Biodiversidad , Sedimentos Geológicos/química , Golfo de México , Metagenoma , Metagenómica , Petróleo/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Agua de Mar/químicaRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.
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Ataxina-3/genética , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Enfermedad de Machado-Joseph/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Anciano de 80 o más Años , Animales , Animales Modificados Genéticamente , Ataxina-3/metabolismo , Encéfalo/patología , Línea Celular , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Drosophila , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Masculino , Ratones , Persona de Mediana Edad , Mutación , Péptidos/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB-mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.
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ADN Glicosilasas/metabolismo , Pulmón/metabolismo , FN-kappa B/metabolismo , Animales , Movimiento Celular , Regulación de la Expresión Génica , Inflamación/metabolismo , Pulmón/patología , Ratones , Transducción de SeñalRESUMEN
The proximity-coupling of a chiral non-collinear antiferromagnet (AFM)1-5 with a singlet superconductor allows spin-unpolarized singlet Cooper pairs to be converted into spin-polarized triplet pairs6-8, thereby enabling non-dissipative, long-range spin correlations9-14. The mechanism of this conversion derives from fictitious magnetic fields that are created by a non-zero Berry phase15 in AFMs with non-collinear atomic-scale spin arrangements1-5. Here we report long-ranged lateral Josephson supercurrents through an epitaxial thin film of the triangular chiral AFM Mn3Ge (refs. 3-5). The Josephson supercurrents in this chiral AFM decay by approximately one to two orders of magnitude slower than would be expected for singlet pair correlations9-14 and their response to an external magnetic field reflects a clear spatial quantum interference. Given the long-range supercurrents present in both single- and mixed-phase Mn3Ge, but absent in a collinear AFM IrMn16, our results pave a way for the topological generation of spin-polarized triplet pairs6-8 via Berry phase engineering15 of the chiral AFMs.
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Salmonella is one among the most versatile and resilient enteric pathogens that is known to have developed various survival strategies within the host system. The ability of the bacteria to circumvent the physiological parameters as well as dodge the antimicrobial stress environment within the host is one of the most crucial steps in establishing an infection. With an alarming rise in multi-drug resistant serovars of non-typhoidal Salmonella and lack of vaccine for combatting the infections, behaviour of the bacteria in the presence of host physiological conditions (NaCl, high and low iron) and antibiotics will help in understanding the survival strategies as well as mechanisms of resistance. Two multi-drug resistant and two sensitive serovars of Salmonella Weltevreden and Salmonella Newport isolated from poultry and seafood were used for growth kinetics and virulence gene expression study. The results obtained revealed that despite similar resistance pattern, effect of individual class of antibiotics on the growth of serovars varied. On the contrary, no significant difference was observed in growth pattern on exposure to these in vitro experimental conditions. Nevertheless, coupling these conditions with antibiotics drastically reduced the minimum inhibitory concentration (MIC) of antibiotics in resistant strains. A first of its kind study that draws attention on the significant effect of antibiotics and physiological conditions on MIC between resistant and sensitive non-typhoidal Salmonella serovars and expression of virulence genes from Salmonella pathogenicity island (SPI) 1 and 2 (invA, hilC, fliC2, sseA and ssrB).