RESUMEN
RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.
Asunto(s)
Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Inestabilidad Genómica/genética , Origen de Réplica/genética , Línea Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Reparación del ADN/genética , ADN de Cadena Simple/genética , Células HEK293 , Células HeLa , Humanos , Unión Proteica/genética , Recombinasa Rad51/genéticaRESUMEN
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN Primasa/metabolismo , Reparación del ADN , Replicación del ADN , ADN de Neoplasias/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Fase G2 , Enzimas Multifuncionales/metabolismo , Neoplasias/metabolismo , Fase S , Antineoplásicos/farmacología , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Primasa/genética , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Inestabilidad Genómica , Células HEK293 , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Enzimas Multifuncionales/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factores de Tiempo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.
Asunto(s)
Roturas del ADN de Cadena Simple , Replicación del ADN , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína-Arginina N-Metiltransferasas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.
Asunto(s)
ADN Primasa/metabolismo , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , ADN/genética , Daño del ADN/genética , Daño del ADN/fisiología , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Primasa/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/fisiología , Células HEK293 , Humanos , Enzimas Multifuncionales/fisiología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.
Asunto(s)
Proteína BRCA1 , Replicación del ADN , Proteínas de Unión al ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Línea Celular Tumoral , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Ubiquitinación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Reparación del ADNRESUMEN
BACKGROUND: Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. METHODS: In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. RESULTS: The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9â µg/mL (158 ± 97.2â µM) and 0.2 ± 0.04â µg/mL (0.5 ± 0.1â µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02â µg/mL (0.2 ± 0.07â µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43â µg/mL (6.0 ± 1.2â µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. CONCLUSIONS: Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.
Asunto(s)
Antibacterianos , Escherichia coli , Staphylococcus aureus Resistente a Meticilina , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Antibacterianos/farmacología , Simulación del Acoplamiento Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Esteroides/farmacología , Esteroides/química , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Grupo Citocromo b , Citocromos/antagonistas & inhibidores , Citocromos/metabolismoRESUMEN
Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants (Capsicum chinense x frutescens), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum. In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.
Asunto(s)
Secuencia de Aminoácidos , Capsicum , Capsicum/química , Capsicum/genética , Antibacterianos/farmacología , Antibacterianos/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/genética , Datos de Secuencia Molecular , Pruebas de Sensibilidad Microbiana , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Modelos MolecularesRESUMEN
The f block is a comparatively understudied group of elements that find applications in many areas. Continued development of technologies involving the lanthanides (Ln) and actinides (An) requires a better fundamental understanding of their chemistry. Specifically, characterizing the electronic structure of the f elements presents a significant challenge due to the spatially core-like but energetically valence-like nature of the f orbitals. This duality led f-block scientists to hypothesize for decades that f-block chemistry is dominated by ionic metal-ligand interactions with little covalency because canonical covalent interactions require both spatial orbital overlap and orbital energy degeneracy. Recent studies on An compounds have suggested that An ions can engage in appreciable orbital mixing between An 5f and ligand orbitals, which was attributed to "energy-degeneracy-driven covalency". This model of bonding has since been a topic of debate because different computational methods have yielded results that support and refute the energy-degeneracy-driven covalency model. In this Viewpoint, literatures concerning the metal- and ligand-edge X-ray absorption near-edge structure (XANES) of five tetravalent f-block systemsâMO2 (M = Ln, An), LnF4, MCl62-, and [Ln(NP(pip)3)4]âare compiled and discussed to explore metal-ligand bonding in f-block compounds through experimental metrics. Based on spectral assignments from a variety of theoretical models, covalency is seen to decrease from CeO2 and PrO2 to TbO2 through weaker ligand-to-metal charge-transfer (LMCT) interactions, while these LMCT interactions are not observed in the trivalent Ln sesquixodes until Yb. In comparison, while XANES characterization of AnO2 compounds is scarce, computational modeling of available X-ray absorption spectra suggests that covalency among AnO2 reaches a maximum between Am and Cm. Moreover, a decrease in covalency is observed upon changing ligands while maintaining an isostructural coordination environment from CeO2 to CeF4. These results could allude to the importance of orbital energy degeneracy in f-block bonding, but there are a variety of data gaps and conflicting results from different modeling techniques that need to be addressed before broad conclusions can be drawn.
RESUMEN
Elucidating the relationship between metal-ligand interactions and the associated conformational change of the ligand is critical for understanding the separation of lanthanides via ion binding. Here we examine DTPA, a multidentate ligand that binds lanthanides, in its free and metal bound conformations using ultrafast polarization dependent vibrational spectroscopy. The polarization dependent pump-probe spectra were analyzed to extract the isotropic and anisotropic response of DTPA's carbonyl groups in the 1550-1650 cm-1 spectral region. The isotropic response reports on the population relaxation of the carbonyl stretching modes. We find that the isotropic response is influenced by the identity of the metal ion. The anisotropy decay of the carbonyl stretching modes reveals a faster decay in the lanthanide-DTPA complexes than in the free DTPA ligand. We attribute the anisotropy decay to energy transfer among the different carbonyl sites - where the conformational change results in an increased coupling between the carbonyl sites of metal-bound DTPA complexes. DFT calculations and theoretical simulations of energy transfer suggest that the carbonyl sites are more strongly coupled in the metal-bound conformations compared to the free DTPA. The stronger coupling in the metal bound DTPA conformation leads to efficient energy transfer among the different carbonyl sites. Comparing the rate of anisotropy decay across the series of metal bound DTPA complexes we find that the anisotropy is sensitive to the charge density of the central metal ion, and thus can serve as a molecular scale reporter for lanthanide ion binding.
RESUMEN
SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Ratones , Staphylococcus aureus/metabolismo , Factores de Transcripción/metabolismo , Infecciones Estafilocócicas/microbiología , Factor sigma/genética , Factor sigma/metabolismo , Estabilidad del ARN , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/metabolismoRESUMEN
Chelators based on hydroxypyridinones have utility in incorporating radioactive metal ions into diagnostic and therapeutic agents used in nuclear medicine. Over the course of our hydroxypyridinone studies, we have prepared two novel chelators, consisting of a cyclen (1,4,7,10-tetraazacyclododecane) ring bearing two pendant hydroxypyridinone groups, appended via methylene acetamide motifs at either the 1,4-positions (L1) or 1,7-positions (L2) of the cyclen ring. In radiolabeling reactions of L1 or L2 with the γ-emitting radioisotope, [111In]In3+, we have observed radiometal-mediated hydrolysis of a single amide group of either L1 or L2. The reaction of either [111In]In3+ or [natIn]In3+ with either L1 or L2, in aqueous alkaline solutions at 80 °C, initially results in formation of [In(L1)]+ or [In(L2)]+, respectively. Over time, each of these species undergoes In3+-mediated hydrolysis of a single amide group to yield species in which In3+ remains coordinated to the resultant chelator, which consists of a cyclen ring bearing a single hydroxypyridinone group and a single carboxylate group. The reactivity toward hydrolysis is higher for the L1 complex compared to that for the L2 complex. Density functional theory calculations corroborate these experimental findings and importantly indicate that the activation energy required for the hydrolysis of L1 is significantly lower than that required for L2. This is the first reported example of a chelator undergoing radiometal-mediated hydrolysis to form a radiometalated complex. It is possible that metal-mediated amide bond cleavage is a source of instability in other radiotracers, particularly those in which radiometal complexation occurs in aqueous, basic solutions at high temperatures. This study highlights the importance of appropriate characterization of radiolabeled products.
RESUMEN
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Asunto(s)
Enzimas AlkB/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Complejos Multiproteicos/metabolismo , Transducción de Señal , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Alquilantes/farmacología , Alquilación , Secuencia de Aminoácidos , Aductos de ADN/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Genes Ligados a X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , ARN Polimerasa II/metabolismo , Empalme del ARN , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patología , UbiquitinaciónRESUMEN
BACKGROUND: Comprehensive Geriatric Assessment (CGA)is a widely accepted intervention for frailty and can be cost-effective within a primary care setting. OBJECTIVE: To explore the feasibility of identifying older adults with frailty and assess the subsequent implementation of a tailored CGA with care and support plan by Advanced Nursing Practitioners (ANPs). METHODS: A mixed-method parallel randomised controlled trial was conducted. Participants were recruited from two General Practice (GP) centres between January and June 2019. Older adults with confirmed frailty, as assessed by practice nurses, were randomised, using a web service, to the intervention or treatment-as-usual (TAU) groups for six months with an interim and a final review. Data were collected on feasibility, health service usage, function, quality of life, loneliness, and participants' experience and perception of the intervention. Non-parametric tests were used to analyse within and between-group differences. P-values were adjusted to account for type I error. Thematic analysis of qualitative data was conducted. RESULTS: One hundred sixty four older adults were invited to participate, of which 44.5% (n = 72) were randomised to either the TAU (n = 37) or intervention (n = 35) groups. All participants in the intervention group were given the baseline, interim and final reviews. Eight participants in each group were lost to post-intervention outcome assessment. The health service use (i.e. hospital admissions, GP/emergency calls and GP/Accident Emergency attendance) was slightly higher in the TAU group; however, none of the outcome data showed statistical significance between-group differences. The TAU group showed a deterioration in the total functional independence and its motor and cognition components post-intervention (p < .05), though the role limitation due to physical function and pain outcomes improved (p < .05). The qualitative findings indicate that participants appreciated the consistency of care provided by ANPs, experienced positive therapeutic relationship and were connected to wider services. DISCUSSION: Frailty identification and intervention delivery in the community by ANPs were feasible. The study shows that older adults with frailty living in the community might benefit from intervention delivered by ANPs. It is suggested to examine the cost-effectiveness of the intervention in sufficiently powered future research. TRIAL REGISTRATIONS: The protocol is available at clinicaltirals.gov, ID: NCT03394534; 09/01/2018.
Asunto(s)
Fragilidad , Evaluación Geriátrica , Humanos , Anciano , Estudios de Factibilidad , Fragilidad/diagnóstico , Fragilidad/terapia , Calidad de Vida , Atención Primaria de SaludRESUMEN
Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.
Asunto(s)
Replicación del ADN , Lamina Tipo A/metabolismo , Modelos Biológicos , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Animales , Células HEK293 , Humanos , Lamina Tipo A/genética , Ratones , Ratones Noqueados , Recombinasa Rad51/genética , Proteína de Replicación A/genéticaRESUMEN
Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings. This resilience is often attributed to a proclivity for biofilm formation, which facilitates a higher tolerance toward external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Here, we performed RNA sequencing (RNA-seq) on biofilm and planktonic populations for the multidrug-resistant isolate AB5075 and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm-forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), had a highly mucoid colony phenotype, enhanced tolerance to polysaccharide degradation, altered antibiotic tolerance, and diminished adherence to abiotic surfaces. RNA-seq of the cspC mutant revealed 201 genes with altered expression, including the downregulation of pili and fimbria genes and the upregulation of multidrug efflux pumps. Using transcriptional arrest assays, it appears that CspC mediates its effects, at least in part, through RNA chaperone activity, influencing the half-life of several important transcripts. Finally, we show that CspC is required for survival during challenge by the human immune system and is key for A. baumannii dissemination and/or colonization during systemic infection. Collectively, our work identifies a cadre of new biofilm-associated genes within A. baumannii and provides unique insight into the global regulatory network of this emerging human pathogen.
Asunto(s)
Acinetobacter baumannii , Humanos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Biopelículas , Proteínas y Péptidos de Choque por Frío/genética , Proteínas y Péptidos de Choque por Frío/metabolismo , Polisacáridos/metabolismo , Proteína C/metabolismo , Proteína C/farmacología , ARN/metabolismo , Virulencia/genéticaRESUMEN
Characterization of transcriptional networks is one of the main strategies used to understand how bacteria interact with their environment. To reveal novel regulatory elements in the human pathogen Staphylococcus aureus, we adapted a traditional transduction protocol to be used in a high-throughput format in combination with the publicly available S. aureus Nebraska Transposon Mutant Library. Specifically, plasmid transductions are performed in 96-well format, so that a single plasmid can be simultaneously transferred into numerous recipient strains. When used in conjunction with bioluminescent reporter constructs, this strategy enables parallel and continuous monitoring of downstream transcriptional effects of hundreds of defined mutations. Here, we use this workflow in a proof-of-concept study to identify novel regulators of the staphylococcal metalloprotease aureolysin. Importantly, this strategy can be utilized with any other bacterium where plasmid transduction is possible, making it a versatile and efficient tool to probe transcriptional regulatory connections.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Biblioteca de Genes , Humanos , Plásmidos/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/enzimología , Lisina/metabolismo , Proteínas de la Membrana/metabolismo , Metilación , Monoéster Fosfórico Hidrolasas/metabolismo , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.
Asunto(s)
Exoma , Enfermedades no Diagnosticadas , Exoma/genética , Pruebas Genéticas , Humanos , Fenotipo , Investigación Biomédica Traslacional , Secuenciación del ExomaRESUMEN
The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL.
Asunto(s)
Drosophila/genética , Regulación de la Expresión Génica , ARN Nuclear Pequeño/genética , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , Animales , Inmunoprecipitación de Cromatina , Drosophila/metabolismo , Técnica del Anticuerpo Fluorescente , Células HCT116 , Humanos , Inmunoprecipitación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Glándulas Salivales/citología , Glándulas Salivales/metabolismoRESUMEN
Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.