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Background: There is limited research on the intra-individual efficacy of ventricular pacing minimization algorithms developed by Biotronik-the Ventricular Pace Suppression algorithm (VpS) and the Intrinsic Rhythm Support plus algorithm (IRSplus) (BIOTRONIK SE & Co. KG, Berlin, Germany). We performed a randomized pilot trial that evaluated the efficacy of two algorithms in patients with symptomatic sinus node dysfunction (SND) who received a dual-chamber pacemaker. Methods: The trial was conducted in 11 tertiary hospitals in South Korea. The patients were randomized to either the VpS or IRSplus algorithm group after a 3-month period of fixed atrioventricular (AV) delay. The primary outcome was the ventricular pacing percentage (Vp%) at each follow-up visit. The secondary outcomes were the occurrence of heart failure (HF) and atrial fibrillation (AF) during the study period. Results: Data from 131 patients were analyzed. Initially, their average Vp% over 3 months with a fixed AV interval was 14.1 ± 19.4%. Patients were randomly assigned to VpS and IRSplus groups, with 66 and 65 in each. Algorithms reduced average Vp% to 4.0 ± 11.3% at 9 months and 6.7 ± 14.9% at 15 months. These algorithms were more effective for patients with paced AV delay (PAVD) ≤300 ms compared to those with PAVD >300 ms. Both algorithms were equally effective in reducing Vp%. Clinical AF or HF hospitalization was not observed during the study period. Conclusion: The VpS and IRSplus algorithms are effective and safe in minimizing unnecessary ventricular pacing in patients with SND.
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Background: Current guidelines consider atrial fibrillation (AF) type as the prognostic factor for a recommendation of catheter ablation. We aimed to determine whether LA and LA appendage (LAA) volumes measured using multislice computed tomography (MSCT) were related to long-term outcomes in AF following radiofrequency catheter ablation (RFCA). Methods: We evaluated 152 consecutive patients with drug-refractory AF (median age, 55.8 ± 9.6 years), including 110 male patients, who underwent RFCA in a single center. All patients underwent MSCT imaging for anatomical assessment. The endpoint of this study was documented AF recurrence after RFCA. Results: The overall procedure success rate was 77.6% (n = 118) during a mean follow-up period of 12.6 months. The LA volume was significantly larger for those who experienced AF recurrence after RFCA than for the patients without recurrent AF after the procedure (153.8 ± 29.9 mL vs. 139.2 ± 34.1 mL, p = 0.025). However, LAA volumes were nearly equivalent between the patients with and without AF recurrence after RFCA (16.2 ± 6.3 mL and 14.7 ± 6.5 mL, respectively; p = 0.235). LA volume ≥ 153.2 mL was the optimal cutoff value for estimating AF recurrence after RFCA, with 94% sensitivity and 66% specificity. LA volume remained an independent predictor of both AF recurrence and permanent AF. Conclusions: LA volume as assessed by MSCT might be helpful for identifying patients likely to achieve successful AF ablation. LA volume ≥ 153.2 mL, but not LAA volume, showed good accuracy in predicting AF recurrence after RFCA.
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OBJECTIVE: Low-density lipoprotein cholesterol (LDL-C) has been commonly calculated by equations, but their performance has not been entirely satisfactory. This study aimed to develop a more accurate LDL-C prediction model using machine learning methods. METHODS: The study involved predicting directly measured LDL-C, using individual characteristics, lipid profiles, and other laboratory results as predictors. The models applied to predict LDL-C values were multiple regression, penalized regression, random forest, and XGBoost. Additionally, a novel 2-step prediction model was developed and introduced. The machine learning methods were evaluated against the Friedewald, Martin, and Sampson equations. RESULTS: The Friedewald, Martin, and Sampson equations had root mean squared error (RMSE) values of 12.112, 8.084, and 8.492, respectively, whereas the 2-step prediction model showed the highest accuracy, with an RMSE of 7.015. The LDL-C levels were also classified as a categorical variable according to the diagnostic criteria of the dyslipidemia treatment guideline, and concordance rates were calculated between the predictive values obtained from each method and the directly measured ones. The 2-step prediction model had the highest concordance rate (85.1%). CONCLUSION: The machine learning method can calculate LDL-C more accurately than existing equations. The proposed 2-step prediction model, in particular, outperformed the other machine learning methods.
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LDL-Colesterol , Aprendizado de Máquina , Humanos , LDL-Colesterol/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , IdosoRESUMO
Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.
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Neurilemoma , Neurofibromatoses , Neurofibromatose 1 , Neurofibromatose 2 , Neoplasias Cutâneas , Animais , Humanos , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Neurofibromatose 2/patologia , Neurofibromatoses/genética , Neurofibromatoses/terapia , Neurofibromatoses/diagnóstico , Neurilemoma/genética , Neurilemoma/terapia , Neurilemoma/diagnósticoRESUMO
Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.
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Cromatina , Estruturas R-Loop , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , CoesinasRESUMO
In the human cell nucleus, dynamically organized chromatin is the substrate for gene regulation, DNA replication, and repair. A central mechanism of DNA loop formation is an ATPase motor cohesin-mediated loop extrusion. The cohesin complexes load and unload onto the chromosome under the control of other regulators that physically interact and affect motor activity. Regulation of the dynamic loading cycle of cohesin influences not only the chromatin structure but also genome-associated human disorders and aging. This review focuses on the recently spotlighted genome organizing factors and the mechanism by which their dynamic interactions shape the genome architecture in interphase.
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Cromatina , Replicação do DNA , Humanos , Cromatina/genética , Interfase/genética , Regulação da Expressão Gênica , Núcleo CelularRESUMO
There have been many attempts to understand the central principle of life mediated by DNA-protein interactions surrounding complex environments. Still, the mechanistic insight of individual protein functions has been lacking in traditional ensemble assays. Thus, techniques visualizing a single molecule have emerged to uncover the discrete roles of DNA-protein interactions and their biophysical properties. This paper will review the advances in single-molecule tools imaging long genomic DNA and their applications in studying dynamic protein interactions. We focus on the three representative techniques, including molecular combing, nanochannel confinement, and DNA curtain assays, which use fluid-driven force to elongate the individual DNA. We provide an integrated perspective and a direction for future use to those who want to observe single DNA molecules along with their cellular factor of interest and employ them for dissecting protein function.
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Cromatina , DNA , Biofísica , Fenômenos BiofísicosRESUMO
Experimental tools and resources, such as animal models, cell lines, antibodies, genetic reagents and biobanks, are key ingredients in biomedical research. Investigators face multiple challenges when trying to understand the availability, applicability and accessibility of these tools. A major challenge is keeping up with current information about the numerous tools available for a particular research problem. A variety of disease-agnostic projects such as the Mouse Genome Informatics database and the Resource Identification Initiative curate a number of types of research tools. Here, we describe our efforts to build upon these resources to develop a disease-specific research tool resource for the neurofibromatosis (NF) research community. This resource, the NF Research Tools Database, is an open-access database that enables the exploration and discovery of information about NF type 1-relevant animal models, cell lines, antibodies, genetic reagents and biobanks. Users can search and explore tools, obtain detailed information about each tool as well as read and contribute their observations about the performance, reliability and characteristics of tools in the database. NF researchers will be able to use the NF Research Tools Database to promote, discover, share, reuse and characterize research tools, with the goal of advancing NF research. Database URL: https://tools.nf.synapse.org/.
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Pesquisa Biomédica , Neurofibromatoses , Animais , Bases de Dados Factuais , Camundongos , Reprodutibilidade dos TestesRESUMO
BACKGROUND AND OBJECTIVES: Atrial tachycardias (ATs) from noncoronary aortic cusp (NCC) uncovered after radiofrequency ablation for atrial fibrillation (AF) are rarely reported. This study was conducted to investigate the prevalence and clinical characteristics of NCC ATs detected during AF ablation and compare their characteristics with de novo NCC ATs without AF. METHODS: Consecutive patients who underwent radiofrequency catheter ablation for AF were reviewed from the multicenter AF ablation registry of 11 tertiary hospitals. The clinical and electrophysiological characteristics of NCC AT newly detected during AF ablation were compared with its comparators (de novo NCC AT ablation cases without AF). RESULTS: Among 10,178 AF cases, including 1,301 redo ablation cases, 8 (0.08%) NCC AT cases were discovered after pulmonary vein isolation (PVI; 0.07% in first ablation and 0.15% in redo ablation cases). All ATs were reproducibly inducible spontaneously or with programmed atrial stimulation without isoproterenol infusion. The P-wave morphological features of tachycardia were variable depending on the case, and most cases exhibited 1:1 atrioventricular conduction. AF recurrence rate after PVI and NCC AT successful ablation was 12.5% (1 of 8). Tachycardia cycle length was shorter than that of 17 de novo ATs from NCC (303 versus 378, p=0.012). No AV block occurred during and after successful AT ablation. CONCLUSIONS: Uncommon NCC ATs (0.08% in AF ablation cases) uncovered after PVI, showing different characteristics compared to de-novo NCC ATs, should be suspected irrespective of P-wave morphologies when AT shows broad propagation from the anterior interatrial septum.
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OBJECTIVES: This study aimed to investigate the prevalence of patients with rheumatoid arthritis (RA) at a high risk of major osteoporosis (OP)-related fractures and the status of OP-related medical treatment for these patients. METHODS: We enrolled 120 patients aged ≥40 years (average, 69.1 years) with RA. The Fracture Risk Assessment Tool (FRAX®) was used to evaluate the fracture risk. Of the 120 patients, the femoral neck bone mineral density (BMD) was evaluated in 102 patients, and their FRAX® scores were calculated alongside the BMD values. Patients observed to be at a high risk of a major OP-related fracture (10-year probability >20% or hip fracture risk >3%), according to FRAX®, were identified as those requiring OP treatment; medication ratio for OP (percentage of patients actually receiving medication among patients requiring OP treatment) was assessed. RESULTS: OP treatment was indicated in 75 (63%) patients; the medication ratio for OP was 49%. The use of biological disease-modifying anti-rheumatic drugs and corticosteroids showed a positive effect; however, the use of methotrexate showed a negative effect on the medication ratio. CONCLUSION: The number of potential patients requiring OP treatment is underestimated. All patients with RA should be assessed to determine their eligibility for OP treatment.
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Artrite Reumatoide , Osteoporose , Absorciometria de Fóton , Adulto , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/epidemiologia , Densidade Óssea , Humanos , Japão/epidemiologia , Osteoporose/induzido quimicamente , Osteoporose/diagnóstico , Osteoporose/tratamento farmacológico , Fraturas por Osteoporose/induzido quimicamente , Fraturas por Osteoporose/epidemiologia , Medição de Risco , Fatores de RiscoRESUMO
The spatial organization of the genome is critical for fundamental biological processes, including transcription, genome replication, and segregation. Chromatin is compacted and organized with defined patterns and proper dynamics during the cell cycle. Aided by direct visualization and indirect genome reconstruction tools, recent discoveries have advanced our understanding of how interphase chromatin is dynamically folded at the molecular level. Here, we review the current understanding of interphase genome organization with a focus on the major regulator of genome structure, the cohesin complex. We further discuss how cohesin harnesses the energy of ATP hydrolysis to shape the genome by extruding chromatin loops.
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Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Genoma , Genômica , Adenosina Trifosfatases/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Genômica/métodos , Humanos , CoesinasRESUMO
Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.
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Candida albicans/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Candida albicans/citologia , Linhagem Celular Tumoral , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Mutação , Fenótipo , Príons/química , Agregados Proteicos , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.
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Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , DNA/química , Conformação de Ácido Nucleico , ATPases Translocadoras de Prótons/química , Humanos , Nucleossomos/química , Multimerização Proteica , Imagem Individual de Molécula , CoesinasRESUMO
Intrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutLα (Mlh1-Pms1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the adenosine triphosphate hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutLα in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.
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Proteínas Intrinsicamente Desordenadas/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Proteínas de Saccharomyces cerevisiae/genética , Catálise , Cromatina/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , Mutação , Proteoma/genética , Saccharomyces cerevisiaeRESUMO
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
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Actinomycetales/enzimologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , DNA Viral/metabolismo , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
BACKGROUND: The pathogenesis of asthma, which is an allergic lung disease, is associated with a variety of allergens such as house dust mite, pollen, and mould, IgE containing serum IgE and allergen-specific-IgE, and inflammatory cytokines including thymus and activation-regulated chemokine (TARC)/CCL17. Because aging is an essential factor in the pathogenesis of asthma, we examined biomarkers related to asthmatic subjects depending on age. RESULTS: Physiological indices such as FEV1(forced expiratory capacity in 1 s), FEV1 (% predicted), and FEV1/FVC(forced vital capacity) (%) in asthmatic subjects were lower than those in normal subjects. Total IgE, Der p1 specific IgE, and Der f1 specific IgE were elevated in serum of asthmatics relative to normal individuals. Regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5 in serum and interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein (MCP)-1/CCL2, RANTES, and macrophage inflammatory protein (MIP)-1α/CCL3 in bronchoalveolar lavage fluid (BALF) of asthmatic subjects were higher than in normal individuals. Upon classification of experimental groups depending on age, physiological indices and Der p1-specific IgE (class) were decreased in middle aged adult and elderly adult groups relative to the young adult group. TARC levels in serum were strongly elevated in the elderly adult group relative to the young adult and the middle aged adult groups. TARC in serum was related to total IgE in serum in the elderly adult group. CONCLUSIONS: Taken together, although TARC in serum and BALF is not different between normal and asthmatic individuals, TARC increases in serum of elderly asthmatic subjects. The level of TARC has a positive effect on the level of IgE in the elderly adult group. These findings may help us better understand the relationship of pathogenesis of allergic diseases and aging.
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DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.
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Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/enzimologia , Reparo de DNA por Recombinação , Imagem Individual de Molécula , Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Difusão , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteína Homóloga a MRE11 , Microscopia de Fluorescência , Proteínas Nucleares/genética , Nucleossomos/genética , Fatores de TempoRESUMO
Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.
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Proteínas de Ligação a DNA/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Ácidos Nucleicos Imobilizados/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Microtecnologia/métodos , Nucleoproteínas/metabolismo , Imagem Óptica/instrumentação , Animais , Proteínas de Ligação a DNA/análise , Difusão , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ácidos Nucleicos Imobilizados/química , Técnicas Analíticas Microfluídicas/métodos , Nucleoproteínas/análise , Imagem Óptica/métodos , Reparo de DNA por Recombinação , Raios UltravioletaRESUMO
Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90-100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5'-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.
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DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Imagem Individual de Molécula/métodos , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , DNA de Cadeia Simples/genética , DNA Viral/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Repetições de TrinucleotídeosRESUMO
The Milken Institute Center for Strategic Philanthropy has launched a Giving Smarter Program in Epilepsy to inform philanthropists on the state of the science for the epilepsy field, key challenges, and solutions to address them. As part of the program, the Milken Institute Center for Strategic Philanthropy hosted a retreat to identify strategic investments that would accelerate epilepsy research to ultimately improve care. The top three prioritized opportunities from the retreat were to 1) invest in data standards and analytical tool development, 2) support young investigators, and 3) promote cross-sector collaborations especially between basic scientists, preclinical researchers, clinicians, and patients.