SEARCH · PubMed Health

Results for “Prime editing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed2023

Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment.

Autophagy regulates the degradation of damaged organelles and protein aggregates, and is critical for neuronal development, homeostasis, and maintenance, yet few neurodevelopmental disorders have been associated with pathogenic variants in genes encoding autophagy-related proteins. We report three individuals from two unrelated families with a neurodevelopmental disorder characterized by speech and motor impairment, and similar facial characteristics. Rare, conserved, bi-allelic variants were identified in ATG4D, encoding one of four ATG4 cysteine proteases important for autophagosome biogenesis, a hallmark of autophagy. Autophagosome biogenesis and induction of autophagy were intact in cells from affected individuals. However, studies evaluating the predominant substrate of ATG4D, GABARAPL1, demonstrated that three of the four ATG4D patient variants functionally impair ATG4D activity. GABARAPL1 is cleaved or "primed" by ATG4D and an in vitro GABARAPL1 priming assay revealed decreased priming activity for three of the four ATG4D variants. Furthermore, a rescue experiment performed in an ATG4 tetra knockout cell line, in which all four ATG4 isoforms were knocked out by gene editing, showed decreased GABARAPL1 priming activity for the two ATG4D missense variants located in the cysteine protease domain required for priming, suggesting that these variants impair the function of ATG4D. The clinical, bioinformatic, and functional data suggest that bi-allelic loss-of-function variants in ATG4D contribute to the pathogenesis of this syndromic neurodevelopmental disorder.

Journal Article↗

PubMed2004

The PCNA-RFC families of DNA clamps and clamp loaders.

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

DNA↗

PubMed2005

Surprisingly high prevalence of anxiety and depression in chronic breathing disorders.

STUDY OBJECTIVES: The objectives of this study were to assess the prevalence, screening, and recognition of depression and anxiety in persons with chronic breathing disorders, including COPD. DESIGN: Cross-sectional study. SETTING: The Michael E. DeBakey Veterans Affairs Medical Center (MEDVAMC). PARTICIPANTS: A large sample of 1,334 persons with chronic breathing disorder diagnoses who received care at the MEDVAMC. MEASUREMENTS: The prevalence of anxiety and depression was measured in a large sample of persons with a chronic breathing disorder diagnosis who received care at the MEDVAMC, using the Primary Care Evaluation of Mental Disorders (PRIME-MD) screening questions. The positive predictive value of the PRIME-MD questions was then determined. The prevalence of anxiety and depressive diagnoses in patients determined to have COPD was then measured, using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (SCID). RESULTS: Of patients screened with the PRIME-MD, 80% screened positive for depression, anxiety, or both. The predictive value of a positive phone screen for either depression or anxiety was estimated to be 80%. In the subsample of patients who had COPD and received a diagnosis using the SCID, 65% received an anxiety and/or depressive disorder diagnosis. Of those patients, only 31% were receiving treatment for depression and/or anxiety. CONCLUSIONS: It is troubling that a mere 31% of COPD patients with depression or anxiety are being treated, particularly given their high prevalence in this population. Practical screening instruments may help increase the recognition of anxiety and depression in medical patients, as suggested by the excellent positive predictive value of the PRIME-MD in our study.

Aged↗

PubMed2026

EndoV does not measurably affect TadA-dependent A-to-I RNA editing in Escherichia coli under exponential-growth conditions in rich medium.

Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information post-transcriptionally and was only recently reported to occur in bacteria. Here, we examined whether endonuclease V (EndoV; encoded by nfi) cleaves inosine-containing RNAs in vivo and thereby influences the abundance and fate of A-to-I-edited mRNAs in Escherichia coli. We generated an nfi loss-of-function mutant carrying a premature stop codon and performed RNA sequencing alongside the isogenic wild-type strain. We observed that global and site-specific editing occurrence or levels in both mRNAs and tRNAs were indistinguishable between strains. Moreover, overexpression of EndoV did not affect the number of edited sites, motif enrichment, or editing levels compared with a control strain overexpressing mCherry. Our findings suggest that, in contrast to human EndoV, bacterial EndoV does not regulate the steady-state pool of edited mRNAs in E. coli under nutrient-rich, exponential-growth conditions in vivo.IMPORTANCEAdenosine-to-inosine (A-to-I) mRNA editing is an emerging regulatory layer in bacteria, but the factors that act on edited transcripts are largely unknown. Endonuclease V (EndoV) was a prime candidate because it cleaves inosine-containing nucleic acids and can act on inosine-containing RNA in vitro. By combining loss-of-function and overexpression of EndoV with genome-wide RNA editing measurements, we show that EndoV does not measurably influence TadA-dependent A-to-I mRNA editing in Escherichia coli under standard laboratory conditions. This negative result is important because it rules out a natural effector candidate and redirects attention to other bacterial pathways that may process edited RNAs. Our work, therefore, sharpens mechanistic models for bacterial RNA editing and helps focus future searches for its regulators and physiological roles.

Escherichia coli↗

PubMed1994

Utility of a new procedure for diagnosing mental disorders in primary care. The PRIME-MD 1000 study.

OBJECTIVE: To assess the validity and utility of PRIME-MD (Primary Care Evaluation of Mental Disorders), a new rapid procedure for diagnosing mental disorders by primary care physicians. DESIGN: Survey; criterion standard. SETTING: Four primary care clinics. SUBJECTS: A total of 1000 adult patients (369 selected by convenience and 631 selected by site-specific methods to avoid sampling bias) assessed by 31 primary care physicians. MAIN OUTCOME MEASURES: PRIME-MD diagnoses, independent diagnoses made by mental health professionals, functional status measures (Short-Form General Health Survey), disability days, health care utilization, and treatment/referral decisions. RESULTS: Twenty-six percent of the patients had a PRIME-MD diagnosis that met full criteria for a specific disorder according to the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition. The average time required of the primary care physician to complete the PRIME-MD evaluation was 8.4 minutes. There was good agreement between PRIME-MD diagnoses and those of independent mental health professionals (for the diagnosis of any PRIME-MD disorder, kappa = 0.71; overall accuracy rate = 88%). Patients with PRIME-MD diagnoses had lower functioning, more disability days, and higher rates of health care utilization than did patients without PRIME-MD diagnoses (for all measures, P < .005). Nearly half (48%) of 287 patients with a PRIME-MD diagnosis who were somewhat or fairly well-known to their physicians had not been recognized to have that diagnosis before the PRIME-MD evaluation. A new treatment or referral was initiated for 62% of the 125 patients with a PRIME-MD diagnosis who were not already being treated. CONCLUSION: PRIME-MD appears to be a useful tool for identifying mental disorders in primary care practice and research.

Adult↗

PubMed2026

The present and future of nonviral delivery-based genome editing for hereditary hearing loss.

PURPOSE OF REVIEW: This review summarizes nonviral genome-editing delivery platforms for hereditary hearing loss, focusing on lipid nanoparticles (LNPs) and engineered virus-like particles (eVLPs), and discusses their advantages over adeno-associated virus-based delivery, as well as the barriers to clinical translation. RECENT FINDINGS: Recent advances have established LNPs as a clinically advanced nonviral platform, although challenges related to inner ear biodistribution, cell type specificity, endosomal escape, and immunogenicity remain to be addressed. In parallel, eVLPs have undergone substantial technical evolution, progressing from early low efficiency systems to advanced base editor- and prime editor-eVLP architectures that enhance cargo loading and editing efficiency. Extracellular vesicle-based genome editing has also emerged as an additional platform, although issues related to reproducibility, loading efficiency, and scalability remain major hurdles. SUMMARY: Nonviral genome editing platforms expand the therapeutic toolkit for hereditary hearing loss by enabling transient delivery of genome editors with potential safety advantages. Future efforts should focus on characterizing biodistribution and immunogenicity, refining cell type-specific tropism, and establishing scalable manufacturing processes to enable successful clinical translation.

Humans↗

PubMed2026

Hormone priming and metabolic engineering of phytohormone crosstalk in rice under combined biotic and abiotic stresses: a multi-omics perspective for climate-resilient crop development.

Rice (Oryza sativa L.) is the caloric backbone for more than half of humanity, yet it remains one of the most vulnerable crops to the simultaneous biotic and abiotic stresses exacerbated by climate change. Phytohormone priming and the complex crosstalk networks governed by transcription factor hubs like WRKY, MYB, and NAC serve as the central adaptive mechanism for stress resilience. This review synthesizes how multi-omics integration, including spatial and single-cell transcriptomics, is resolving the molecular architecture of hormonal priming and epigenetic stress memory. We critically evaluate advanced metabolic engineering and genome-editing strategies such as CRISPR-Cas9, base/prime editing, and synthetic gene circuits that enable precision modifications to decouple stress tolerance from historical yield penalties. Furthermore, we discuss the emerging roles of microbiome-assisted priming via synthetic consortia and the application of artificial intelligence and digital twins (continuously updated computational models of crop physiology) for predictive stress management. By integrating these diverse technological pillars, we propose a systems-level roadmap for developing climate-resilient rice cultivars capable of maintaining yield stability across a volatile combinatorial stress landscape. This synthesis provides a framework for translating mechanistic hormonal insights into field-applicable cultivars to ensure global food security.

CRISPR↗

PubMed2026

Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.

Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A-a key protein that binds and protects ssDNA-to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1-2 weeks, making it a rapid solution for assessing genome-wide off-target activity.

CRISPR-Cas Systems↗

PubMed2026

A voyage of reprogrammable metabolic bioengineering reshapes plant defense: from editing tools to synthetic systems.

Metabolic bioengineering has emerged as a transformative approach for reshaping plant defense by targeting intrinsic biosynthetic pathways to enhance immunity in modern agriculture. Moving beyond proof-of-concept metabolomics to broad-spectrum programmable pathway engineering addresses gaps in plant rational design and optimizes resilience in response to diverse environmental cues. This review aims to comprehensively highlight the transition of innovative approaches to phenolics, alkaloids, flavonoids, terpenoids, and benzoxazinoids, inferring adaptive reprogramming that mediates the growth-defense balance and functions as molecular sentinels in plants. Furthermore, decoding the volatile metabolome reveals a dynamic signaling interface that influences defense responses and stress-induced plant-microbe interactions, with the shikimate, jasmonate, and salicylate pathways functioning as central hubs for microbial deterrence and priming immune memory. Recent developments in multi-scalar genome-editing strategies, including CRISPR-driven combinatorial edits, enzyme orthogonalization, fluxomics, and spatially resolved multi-omics, reconfigure central and specialized metabolic fluxes toward improved defense function and regulation. Additionally, emerging tools, such as WUSCHEL2 and BABY BOOM transcriptional modules, and artificial engineering strategies integrating deep learning model-driven predictions facilitate rapid development of synthetic genetic circuits and support a predictive engineering of plants. Moreover, Mass spectrometry imaging (MSI) in spatial metabolomics enables to obtain structures and locations of unidentified endogenous metabolites within cells and tissues. Overall, this review emphasizes a diverse array of primary and secondary metabolites, spanning molecular concepts to recent advances in plant immune mechanisms. It also illustrates new frontiers in programmable metabolic engineering that accelerate the understanding of plant-microbe-metabolite cross-talks, offering strategies to improve plant resistance and advance sustainable agricultural solutions.

metabolic bioengineering↗

PubMed1998

The value of screening for psychiatric disorders in rheumatology referrals.

BACKGROUND: Musculoskeletal complaints are common and often unexplained and often lead to rheumatology referrals. The prevalence of psychiatric disease in patients with musculoskeletal complaints is unknown. OBJECTIVES: To determine the prevalence of common psychiatric disorders among patients referred to a rheumatology clinic and the likelihood of establishing a rheumatic diagnosis if a psychiatric disorder is present. DESIGN: Prospective diagnostic survey. SETTING: Two hospital-based rheumatology clinics and a general medicine clinic. PARTICIPANTS: A consecutive sample of newly referred patients (n = 185) and their rheumatologists (n = 9). INTERVENTION: Before their visit, all patients filled out a self-administered version of PRIME-MD (Primary Care Evaluation of Mental Disorders), a questionnaire that makes Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition: Primary Care Version, diagnoses of depressive, anxiety, and somatoform disorders. After the visit, the study rheumatologists, who were unaware of the PRIME-MD results, completed a questionnaire regarding their diagnostic assessment. These patients were compared with 210 patients with musculoskeletal complaints who were cared for in a general medicine clinic. MAIN OUTCOME MEASURES: Psychiatric and rheumatic disorders. RESULTS: Compared with patients with musculoskeletal complaints in a general medicine clinic, patients referred to a rheumatology clinic had a higher prevalence of psychiatric disease (40% vs 29%; P = .008), had an almost 2-fold higher prevalence of anxiety disorders, and were more likely to have multiple psychiatric disorders (odds ratio = 2.70, 95% confidence interval = 1.50-5.00). The likelihood of a psychiatric disorder differed among patients with connective tissue disease, nonsystemic articular or periarticular disorders, and nonarticular disorders (27%, 38%, 55%, respectively; P = .006). In a best-fitting logistic regression model, psychiatric disorders markedly decreased the likelihood of a connective tissue disease (odds ratio = 0.24, 95% confidence interval = 0.09-0.62). CONCLUSIONS: Forty percent of patients referred to a rheumatology clinic in this study had a psychiatric disorder, and its presence predicted a lower likelihood of a connective tissue disease. Prospective studies are needed to determine if screening for psychiatric disease before referring patients with unexplained musculoskeletal complaints would reduce costs or improve recognition of potentially treatable psychiatric disorders.

Adolescent↗

PubMedDate not supplied

Using the Hospital Anxiety and Depression Scale to screen for depression in cardiac patients.

OBJECTIVE: There is an increasing interest in trying to identify patients with chronic physical illness who would benefit from interventions to decrease psychological distress. The Hospital Anxiety and Depression Scale (HADS) is one measure that can be effectively used to identify patients struggling with anxiety and/or depression in addition to comorbid medical illness such as cardiac disease. The aim of this study is to determine if the HADS correctly identified patients with depression according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition [determined using the Primary Care Evaluation of Mental Disorders (PRIME-MD), a gold-standard diagnostic tool], as depressed and to determine which items on the HADS most strongly predicted the PRIME-MD major depression diagnosis among cardiac patients. METHOD: Patient data were obtained from a randomized controlled trial of treatment for psychological distress in patients surviving recent myocardial infarction or life-threatening ischemic heart disease. The HADS was used to evaluate depression and/or anxiety among patients hospitalized for heart disease, and the PRIME-MD was used to diagnose major depressive disorder (MDD) in these patients. We used receiver-operating characteristic (ROC) curves and logistic regression to analyze patient responses to both of these measures. RESULTS: Among the 79 study patients, the area under the ROC curve (AUC) for the HADS depression subscale was 0.81 with an S.E. of 0.05, and the AUC was 0.70 for the HADS anxiety subscale with an S.E. of 0.06. Using a cutoff of 7 on each HADS subscale to determine PRIME-MD diagnosis of MDD, the sensitivity and specificity were 81% and 54%, respectively, for the depression subscale and 81% and 40%, respectively, for the anxiety subscale. In addition, a few individual HADS items predicted PRIME-MD diagnosis of MDD, namely, concentration and positive anticipation (depression items) and relaxed and worry (anxiety items), each of which were strongly correlated with PRIME-MD diagnosis of MDD. Finally, items positive anticipation and relaxed provided reasonable approximations to the results found from the HADS depression subscale measure, which performed better than the HADS anxiety subscale in predicting PRIME-MD diagnosis of depression. CONCLUSIONS: Using the HADS to detect mental disorders in primary care populations can save valuable physician time, and perhaps ensure that patients are treated for their mental distress as well as medical disease.

Aged↗

PubMed1992

The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.

The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme. A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors. Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.

Bacteriophages↗

PubMed1997

A computer-administered telephone interview to identify mental disorders.

CONTEXT: Common mental disorders are often not identified in primary care settings. OBJECTIVE: To evaluate the validity and clinical utility of a telephone-assisted computer-administered version of Primary Care Evaluation of Mental Disorders (PRIME-MD), a brief questionnaire and interview instrument designed to identify psychiatric disorders in primary care patients. DESIGN: Comparison of diagnoses obtained by computer over the telephone using interactive voice response (IVR) technology vs those obtained by a trained clinician over the telephone using the Structured Clinical Interview for DSM-IV [Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition] Diagnosis (SCID). A subsample also received the clinician-administered version of PRIME-MD. PATIENTS: Outpatients (N=200) from 4 primary care clinics, an eating disorders clinic, an alcohol treatment facility, psychiatric outpatients, and community controls. SETTING: Interviews conducted by telephone, except for face-to-face administration of PRIME-MD, which was conducted at either the primary care clinic or a research clinic. MEASUREMENTS AND MAIN RESULTS: Prevalence rates for any psychiatric disorder were similar between diagnoses made by the computer and those made by a mental health professional using the SCID (60.0% vs 58.5%). Prevalence rates for individual diagnoses were generally similar across versions. However, primary care patients reported twice as much alcohol abuse on the computer (15.0%) as on either the SCID (7.5%) or the clinician-administered PRIME-MD (7.5%). Using the SCID as the criterion, both the computer- and clinician-administered versions of PRIME-MD demonstrated high and roughly equivalent levels of sensitivity and specificity. Overall agreement (K) for any diagnosis was 0.67 for the computer-administered PRIME-MD and 0.70 for the clinician-administered PRIME-MD. CONCLUSIONS: The computer-administered PRIME-MD appears to be a valid instrument for assessing psychopathology in primary care patients. Interactive voice response technology allows for increased availability, and provides primary care physicians with information that will increase the quality of patient care without additional physician time and at minimal expense.

Adult↗

PubMed2006

Multi-directional cross-regulation of NK cell function during innate immune responses.

In recent years a number of studies allowed major advances in our understanding of different cell types of the innate immunity and of the role they play during the early phases of infections. Some of these cells, such as mast cells, endothelial cells and certain immature dendritic cells (iDCs), are resident within peripheral tissues, while others, including NK cells, are rapidly recruited from blood stream. These studies indicated that innate immunity cells interact each other in inflamed tissues and in secondary lymphoid organs leading to modulation or amplification of different innate effector mechanisms and that a large array of microbial products can directly activate different effector cells of the innate immunity, also including NK cells. The final outcome of these cellular interactions may have dramatic impact on the quality and strength of down-stream adaptive immune responses. Noticeably, the effect on the adaptive immunity can result not only from the action of polarizing cytokines such as IL12 or IL4, but also from the NK-mediated "DC editing" leading to the selection of the most suitable DCs for subsequent priming of Th1 cell responses. Thus classical innate effector cells can also be viewed as regulatory cells that play a pivotal role in defenses against pathogens.

Cytokines↗

PubMed2002

Improved detection of depression in primary care through severity evaluation.

OBJECTIVES: To determine whether the use of a symptom severity measure to augment an existing Diagnostic and Statistical Manual of Mental Disorders-Third Edition, Revised (DSM-III-R) criteria-based depression screener (PRIME-MD) would decrease the difficulties associated with depression screening in primary care by filtering out patients with minimal impairment. STUDY DESIGN: The study design was secondary data analysis. POPULATION: The study sample comprised 1317 patients, with intentional oversampling by ethnicity and sex, presenting for routine care at a university family practice center in Galveston, Texas. OUTCOMES MEASURED: The primary outcomes were cross-sectional, health-related quality-of-life outcomes of subjects who met symptom severity criteria as well as criteria for a DSM-III-R mood disorder. Health care utilization outcomes were examined as secondary outcomes. RESULTS: The combination of a 6-item depression severity instrument and the PRIME-MD resulted in 71% of depressed subjects being categorized as severely symptomatic and 29% as minimally symptomatic. Severely symptomatic subjects had significantly worse SF-36 Mental Health Component Summary scale (MCS) scores than did minimally symptomatic subjects (32.8 vs 43.5, P <.05). Minimally symptomatic subjects had MCS scores similar to those of a third group of subjects who did not meet DSM-III-R "threshold" criteria for mood disorder but who were severely symptomatic. Adjusted health care utilization was higher for the initial 3-month charge period in the severely symptomatic depressed subjects compared with minimally symptomatic depressed subjects ($679.20 vs $462.38, P <.05). CONCLUSIONS: The 6-item depression severity measure effectively separated patients meeting DSM-III-R "threshold" depression criteria into 2 groups: one presenting with severe symptoms and impairment and the other presenting with mild symptoms and significantly less impairment. A strategy of initial screening using a brief depression severity instrument, followed with a DSM criteria-based instrument, could decrease the immediate clinician workload by one third and focus treatment on those most likely to benefit.

Adult↗

PubMed2026

Epigenetic priming and locus-specific demethylation enhance cell-death susceptibility in liver cancer.

Liver cancer treatment with epigenetic drugs remains challenging because demethylating agents such as 5-azacytidine (5-AZA) induce genome-wide toxicity and may activate oncogenes. We hypothesized that a low, nontoxic dose of 5-AZA could prime liver cancer cells by partially relaxing chromatin at selected loci to restore silenced cell-death regulators. HepG2 cells treated with 2 μM 5-AZA underwent ATAC-seq and RNA-seq to identify genes with promoter opening and increased expression. Among ten candidates, BFL-1 and SQOR were prioritized for roles in cell death and redox control. Forced expression of either gene increased sensitivity to TNF-α/cycloheximide (CHX) and sorafenib, both of which elevated mitochondrial reactive oxygen species. To establish causality in a physiological context, we used CRISPR-dCas9-TET1 to demethylate CpG-rich promoter regions of BFL-1 or SQOR. Locus-specific editing sensitized cells to TNF-α/CHX more rapidly than conventional overexpression and reproduced the heightened death response elicited by low-dose 5-AZA without baseline toxicity. Analysis of the cancer cell line encyclopedia and The Cancer Genome Atlas datasets showed consistent BFL-1 downregulation in liver cancer, variable SQOR expression across cancers, and positive correlations of both genes with tumor-suppression markers and immune-cell infiltration. These results indicate that targeted reactivation of BFL-1 and SQOR increases cell-death susceptibility in liver cancer cells. Integrating low-dose pharmacologic priming with precise epigenetic editing may preserve genome-wide methylation while restoring cell-death competence, providing proof-of-concept for locus-specific epigenetic therapy in liver cancer.

Humans↗

PubMed2026

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of capping protein and VASP.

Cell migration frequently employs protrusions termed lamellipodia, constituting the prime model system for generation of branched actin filament networks. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and Ena/VASP family polymerases in lamellipodial actin assembly. We show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. While Pfn promotes Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal vanishes in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, differentially regulating VASP and its antagonizer CP. Mathematical modeling of our data suggest Ena/VASP and CP to compete for recruitment to lamellipodial edges. Our work provides critical insights into the molecular logic of branched actin network assembly in protrusion and force generation.

Profilins↗

PubMed2005

The relationship between immunodominance, DM editing, and the kinetic stability of MHC class II:peptide complexes.

Immunodominance refers to the restricted antigen specificity of T cells detected in the immune response after immunization with complex antigens. Despite the presence of many potential peptide epitopes within these immunogens, the elicited T-cell response apparently focuses on a very limited number of peptides. Over the last two decades, a number of distinct explanations have been put forth to explain this very restricted specificity of T cells, many of which suggest that endosomal antigen processing restricts the array of peptides available to recruit CD4 T cells. In this review, we present evidence from our laboratory that suggest that immunodominance in CD4 T-cell responses is primarily due to an intrinsic property of the peptide:class II complexes. The intrinsic kinetic stability of peptide:class II complexes controls DM editing within the antigen-presenting cells and thus the initial epitope density on priming dendritic cells. Additionally, we hypothesize that peptides that possess high kinetic stability interactions with class II molecules display persistence at the cell surface over time and will more efficiently promote T-cell signaling and differentiation than competing, lower-stability peptides contained within the antigen. We discuss this model in the context of the existing data in the field of immunodominance.

Animals↗