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1.
Pacing Clin Electrophysiol ; 47(2): 211-221, 2024 02.
Article in English | MEDLINE | ID: mdl-38265169

ABSTRACT

BACKGROUND: Conduction system pacing (CSP) is increasingly utilized to prevent and correct dyssynchrony. Barriers to CSP adoption include limited training, methodologic variability, laboratory slot allocation, and few data on learning curves. We report learning curves/clinical outcomes from a single experienced electrophysiologist who was new to CSP, and share gained insights. METHODS: Retrospective analysis of all patients who underwent attempted CSP implantation (2016-2023). Patient characteristics, ECGs, echocardiograms, fluoroscopy/procedure times, lead data were recorded at implant and follow-up. RESULTS: CSP leads were implanted successfully in 167/191(87.4%) patients with a follow-up of 278 ± 378 days. His-bundle pacing (HBP = 59) and left-bundle-area pacing (LBAP = 108) had similar procedure/fluoroscopy times, QRS duration decreases, and ejection fraction improvements (all p > NS). Eight HBP lead revisions were required for high capture thresholds LBAP demonstrated lower pacing thresholds, higher lead impedances, and greater R-wave amplitudes at implant and follow-up. After 25 HBP cases, implant pacing thresholds, fluoroscopy, procedural times did not decrease. After 25 LBAP cases, there were significant decreases in all these parameters (p < 0.05). A separate analysis in LBAP patients with recorded Purkinje signals showed no differences in paced ECG characteristics between patients with pre- QRS Purkinje signals versus patients with Purkinje signals post-QRS onset. CONCLUSIONS: Experienced implanters who are new to CSP can achieve steady-state procedural/fluoroscopy times after a learning curve of 25 implants. LBAP showed lower capture thresholds and higher success rates. Adequate depth of lead deployment (as determined by published parameters) does not require Purkinje potential to be pre-QRS. Operators new to CSP.can forego HBP and directly implement LBAP.


Subject(s)
Bundle of His , Learning Curve , Humans , Retrospective Studies , Cardiac Pacing, Artificial/methods , Cardiac Conduction System Disease , Electrocardiography/methods , Treatment Outcome
2.
Leukemia ; 37(8): 1698-1708, 2023 08.
Article in English | MEDLINE | ID: mdl-37391485

ABSTRACT

Many inherited bone marrow failure syndromes (IBMFSs) present a high risk of transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). During transformation of IBMFSs, hematopoietic stem and progenitor cells (HSPCs) with poor fitness gain ectopic, dysregulated self-renewal secondary to somatic mutations via undefined mechanisms. Here, in the context of the prototypical IBMFS Fanconi anemia (FA), we performed multiplexed gene editing of mutational hotspots in MDS-associated genes in human induced pluripotent stem cells (iPSCs) followed by hematopoietic differentiation. We observed aberrant self-renewal and impaired differentiation of HSPCs with enrichment of RUNX1 insertions and deletions (indels), generating a model of IBMFS-associated MDS. We observed that compared to the failure state, FA MDS cells show mutant RUNX1-mediated blunting of the G1/S cell cycle checkpoint that is normally activated in FA in response to DNA damage. RUNX1 indels also lead to activation of innate immune signaling, which stabilizes the homologous recombination (HR) effector BRCA1, and this pathway can be targeted to abrogate viability and restore sensitivity to genotoxins in FA MDS. Together, these studies develop a paradigm for modeling clonal evolution in IBMFSs, provide basic understanding of the pathogenesis of MDS, and uncover a therapeutic target in FA-associated MDS.


Subject(s)
Fanconi Anemia , Induced Pluripotent Stem Cells , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Humans , Fanconi Anemia/genetics , Fanconi Anemia/pathology , Fanconi Anemia/therapy , Congenital Bone Marrow Failure Syndromes/complications , Core Binding Factor Alpha 2 Subunit/genetics , Induced Pluripotent Stem Cells/pathology , Myelodysplastic Syndromes/pathology , Mutation , Leukemia, Myeloid, Acute/pathology
3.
Cell Rep ; 39(4): 110752, 2022 04 26.
Article in English | MEDLINE | ID: mdl-35476984

ABSTRACT

High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.


Subject(s)
Leukemia, Myeloid, Acute , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Glycolysis , Humans , Hypoxia , Leukemia, Myeloid, Acute/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
4.
Blood Adv ; 4(19): 4679-4692, 2020 10 13.
Article in English | MEDLINE | ID: mdl-33002135

ABSTRACT

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.


Subject(s)
Fanconi Anemia , Induced Pluripotent Stem Cells , Animals , Cell Differentiation , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group A Protein/genetics , Humans , Mice , Tumor Suppressor Protein p53/genetics
5.
Proc Natl Acad Sci U S A ; 117(9): 4653-4663, 2020 03 03.
Article in English | MEDLINE | ID: mdl-32060122

ABSTRACT

The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3' ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.


Subject(s)
DNA-Binding Proteins/immunology , MicroRNAs/metabolism , RNA 3' End Processing , Single-Domain Antibodies/immunology , Animals , Binding Sites , DNA-Binding Proteins/chemistry , HEK293 Cells , HeLa Cells , Humans , Mice , MicroRNAs/genetics , Protein Binding , RNA Stability , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Sf9 Cells , Spodoptera
6.
J Exp Med ; 216(3): 527-538, 2019 03 04.
Article in English | MEDLINE | ID: mdl-30728174

ABSTRACT

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.


Subject(s)
Hematopoiesis/physiology , Leukemia/pathology , Oncogene Proteins, Fusion/genetics , Aging , Animals , Animals, Newborn , B-Lymphocytes/pathology , Chemokine CCL5/genetics , Chemokine CCL5/metabolism , Female , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Histone-Lysine N-Methyltransferase/genetics , Leukemia/genetics , Leukocyte Common Antigens/metabolism , Male , Mice, Inbred C57BL , Myeloid-Lymphoid Leukemia Protein/genetics , Stromal Cells/pathology , Tumor Microenvironment
7.
Cell Rep ; 23(10): 3091-3101, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29874593

ABSTRACT

LIN28 is an RNA-binding protein that regulates the maturation of the let-7 family of microRNAs by bipartite interactions with let-7 precursors through its two distinct cold shock and zinc-knuckle domains. Through inhibition of let-7 biogenesis, LIN28 functions as a pluripotency factor, as well as a driver of tumorigenesis. Here, we report a fluorescence polarization assay to identify small-molecule inhibitors for both domains of LIN28 involved in let-7 interactions. Of 101,017 compounds screened, six inhibit LIN28:let-7 binding and impair LIN28-mediated let-7 oligouridylation. Upon further characterization, we demonstrate that the LIN28 inhibitor TPEN destabilizes the zinc-knuckle domain of LIN28, while LI71 binds the cold shock domain to suppress LIN28's activity against let-7 in leukemia cells and embryonic stem cells. Our results demonstrate selective pharmacologic inhibition of individual domains of LIN28 and provide a foundation for therapeutic inhibition of the let-7 biogenesis pathway in LIN28-driven diseases.


Subject(s)
MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/metabolism , Small Molecule Libraries/pharmacology , Uridine/metabolism , Binding Sites , Cell Line, Tumor , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Fluorescence Polarization , High-Throughput Screening Assays , Humans , MicroRNAs/genetics , Models, Molecular , Niacin/chemistry , Small Molecule Libraries/chemistry
8.
Malar J ; 13: 158, 2014 Apr 25.
Article in English | MEDLINE | ID: mdl-24767409

ABSTRACT

BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/µL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/µL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.


Subject(s)
Malaria/diagnosis , Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Parasitology/methods , Plasmodium/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Blood/parasitology , Coinfection/diagnosis , Coinfection/parasitology , Freeze Drying/methods , Humans , Malaria/parasitology , Plasmodium/classification , Plasmodium/genetics , Sensitivity and Specificity , Temperature
9.
EMBO J ; 24(12): 2087-95, 2005 Jun 15.
Article in English | MEDLINE | ID: mdl-15933718

ABSTRACT

The tail of the bacteriophage P22 is composed of multiple protein components and integrates various biological functions that are crucial to the assembly and infection of the phage. The three-dimensional structure of the P22 tail machine determined by electron cryo-microscopy and image reconstruction reveals how the five types of polypeptides present as 51 subunits are organized into this molecular machine through twelve-, six- and three-fold symmetry, and provides insights into molecular events during host cell attachment and phage DNA translocation.


Subject(s)
Bacteriophage P22/chemistry , Viral Tail Proteins/chemistry , Amino Acid Sequence , Bacteriophage P22/genetics , Cryoelectron Microscopy , Crystallography, X-Ray , Fourier Analysis , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Viral Tail Proteins/genetics
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