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Biology subjects

Harrison, D.

Publications and source records attributed to Harrison, D..

9 recordsLinked to original sources

Treatment of murine autoimmune myocarditis with a novel monoclonal antibody that targets multiple inflammatory pathways

Severe forms of inflammation-induced acute and chronic myocarditis have a poor prognosis. Promising therapeutic efforts focused on monoclonal antibodies (mAbs) inhibiting inflammation-inducing molecules. However, most mAbs target only one or a limited number of such molecules. Since inflammation involves multiple redundant pathways, we postulated that an mAb inhibiting multiple inflammatory pathways would be a potent therapeutic agent. We initially tested the commercially available anti-natural killer (NK) cell mAb (anti-NK1.1), which binds a receptor expressed on NK cells and depletes them. Since NK cells are key cellular orchestrators of inflammation, by reducing their number, we aimed to inhibit multiple inflammatory pathways. Our initial studies demonstrated that administration of this antibody significantly improved myocardial outcomes in mouse models of acute myocardial infarction and of heart failure. Since NK1.1 is not expressed in human cells, we built on these promising preclinical results by developing a novel mAb targeting CD160 on human NK cells for evaluation as an immunosuppressive therapy. We found that the anti-CD160 mAb depletes both murine and human NK cells. We also found that, while CD160+ cells were largely present in the NK population, they also occurred among CD8+ and {gamma}/{delta} T cell subsets in human cells. Anti-CD160 therapy entirely prevented the deterioration of the myocardial function of mice with autoimmune-induced acute myocarditis. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases. Key PointsO_ST_ABSQuestionC_ST_ABSCan the depletion of CD160+ cells prevent autoimmune-induced myocarditis? FindingsIn this study we found that CD160 is expressed by mouse and human natural killer cells and other subtypes of cytotoxic T cells, and that a monoclonal antibody targeting CD160 depletes NK cells. In a preclinical model of experimental autoimmune myocarditis, administration of the anti-CD160 monoclonal antibody prevented myocardial dysfunction and systemic inflammation. MeaningOur results are compatible with the hypothesis that early autoimmune-induced myocardial dysfunction is promoted by CD160+ cells, which elevate inflammation-induced circulating factors (or factors released by tissue-resident cytotoxic immune cells) that cause myocardial dysfunction in the absence of myocardial necrosis or fibrosis, and further, that targeting CD160+cells with a mAb that depletes NK cells (and probably CD160 expressing cytotoxic T cells) entirely prevents the deterioration of myocardial function in such mice. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases.

systems biology↗

Highly Efficient Multiplexed Genome Engineering and Clone Selection to Enable Next Generation Induced Pluripotent Stem Cell (iPSC)-based Cell Therapies

Human induced pluripotent stem cells (iPSCs) have revolutionized regenerative medicine and cellular therapies. To improve the functionality and safety of iPSC-based therapies, genome engineering has been employed to disrupt gene expression and introduce therapeutic transgenes. However, current genome editing methods face significant challenges, including labor-intensive procedures, low efficiency, and safety concerns. Here, we report a novel and efficient approach for multiplex genome engineering in iPSCs using Integrase-mediated Programmable Genomic Integration (I-PGI) technology. I-PGI combines CRISPR-mediated genome editing and site-specific integrases, enabling precise gene insertion without DNA double-strand breaks. By optimizing I-PGI components and protocols, we achieved multiple gene knockouts and knock-ins of up to five transgenes in a single process. Additionally, we developed a streamlined workflow for enrichment, deposition, and screening of single-cell clones with the desired genome edits. This approach significantly accelerates and improves the precision of multiplex iPSC engineering, paving the way for next-generation iPSC-based therapies.

genomics↗

A nuclease-driven mechanism of post-replicative ssDNA gap suppression.

During replication stress, failure to resolve post-replicative ssDNA gaps generated by PRIMPOL-mediated replication repriming is linked with chemosensitivity, and in all models reported to date the nuclease MRE11 has been implicated as a gap-promoting factor. We have dissected ssDNA gap dynamics following nucleoside analogue-mediated nascent strand termination and report a novel mechanism via which loss of the MRE11 negative regulator MRN-Interacting Protein (MRNIP) leads to MRE11 exonuclease-dependent suppression of post-replicative ssDNA gaps. This process is driven by UBC13 and the B-family TLS polymerase REV3L, suggesting that in the absence of MRNIP, dysregulated MRE11 activity at chain termination sites licenses gap filling via template switching. In the absence of PRIMPOL-dependent repriming, MRNIP loss leads to paradoxical SMARCAL1, MRE11 and primosome-dependent ssDNA gaps, suggesting that nucleolytic digestion of reversed forks generates DNA intermediates that platform primase redundancy in the context of chain termination. We also highlight novel site-specific roles for the anti-resection factor 53BP1 in enabling primase redundancy via suppression of the nuclease EXO1, and in limiting the EXO1-dependent processing of post-replicative ssDNA gaps. Finally, we demonstrate that CDK-dependent MRNIP phosphorylation is required for MRNIP functionality in the regulation of ssDNA gaps and sensitivity to chain terminators. This work represents the first report of nuclease-driven post-replicative gap filling, illuminates an additional level of versatility in the replication stress response, and expands our understanding of the context-dependent links between nuclease regulation and chemoresistance.

cancer biology↗

Mechanosensitive Endothelial METTL7A Regulates Internal m7G mRNA Methylation and Protects Against Atherosclerosis

BACKGROUNDInternal N7-methylguanosine (m7G) is a recently identified chemical modification of mammalian mRNA and a component of the epitranscriptome. While the epitranscriptome plays a key role in regulating RNA metabolism and cellular function, the specific contribution of internal m7G to cardiovascular health and disease remains unknown. Atherosclerosis preferentially develops at sites where disturbed blood flow activates endothelial cells, but whether internal m7G and its regulatory machinery influence endothelial mechanotransduction and atherogenesis is unclear. METHODSWe integrated epitranscriptomic profiling, human tissue analysis, genetically modified mouse models, and targeted nanomedicine approaches to investigate the role of Methyltransferase-like protein 7A (METTL7A), a putative internal m7G methyltransferase, in regulating the flow-sensitive endothelial transcriptome and atherosclerosis. Vascular endothelial cells were subjected to well-defined athero-protective and athero-prone flow waveforms in vitro and in vivo. METTL7A function was assessed using RNA sequencing (RNA-seq), liquid chromatography-tandem mass spectrometry (LC-MS/MS), crosslinking immunoprecipitation sequencing (CLIP-seq), RNA stability assays, and a CRISPR-Cas-inspired RNA targeting system (CIRTS). METTL7A expression in human coronary arteries with and without atherosclerosis was evaluated by RNA-seq and immunostaining. In vivo atherosclerosis studies were conducted in both global and endothelial-specific Mettl7a1 knockout mice. Endothelial METTL7A expression was restored using cationic polymer-based nanoparticles delivering CDH5 promoter-driven METTL7A plasmids or VCAM1-targeted lipid nanoparticles delivering N1-methylpseudouridine (m{superscript 1}{Psi})-modified METTL7A mRNA. RESULTSAthero-protective unidirectional flow significantly induced METTL7A expression, which promoted internal m7G methylation of endothelial transcripts, while other major epitranscriptomic marks and cap-associated m7G were not affected by METTL7A. METTL7A preferentially binds to AG-enriched motifs in protein-coding mRNAs and plays a key role in regulating KLF4 and NFKBIA transcripts, enhancing their internal m7G and stability and supporting vascular homeostasis. In contrast, endothelial METTL7A expression was significantly reduced by disturbed blood flow and in human atherosclerotic lesions. Global or endothelial-specific loss of METTL7A exacerbated disturbed flow-induced atherosclerosis in mice, independent of serum lipid levels. Restoration of endothelial METTL7A, via nanoparticle-mediated plasmid or m1{Psi} mRNA delivery, markedly reduced lesion formation in Mettl7a1-/- and ApoE-/- mice. CONCLUSIONSThese findings establish METTL7A as a previously unrecognized mechanosensitive methyltransferase that maintains endothelial homeostasis by stabilizing key anti-inflammatory transcripts, KLF4 and NFKBIA, through internal m7G methylation. Loss of METTL7A disrupts endothelial function and accelerates atherogenesis in response to disturbed flow. Therapeutic restoration of endothelial METTL7A, via targeted nanoparticle-mediated gene or m1{Psi} mRNA delivery, significantly lessens atherosclerosis. Collectively, these results uncover a novel epitranscriptomic mechanism governing vascular health and position METTL7A as a promising target for precision nanomedicine in atherosclerotic cardiovascular disease.

molecular biology↗

Clonal dynamics and somatic evolution of haematopoiesis in mouse

Haematopoietic stem cells maintain blood production throughout life. While extensively characterised using the laboratory mouse, little is known about how the population is sustained and evolves with age. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1845 single cell-derived colonies, and used phylogenetic analysis to infer the ontogeny and population dynamics of the stem cell pool. Mouse stem cells and progenitors accrue [~]45 somatic mutations per year, a rate only about 2-fold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns reveal that stem and multipotent progenitor cell pools are both established during embryogenesis, after which they independently self-renew in parallel over life. The stem cell pool grows steadily over the mouse lifespan to approximately 70,000 cells, self-renewing about every six weeks. Aged mice did not display the profound loss of stem cell clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing revealed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations and exhibited a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse.

genomics↗

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice

This study investigated the prevalence and progression of primary osteoarthritis (OA) in aged UM-HET3 mice. Using the Osteoarthritis Research Society International (OARSI) scoring system, we assessed articular cartilage (AC) integrity in 182 knee joints of 22-25 months old mice. Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and the NLR family pyrin domain containing-3 (NLRP3) inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and {beta}-galactosidase, also correlated with AC scores. Using micro-CT, we examined the correlations between subchondral bone (SCB) morphology traits and AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Finally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Notably, MB and MitoQ treatments influenced the diseases progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. In conclusion, our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

pathology↗

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF{kappa}B1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF{kappa}B1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

systems biology↗

Targeting mitochondrial dysfunction using methylene blue or mitoquinone to improve skeletal aging

Declines in mitochondrial content and impaired cytochrome c oxidase activity (complex IV) can result in reduced energy metabolism and increased levels of oxidants. Methylene blue (MB) is a well-established antioxidant that has been shown to improve mitochondrial function in both in vitro and in vivo settings. Mitoquinone (MitoQ) is a selective antioxidant that specifically targets mitochondria and effectively reduces the accumulation of reactive oxygen species (ROS) within cells. To investigate the effect of acute and long-term administration of MB on skeletal morphology, we administered MB to aged (18 months old) female C57BL/J6 mice, as well as to adult male and female mice with a genetically diverse background (UM-HET3). Additionally, we used MitoQ as an alternative approach to target mitochondrial oxidative stress during aging in adult female and male UM-HET3 mice. Although we observed some beneficial effects of MB and MitoQ in vitro, the administration of these compounds in vivo did not alter the progression of age-induced bone loss. Specifically, treating 18-month-old female mice with MB for 6 or 12 months did not have an effect on age-related bone loss. Similarly, long-term treatment with MB from 7 to 22 months or with MitoQ from 4 to 22 months of age did not affect the morphology of cortical bone at the mid-diaphysis of the femur, trabecular bone at the distal-metaphysis of the femur, or trabecular bone at the lumbar vertebra-5 (L5) in UM-HET3 mice. Based on our findings, it appears that long-term treatment with MB or MitoQ alone, as a means to reduce skeletal oxidative stress, is insufficient to inhibit age-associated bone loss. This supports the notion that interventions solely with antioxidants may not provide adequate protection against skeletal aging.

pharmacology and toxicology↗

The impact of coregistration of gradient recalled echo images on quantitative susceptibility and R2* mapping at 7T

IntroductionWhile image coregistration is inevitable and quantitative parametric maps, such as R2* and QSM, are increasingly used in multi-parametric studies of the brain, there is a lack of investigations on the reliability of quantitative metrics after coregistration. The purposes of this study were 1) to evaluate the reliability of R2* and QSM at 7T and 2) to assess the statistical agreement in the quantitative metrics obtained by two different coregistration approaches. MethodsWe compared the reliability of R2* and quantitative susceptibility maps obtained from brains of eight healthy participants by two coregistration approaches: 1) transformation of pre-processed quantitative maps and 2) processing quantitative maps after transformation using pixel- and ROI-based analyses. Two-sample Kolmogorov-Smirnov, Mann-Whitney U, Paired T, Intraclass-correlation tests were performed appropriately. ResultsR2* remained invariant regardless of the cogeneration timing. However, magnetic susceptibility was significantly altered when processed in transformed space, whereas it remained invariant in all KS-tests and pixel values were only different in 2 out of 64 U-tests for direct QSM coregistration. Paired t-test revealed that ROI-based group-mean R2* was invariant to both approaches, while group-mean susceptibility was invariant to direct coregistration but differed in one structure processed in transformed space. For all pairs of measurements of R2*, ICCs were excellent. ICCs for magnetic susceptibility were excellent when processed in its native space while the ICCs were lower than 0.9 or poor when processed in transformed space. Further analysis revealed that the choice of interpolation approach affected the resultant QSM. ConclusionsOur study shows that R2* could be safely processed in a transformed space, whereas QSM was less reliable when processed in the transformed space. Hence, caution is advised when using QSM in a multi-parametric study, and it is strongly recommended to process QSM in its native space prior to any coregistration or spatial transformation.

bioengineering↗