PubMed

Recent Publications

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells

Sci Rep. 2021 Feb 25;11(1):4523. doi: 10.1038/s41598-021-84071-6.

ABSTRACT

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

PMID:33633238 | PMC:PMC7907388 | DOI:10.1038/s41598-021-84071-6



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netDx: Software for building interpretable patient classifiers by multi-'omic data integration using patient similarity networks

F1000Res. 2020 Oct 15;9:1239. doi: 10.12688/f1000research.26429.2. eCollection 2020.

ABSTRACT

Patient classification based on clinical and genomic data will further the goal of precision medicine. Interpretability is of particular relevance for models based on genomic data, where sample sizes are relatively small (in the hundreds), increasing overfitting risk netDx is a machine learning method to integrate multi-modal patient data and build a patient classifier. Patient data are converted into networks of patient similarity, which is intuitive to clinicians who also use patient similarity for medical diagnosis. Features passing selection are integrated, and new patients are assigned to the class with the greatest profile similarity. netDx has excellent performance, outperforming most machine-learning methods in binary cancer survival prediction. It handles missing data - a common problem in real-world data - without requiring imputation. netDx also has excellent interpretability, with native support to group genes into pathways for mechanistic insight into predictive features. The netDx Bioconductor package provides multiple workflows for users to build custom patient classifiers. It provides turnkey functions for one-step predictor generation from multi-modal data, including feature selection over multiple train/test data splits. Workflows offer versatility with custom feature design, choice of similarity metric; speed is improved by parallel execution. Built-in functions and examples allow users to compute model performance metrics such as AUROC, AUPR, and accuracy. netDx uses RCy3 to visualize top-scoring pathways and the final integrated patient network in Cytoscape. Advanced users can build more complex predictor designs with functional building blocks used in the default design. Finally, the netDx Bioconductor package provides a novel workflow for pathway-based patient classification from sparse genetic data.

PMID:33628435 | PMC:PMC7883323 | DOI:10.12688/f1000research.26429.2



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A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth

ACS Chem Biol. 2021 Mar 19;16(3):548-556. doi: 10.1021/acschembio.1c00017. Epub 2021 Feb 23.

ABSTRACT

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

PMID:33621466 | DOI:10.1021/acschembio.1c00017



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Understanding right ventricular dyssynchrony: Its myriad determinants and clinical relevance

Exp Physiol. 2021 Apr;106(4):797-800. doi: 10.1113/EP089366. Epub 2021 Mar 2.

NO ABSTRACT

PMID:33599973 | DOI:10.1113/EP089366



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Biological and therapeutic implications of a unique subtype of NPM1 mutated AML

Nat Commun. 2021 Feb 16;12(1):1054. doi: 10.1038/s41467-021-21233-0.

ABSTRACT

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.

PMID:33594052 | PMC:PMC7886883 | DOI:10.1038/s41467-021-21233-0



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ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping

NPJ Genom Med. 2020 Apr 8;5(1):16. doi: 10.1038/s41525-020-0123-6.

ABSTRACT

Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3 :c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

PMID:33579975 | DOI:10.1038/s41525-020-0123-6



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PRMT5 inhibition disrupts splicing and stemness in glioblastoma

Nat Commun. 2021 Feb 12;12(1):979. doi: 10.1038/s41467-021-21204-5.

ABSTRACT

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

PMID:33579912 | PMC:PMC7881162 | DOI:10.1038/s41467-021-21204-5



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Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis

Mol Metab. 2021 Feb 6;47:101185. doi: 10.1016/j.molmet.2021.101185. Online ahead of print.

ABSTRACT

OBJECTIVE: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology.

METHODS: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre+ UVRAGfl/fl (M-UVRAG-/-) mice were compared to littermate Myf6-Cre+ UVRAG+/+ (M-UVRAG+/+) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age.

RESULTS: M-UVRAG-/- mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG+/+ littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG-/- mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis.

CONCLUSIONS: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.

PMID:33561544 | PMC:PMC7921879 | DOI:10.1016/j.molmet.2021.101185



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Diverse Eukaryotic CGG Binding Proteins Produced by Independent Domestications of hAT Transposons

Mol Biol Evol. 2021 Feb 9:msab007. doi: 10.1093/molbev/msab007. Online ahead of print.

ABSTRACT

The human transcription factor (TF) CGGBP1 ("CGG Binding Protein") is conserved only in amniotes, and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a hAT DNA transposon. Here, we show that sequence-specific DNA binding proteins with this bipartite domain structure have resulted from dozens of independent hAT domestications in different eukaryotic lineages. CGGBPs display a wide range of sequence specificity, usually including preferences for CGG or CGC trinucleotides, while some bind AT-rich motifs. The CGGBPs are almost entirely non-syntenic, and their protein sequences, DNA binding motifs, and patterns of presence or absence in genomes are uncharacteristic of ancestry via speciation. At least eight CGGBPs in the coelacanth Latimeria chalumnae bind distinct motifs, and the expression of the corresponding genes varies considerably across tissues, suggesting tissue-restricted function.

PMID:33561217 | DOI:10.1093/molbev/msab007



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Crystal structure of the BRPF2 PWWP domain in complex with DNA reveals a different binding mode than the HDGF family of PWWP domains

Biochim Biophys Acta Gene Regul Mech. 2021 Mar;1864(3):194688. doi: 10.1016/j.bbagrm.2021.194688. Epub 2021 Feb 6.

ABSTRACT

The PWWP domain was first identified in the HDGF protein family and named after the conserved Proline-Tryptophan-Tryptophan-Proline motif in WHSC1. The PWWP domain-containing proteins play important roles in different biological processes, such as DNA replication, transcription, DNA repair, pre-mRNA processing by recognizing methylated histone and dsDNA simultaneously. Recently, how the HDGF family of PWWP domains recognize histone H3K36me3-modified nucleosome has been reported. In order to better understand the interactions between the PWWP domain and dsDNA, we carried out family-wide characterization of dsDNA binding abilities of human PWWP domains. Our binding assays confirmed that PWWP domains bind to dsDNA without sequence selectivity. Our crystal structure of the BRPF2 PWWP domain in complex with a 12-mer dsDNA reveals that the PWWP domain interacts with dsDNA by binding to its major groove, instead of the minor groove observed in the HDGF family of PWWP domains. Our study indicates that PWWP domains could bind to dsDNA in different modes.

PMID:33556623 | DOI:10.1016/j.bbagrm.2021.194688



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