PubMed

Recent Publications

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

J Vis Exp. 2021 Feb 18;(168). doi: 10.3791/62307.

ABSTRACT

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

PMID:33682863 | DOI:10.3791/62307



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A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2

Nat Commun. 2021 Mar 3;12(1):1405. doi: 10.1038/s41467-021-21653-y.

ABSTRACT

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

PMID:33658502 | DOI:10.1038/s41467-021-21653-y



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Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism

Elife. 2021 Mar 1;10:e64806. doi: 10.7554/eLife.64806. Online ahead of print.

ABSTRACT

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologues in snake venoms (LAAO, L-amino acid oxidases), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell productive gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

PMID:33646117 | DOI:10.7554/eLife.64806



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Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

Genomics Proteomics Bioinformatics. 2021 Feb 23:S1672-0229(21)00037-1. doi: 10.1016/j.gpb.2020.06.020. Online ahead of print.

ABSTRACT

Synechocystis sp. PCC 6803 (hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Though known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, we used co-fractionation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to define 291 multi-protein complexes, encompassing 24,092 protein-protein interactions (PPIs) among 2062 distinct gene products. This information not only revealed the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also showed how protein functions vary from bacteria to higher plants due to changes in interaction partners. It also allowed us to uncover the functions of hypothetical proteins, such as Sll0445, Sll0446, and Sll0447, participating in photosynthesis and cell motility, and Sll1334 in regulating fatty acid biogenesis. Here we present the most extensive protein interaction data for Synechocystis so far, which provides critical insights into fundamental molecular mechanisms in Cyanobacterium.

PMID:33636367 | DOI:10.1016/j.gpb.2020.06.020



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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 Feb 23. doi: 10.1021/acschembio.1c00017. Online ahead of print.

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 Feb 18. doi: 10.1113/EP089366. Online ahead of print.

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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