Significance: Telomerase, which extends DNA at chromosome ends, is composed of an RNA template and a catalytic protein subunit, telomerase reverse transcriptase (TERT). TERT gene expression has been of great interest because it is required for proliferation of most cancers, but expression investigations have been limited due to low endogenous mRNA levels. Here, we employ single-molecule RNA fluorescent in situ hybridization (FISH) in 10 human cancer cell lines and make findings that are unanticipated based upon bulk TERT mRNA measurements. For example, there is great cell-to-cell variation in the number of transcription sites, and spliced TERT mRNA has greater nuclear localization than cytoplasmic. Overall, our findings provide insights regarding TERT expression, localization patterns, and variability in cancer on a single-cell level.

Telomerase reverse transcriptase (TERT) is pathologically expressed in the vast majority of human cancers, but the epigenetic regulation of its expression is only beginning to be understood. In particular, the active TERT gene in cancer cells has been characterized as having a hypermethylated CpG island, opposite to the general association of DNA methylation with gene repression. Here, we analyzed TERT promoter CpG methylation in 833 human cancer cell lines representing 23 different tissue types and found hypermethylation of the upstream portion of the CpG island and more conserved hypomethylation of a region including the proximal TERT promoter and exon 1. In cell lines with monoallelic expression of TERT, we found allelic methylation of the proximal TERT promoter. This included cell lines with the −124 or −146 activating promoter mutation as well as wild‐type TERT cancer lines. In these cell line types, decreased proximal promoter methylation is associated with the active allele. Compared to cells with monoallelic expression of TERT, lines with biallelic expression of TERT had even lower methylation in the proximal TERT promoter. Thus, in cell lines from cancers of many different tissues, the TERT proximal promoter has canonical DNA methylation, with low methylation correlating with increased TERT expression.

Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes’ energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide–thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

Although it is estimated that more than one million Americans have celiac disease (CD), it remains challenging to diagnose. CD, an autoimmune and inflammatory response following the ingestion of gluten-containing foods, has symptoms overlapping with other diseases and requires invasive diagnostics. The gold standard for CD diagnosis involves serologic blood tests followed by invasive confirmatory biopsies. Here, we propose a less invasive method using an electrochemical DNA (E-DNA) biosensor for CD-specific autoantibodies (AABs) circulating in blood. In our approach, CD-specific AABs bind a synthetic neoepitope, causing a conformational change in the biosensor, as well as a change in the environment of an attached redox reporter, producing a measurable current reduction. We assessed the biosensor’s ability to detect CD-specific patient-derived AABs in physiological buffer as well as buffer supplemented with bovine serum. Our biosensor was able to detect AABs in a dose-dependent manner; increased signal change correlated with increased AAB concentration with an apparent dissociation constant of 0.09 ± 0.03 units/mL of AABs. Furthermore, we found our biosensor to be target-specific, with minimal off-target binding of multiple unrelated biomarkers. Future efforts aimed at increasing sensitivity in complex media may build upon the biosensor design presented here to further improve CD AAB detection and CD diagnostic tools.