No abstract

Single-molecule DNA sequencing based on measuring the physical properties of bases as they pass through a nanopore1,2 eliminates the need for the enzymes and reagents used in other approaches. Theoretical calculations indicate that electron tunneling could identify bases in single-stranded DNA, yielding long reads and eliminating enzymatic processing.3–5 It was shown recently that tunneling can sense individual nucleotides6 and nucleosides.7 Here, we show that tunneling electrodes functionalized with recognition reagents can identify a single base flanked by other bases in a short DNA oligomer. The residence time of a single base in a recognition junction is on the order of a second, but pulling the DNA through the junction with a force of tens of piconewtons would yield reading speeds of tens of bases per second.

The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts.

The mechanical stiffness of individual cells is important in tissue homeostasis, cell growth, division, and motility, and the epithelial-mesenchymal transition in the initiation of cancer. In this work, a normal squamous cell line (EPC2) and metaplastic (CP-A) as well as dysplastic (CP-D) Barrett’s Esophagus columnar cell lines are studied as a model of pre-neoplastic progression in the human esophagus. We used the combination of an atomic force microscope (AFM) with a scanning confocal fluorescence lifetime imaging microscope (FLIM) to study the mechanical properties of single adherent cells. 64 force indentation curves were taken over the nucleus of each cell in an 8×8 grid pattern. Analyzing the force indentation curves, indentation depth dependent Young’s moduli were found for all cell lines. Stiffness tomograms demonstrate distinct differences between the mechanical properties of the studied cell lines. Comparing the stiffness for indentation forces of 1 nN, most probable Young’s moduli were calculated to 4.7 kPa for EPC2 (n=18 cells), 3.1 kPa for CP-A (n=10), and 2.6 kPa for CP-D (n=19). We also tested the influence of nuclei and nucleoli staining organic dyes on the mechanical properties of the cells. For stained EPC2 cells (n=5), significant stiffening was found (9.9 kPa), while CP-A cells (n=5) showed no clear trend (2.9 kPa) and a slight softening was observed (2.1 kPa) in the case of CP-D cells (n=16). Some force-indentation curves show non-monotonic discontinuities with segments of negative slope, resembling a sawtooth pattern. We found the incidence of these ‘breakthrough events’ to be highest in the dysplastic CP-D cells, intermediate in the metaplastic CP-A cells, and lowest in the normal EPC2 cells. This observation suggests that the microscopic explanation for the increased compliance of cancerous and pre-cancerous cells may lie in their susceptibility to ‘crumble and yield’ rather than their ability to ‘bend and flex’.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate. STEM CELLS