Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s-1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.

Trait Mindfulness and Social Support Predict Lower Perceived Stress Burden in Patients Undergoing Radiation Therapy.

Purpose: Cancer diagnosis and treatment, including radiation therapy (RT), cause significant patient stress. Mindfulness and social support have been shown to help manage the psychological effects of cancer treatment. The objective of our study was to determine the sociodemographic and clinical factors associated with stress burden in patients receiving RT. Methods and Materials: Patients receiving RT for cancer at a single institution were given a 3-section survey to complete during the first on-treatment visit. The survey included the Perceived Stress Scale, Medical Outcomes Study Social Support Survey, and Mindfulness Attention Awareness Scale, which were used to measure stress, social support, and trait mindfulness, respectively. Linear regression analysis was performed to determine associations between perceived stress and age, patient sex, race and ethnicity, treatment intent, disease site, trait mindfulness, and social support. Factors significant in univariable analysis were analyzed with a multivariable analysis. Results: A total of 93 patients undergoing RT at a tertiary care academic institution were recruited from July to September 2019. Median scores for Perceived Stress Scale, Medical Outcomes Study Social Support Survey, and Mindfulness Attention Awareness Scale were 14.6 (range, 0-31; SD, 6.9), 4.2 (range, 1-5; SD, 1.0), and 5.1 (range, 3.1-6.0; SD, 0.8), respectively. On univariable analysis, mindfulness and social support were associated with decreased stress burden, and female sex and palliative intent were associated with increased stress burden. These factors all maintained significance in multivariable analysis. Conclusions: These results suggest measures to improve mindfulness and perceived social support, such as mindfulness meditation and psychoeducational approaches, may lessen the stress burden and improve quality of life for patients undergoing RT. Future studies should analyze the longitudinal impact of individual patient characteristics, including patient sex and treatment intent, to better understand their effects on psychological maladjustment during cancer care.

Measuring integrin force loading rates using a two-step DNA tension sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (pN) forces exerted by cells have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrins receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop a LR probe which is comprised of an engineered DNA structures that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN corresponding to hairpin unfolding and a high force threshold at 56 pN triggered through duplex shearing. These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live-cells. Automated analysis of tens of thousands of events from 8 cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 sec. A small subset of these events (<10%) mature in magnitude to >56pN with a median loading rate of 1.3 pNs-1 with these mechanical ramp events localizing at the periphery of the cell-substrate junction. Importantly, the LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of mechanotransduction.

An Endosomal Escape Trojan Horse Platform to Improve Cytosolic Delivery of Nucleic Acids.

Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers.

Force-Triggered Self-Destructive Hydrogels.

Self-destructive polymers (SDPs) are defined as a class of smart polymers that autonomously degrade upon experiencing an external trigger, such as a chemical cue or optical excitation. Because SDPs release the materials trapped inside the network upon degradation, they have potential applications in drug delivery and analytical sensing. However, no known SDPs that respond to external mechanical forces have been reported, as it is fundamentally challenging to create mechano-sensitivity in general and especially so for force levels below those required for classical force-induced bond scission. To address this challenge, the development of force-triggered SDPs composed of DNA crosslinked hydrogels doped with nucleases is described here. Externally applied piconewton forces selectively expose enzymatic cleavage sites within the DNA crosslinks, resulting in rapid polymer self-degradation. The synthesis and the chemical and mechanical characterization of DNA crosslinked hydrogels, as well as the kinetics of force-triggered hydrolysis, are described. As a proof-of-concept, force-triggered and time-dependent rheological changes in the polymer as well as encapsulated nanoparticle release are demonstrated. Finally, that the kinetics of self-destruction are shown to be tuned as a function of nuclease concentration, incubation time, and thermodynamic stability of DNA crosslinkers.

Cytomegalovirus infection in heart transplant patient presenting as appendicitis.

Cytomegalovirus (CMV) may manifest in various ways. While immunocompetent hosts may be asymptomatic or present with a mononucleosis-like illness, immunocompromised patients can have organ-specific disease capable of significant morbidity and mortality. CMV appendicitis is a particularly rare presentation. A 22-year-old female with a history of orthotopic heart transplantation presented to our hospital with a three-day history of worsening abdominal pain. A computed tomography scan of her abdomen was consistent with acute uncomplicated appendicitis, and she underwent laparoscopic appendectomy. Pathology revealed acute appendicitis with numerous large cells with intranuclear "owl's eye" inclusions characteristic of CMV. Her CMV viral load was elevated at 327,018 IU/ml. She was started on ganciclovir which resulted in improvement of her CMV level to 30,118 IU/ml within three weeks. CMV is a frequent cause of opportunistic infection in solid organ transplant patients and commonly involves the gastrointestinal tract. Acute appendicitis is a rarely reported complication to consider in the differential diagnosis of abdominal pain in immunocompromised patients. Learning objective: Heart transplant recipients are at increased risk for opportunistic infections. Cytomegalovirus (CMV) is a frequent culprit and can present with a broad range of disease. A particularly rare presentation is that of acute appendicitis. We describe a case of a young woman with CMV appendicitis following orthotopic heart transplant.

Diagnosis of Alzheimer's disease in an exhumed decomposed brain after twenty months of burial in a deep grave.

After 20 months of interment in a deep grave, the decomposed body of the 81-year old testator of a will was exhumed to sustain the burden of proof that he lacked testamentary capacity when the will was rewritten two days prior to his death. The brain was mushy and pulverized with complete disappearance of the brainstem, cerebellum and subcortical ganglia. Small foci of relatively intact dorsal frontal neocortex were identified. Sections from these foci were stained with hematoxylin and eosin, bielchowsky silver stain and immunostains for beta amyloid peptide (betaA4), tau and alpha-synuclein. Despite severe autolysis and decomposition, the bielchowsky stain and the betaA4 immunostains showed preserved frequent neuritic amyloid plaques with very few residual neurofibrillary tangles. Cerebral Amyloid Angiopathy was present. At the present time this case represents the first documented and reported case of direct tissue diagnosis of Alzheimer's Disease pathology in a decomposed brain following long term burial in a deep grave.