We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.he study of RNA is a critical component of biological research and in the diagnosis and treatment of disease. Recently, the localization of mRNA has been identified as an essential process for a number of cellular functions, including restricting the production of certain proteins to specific compartments within cells (1). For instance, synaptic potentiation, the basis of learning and memory, relies on the local translation of specific mRNAs in pre-and postsynaptic compartments (2). Likewise, the misregulation of RNA distribution is associated with many disorders, including mental retardation, autism, and cancer metastasis (3-5). However, despite the significant role of mRNA transport and localization in cellular function, the available methods to visualize these phenomena are severely limited. For example, FISH, the most commonly used technique to analyze spatial distribution of RNA, requires fixation and permeabilization of cells before analysis (6). As a result, analysis of dynamic RNA distribution is restricted to a single snapshot in time (7,8). With such a limitation, understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult if not impossible. Furthermore, fixed cell analysis is a lengthy and highly specialized procedure due to the number of steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases. Likewise, live cell analysis platforms such as molecular beacons require toxic transfection techniques, such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus on cellular entry (9, 10). Recently more sophisticated live cell analyses have been developed that use genetic engineering to introduce exogenous hybrid gene...
Objectives Recent research suggests that brown adipose tissue (BAT) plays a functional role in non‐shivering thermogenesis; however, few studies have examined population variation in BAT or its relationship with other mechanisms of adaptation to cold stress. This study characterized BAT thermogenesis and other adaptive responses to low temperatures among Indigenous Siberian young adults and young adults living near Chicago, IL. Materials and methods We recruited 72 Yakut participants (42 females; 30 males) and 54 participants in Evanston, IL (40 females; 14 males). Anthropometric dimensions and resting metabolic rate (RMR) were measured, and we calculated percent divergence in RMR from expected values (divRMR). We also quantified change in supraclavicular temperature, sternum temperature, and energy expenditure after a mild cooling condition. Results Participants in Yakutia were less likely to shiver during the cooling condition (p < .05) and exhibited significantly greater evidence of BAT thermogenesis, warmer sternum temperatures, and higher divRMR than participants in Evanston (p < .05). Additionally, the relationship between change in supraclavicular temperature and energy expenditure differed between the two samples. Conclusions Yakut young adults displayed greater evidence of BAT thermogenesis in response to mild cooling compared with young adults living near Chicago, IL. Furthermore, the relationship between BAT thermogenesis and change in energy expenditure appears to be stronger among Yakut adults. Adults that exhibited greater metabolic response to cold stress, such as higher BAT thermogenesis and divRMR, maintained warmer sternum temperatures. These results highlight the degree to which adaptation to cold climates involves multiple integrated biological pathways.
We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.
Objectives: Telomeres are the protective caps of chromosomes. They shorten with cell replication, age, and possibly environmental stimuli (e.g., infection and stress). Short telomere length (TL) predicts subsequent worse health. Although venous whole blood (VWB) is most commonly used for TL measurement, other, more minimally-invasive, sampling techniques are becoming increasingly common due to their field-friendliness, allowing for feasible measurement in low-resource contexts. We conducted validation work for measuring TL in dried blood spots (DBS) and incorporated our results into a meta-analysis evaluating minimally-invasive sampling techniques to measure TL.Methods: We isolated DNA extracts from DBS using a modified extraction protocol and tested how they endured different shipping conditions and long-term cryostorage. We then included our in-house DBS TL validation statistics (correlation values with VWB TL and age) in a series of meta-analyses of results from 24 other studies that published similar associations for values between TL measured in DBS, saliva, and buccal cells (BC). Results: Our modified DBS extraction technique produced DNA yields that were roughly twice as large as previously recorded. Partially extracted DBS DNA was stable for seven days at room temperature, and still provided reliable TL measurements, as determined by external validation statistics. In our meta-analysis, DBS TL had the highest external validity (i.e., strongest overall association with both VWB TL and age), followed by saliva, and then BC. Conclusions: Measuring TL using DNA isolated from DBS is the best option for minimally-invasively collected samples, which can be used to expand research to diverse settings and populations.
Objectives Telomeres are the protective caps of chromosomes. They shorten with cell replication, age, and possibly environmental stimuli (eg, infection and stress). Short telomere length (TL) predicts subsequent worse health. Although venous whole blood (VWB) is most commonly used for TL measurement, other, more minimally invasive, sampling techniques are becoming increasingly common due to their field‐friendliness, allowing for feasible measurement in low‐resource contexts. We conducted statistical validation work for measuring TL in dried blood spots (DBS) and incorporated our results into a meta‐analysis evaluating minimally invasive sampling techniques to measure TL. Methods We isolated DNA extracts from DBS using a modified extraction protocol and tested how they endured different shipping conditions and long‐term cryostorage. We then included our in‐house DBS TL validation statistics (correlation values with VWB TL and age) in a series of meta‐analyses of results from 24 other studies that published similar associations for values between TL measured in DBS, saliva, and buccal cells. Results Our modified DBS extraction technique produced DNA yields that were roughly twice as large as previously recorded. Partially extracted DBS DNA was stable for 7 days at room temperature, and still provided reliable TL measurements, as determined by external validation statistics. In our meta‐analysis, DBS TL had the highest external validity, followed by saliva, and then buccal cells—possibly reflecting similarities/differences in cellular composition vs VWB. Conclusions DBS DNA is the best proxy for VWB from the three minimally‐invasively specimen types evaluated and can be used to expand TL research to diverse settings and populations.
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