Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

Extending the dynamic range of biomarker quantification through molecular equalization.

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved-in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two-pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our 'EVROS' strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 µl sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.

Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

Sensitivity of leaf size and shape to climate: global patterns and paleoclimatic applications.

• Paleobotanists have long used models based on leaf size and shape to reconstruct paleoclimate. However, most models incorporate a single variable or use traits that are not physiologically or functionally linked to climate, limiting their predictive power. Further, they often underestimate paleotemperature relative to other proxies. • Here we quantify leaf-climate correlations from 92 globally distributed, climatically diverse sites, and explore potential confounding factors. Multiple linear regression models for mean annual temperature (MAT) and mean annual precipitation (MAP) are developed and applied to nine well-studied fossil floras. • We find that leaves in cold climates typically have larger, more numerous teeth, and are more highly dissected. Leaf habit (deciduous vs evergreen), local water availability, and phylogenetic history all affect these relationships. Leaves in wet climates are larger and have fewer, smaller teeth. Our multivariate MAT and MAP models offer moderate improvements in precision over univariate approaches (± 4.0 vs 4.8°C for MAT) and strong improvements in accuracy. For example, our provisional MAT estimates for most North American fossil floras are considerably warmer and in better agreement with independent paleoclimate evidence. • Our study demonstrates that the inclusion of additional leaf traits that are functionally linked to climate improves paleoclimate reconstructions. This work also illustrates the need for better understanding of the impact of phylogeny and leaf habit on leaf-climate relationships.

Lipid Biomarker Record of the Serpentinite-Hosted Ecosystem of the Samail Ophiolite, Oman and Implications for the Search for Biosignatures on Mars.

Serpentinization is a weathering process in which ultramafic rocks react with water, generating a range of products, including serpentine and other minerals, in addition to H2 and low-molecular-weight hydrocarbons that are capable of sustaining microbial life. Lipid biomarker analyses of serpentinite-hosted ecosystems hold promise as tools for investigating microbial activity in ancient Earth environments and other terrestrial planets such as Mars because lipids have the potential for longer term preservation relative to DNA, proteins, and other more labile organic molecules. Here, we report the first lipid biomarker record of microbial activity in the mantle section of the Samail Ophiolite, in the Sultanate of Oman, a site undergoing active serpentinization. We detected isoprenoidal (archaeal) and branched (bacterial) glycerol dialkyl glycerol tetraether (GDGT) lipids, including those with 0-3 cyclopentane moieties, and crenarchaeol, an isoprenoidal GDGT containing four cyclopentane and one cyclohexane moieties, as well as monoether lipids and fatty acids indicative of sulfate-reducing bacteria. Comparison of our geochemical data and 16S rRNA data from the Samail Ophiolite with those from other serpentinite-hosted sites identifies the existence of a common core serpentinization microbiome. In light of these findings, we also discuss the preservation potential of serpentinite lipid biomarker assemblages on Earth and Mars. Continuing investigations of the Samail Ophiolite and other terrestrial analogues will enhance our understanding of microbial habitability and diversity in serpentinite-hosted environments on Earth and elsewhere in the Solar System.

High density DNA data storage library via dehydration with digital microfluidic retrieval.

DNA promises to be a high density data storage medium, but physical storage poses a challenge. To store large amounts of data, pools must be physically isolated so they can share the same addressing scheme. We propose the storage of dehydrated DNA spots on glass as an approach for scalable DNA data storage. The dried spots can then be retrieved by a water droplet using a digital microfluidic device. Here we show that this storage schema works with varying spot organization, spotted masses of DNA, and droplet retrieval dwell times. In all cases, the majority of the DNA was retrieved and successfully sequenced. We demonstrate that the spots can be densely arranged on a microfluidic device without significant contamination of the retrieval. We also demonstrate that 1 TB of data could be stored in a single spot of DNA and successfully retrieved using this method.

Decreasing Unplanned Office Visits Due to Cast Problems in the Pediatric Population.

INTRODUCTION: Unplanned office visits due to cast-related problems in the pediatric orthopedic office are common. Decreasing problems associated with the use of a cast would improve patient safety, increase office productivity, and decrease inconvenience to the child and family. METHODS: Pediatric patients treated with a cast in our office were included in the study if they returned for an unplanned office visit due to a cast-related problem. Group 1 received verbal cast care instruction. Group 2 had the same verbal instruction in addition to a written handout identical to the verbal instructions. Group 3 was provided the same verbal instructions and a revised handout limiting the number of instructions and focused on keeping the cast away from water. RESULTS: The study included 550 patients with 146 in group 1, 124 in group 2, and 280 in group 3. Comparing group 1 (10.3%) and group 2 (10.5%), there was almost no difference in the rate of unplanned office visits due to cast-related problems. Combining the revised handout with verbal instructions in group 3, the percentage of patients returning for an unplanned visit was 6%. There was a relative decrease in office visits by 55% and an absolute decrease of 4.5% when comparing group 2 and group 3. CONCLUSIONS: There was a decrease in the number of unplanned office visits due to cast problems utilizing a handout focused on keeping the cast dry in collaboration with verbal cast care instructions. However, the decrease was not statistically significant.

Random access in large-scale DNA data storage.

Synthetic DNA is durable and can encode digital data with high density, making it an attractive medium for data storage. However, recovering stored data on a large-scale currently requires all the DNA in a pool to be sequenced, even if only a subset of the information needs to be extracted. Here, we encode and store 35 distinct files (over 200 MB of data), in more than 13 million DNA oligonucleotides, and show that we can recover each file individually and with no errors, using a random access approach. We design and validate a large library of primers that enable individual recovery of all files stored within the DNA. We also develop an algorithm that greatly reduces the sequencing read coverage required for error-free decoding by maximizing information from all sequence reads. These advances demonstrate a viable, large-scale system for DNA data storage and retrieval.