Probing excited conformational states involved in microRNA biogenesis

MicroRNAs are evolutionarily conserved small, non-coding RNAs, ~20-22 nucleotides in length, which serve as critical gene regulators in many aspects of biological processes, including development, cellular proliferation, differentiation, and apoptosis. Due to this central regulatory role of microRNAs, their biogenesis is tightly regulated through the binding of multiple protein activators/suppressors and sequence-specific recognition motifs that are utilized by the biogenesis machineries. While the importance of these microRNA sequences in their self-regulation of biogenesis has just begun to be unveil, most of these RNA elements are highly dynamic and have evaded high-resolution structural studies by conventional structural biology techniques. In my thesis work, I have developed and applied state-of-the-art solution-state nuclear magnetic resonance (NMR) techniques, which enabled me to overcome technical challenges in high-resolution structural and dynamic characterization of these flexible RNAs. Together with mutagenesis and functional assays, I have first characterized structure, dynamics, and function of the precursor of microRNA-21 (pre-miR-21), one of the first identified human microRNAs, which also functions as an oncogene involved in tumorigenesis, progression, and metastasis. My results unveiled a pH-dependent conformational ensemble in pre-miR-21 that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, where the alternative excited conformation transiently sequesters the bulged adenine into a non-canonical protonated A+–G mismatch and confers a two-fold enhancement in Dicer processing. Next, I have studied the mismatch GHG (mGHG) motif in primary microRNAs, an essential RNA recognition site for the microprocessor (Drosha/DGCR8). Remarkably, my results revealed that the mGHG motif also exists as a pH-dependent conformational ensemble that involves a sparsely populated and short-lived protonated state. Furthermore, I showed that these inherent excited-state-mediated dynamics correlate with microRNA recognition and/or productive cleavage by the microprocessor complex. Together, these studies suggest that microRNA can encode a complex conformational landscape to direct functional outcomes, adopting multiple conformations that result in differential enzymatic fitness for each cleavage step of the canonical microRNA biogenesis pathway and exemplifying a novel RNA-centric regulation of microRNA biogenesis.Doctor of Philosoph

High-resolution continuous-flow analysis setup for water isotopic measurement from ice cores using laser spectroscopy

Here we present an experimental setup for water stable isotope (&delta;¹⁸O and δD) continuous-flow measurements and provide metrics defining the performance of the setup during a major ice core measurement campaign (Roosevelt Island Climate Evolution; RICE). We also use the metrics to compare alternate systems. Our setup is the first continuous-flow laser spectroscopy system that is using off-axis integrated cavity output spectroscopy (OA-ICOS; analyzer manufactured by Los Gatos Research, LGR) in combination with an evaporation unit to continuously analyze water samples from an ice core. <br><br> A Water Vapor Isotope Standard Source (WVISS) calibration unit, manufactured by LGR, was modified to (1) enable measurements on several water standards, (2) increase the temporal resolution by reducing the response time and (3) reduce the influence from memory effects. While this setup was designed for the continuous-flow analysis (CFA) of ice cores, it can also continuously analyze other liquid or vapor sources. <br><br> The custom setups provide a shorter response time (~ 54 and 18 s for 2013 and 2014 setup, respectively) compared to the original WVISS unit (~ 62 s), which is an improvement in measurement resolution. Another improvement compared to the original WVISS is that the custom setups have a reduced memory effect. <br><br> Stability tests comparing the custom and WVISS setups were performed and Allan deviations (&sigma;_Allan) were calculated to determine precision at different averaging times. For the custom 2013 setup the precision after integration times of 10³ s is 0.060 and 0.070 &permil; for δ¹⁸O and δD, respectively. The corresponding &sigma;_Allan values for the custom 2014 setup are 0.030, 0.060 and 0.043 &permil; for &delta;¹⁸O, δD and &delta;¹⁷O, respectively. For the WVISS setup the precision is 0.035, 0.070 and 0.042 &permil; after 10³ s for δ¹⁸O, δD and &delta;¹⁷O, respectively. Both the custom setups and WVISS setup are influenced by instrumental drift with δ¹⁸O being more drift sensitive than δD. The &sigma;_Allan values for δ¹⁸O are 0.30 and 0.18 &permil; for the custom 2013 and WVISS setup, respectively, after averaging times of 10⁴ s (2.78 h). Using response time tests and stability tests, we show that the custom setups are more responsive (shorter response time), whereas the University of Copenhagen (UC) setup is more stable. More broadly, comparisons of different setups address the challenge of integrating vaporizer/spectrometer isotope measurement systems into a CFA campaign with many other analytical instruments

Effect of inhaled tacrolimus on ischemia reperfusion injury in rat lung transplant model

ObjectiveSystemic tacrolimus therapy has been shown to protect against lung ischemia-reperfusion injury in animal models. We sought to investigate on a functional and cellular level if inhaled nanoparticle tacrolimus administered to the donor lung before procurement could similarly attenuate ischemia-reperfusion injury after lung transplant.MethodsAn isogenic orthotopic rat model of single left lung transplant was used. Donor animals were pretreated with inhaled tacrolimus (treatment group) or inhaled lactose (controls) before lung procurement. Lung grafts were subjected to 3 hours of cold ischemia followed by 4 hours of reperfusion after graft implantation. Recipient animal arterial blood gas measurement and isograft wet to dry weight ratios were obtained. Macrophage, neutrophil, and T-cell accumulation and activation in lung isografts, including γδ T-cell, T-helper, and cytotoxic T-cell subtypes were analyzed by flow cytometry. Tacrolimus levels were measured in the lung isograft using liquid chromatography/mass spectrometry. Isograft cytokine levels were measured with commercial enzyme-linked immunosorbent assay and microbead array kits.ResultsOxygenation in treatment group animals was significantly higher than in controls. The presence of macrophages, neutrophils, and all T-cell subtypes in the isografts as well as isograft levels of inflammatory cytokines were all less in the treatment group versus controls, although no single variable achieved statistical significance.ConclusionsInhaled nanoparticle tacrolimus treatment of lung donors is associated with an attenuation of ischemia-reperfusion injury on a functional and cellular level in lung transplant

Calculating uncertainty for the RICE ice core continuous flow analysis water isotope record

We describe a systematic approach to the calibration and uncertainty estimation of a high-resolution continuous flow analysis (CFA) water isotope (δ2H, δ18O) record from the Roosevelt Island Climate Evolution (RICE) Antarctic ice core. Our method establishes robust uncertainty estimates for CFA δ2H and δ18O measurements, comparable to those reported for discrete sample δ2H and δ18O analysis. Data were calibrated using a time-weighted two-point linear calibration with two standards measured both before and after continuously melting 3 or 4&thinsp;m of ice core. The error at each data point was calculated as the quadrature sum of three factors: Allan variance error, scatter over our averaging interval (error of the variance) and calibration error (error of the mean). Final mean total uncertainty for the entire record is δ2H = 0.74 ‰ and δ18O = 0.21 ‰. Uncertainties vary through the data set and were exacerbated by a range of factors, which typically could not be isolated due to the requirements of the multi-instrument CFA campaign. These factors likely occurred in combination and included ice quality, ice breaks, upstream equipment failure, contamination with drill fluid and leaks or valve degradation. We demonstrate that our methodology for documenting uncertainty was effective across periods of uneven system performance and delivered a significant achievement in the precision of high-resolution CFA water isotope measurements.</p

Diverse soil carbon dynamics expressed at the molecular level

The stability and potential vulnerability of soil organic matter (SOM) to global change remains incompletely understood due to the complex processes involved in its formation and turnover. Here we combine compound-specific radiocarbon analysis with fraction-specific and bulk-level radiocarbon measurements in order to further elucidate controls on SOM dynamics in a temperate and sub-alpine forested ecosystem. Radiocarbon contents of individual organic compounds isolated from the same soil interval generally exhibit greater variation than those among corresponding operationally-defined fractions. Notably, markedly older ages of long-chain plant leaf wax lipids (n-alkanoic acids) imply that they reflect a highly stable carbon pool. Furthermore, marked 14C variations among shorter- and longer-chain n-alkanoic acid homologues suggest that they track different SOM pools. Extremes in SOM dynamics thus manifest themselves within a single compound class. This exploratory study highlights the potential of compound-specific radiocarbon analysis for understanding SOM dynamics in ecosystems potentially vulnerable to global change

The Ross Sea Dipole-temperature, snow accumulation and sea ice variability in the Ross Sea region, Antarctica, over the past 2700 years

High-resolution, well-dated climate archives provide an opportunity to investigate the dynamic interactions of climate patterns relevant for future projections. Here, we present data from a new, annually dated ice core record from the eastern Ross Sea, named the Roosevelt Island Climate Evolution (RICE) ice core. Comparison of this record with climate reanalysis data for the 1979-2012 interval shows that RICE reliably captures temperature and snow precipitation variability in the region. Trends over the past 2700 years in RICE are shown to be distinct from those in West Antarctica and the western Ross Sea captured by other ice cores. For most of this interval, the eastern Ross Sea was warming (or showing isotopic enrichment for other reasons), with increased snow accumulation and perhaps decreased sea ice concentration. However, West Antarctica cooled and the western Ross Sea showed no significant isotope temperature trend. This pattern here is referred to as the Ross Sea Dipole. Notably, during the Little Ice Age, West Antarctica and the western Ross Sea experienced colder than average temperatures, while the eastern Ross Sea underwent a period of warming or increased isotopic enrichment. From the 17th century onwards, this dipole relationship changed. All three regions show current warming, with snow accumulation declining in West Antarctica and the eastern Ross Sea but increasing in the western Ross Sea. We interpret this pattern as reflecting an increase in sea ice in the eastern Ross Sea with perhaps the establishment of a modern Roosevelt Island polynya as a local moisture source for RICE

Regenerative agriculture in Aotearoa New Zealand - research pathways to build science-based evidence and national narratives.

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