Influence of RNA Polymerase II Catalytic Activity on Transcription Start Site Selection

RNA Polymerase II (Pol II) is responsible for expression of all protein-coding genes in eukaryotes. To understand gene expression at the molecular level, it is essential to understand the mechanisms of Pol II function. I investigate how Pol II catalytic activity influences the first step of gene expression, transcription initiation, in Saccharomyces cerevisiae. My dissertation focuses on the mechanisms by which Pol II activity defects contribute to transcription start site (TSS) selection/utilization and promoter output. I utilize mutants with substitutions in the Pol II active site that have different elongation rates in vitro and show varied growth phenotypes in vivo that correlate with the observed alterations in elongation rates. I employ genetic and biochemical approaches to investigate the relationships between the TSS phenotypes of Pol II activity/General Transcription Factor (GTF) mutants and conditional and general growth phenotypes. I show that while some conditional growth phenotypes correlate closely with TSS defects, TSS defects are not likely the main determinant for general growth defects. I further explore growth phenotypes and TSS defects between Pol II genetic interactors and GTF mutants combined with Pol II activity mutants and present different models for the relationships discovered so far. I discover a novel function of Sub1, which is suggested to play positive roles in transcription as an initiation factor, altering TSS utilization on its own and modifying TSS defects conferred by Pol II activity mutants. I take a genome wide approach to map TSSs, general transcription factor occupancies, and nucleosome positions to investigate the mechanism of Pol II activity control over initiation, and determine how promoter architecture influences TSS selection/utilization and nucleosome positioning. I show that promoter architecture is a critical parameter in determination of various initiation properties including transcription output (gene expression) levels

Validity of oral fluid test for Delta-9-tetrahydrocannabinol in drivers using the 2013 National Roadside Survey Data

Background Driving under the influence of marijuana is a serious traffic safety concern in the United States. Delta 9-tetrahydrocannabinol (THC) is the main active compound in marijuana. Although blood THC testing is a more accurate measure of THC-induced impairment, measuring THC in oral fluid is a less intrusive and less costly method of testing. Methods We examined whether the oral fluid THC test can be used as a valid alternative to the blood THC test using a sensitivity and specificity analysis and a logistic regression, and estimate the quantitative relationship between oral fluid THC concentration and blood THC concentration using a correlation analysis and a linear regression on the log-transformed THC concentrations. We used data from 4596 drivers who participated in the 2013 National Roadside Survey of Alcohol and Drug Use by Drivers and for whom THC testing results from both oral fluid and whole blood samples were available. Results Overall, 8.9% and 9.4% of the participants tested positive for THC in oral fluid and whole blood samples, respectively. Using blood test as the reference criterion, oral fluid test for THC positivity showed a sensitivity of 79.4% (95% CI: 75.2%, 83.1%) and a specificity of 98.3% (95% CI: 97.9%, 98.7%). The log-transformed oral fluid THC concentration accounted for about 29% of the variation in the log-transformed blood THC concentration. That is, there is still 71% of the variation in the log-transformed blood THC concentration unexplained by the log-transformed oral fluid THC concentration. Back-transforming to the original scale, we estimated that each 10% increase in the oral fluid THC concentration was associated with a 2.4% (95% CI: 2.1%, 2.8%) increase in the blood THC concentration. Conclusions The oral fluid test is a highly valid method for detecting the presence of THC in the blood but cannot be used to accurately measure the blood THC concentration

High drug-loaded microspheres enabled by controlled in-droplet precipitation promote functional recovery after spinal cord injury

High drug loading improves therapeutic efficacy and reduces side effects in drug delivery. Here, the authors use controlled diffusion of solvents to precipitate drug nanoparticles in polymer particles while the polymer is solidifying and demonstrate the particles for drug delivery in a spinal cord injury model. Drug delivery systems with high content of drug can minimize excipients administration, reduce side effects, improve therapeutic efficacy and/or promote patient compliance. However, engineering such systems is extremely challenging, as their loading capacity is inherently limited by the compatibility between drug molecules and carrier materials. To mitigate the drug-carrier compatibility limitation towards therapeutics encapsulation, we developed a sequential solidification strategy. In this strategy, the precisely controlled diffusion of solvents from droplets ensures the fast in-droplet precipitation of drug molecules prior to the solidification of polymer materials. After polymer solidification, a mass of drug nanoparticles is embedded in the polymer matrix, forming a nano-in-micro structured microsphere. All the obtained microspheres exhibit long-term storage stability, controlled release of drug molecules, and most importantly, high mass fraction of therapeutics (21.8-63.1 wt%). Benefiting from their high drug loading degree, the nano-in-micro structured acetalated dextran microspheres deliver a high dose of methylprednisolone (400 mu g) within the limited administration volume (10 mu L) by one single intrathecal injection. The amount of acetalated dextran used was 1/433 of that of low drug-loaded microspheres. Moreover, the controlled release of methylprednisolone from high drug-loaded microspheres contributes to improved therapeutic efficacy and reduced side effects than low drug-loaded microspheres and free drug in spinal cord injury therapy.Peer reviewe

Enhanced Bio-Barcode Immunoassay Using Droplet Digital PCR for Multiplex Detection of Organophosphate Pesticides

A bio-barcode immunoassay based on droplet digital polymerase chain reaction (ddPCR) was developed to simultaneously quantify triazophos, parathion, and chlorpyrifos in apple, cucumber, cabbage, and pear. Three gold nanoparticle (AuNP) probes and magnetic nanoparticle (MNP) probes were prepared, binding through their antibodies with the three pesticides in the same tube. Three groups of primers, probes, templates, and three antibodies were designed to ensure the specificity of the method. Under the optimal conditions, the detection limits (expressed as IC10) of triazophos, parathion, and chlorpyrifos were 0.22, 0.45, and 4.49 ng mL–1, respectively. The linear ranges were 0.01–20, 0.1–100, and 0.1–500 ng mL–1, and the correlation coefficients (R2) were 0.9661, 0.9834, and 0.9612, respectively. The recoveries and relative standard deviations (RSDs) were in the ranges of 75.5–98.9 and 8.3–16.7%. This study provides the first insights into the ddPCR for the determination of organophosphate pesticides. It also laid the foundation for high-throughput detection of other small molecules.This study was financially supported by the National Key Research Program of China (No. 2019YFC1604503), the NIEHS Superfund Research Program (No. P42 ES04699), the Central Public-interest Scientific Institution Basal Research Fund for the Chinese Academy of Agricultural Sciences (No. Y2021PT05), and the Ningbo Innovation Project for Agro-Products Quality and Safety (No. 2019CXGC007).Peer reviewe

Gray matter density reduction associated with adjuvant chemotherapy in older women with breast cancer

PURPOSE: The purpose of this study was to evaluate longitudinal changes in brain gray matter density (GMD) before and after adjuvant chemotherapy in older women with breast cancer. METHODS: We recruited 16 women aged ≥ 60 years with stage I-III breast cancers receiving adjuvant chemotherapy (CT) and 15 age- and sex-matched healthy controls (HC). The CT group underwent brain MRI and the NIH Toolbox for Cognition testing prior to adjuvant chemotherapy (time point 1, TP1) and within 1 month after chemotherapy (time point 2, TP2). The HC group underwent the same assessments at matched intervals. GMD was evaluated with the voxel-based morphometry. RESULTS: The mean age was 67 years in the CT group and 68.5 years in the HC group. There was significant GMD reduction within the chemotherapy group from TP1 to TP2. Compared to the HC group, the CT group displayed statistically significantly greater GMD reductions from TP1 to TP2 in the brain regions involving the left anterior cingulate gyrus, right insula, and left middle temporal gyrus (pFWE(family-wise error)-corrected < 0.05). The baseline GMD in left insula was positively correlated with the baseline list-sorting working memory score in the HC group (pFWE-corrected < 0.05). No correlation was observed for the changes in GMD with the changes in cognitive testing scores from TP1 to TP2 (pFWE-corrected < 0.05). CONCLUSIONS: Our findings indicate that GMD reductions were associated with adjuvant chemotherapy in older women with breast cancer. Future studies are needed to understand the clinical significance of the neuroimaging findings. This study is registered on ClinicalTrials.gov (NCT01992432)

Effects of Badminton Expertise on Representational Momentum: A Combination of Cross-Sectional and Longitudinal Studies

Representational momentum (RM) has been found to be magnified in experts (e.g., sport players) with respect to both real and implied motion in expert-familiar domains. However, it remains unclear whether similar effects can be achieved in expert-unfamiliar domains, especially within the context of implied motion. To answer this question, we conducted two independent experiments using an implied motion paradigm and examined the expert effects of badminton training on RM in both adult and child players. In Experiment 1, we used a cross-sectional design and compared RM between adult professional badminton players and matched controls. The results revealed significantly enhanced RM for adult players, supporting the expert effect in expert-unfamiliar domains for implied motion. However, cross-sectional studies could not ascertain whether the observed expert effect was due to innate factors or expertise acquirement. Therefore, in Experiment 2, we used a longitudinal design and compared RM between two groups of child participants, naming child players who had enrolled professional badminton training program at a sports school and age-matched peer non-players who attended an ordinary primary school without sports training. Before training, there were no differences in RM among child players, their non-player peers, and adult non-players. However, after 4 years of badminton training, child players demonstrated significantly enhanced RM compared to themselves prior to training. The increased RM observed in both adult and child players suggests that badminton expertise modulates implied motion RM

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01–25, 0.01–50, and 0.1–50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%–17.6%, which correlate well with results obtained by LC-MS/MS. The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.This work was supported by the Central Public Interest Scientific Institution Basal Research Fund for the Chinese Academy of Agricultural Sciences (Grant No.: Y2021PT05), National Institute of Environmental Health Science Superfund Research Program (Grant No.: P42 ES004699), National Academy of Sciences (Subaward No.: 2000009144), and Ningbo Innovation Project for Agro-Products Quality and Safety (Grant No.: 2019CXGC007).Peer reviewe

Intrinsic brain activity changes associated with adjuvant chemotherapy in older women with breast cancer: a pilot longitudinal study

Purpose Older cancer patients are at increased risk of cancer-related cognitive impairment. The purpose of this study was to assess the alterations in intrinsic brain activity associated with adjuvant chemotherapy in older women with breast cancer. Methods Chemotherapy treatment (CT) group included sixteen women aged ≥ 60 years (range 60–82 years) with stage I-III breast cancers, who underwent both resting-state functional magnetic resonance imaging (rs-fMRI) and neuropsychological testing with NIH Toolbox for Cognition before adjuvant chemotherapy, at time point 1 (TP1), and again within 1 month after completing chemotherapy, at time point 2 (TP2). Fourteen age- and sex-matched healthy controls (HC) underwent the same assessments at matched intervals. Three voxel-wise rs-fMRI parameters: amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF), and regional homogeneity (ReHo), were computed at each time point. The changes in rs-fMRI parameters from TP1 to TP2 for each group, the group differences in changes (the CT group vs. the HC group), and the group difference in the baseline rs-fMRI parameters were assessed. In addition, correlative analysis between the rs-fMRI parameters and neuropsychological testing scores was also performed. Results In the CT group, one brain region, which included parts of the bilateral subcallosal gyri and right anterior cingulate gyrus, displayed increased ALFF from TP1 to TP2 (cluster p-corrected=0.024); another brain region in the left precuneus displayed decreased fALFF from TP1 to TP2 (cluster level p-corrected=0.025). No significant changes in the rs-fMRI parameters from TP1 to TP2 were observed in the HC group. Although ALFF and fALFF alterations were observed only in the CT group, none of the between-group differences in rs-fMRI parameter changes reached statistical significance. Conclusions Our study results of ALFF and fALFF alterations in the chemotherapy-treated women suggest that adjuvant chemotherapy may affect intrinsic brain activity in older women with breast cancer

Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process