Regulation of transcription factor binding specificity: from binding motifs to local DNA context

Regulation of transcription factor (TF) binding specificity lies at the heart of transcriptional control which governs how cells divide, differentiate, and respond to their environments. TFs are known to bind to DNA in a sequence specific manner, and such short sequence is known as transcription factor binding site (TFBS). However, the in vivo TF bound regions do not always contain a TFBS, and additionally, there are often excessive non-functional TFBSs with binding potential in the regulatory regions that are unbound for a given TF. This dissertation focuses on understanding the principles of TF binding specificity and is divided into two chapters: 1) developing a novel high throughput method that would facilitate the study of TF binding regulations and the resulting functional output; 2) analyzing the roles of local DNA context around TFBS in specifying TF localization. In the first chapter of this dissertation, we report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned “out of phase”. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p, and Gcr2p, that act together as a “TF collective”, an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function. In the second chapter of this dissertation, we seek out to investigate if predictive information is embedded in local DNA context (LDC) on a large collection of TFs in Saccharomyces cerevisiae. We identify there is a general preference for TFs to bind at CG rich sequences; we then analyze whether such preference is linked to intrinsic nucleosome binding preference and found the CG preference in LDC for TF binding was independent of nucleosome regulation. We next examine the possible mechanism by which LDC influence TFs binding site selection, through recruiting ‘licensing’ factors or kinetically assisting TF search for a target site. We show high CG LDC is preferred by TFs in vitro condition, which suggests such preference only involves TFs and DNA and directs us to TF search kinetics mechanism. CG rich feature in LDC may act as an energetical funnel to facilitate TF recognizing a target binding site, and we verify the theoretical validity of this hypothesis with Gillespie simulation. In the end, we reveal CG preference was also present in a large group of human TFs, indicating the usage of LDC is a general mechanism for TF binding specificity

Method Validation of Functional Magnetic Resonance Imaging and Electrophysiological Recording to Investigate Mechanisms of Vagus Nerve

Vagus nerve stimulation (VNS) is used clinically to treat epilepsy and depression, but its mechanism of action is unknown. Useful techniques to study this are functional magnetic resonance imaging (fMRI) and the local field potential (LFP). fMRI relies on oxygen use in the brain to show areas where neurons are active. The LFP is an electrical signal created by neuron action potentials and other current moving across cell membranes. The most information can be gained when the two methods are used simultaneously, however, this is difficult to do. This study seeks to validate the technique of fMRI-LFP as applied to study the mechanism of VNS. The rat is used as an animal model. Previously collected data is analyzed to determine effects of stimulation on respiration, since this will affect oxygen levels in the blood. Recording electrodes of different materials are tested to find the artifact size created in an MRI environment. Iridium electrodes were found to have the smallest artifact and therefore the best performance. It is unclear whether the stimulation used affects respiration, so a simultaneous fMRI-LFP experiment is needed to interpret fMR images. More work needs to be done before fMRI-LFP recordings can be taken during VNS

Quantitative analysis of transcription factor binding and expression using Calling Cards Reporter Arrays

We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned \u27out of phase\u27. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a \u27TF collective\u27, an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function

Near-Infrared Alcohol Detection Circuit Based On Multisim

Because the number of private cars has expanded, drunk driving has become more and more frequent. The detection of a driver’s alcohol concentration has become the focus of attention. Therefore, infrared alcohol detection was studied. The principle of infrared blood glucose noninvasive detection was investigated, and it was compared with infrared spectrum detection. Finally, using transmission technology and an infrared emitter and receiver, an infrared alcohol identification circuit was designed by NBohr’s Law and the Correcting Beer-Lambert Law. It was composed of an infrared acquisition circuit, an infrared electronic filter circuit, and an infrared amplifier circuit. And the infrared alcohol identification circuit was composed of multiple circuits in series and parallel. At various pins on the first AD844AN, the infrared electronic filter circuit receives an alternating current source voltage of 1000V with a basic signal frequency of 60 Hz. At the input end, the infrared amplifier circuit receives a current signal with a frequency of 1 Hz and an amplitude of 5 uA and performs the reproduction experiment using Multisim. As a result of the signal being upgraded to fulfill the objective of recognition, distinct information reappears and exhibits different waveforms

Acute effects of vagus nerve stimulation parameters on gastric motility assessed with magnetic resonance imaging

BackgroundVagus nerve stimulation (VNS) is an emerging bioelectronic therapy for regulating food intake and controlling gastric motility. However, the effects of different VNS parameters and polarity on postprandial gastric motility remain incompletely characterized.MethodsIn anesthetized rats (N = 3), we applied monophasic electrical stimuli to the left cervical vagus and recorded compound nerve action potential (CNAP) as a measure of nerve response. We evaluated to what extent afferent or efferent pathway could be selectively activated by monophasic VNS. In a different group of rats (N = 13), we fed each rat a gadolinium- labeled meal and scanned the rat stomach with oral contrast- enhanced magnetic resonance imaging (MRI) while the rat was anesthetized. We evaluated the antral and pyloric motility as a function of pulse amplitude (0.13, 0.25, 0.5, 1 mA), width (0.13, 0.25, 0.5 ms), frequency (5, 10 Hz), and polarity of VNS.Key ResultsMonophasic VNS activated efferent and afferent pathways with about 67% and 82% selectivity, respectively. Primarily afferent VNS increased antral motility across a wide range of parameters. Primarily efferent VNS induced a significant decrease in antral motility as the stimulus intensity increased (R = - .93, P < .05 for 5 Hz, R = - .85, P < .05 for 10 Hz). The VNS with either polarity tended to promote pyloric motility to a greater extent given increasing stimulus intensity.Conclusions and InferencesMonophasic VNS biased toward the afferent pathway is potentially more effective for facilitating occlusive contractions than that biased toward the efferent pathway.We investigated a possible differential effect of primarily afferent versus efferent cervical VNS on gastric motility under a range of VNS parameters. Gastric MRI data revealed that primarily afferent VNS induced stronger antral contractions relative to primarily efferent VNS. These results could serve as an index for optimizing VNS parameters for promoting gastric motility. Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155957/1/nmo13853_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155957/2/nmo13853.pd

In vivo tracing of the ascending vagal projections to the brain with manganese enhanced magnetic resonance imaging

IntroductionThe vagus nerve, the primary neural pathway mediating brain-body interactions, plays an essential role in transmitting bodily signals to the brain. Despite its significance, our understanding of the detailed organization and functionality of vagal afferent projections remains incomplete.MethodsIn this study, we utilized manganese-enhanced magnetic resonance imaging (MEMRI) as a non-invasive and in vivo method for tracing vagal nerve projections to the brainstem and assessing their functional dependence on cervical vagus nerve stimulation (VNS). Manganese chloride solution was injected into the nodose ganglion of rats, and T1-weighted MRI scans were performed at both 12 and 24 h after the injection.ResultsOur findings reveal that vagal afferent neurons can uptake and transport manganese ions, serving as a surrogate for calcium ions, to the nucleus tractus solitarius (NTS) in the brainstem. In the absence of VNS, we observed significant contrast enhancements of around 19–24% in the NTS ipsilateral to the injection side. Application of VNS for 4 h further promoted nerve activity, leading to greater contrast enhancements of 40–43% in the NTS.DiscussionThese results demonstrate the potential of MEMRI for high-resolution, activity-dependent tracing of vagal afferents, providing a valuable tool for the structural and functional assessment of the vagus nerve and its influence on brain activity

VastGaussian: Vast 3D Gaussians for Large Scene Reconstruction

Existing NeRF-based methods for large scene reconstruction often have limitations in visual quality and rendering speed. While the recent 3D Gaussian Splatting works well on small-scale and object-centric scenes, scaling it up to large scenes poses challenges due to limited video memory, long optimization time, and noticeable appearance variations. To address these challenges, we present VastGaussian, the first method for high-quality reconstruction and real-time rendering on large scenes based on 3D Gaussian Splatting. We propose a progressive partitioning strategy to divide a large scene into multiple cells, where the training cameras and point cloud are properly distributed with an airspace-aware visibility criterion. These cells are merged into a complete scene after parallel optimization. We also introduce decoupled appearance modeling into the optimization process to reduce appearance variations in the rendered images. Our approach outperforms existing NeRF-based methods and achieves state-of-the-art results on multiple large scene datasets, enabling fast optimization and high-fidelity real-time rendering.Comment: Accepted to CVPR 2024. Project website: https://vastgaussian.github.i

Infrared anomalies in ultrathin Ti3C2Tx MXene films

Visible transparent but infrared reflective materials are ideal candidates for both transparent conductive films and low-emissivity glass, which are highly desired in a broad variety of areas such as touchscreens and displays, photovoltaics, smart windows, and antistatic coatings. Ultrathin Ti3C2Tx MXene films are emerging as promising low-emissivity transparent candidates. However, the fundamental IR properties of Ti3C2Tx has not been revealed experimentally due to daunting challenges in the preparation of continuous, large-area, and ultrathin films of optical quality on flat substrates. Herein, we proposed a tape-free transfer method that can help prepare centimeter-size and ultrathin (down to 8 nm) Ti3C2Tx films on diverse optical substrates. Benefitting from this method, the refractive index and permittivity for Ti3C2Tx were successfully measured. Ti3C2Tx films exhibit large in-plane permittivity in the IR region, yielding maximum IR reflectance of 88% for bulk films. Interestingly, three anomalies were found in ultrathin Ti3C2Tx films: strong dispersion in the permittivity, interlayer space-dependent optical properties, and abnormally high IR absorption for a 15-nm-thick film. These anomalies are important guidelines in the design of Ti3C2Tx-based low-emissivity transparent films and other related devices, and may inspire other intriguing applications such as ultrathin IR absorption coatings and tunable IR optical devices