Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Assessing an aflatoxin exposure biomarker: Exploring the interchangeability and correlation between venous and capillary blood samples.

Exposure to dietary aflatoxins has been recognized as a potential threat to child nutrition and growth, in addition to being a known carcinogen. The ability to accurately assess concentration of aflatoxin in the blood of at-risk individuals is therefore very important to inform public health policies and on-the-ground programs around the world. Venous blood is frequently used to quantify biomarkers of exposure such as AFB1-lysine adducts. However, venous blood collection methods are invasive, requiring highly trained staff, which makes this method challenging to implement, especially in resource-limited settings. In contrast, capillary blood collection by fingerprick is less invasive and has the potential for application in point-of-need monitoring. The aim of this exploratory study was to investigate the correlation and interchangeability of capillary and venous human blood samples in the quantification of AFB1-lysine adduct concentration. A total of 72 venous and capillary blood samples were collected from 36 women of reproductive age (16-49 years) in northern Uganda. All sample specimens were analyzed using high-performance liquid chromatography with fluorescence detection. Regression analysis and Bland-Altman analysis were performed to compare AFB1-lysine concentrations between venous and capillary sample pairs. Bland-Altman analysis of albumin-normalized AFB1-lysine data-bias was -0.023 pg/mg-albumin and the 95% limits of agreement were 0.51 to -0.56 pg/mg-albumin for log-transformed data. There was a positive correlation between albumin-normalized venous and capillary AFB1-lysine concentrations with r of 0.71 (p < .0001). A lack of any accepted clinical cutoff for aflatoxin exposure makes definition of an 'acceptable' limit for statistical analysis and comparison of methods challenging. Our data suggests a positive correlation between albumin-normalized AFB1-lysine concentrations in venous and capillary sample pairs, but relatively weak agreement and interchangeability based on Bland-Altman analysis.

Measurement accuracy enhancement with multi-event detection using the deep learning approach in Raman distributed temperature sensors.

In this work, we present a novel deep learning framework for multi-event detection with enhanced measurement accuracy from the measured data of a Raman Optical Time Domain Reflectometer (Raman-OTDR). We demonstrate the utility of a deep learning-based approach by comparing the results from three popular neural networks, i.e. vanilla recurrent neural network (RNN), long short-term memory (LSTM), and gated recurrent unit (GRU). Before feeding the experimentally obtained data to the neural network, we sanitize our data through a correlation filtering operation to suppress outlier noise spikes. Based on experiments with Raman-OTDR traces consisting of single temperature event, we show that the GRU is able to provide better performance compared to RNN and LSTM models. Specifically, a bidirectional-GRU (bi-GRU) architecture is found to outperform other architectures owing to its use of data from both previous as well as later time steps. Although this feature is similar to that used recently in one dimension convolutional neural network (1D-CNN), the bi-GRU is found to be more effective in providing enhanced measurement accuracy while maintaining good spatial resolution. We also propose and demonstrate a threshold-based algorithm for accurate and fast estimation of multiple events. We demonstrate a 4x improvement in the spatial resolution compared to post-processing using conventional total variational denoising (TVD) filters, while the temperature accuracy is maintained within ± 0.5 oC of the set temperature.

Stimulated Brillouin scattering mitigation using optimized phase modulation waveforms in high power narrow linewidth Yb-doped fiber amplifiers.

We demonstrate the mitigation of stimulated Brillouin scattering (SBS) in a double-clad single mode Yb-doped optical fiber amplifier through external phase modulation of narrow linewidth laser radiation using optimized periodic waveforms from an arbitrary waveform generator. Such optimized phase modulation waveforms are obtained through a multi-objective Pareto optimization based on a comprehensive model for SBS in high power narrow linewidth fiber amplifiers using Brillouin parameters determined from controlled measurements. The ability of our approach to mitigate SBS is tested experimentally as a function of RMS linewidth of the modulated optical radiation, and we measure an enhancement in SBS threshold with respect to optical linewidth of ∼ 10 GHz-1. Furthermore, we discuss the dependence of the SBS threshold enhancement on key parameters such as the amplifier length and the period of the optimized waveforms. Through simulations we find that waveforms of sufficiently long periods and optimized for a relatively long fiber (10 m) are effective for SBS suppression for shorter fibers as well. We also investigate the effect of increase in the bandwidth and amplitude of the modulation waveform on the SBS threshold enhancement observed at higher optical linewidth.

A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment.

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.

Rapid diagnostic testing platform for iron and vitamin A deficiency.

Micronutrient deficiencies such as those of vitamin A and iron affect a third of the world's population with consequences such as night blindness, higher child mortality, anemia, poor pregnancy outcomes, and reduced work capacity. Many efforts to prevent or treat these deficiencies are hampered by the lack of adequate, accessible, and affordable diagnostic methods that can enable better targeting of interventions. In this work, we demonstrate a rapid diagnostic test and mobile enabled platform for simultaneously quantifying iron (ferritin), vitamin A (retinol-binding protein), and inflammation (C-reactive protein) status. Our approach, enabled by combining multiple florescent markers and immunoassay approaches on a single test, allows us to provide accurate quantification in 15 min even though the physiological range of the markers of interest varies over five orders of magnitude. We report sensitivities of 88%, 100%, and 80% and specificities of 97%, 100%, and 97% for iron deficiency (ferritin <15 ng/mL or 32 pmol/L), vitamin A deficiency (retinol-binding protein <14.7 µg/mL or 0.70 µmol/L) and inflammation status (C-reactive protein >3.0 µg/mL or 120 nmol/L), respectively. This technology is suitable for point-of-care use in both resource-rich and resource-limited settings and can be read either by a standard laptop computer or through our previously developed NutriPhone technology. If implemented as either a population-level screening or clinical diagnostic tool, we believe this platform can transform nutritional status assessment and monitoring globally.

Multi-point dynamic strain sensing using external modulation-based Brillouin optical correlation domain analysis.

We report a novel technique to detect dynamic strain variations simultaneously at multiple locations. Our technique is based on Brillouin optical correlation domain analysis implemented through external modulation to generate multiple independently-accessible correlation peaks within the sensing fiber. Experiments are carried out to demonstrate the precise determination of Brillouin frequency shift (BFS) from multiple locations independently. As a proof of principle, two correlation peaks are generated within a 1 km long fiber and their independent tunability is verified experimentally by mapping the spatial profile of the two correlations. We also experimentally demonstrate the detection of dynamic strain variations at two locations simultaneously, each with a spatial resolution of 60 cm over 100 m long fiber.

Raman amplification of optical beam carrying orbital angular momentum in a multimode step-index fiber.

We experimentally demonstrate 15 dB of Raman amplification of 1115 nm pulses in an orbital angular momentum mode (OAMM) with charge l=+2, S=+1 in 5 m of step-index 25 µm-diameter-core fiber. The total output reaches 4.5 kW of peak power and 68.5 µJ of energy in ∼15 ns pulses at 4 kHz repetition rate. An Yb-doped fiber source pumps the Raman amplifier at 1060 nm with 60 ns pulses. Using a spatial light modulator for modal decomposition, we measure 83% purity for the amplified target OAMM of selected polarization. To the best of our knowledge, this is the first time high energy, peak power, gain, and purity are achieved in a fiber Raman amplifier for a single OAMM.

Investigation of temporal dynamics due to stimulated Brillouin scattering using statistical correlation in a narrow-linewidth cw high power fiber amplifier.

We experimentally investigate the dynamics of backscattered light due to stimulated Brillouin scattering (SBS) in a narrow-linewidth continuous wave (CW) fiber amplifier. We observe the onset of sharp intensity variations in the backscattered radiation as we increase the pump power, which when analyzed using Karl-Pearson's correlation coefficient reveals a distinct structure. We find that such structure is associated with the onset of stimulated Brillouin scattering (SBS) in the fiber amplifier. Moreover, at higher pump power levels we observe a periodic signature in the Karl-Pearson correlation trace that precedes an observation of kW pulses in the backscattered radiation. Based on controlled experiments, we conclude that the formation of the above kW pulses in our system is preceded by the onset of SBS.

Performance analysis of frequency shift estimation techniques in Brillouin distributed fiber sensors.

The performance of post-processing techniques carried out on the Brillouin gain spectrum to estimate the Brillouin frequency shift (BFS) in standard Brillouin distributed sensors is evaluated. Curve fitting methods with standard functions such as polynomial and Lorentzian, as well as correlation techniques such as Lorentzian Cross-correlation and Cross Reference Plot Analysis (CRPA), are considered for the analysis. The fitting procedures and key parameters for each technique are optimized, and the performance in terms of BFS uncertainty, BFS offset error and processing time is compared by numerical simulations and through controlled experiments. Such a quantitative comparison is performed in varying conditions including signal-to-noise ratio (SNR), frequency measurement step, and BGS truncation. It is demonstrated that the Lorentzian cross-correlation technique results in the largest BFS offset error due to truncation, while exhibiting the smallest BFS uncertainty and the shortest processing time. A novel approach is proposed to compensate such a BFS offset error, which enables the Lorentzian cross-correlation technique to completely outperform other fitting methods.

Multimode-pumped Raman amplification of a higher order mode in a large mode area fiber.

We report the first demonstration of Raman amplification in a fiber of a single Bessel-like higher order mode using a multimode pump source. We amplify the LP08-mode with a 559-µm2 effective mode area at a signal wavelength of 1115 nm in a pure-silica-core step-index fiber. A maximum of 18 dB average power gain is achieved in a 9-m long gain fiber, with output pulse energy of 115 µJ. The Raman pump source comprises a pulsed 1060 nm ytterbium-doped fiber amplifier with V-value ~30, which is matched to the Raman gain fiber. The pump depletion as averaged over the signal pulses reaches 36.7%. The conversion of power from the multimode pump into the signal mode demonstrates the potential for efficient brightness enhancement with low amplification-induced signal mode purity degradation.

A cell-type-specific protein-protein interaction modulates transcriptional activity of a master regulator in Caulobacter crescentus.

Progression through the Caulobacter cell cycle is driven by the master regulator CtrA, an essential two-component signaling protein that regulates the expression of nearly 100 genes. CtrA is abundant throughout the cell cycle except immediately prior to DNA replication. However, the expression of CtrA-activated genes is generally restricted to S phase. We identify the conserved protein SciP (small CtrA inhibitory protein) and show that it accumulates during G1, where it inhibits CtrA from activating target genes. The depletion of SciP from G1 cells leads to the inappropriate induction of CtrA-activated genes and, consequently, a disruption of the cell cycle. Conversely, the ectopic synthesis of SciP is sufficient to inhibit CtrA-dependent transcription, also disrupting the cell cycle. SciP binds directly to CtrA without affecting stability or phosphorylation; instead, SciP likely prevents CtrA from recruiting RNA polymerase. CtrA is thus tightly regulated by a protein-protein interaction which is critical to cell-cycle progression.

Extracellular 4'-phosphopantetheine is a source for intracellular coenzyme A synthesis.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Coenzyme A, more than 'just' a metabolic cofactor.

In all organisms biomolecules play a vital role to enable proper cellular metabolism. Alteration of metabolite homoeostasis disrupts the physiology of cells, leading to various diseases [DeBerardinis and Thompson (2012) Cell, 148, 1132-1144]. Recent studies advances our understanding that some metabolites are not only involved in cellular metabolism, but also have other molecular functions. It has become evident that similar to multifunctional 'moonlighting proteins', 'moonlighting metabolites' also exists. One clear example is nicotinamide adenine dinucleotide (NAD). NAD is a ubiquitous molecule with a well-known function in many metabolic reactions, but it also has become clear that NAD is involved in the regulation of sirtuins. Sirtuins play a role in cancer, diabetes, and cardiovascular, neurodegenerative and other diseases [Donmez and Outeiro (2013) EMBO Mol. Med. 5, 344-352] and the deacetylation capacity of sirtuin proteins is NAD-dependent. This direct role of NAD in age-related diseases could not be anticipated when NAD was initially discovered as a metabolic cofactor [Donmez and Outeiro (2013) EMBO Mol. Med. 5, 344-352; Mouchiroud et al. (2013) Crit. Rev. Biochem. Mol. Biol. 48, 397-408]. Recent findings now also indicate that CoA (coenzyme A), another metabolic cofactor, can be considered as being more than 'just' a metabolic cofactor, and altered CoA levels lead to severe and complex effects.

Physics informed contour selection for rapid image segmentation.

Effective training of deep image segmentation models is challenging due to the need for abundant, high-quality annotations. To facilitate image annotation, we introduce Physics Informed Contour Selection (PICS)-an interpretable, physics-informed algorithm for rapid image segmentation without relying on labeled data. PICS draws inspiration from physics-informed neural networks (PINNs) and an active contour model called snake. It is fast and computationally lightweight because it employs cubic splines instead of a deep neural network as a basis function. Its training parameters are physically interpretable because they directly represent control knots of the segmentation curve. Traditional snakes involve minimization of the edge-based loss functionals by deriving the Euler-Lagrange equation followed by its numerical solution. However, PICS directly minimizes the loss functional, bypassing the Euler Lagrange equations. It is the first snake variant to minimize a region-based loss function instead of traditional edge-based loss functions. PICS uniquely models the three-dimensional (3D) segmentation process with an unsteady partial differential equation (PDE), which allows accelerated segmentation via transfer learning. To demonstrate its effectiveness, we apply PICS for 3D segmentation of the left ventricle on a publicly available cardiac dataset. We also demonstrate PICS's capacity to encode the prior shape information as a loss term by proposing a new convexity-preserving loss term for left ventricle. Overall, PICS presents several novelties in network architecture, transfer learning, and physics-inspired losses for image segmentation, thereby showing promising outcomes and potential for further refinement.

An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: results from an ethnically diverse clinical sample of 23,453 individuals.

PURPOSE: Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern. METHODS: A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study. RESULTS: Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease. CONCLUSION: To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of "racial" categories by the physician, as recommended by recent guidelines.

Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

Instability of mixing layers: Momentum and thermal transport in the continuum breakdown regime.

We examine the momentum and thermal transport in the continuum breakdown regime of a mixing layer flow, which exhibits Kelvin-Helmholtz instability under ideal continuum conditions. The Grad 13 moment model is used as it provides an adequate description of the flow physics (second-order accurate in Knudsen number) in the transition regime. Analytical solutions are developed under breakdown conditions for two-dimensional, compressible, parallel shear flows. It is shown that the deviation of viscous stress and heat flux from the Navier-Stokes-Fourier system follows two different scaling regimes depending upon the Mach number. At low Mach numbers, the departure of all stress and heat-flux components depends only upon the Knudsen number. At high Mach number, the scaling of shear stress and transverse heat flux depends on the product of the Knudsen and Mach numbers. The normal stresses depend individually on the Knudsen and Mach number. The scaling results are verified against numerical simulations of compressible mixing layers performed using the unified gas kinetic scheme for various degrees of rarefaction.

Continuum breakdown in compressible mixing layers.

Gas-kinetic simulations of rarefied and compressible mixing layers are performed to characterize continuum breakdown and the effect on the Kelvin-Helmholtz instability. The unified gas-kinetic scheme (UGKS) is used to perform the simulations at different Mach and Knudsen numbers. The UGKS stress tensor and heat-flux vector fields are compared against those given by the Navier-Stokes-Fourier constitutive equations. The most significant difference is seen in the shear stress and transverse heat flux. The study demonstrates the existence of two distinct continuum breakdown regimes, one at low and the other at high convective Mach numbers. Overall, at low convective Mach numbers, the deviation from continuum stress and heat flux appears to scale exclusively with the micro-macro length scale ratio given by the Knudsen number. On the other hand, at high convective Mach numbers, the deviation depends on the global micro-macro timescale ratio given by the product of Mach and Knudsen numbers. We further demonstrate that, unlike shear stresses and transverse heat flux, the deviations in normal stresses and the streamwise heat flux depend separately on Knudsen and Mach numbers. A local parameter called the gradient Knudsen number is proposed to characterize the rarefaction effects on the local momentum and thermal transport. Noncontinuum aspects of gas-kinetic stress-tensor and heat-flux behavior that Grad's 13-moment equation model reasonably captures are identified.

A diagnostic platform for rapid, simultaneous quantification of procalcitonin and C-reactive protein in human serum.

BACKGROUND: Early and accurate determination of bacterial infections as a potential cause for a patient's systemic inflammatory response is required for timely administration of appropriate treatment and antibiotic stewardship. Procalcitonin (PCT) and C-reactive protein (CRP) have both been used as biomarkers to infer bacterial infections, particularly in the context of sepsis. There is an urgent need to develop a platform for simultaneous quantification of PCT and CRP, to enable the potential use of these biomarkers at the point-of-care. METHODS: A multiplexed lateral flow assay (LFA) and a fluorescence optical reader were developed. Assay performance was validated by testing spiked antigens in the buffer, followed by a validation study comparing results with conventional assays (Roche Cobas e411 Elecsys PCT and Siemens ADVIA XPT CRP) in 25 archived remnant human serum samples. FINDINGS: A linear regression correlation of 0·97 (P < 0·01) was observed for PCT, and a correlation of 0·95 (P < 0·01) was observed for CRP using direct patient samples. We also validated our platform's ability to accurately quantify high-dose CRP in the hook effect range where excess unlabeled analytes occupy binding sites at test lines. INTERPRETATION: A fluorescence reader-based duplex LFA for simultaneous quantification of PCT and CRP was developed and successfully validated with clinical samples. The rapid, portable, and low-cost nature of the platform offers potential for differentiation of bacterial and viral infections in emergency and low-resource settings at the point-of-care. FUNDING: NIH/NIBIB Award 1R01EB021331, and Academic Venture Fund from the Atkinson Center for a Sustainable Future at Cornell University.