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Abstract CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. The RALA cell penetrating peptide is an amphiphilic peptide that self‐assembles into nanoparticles through electrostatic interactions with anionic molecules and delivers them across the cell membrane. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA effectively encapsulated and delivered CRISPR DNAs, RNAs, and ribonucleic proteins (RNPs) to primary mesenchymal stem cells (MSCs), outperforming commercially available reagents. We then used the RALA peptide for the knock‐in and knock‐out of reporter genes into primary MSCs and the transcriptional activation of therapeutically relevant genes. Finally, we demonstrate in vivo gene editing using RALA to knock‐out luciferase and GFP in a reporter mouse model. In summary, we establish RALA as a powerful tool for safer and more effective delivery of CRISPR machinery in multiple cargo formats for a wide range of ex vivo and in vivo gene editing strategies. 

ABSTRACT Diacylglycerol O‐acyltransferase 1 (DGAT1) is an integral membrane protein that uses acyl‐coenzyme A (acyl‐CoA) and diacylglycerol (DAG) to catalyze the formation of triacylglycerides (TAGs). The acyl transfer reaction occurs between the activated carboxylate group of the fatty acid and the free hydroxyl group on the glycerol backbone of DAG. However, how the two substrates enter DGAT1's catalytic reaction chamber and interact with DGAT1 remains elusive. This study aims to explore the structural basis of DGAT1's substrate recognition by investigating each substrate's pathway to the reaction chamber. Using a human DGAT1 cryo‐EM structure in complex with an oleoyl‐CoA substrate, we designed two different all‐atom molecular dynamics (MD) simulation systems: DGAT1^away(both acyl‐CoA and DAG away from the reaction chamber) and DGAT1^bound(acyl‐CoA bound in and DAG away from the reaction chamber). Our DGAT1^awaysimulations reveal that acyl‐CoA approaches the reaction chamber via interactions with positively charged residues in transmembrane helix 7. DGAT1^boundsimulations show DAGs entering into the reaction chamber from the cytosol leaflet. The bound acyl‐CoA's fatty acid lines up with the headgroup of DAG, which appears to be competent to TAG formation. We then converted them into TAG and coenzyme (CoA) and used adaptive biasing force (ABF) simulations to explore the egress pathways of the products. We identify their escape routes, which are aligned with their respective entry pathways. Visualization of the substrate and product pathways and their interactions with DGAT1 is expected to guide future experimental design to better understand DGAT1 structure and function. 

Abstract Taking place naturally in a gas subject to a given wall temperature distribution, the “ghost effect” exhibits a rare kinetic effect beyond the prediction of classical fluid theory and Fourier law in such a classical problem in physics. As the Knudsen number goes to zero, the finite variation of temperature in the bulk is determined by an infinitesimal, ghost‐like velocity field, created by a givenfinitevariation of the tangential wall temperature as predicted by Maxwell's slip boundary condition. Mathematically, such a finite variation leads to the presence of a severe singularity and a Knudsen layer approximation in the fundamental energy estimate. Neither difficulty is within the reach of any existing PDE theory on the steady Boltzmann equation in a general 3D bounded domain. Consequently, in spite of the discovery of such a ghost effect from temperature variation in as early as 1960s, its mathematical validity has been a challenging and intriguing open question, causing confusion and suspicion. We settle this open question in affirmative if the temperature variation is small but finite, by developing a new framework with four major innovations as follows: (1) a key ‐Hodge decomposition and its corresponding local ‐conservation law eliminate the severe bulk singularity, leading to a reduced energy estimate; (2) a surprising gain in via momentum conservation and a dual Stokes solution; (3) the ‐conservation, energy conservation, and a coupled dual Stokes–Poisson solution reduces to an boundary singularity; (4) a crucial construction of ‐cutoff boundary layer eliminates such boundary singularity via new Hardy's and BV estimates. 

ABSTRACT Peptides are widely used in biomaterials due to their ease of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell‐secreted enzymes, which creates the possibility of utilizing cell‐secreted enzymes for tuning cell–material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography–mass spectrometry (LC–MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC–MS for the multiplexed quantification of cell‐induced peptide degradation, including the need for nondegradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. Removing these constituents can be expensive, time‐consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified samples onto the LC–MS without any purification enables rapid and accurate quantification of peptide concentration and that hundreds of LC–MS runs can be done on a single column without significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC–MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost‐effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide‐based therapies. 

Abstract This work demonstrates the capability to crystallize YAG via femtosecond pulsed laser. Challenges in using melt‐quench glass are shown to restrict glass composition and have not yielded YAG via femtosecond laser crystallization. An alternative glass‐making technique was used to fabricate a range of compositions not otherwise possible. Glasses of YAG with added silica in the range of 0–20 mol% were tested under the laser to explore the allowable deviation from stoichiometric YAG. Raman spectroscopy and Electron backscatter diffraction indicated successful fabrication of YAG, and usage of combined excitation emission spectroscopy (CEES) allowed probing of erbium doped compositions. 

ABSTRACT This paper presents a comprehensive examination of finite element modeling (FEM) approaches for seismocardiography (SCG), a non‐invasive method for assessing cardiac function through chest surface vibrations. The paper provides a comparative analysis of existing FEM approaches, exploring the strengths and challenges of various modeling choices in the literature. Additionally, we introduce a sample framework for developing FEM models of SCG, detailing key methodologies from governing equations and meshing techniques to boundary conditions and material property selection. This framework serves as a guide for researchers aiming to create accurate models of SCG signal propagation and offers insights into capturing complex cardiac mechanics and their transmission to the chest surface. By consolidating the current methodologies, this paper aims to establish a reference point for advancing FEM‐based SCG modeling, ultimately improving our understanding of SCG waveforms and enhancing their reliability and applicability in cardiovascular health assessment.