Perturb-tracing enables high-content screening of multi-scale 3D genome regulators.

Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells.

Dual Monitoring of Blood Acetylcholinesterase Content and Catalytic Activity Utilizing Fluorometry-Integrated Surface Plasmon Resonance.

Acetylcholinesterase inhibitors (AChEIs), particularly donepezil, are commonly used to treat mild-to-moderate Alzheimer's disease (AD). However, drug accumulation during long-term use could change AChE activity and content, leading to peripheral side effects and prompting medication discontinuation. However, there are a lack of methods to simultaneously determine the content and catalytic activity of AChE.

G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration.

The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, β-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) to support localized β-actin synthesis, crucial for cell migration. However, whether other mRNAs and RBPs also localize at FAs remains unclear.

Islatravir Dimer: Crystal Form Dependence of a Radiation-Induced Degradation Product.

A rare example of crystal form-dependent, gamma radiation-induced degradation is presented. Islatravir is known to exist in several polymorphic forms, but only one of these forms shows the generation of a specific dimer degradation product under gamma irradiation. Extended gamma irradiation studies demonstrated that only one of the known crystalline forms shows an appreciable rate of dimer formation.

Perturb-tracing enables high-content screening of multiscale 3D genome regulators.

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multiscale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screen, a new cellular barcode readout method (BARC-FISH), and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their genome organization from 13,000 imaging target-perturbation combinations, alongside perturbation-paired barcode readout in the same single cells.

Effects of boiling and steaming process on dietary fiber components and in vitro fermentation characteristics of 9 kinds of whole grains.

Whole grains (WGs) are considered as the representative sources of dietary fiber (DF). Thermal treatments can change the properties of DF, and potentially affecting the gut microbiota as well as human health. In this study, DF content and in vitro fermentation characteristics of 9 kinds of WGs (highland barley, barley, buckwheat, proso millet, quinoa, sorghum, coix seed, foxtail millet, and oats) after boiling and steaming treatments were compared.

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations.

Background: Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies.

DFT studies on Ni-catalyzed intermolecular cycloaddition of diynes with methyleneaziridines.

The mechanisms of nickel-catalyzed intermolecular cycloaddition of diynes with methyleneaziridines to form substituted pyrroles have been investigated with DFT calculations. The DFT results don't support the originally proposed mechanisms, which involve β-C elimination or α-C elimination. Detailed calculations revealed that the preferred catalytic cycle is a combination of the cod dissociative mechanism and the cod associative mechanism, which is comprised of four stages: oxidative addition, ligand substitution of the diyne by cod, alkyne insertion and reductive elimination.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics.

Chromatin tracing and multiplexed imaging of nucleome architectures (MINA) and RNAs in single mammalian cells and tissue.

The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue and how organization affects gene expression remains a major challenge.

ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH.

Chromatin Tracing: Imaging 3D Genome and Nucleome.

Correct 3D genome organization is essential for the proper functioning of the genome. Recent advances in image-based 3D genomics techniques have enabled direct tracing of chromatin folding and multiplexed imaging of nucleome architectures in single cells of several important biological systems. Here, we discuss these advances and the future directions of image-based 3D genomics.

Multiplexed imaging of nucleome architectures in single cells of mammalian tissue.

The three-dimensional architecture of the genome affects genomic functions. Multiple genome architectures at different length scales, including chromatin loops, domains, compartments, and lamina- and nucleolus-associated regions, have been discovered. However, how these structures are arranged in the same cell and how they are mutually correlated in different cell types in mammalian tissue are largely unknown.

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle.

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets.

Assessing the Utility of In Vitro Screening Tools for Predicting Bio-Performance of Oral Peptide Delivery.

Purpose: In this study we evaluated the utility of in-vitro screening tools for predicting the in-vivo behavior of six cyclic peptides with different solubility and permeability properties (BCS class II and III), intended for oral delivery in presence of permeation enhancer Labrasol.

Methods: An in vitro flux assay was used to assess peptide permeation across a biomimetic, lipid-based membrane and in vivo studies in rats were used to determine oral peptide bioavailability in the presence of Labrasol.

Results: The in vitro flux was significantly increased for BCS class III peptides, while it significantly decreased or remained unchanged for BCS class II peptides with increasing Labrasol concentrations.

Cross 1,3-dipolar cycloadditions of C,N-cyclic azomethine imines with an N-benzyl azomethine ylide: facile access to fused tricyclic 1,2,4-hexahydrotriazines.

A cross 1,3-dipolar cycloaddition of two different ylides between C,N-cyclic azomethine imines with an in situ-generated nonstabilized azomethine ylide from an N-benzyl precursor was realized. The reactions afforded a clean and facile access to diverse fused tricyclic 1,2,4-hexahydrotriazines in high yields (up to 96%). The chemical structures of the typical compounds were confirmed by X-ray single-crystal structure analysis.

Inhibition of MD2-dependent inflammation attenuates the progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) can progress to the more serious non-alcoholic steatohepatitis (NASH), characterized by inflammatory injury and fibrosis. The pathogenic basis of NAFLD progressing to NASH is currently unknown, but growing evidence suggests MD2 (myeloid differentiation factor 2), an accessory protein of TLR4, is an important signalling component contributing to this disease. We evaluated the effectiveness of the specific MD2 inhibitor, L6H21, in reducing inflammatory liver injury in a relevant high-fat diet (HFD) mouse model of NASH and in the palmitic acid (PA)-stimulated human liver cell line (HepG2).

Development of a prodrug of hydantoin based TACE inhibitor.

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone.

A canine biorelevant dissolution method for predicting in vivo performance of orally administered sustained release matrix tablets.

Preclinical species are a crucial component of drug development, but critical differences in physiology and anatomy need to be taken into account when attempting to extrapolate to humans or between species. The same is true when trying to develop oral formulations for preclinical species, especially unconventional formulations, such as sustained release tablets. During the evaluation of such specialized dosage forms, dissolution can be a critical in vitro tool used to rank-order formulations and ultimately choose the desired release rate.

Discovery of a Potent, Injectable Inhibitor of Aurora Kinases Based on the Imidazo-[1,2-a]-Pyrazine Core.

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 μM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.