A comprehensive AI model development framework for consistent Gleason grading.

BACKGROUND: Artificial Intelligence(AI)-based solutions for Gleason grading hold promise for pathologists, while image quality inconsistency, continuous data integration needs, and limited generalizability hinder their adoption and scalability. METHODS: We present a comprehensive digital pathology workflow for AI-assisted Gleason grading. It incorporates A!MagQC (image quality control), A!HistoClouds (cloud-based annotation), Pathologist-AI Interaction (PAI) for continuous model improvement, Trained on Akoya-scanned images only, the model utilizes color augmentation and image appearance migration to address scanner variations. We evaluate it on Whole Slide Images (WSI) from another five scanners and conduct validations with pathologists to assess AI efficacy and PAI. RESULTS: Our model achieves an average F1 score of 0.80 on annotations and 0.71 Quadratic Weighted Kappa on WSIs for Akoya-scanned images. Applying our generalization solution increases the average F1 score for Gleason pattern detection from 0.73 to 0.88 on images from other scanners. The model accelerates Gleason scoring time by 43% while maintaining accuracy. Additionally, PAI improve annotation efficiency by 2.5 times and led to further improvements in model performance. CONCLUSIONS: This pipeline represents a notable advancement in AI-assisted Gleason grading for improved consistency, accuracy, and efficiency. Unlike previous methods limited by scanner specificity, our model achieves outstanding performance across diverse scanners. This improvement paves the way for its seamless integration into clinical workflows.

Gleason grading is a well-accepted diagnostic standard to assess the severity of prostate cancer in patients' tissue samples, based on how abnormal the cells in their prostate tumor look under a microscope. This process can be complex and time-consuming. We explore how artificial intelligence (AI) can help pathologists perform Gleason grading more efficiently and consistently. We build an AI-based system which automatically checks image quality, standardizes the appearance of images from different equipment, learns from pathologists' feedback, and constantly improves model performance. Testing shows that our approach achieves consistent results across different equipment and improves efficiency of the grading process. With further testing and implementation in the clinic, our approach could potentially improve prostate cancer diagnosis and management.

Design and Characterization of Prodrug-like Inhibitors for Preventing Glutamate Efflux through Reverse Transport.

Glutamate transporters are responsible for active transport of the major excitatory neurotransmitter glutamate across the cell membrane, regulating the extracellular glutamate concentration in the mammalian brain. Extracellular glutamate levels in the brain are usually in the submicromolar range but can increase by exocytosis, inhibition of cellular uptake, or through glutamate release by reverse transport, as well as other mechanisms, which can lead to neurodegeneration and neuronal cell death. Such conditions can be encountered upon energy deprivation during an ischemic stroke. Here, we developed acetoxymethyl (AM) ester prodrug-like derivatives of excitatory amino acid transporter (EAAT) inhibitors that permeate the cell membrane and are activated, most likely through hydrolysis by endogenous cellular esterases, to form the active EAAT inhibitor. Upon increase in external K+ concentration, the inhibitors block glutamate efflux by EAAT reverse transport. Using a novel high-affinity fluorescent prodrug-like inhibitor, dl-threo-9-anthracene-methoxy-aspartate (TAOA) AM ester, we demonstrate that the precursor rapidly accumulates inside cells. Electrophysiological methods and fluorescence assays utilizing the iGluSnFR external glutamate sensor were used to demonstrate the efficacy of AM ester-protected inhibitors in inhibiting K+-mediated glutamate release. Together, our results provide evidence for a novel method to potentially prevent glutamate release by reverse transport under pathophysiological conditions in a model cell system, as well as in human astrocytes, while leaving glutamate uptake under physiological conditions operational. This method could have wide-ranging applications in the prevention of glutamate-induced neuronal cell death.

Conserved allosteric inhibition mechanism in SLC1 transporters.

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, non-competitive inhibitor of EAAT1. Alanine serine cysteine transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine between intracellular and extracellular compartments . ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here, we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2. Guided by computational modeling using ligand docking and molecular dynamics simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double-mutant transporter, 100% of the inhibitory effect of UCPH-101 could be restored, and the apparent affinity was increased (Ki = 4.3 µM), much closer to the EAAT1 value of 0.6 µM. Finally, we identify a novel non-competitive ASCT2 inhibitor, through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective ASCT2 allosteric inhibitors can be designed .

Observing spontaneous, accelerated substrate binding in molecular dynamics simulations of glutamate transporters.

Glutamate transporters are essential for removing the neurotransmitter glutamate from the synaptic cleft. Glutamate transport across the membrane is associated with elevator-like structural changes of the transport domain. These structural changes require initial binding of the organic substrate to the transporter. Studying the binding pathway of ligands to their protein binding sites using molecular dynamics (MD) simulations requires micro-second level simulation times. Here, we used three methods to accelerate aspartate binding to the glutamate transporter homologue Gltph and to investigate the binding pathway. 1) Two methods using user-defined forces to prevent the substrate from diffusing too far from the binding site. 2) Conventional MD simulations using very high substrate concentrations in the 0.1 M range. The final, substrate bound states from these methods are comparable to the binding pose observed in crystallographic studies, although they show more flexibility in the side chain carboxylate function. We also captured an intermediate on the binding pathway, where conserved residues D390 and D394 stabilize the aspartate molecule. Finally, we investigated glutamate binding to the mammalian glutamate transporter, excitatory amino acid transporter 1 (EAAT1), for which a crystal structure is known, but not in the glutamate-bound state. Overall, the results obtained in this study reveal new insights into the pathway of substrate binding to glutamate transporters, highlighting intermediates on the binding pathway and flexible conformational states of the side chain, which most likely become locked in once the hairpin loop 2 closes to occlude the substrate.

RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells.

BACKGROUND: Human hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved. METHODS: Floating cell was separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot. A regulatory network was constructed to figure out the relationship between cell adhesin, cytoskeleton, pluripotency, and hematopoiesis. RESULTS: The overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions, and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated that he TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way. CONCLUSIONS: Regulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.

An engineered palette of metal ion quenchable fluorescent proteins.

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.

Accurate high-throughput structure mapping and prediction with transition metal ion FRET.

Mapping the landscape of a protein's conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein maltose binding protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells.

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism.

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/ß fold family of hydrolases, of which this CE is a member.

In Silico Design and Evaluation of Carboxylesterase Inhibitors.

Carboxylesterases (CEs) are important enzymes that catalyze biological detoxification, hydrolysis of certain pesticides, and metabolism of many esterified drugs. The development of inhibitors for CE has many potential uses, including increasing drug lifetime and altering biodistrubution; reducing or abrogating toxicity of metabolized drugs; and reducing pest resistance to insecticides. In this review, we discuss the major classes of known mammalian CE inhibitors and describe our computational efforts to design new scaffolds for development of novel, selective inhibitors. We discuss several strategies for in silico inhibitor development, including structure docking, database searching, multidimensional quantitative structure activity analysis (QSAR), and a newly-used approach that uses QSAR combined with de novo drug design. While our research is focused on design of specific inhibitors for human intestinal carboxylesterase (hiCE), the methods described are generally applicable to inhibitors of other enzymes, including CE from other tissues and organisms.