Controlling human organoid symmetry breaking reveals signaling gradients drive segmentation clock waves.

Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

TRKing down drug resistance in NTRK fusion-positive cancers†.

In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

JC virus spread is potentiated by glial replication and demyelination-linked glial proliferation.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating infection of the immunosuppressed brain, mediated by the gliotropic polyomavirus JCV. JCV replicates in human glial progenitor cells and astrocytes, which undergo viral T antigen-triggered mitosis, enabling viral replication. We asked if JCV spread might therefore be accelerated by glial proliferation. Using both in vitro analysis and a human glial chimeric mouse model of JCV infection, we found that dividing human astrocytes supported JCV propagation to a substantially greater degree than did mitotically quiescent cells. Accordingly, bulk and single cell RNA-sequence analysis revealed that JCV-infected glia differentially manifested cell cycle-linked disruption of both DNA damage response and transcriptional regulatory pathways. In vivo, JCV infection of humanized glial chimeras was greatly accentuated by cuprizone-induced demyelination and its associated mobilization of GPCs. Importantly, in vivo infection triggered the death of uninfected as well as infected glia, reflecting significant bystander death. Together, these data suggest that JCV propagation in PML may be accelerated by glial cell division. As such, the accentuated glial proliferation attending disease-associated demyelination may provide an especially favorable environment for JCV propagation, thus potentiating oligodendrocytic bystander death and further accelerating demyelination in susceptible hosts.

Functional consequences of changes in the distribution of Ca2+ extrusion pathways between t-tubular and surface membranes in a model of human ventricular cardiomyocyte.

The sarcolemmal Ca2+ efflux pathways, Na+-Ca2+-exchanger (NCX) and Ca2+-ATPase (PMCA), play a crucial role in the regulation of intracellular Ca2+ load and Ca2+ transient in cardiomyocytes. The distribution of these pathways between the t-tubular and surface membrane of ventricular cardiomyocytes varies between species and is not clear in human. Moreover, several studies suggest that this distribution changes during the development and heart diseases. However, the consequences of NCX and PMCA redistribution in human ventricular cardiomyocytes have not yet been elucidated. In this study, we aimed to address this point by using a mathematical model of the human ventricular myocyte incorporating t-tubules, dyadic spaces, and subsarcolemmal spaces. Effects of various combinations of t-tubular fractions of NCX and PMCA were explored, using values between 0.2 and 1 as reported in animal experiments under normal and pathological conditions. Small variations in the action potential duration (≤ 2%), but significant changes in the peak value of cytosolic Ca2+ transient (up to 17%) were observed at stimulation frequencies corresponding to the human heart rate at rest and during activity. The analysis of model results revealed that the changes in Ca2+ transient induced by redistribution of NCX and PMCA were mainly caused by alterations in Ca2+ concentrations in the subsarcolemmal spaces and cytosol during the diastolic phase of the stimulation cycle. The results suggest that redistribution of both transporters between the t-tubular and surface membranes contributes to changes in contractility in human ventricular cardiomyocytes during their development and heart disease and may promote arrhythmogenesis.

FoxO1 silencing in Atp7b-/- neural stem cells attenuates high copper-induced apoptosis via regulation of autophagy.

Wilson disease (WD) is a severely autosomal genetic disorder triggered by dysregulated copper metabolism. Autophagy and apoptosis share common modulators that process cellular death. Emerging evidences suggest that Forkhead Box O1 over-expression (FoxO1-OE) aggravates abnormal autophagy and apoptosis to induce neuronal injury. However, the underlying mechanisms remain undetermined. Herein, the aim of this study was to investigate how regulating FoxO1 affects cellular autophagy and apoptosis to attenuate neuronal injury in a well-established WD cell model, the high concentration copper sulfate (CuSO4, HC)-triggered Atp7b-/- (Knockout, KO) neural stem cell (NSC) lines. The FoxO1-OE plasmid, or siRNA-FoxO1 (siFoxO1) plasmid, or empty vector plasmid was stably transfected with recombinant lentiviral vectors into HC-induced Atp7b-/- NSCs. Toxic effects of excess deposited copper on wild-type (WT), Atp7b-/- WD mouse hippocampal NSCs were tested by Cell Counting Kit-8 (CCK-8). Subsequently, the FoxO1 expression was evaluated by immunofluorescence (IF) assay, western blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Meanwhile, the cell autophagy and apoptosis were evaluated by flow cytometry (FC), TUNEL staining, 2,7-dichlorofluorescein diacetate (DCFH-DA), JC-1, WB, and qRT-PCR. The current study demonstrated a strong rise in FoxO1 levels in HC-treated Atp7b-/- NSCs, accompanied with dysregulated autophagy and hyperactive apoptosis. Also, it was observed that cell viability was significantly decreased with the over-expressed FoxO1 in HC-treated Atp7b-/- WD model. As intended, silencing FoxO1 effectively inhibited abnormal autophagy in HC-treated Atp7b-/- NSCs, as depicted by a decline in LC3II/I, Beclin-1, ATG3, ATG7, ATG13, and ATG16, whereas simultaneously increasing P62. In addition, silencing FoxO1 suppressed apoptosis via diminishing oxidative stress (OS), and mitochondrial dysfunction in HC-induced Atp7b-/- NSCs. Collectively, these results clearly demonstrate the silencing FoxO1 has the neuroprotective role of suppressing aberrant cellular autophagy and apoptosis, which efficiently attenuates neuronal injury in WD.

Toolbox for creating three-dimensional liver models.

Research within the hepato-biliary system and hepatic function is currently experiencing heightened interest, this is due to the high frequency of relapse rates observed in chronic conditions, as well as the imperative for the development of innovative therapeutic strategies to address both inherited and acquired diseases within this domain. The most commonly used sources for studying hepatocytes include primary human hepatocytes, human hepatic cancer cell lines, and hepatic-like cells derived from induced pluripotent stem cells. However, a significant challenge in primary hepatic cell culture is the rapid decline in their phenotypic characteristics, dedifferentiation and short cultivation time. This limitation creates various problems, including the inability to maintain long-term cell cultures, which can lead to failed experiments in drug development and the creation of relevant disease models for researchers' purposes. To address these issues, the creation of a powerful 3D cell model could play a pivotal role as a personalized disease model and help reduce the use of animal models during certain stages of research. Such a cell model could be used for disease modelling, genome editing, and drug discovery purposes. This review provides an overview of the main methods of 3D-culturing liver cells, including a discussion of their characteristics, advantages, and disadvantages.

Data integration strategies for whole-cell modeling.

Data makes the world go round-and high quality data is a prerequisite for precise models, especially for whole-cell models (WCM). Data for WCM must be reusable, contain information about the exact experimental background, and should-in its entirety-cover all relevant processes in the cell. Here, we review basic requirements to data for WCM and strategies how to combine them. As a species-specific resource, we introduce the Yeast Cell Model Data Base (YCMDB) to illustrate requirements and solutions. We discuss recent standards for data as well as for computational models including the modeling process as data to be reported. We outline strategies for constructions of WCM despite their inherent complexity.

Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of ß-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of ß-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

Prinsepia utilis Royle polysaccharides promote skin barrier repair through the Claudin family.

BACKGROUND: Plant polysaccharides have various biological activities. However, few studies have been conducted on the skin barrier of Prinsepia utilis Royle polysaccharide extract (PURP). MATERIALS AND METHODS: The proportions of polysaccharides, monosaccharides and proteins were determined by extracting polysaccharides from fruit meal using water. The healing rate was measured by cell scratch assays. SDS-damaged reconstructed human epidermal models, an acetone-ether-induced mouse model and an IL-4-induced cellular inflammation model were used to detect the effects of polysaccharides on the phenotype, HA, TEWL, and TEER, with further characterizations performed using QRT-PCR, Western blotting, immunofluorescence (IF) assays. RESULTS: PURP contained 35.73% polysaccharides and 11.1% proteins. PURP promoted cell migration and increased skin thickness in a reconstructed human epidermis model. The TEWL significantly decreased, and the HA content significantly increased. PURP significantly increased the TEER and decreased the permeability of the SDS-damaged reconstructed human epidermis model. Claudin-3, Claudin-4, and Claudin-5 were significantly upregulated. IF and Western blot analysis revealed that the Claudin-4 level significantly increased after treatment with PURP. Claudin-1, Claudin-3, Claudin-4, and Claudin-5 gene expression and IF and immunohistochemical staining were significantly increased in mice treated with acetone-ether. PURP promoted the expression of Claudin-1, Claudin-3, Claudin-4, and Claudin-5 after treatment with 100 ng/mL IL-4. PURP also downregulated the expression of NO, IL6, TNFα and NFκB in Raw 264.7 cells and in a mouse model. CONCLUSION: We hypothesize that PURP may repair the skin barrier by promoting the expression of the claudin family and can assist in skin therapy.

Assessment of osteopontin as an early nephrotoxicity indicator in human renal proximal tubule cells and its application in evaluating lanthanum-induced nephrotoxicity.

Nephrotoxicity is a common adverse effect induced by various chemicals, necessitating the development of reliable toxicity screening models for nephrotoxicity assessment. In this study, we assessed a group of nephrotoxicity indicators derived from different toxicity pathways, including conventional endpoints and kidney tubular injury biomarkers such as clusterin (CLU), kidney injury molecule-I (KIM-1), osteopontin (OPN), and neutrophil gelatinase-associated lipocalin (NGAL), using HK-2 and induced pluripotent stem cells (iPSCs)-derived renal proximal tubular epithelial-like cells (PTLs). Among the biomarkers tested, OPN emerged as the most discerning and precise marker. The predictive potential of OPN was tested using a panel of 10 nephrotoxic and 5 non-nephrotoxic compounds. The results demonstrated that combining OPN with the half-maximal inhibitory concentration (IC50) enhanced the diagnostic accuracy in both cellular models. Additionally, PTLs cells showed superior predictive efficacy for nephrotoxicity compared to HK-2 cells in this investigation. The two cellular models were utilized to evaluate the nephrotoxicity of lanthanum. The findings indicated that lanthanum possesses nephrotoxic properties; however, the degree of nephrotoxicity was relatively low, consistent with the outcomes of in vivo experiments.

In Vitro Modelling of Osteogenesis Imperfecta with Patient-Derived Induced Mesenchymal Stem Cells.

(1) Mesenchymal stem cells (MSCs) are a valuable cell model to study the bone pathology of Osteogenesis Imperfecta (OI), a rare genetic collagen-related disorder characterized by bone fragility and skeletal dysplasia. We aimed to generate a novel OI induced mesenchymal stem cell (iMSC) model from induced pluripotent stem cells (iPSCs) derived from human dermal fibroblasts. For the first time, OI iMSCs generation was based on an intermediate neural crest cell (iNCC) stage. (2) Skin fibroblasts from healthy individuals and OI patients were reprogrammed into iPSCs and subsequently differentiated into iMSCs via iNCCs. (3) Successful generation of iPSCs from acquired fibroblasts was confirmed with changes in cell morphology, expression of iPSC markers SOX2, NANOG, and OCT4 and three germ-layer tests. Following differentiation into iNCCs, cells presented increased iNCC markers including P75NTR, TFAP2A, and HNK-1 and decreased iPSC markers, shown to reach the iNCC stage. Induction into iMSCs was confirmed by the presence of CD73, CD105, and CD90 markers, low expression of the hematopoietic, and reduced expression of the iNCC markers. iMSCs were trilineage differentiation-competent, confirmed using molecular analyses and staining for cell-type-specific osteoblast, adipocyte, and chondrocyte markers. (4) In the current study, we have developed a multipotent in vitro iMSC model of OI patients and healthy controls able to differentiate into osteoblast-like cells.

Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators.

Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.

Cell-Penetrating Peptide-Mediated Biomolecule Transportation in Artificial Lipid Vesicles and Living Cells.

Signal transduction and homeostasis are regulated by complex protein interactions in the intracellular environment. Therefore, the transportation of impermeable macromolecules (nucleic acids, proteins, and drugs) that control protein interactions is essential for modulating cell functions and therapeutic applications. However, macromolecule transportation across the cell membrane is not easy because the cell membrane separates the intra/extracellular environments, and the types of molecular transportation are regulated by membrane proteins. Cell-penetrating peptides (CPPs) are expected to be carriers for molecular transport. CPPs can transport macromolecules into cells through endocytosis and direct translocation. The transport mechanism remains largely unclear owing to several possibilities. In this review, we describe the methods for investigating CPP conformation, translocation, and cargo transportation using artificial membranes. We also investigated biomolecular transport across living cell membranes via CPPs. Subsequently, we show not only the biochemical applications but also the synthetic biological applications of CPPs. Finally, recent progress in biomolecule and nanoparticle transportation via CPPs into specific tissues is described from the viewpoint of drug delivery. This review provides the opportunity to discuss the mechanism of biomolecule transportation through these two platforms.

Differential effects of OATP2B1 on statin accumulation and toxicity in a beta cell model.

An increased risk of new-onset diabetes mellitus has been recently reported for statin therapy, and experimental studies have shown reduced glucose-stimulated insulin secretion (GSIS) and mitochondrial dysfunction in beta cells with effects differing among agents. Organic anion transporting polypeptide (OATP) 2B1 contributes to hepatic uptake of rosuvastatin, atorvastatin and pravastatin, three known substrates. Since OATP2B1 is present in beta cells of the human pancreas, we investigated if OATP2B1 facilitates the local accumulation of statins in a rat beta cell model INS-1 832/13 (INS-1) thereby amplifying statin-induced toxicity. OATP2B1 overexpression in INS-1 cells via adenoviral transduction showed 2.5-, 1.8- and 1.4-fold higher cellular retention of rosuvastatin, atorvastatin and pravastatin, respectively, relative to LacZ control, while absolute intracellular concentration was about twice as high for the lipophilic atorvastatin compared to the more hydrophilic rosuvastatin and pravastatin. After 24 h statin treatment at high concentrations, OATP2B1 enhanced statin toxicity involving activation of intrinsic apoptosis (caspase 3/7 activation) and mitochondrial dysfunction (NADH dehydrogenase activity) following rosuvastatin and atorvastatin, which was partly reversed by isoprenoids. OATP2B1 had no effect on statin-induced reduction in GSIS, mitochondrial electron transport chain complex expression or caspase 9 activation. We confirmed a dose-dependent reduction in insulin secretion by rosuvastatin and atorvastatin in native INS-1 with a modest change in cellular ATP. Collectively, our results indicate a role of OATP2B1, which is abundant in human beta cells, in statin accumulation and statin-induced toxicity but not insulin secretion of rosuvastatin and atorvastatin in INS-1 cells.

Leveraging DNA-Encoded Cell-Mimics for Environment-Adaptive Transmembrane Channel Release-Induced Cell Death.

The advancement of cell-mimic materials, which can forge sophisticated physicochemical dialogues with living cells, has unlocked a realm of intriguing prospects within the fields of synthetic biology and biomedical engineering. Inspired by the evolutionarily acquired ability of T lymphocytes to release perforin and generate transmembrane channels on targeted cells for killing, herein we present a pioneering DNA-encoded artificial T cell mimic model (ARTC) that accurately mimics T-cell-like behavior. ARTC responds to acidic conditions similar to those found in the tumor microenvironment and then selectively releases a G-rich DNA strand (LG4) embedded with C12 lipid and cholesterol molecules. Once released, LG4 effectively integrates into the membranes of neighboring live cells, behaving as an artificial transmembrane channel that selectively transports K+ ions and disrupts cellular homeostasis, ultimately inducing apoptosis. We hope that the emergence of ARTC will usher in new perspectives for revolutionizing future disease treatment and catalyzing the development of advanced biomedical technologies.

Polycystin-2-dependent transcriptome reveals early response of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin genes, Pkd1 and Pkd2, but the underlying pathogenic mechanisms are poorly understood. To identify genes and pathways that operate downstream of polycystin-2 (PC2), a comprehensive gene expression database was created, cataloging changes in the transcriptome immediately following PC2 protein depletion. To explore cyst initiation processes, an immortalized mouse inner medullary collecting duct line was developed with the ability to knock out the Pkd2 gene conditionally. Genome-wide transcriptome profiling was performed using RNA sequencing in the cells immediately after PC2 was depleted and compared with isogenic control cells. Differentially expressed genes were identified, and a bioinformatic analysis pipeline was implemented. Altered expression of candidate cystogenic genes was validated in Pkd2 knockout mice. The expression of nearly 900 genes changed upon PC2 depletion. Differentially expressed genes were enriched for genes encoding components of the primary cilia, the canonical Wnt pathway, and MAPK signaling. Among the PC2-dependent ciliary genes, the transcription factor Glis3 was significantly downregulated. MAPK signaling formed a key node at the epicenter of PC2-dependent signaling networks. Activation of Wnt and MAPK signaling, concomitant with the downregulation of Glis3, was corroborated in Pkd2 knockout mice. The data identify a PC2 cilia-to-nucleus signaling axis and dysregulation of the Gli-similar subfamily of transcription factors as a potential initiator of cyst formation in ADPKD. The catalog of PC2-regulated genes should provide a valuable resource for future ADPKD research and new opportunities for drug development.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Mutations in polycystin genes cause the disease, but the underlying mechanisms of cystogenesis are unknown. To help fill this knowledge gap, we created an inducible cell model of ADPKD and assembled a catalog of genes that respond in immediate proximity to polycystin-2 depletion using transcriptomic profiling. The catalog unveils a ciliary signaling-to-nucleus axis proximal to polycystin-2 dysfunction, highlighting Glis, Wnt, and MAPK signaling.

Screening of Natural Compounds for CYP11A1 Stimulation Against Cell Renal Cell Carcinoma.

BACKGROUND: Renal cancer therapies are challenging owing to the extensive spreading of this cancer to other organs and its ability to pose resistance to current medications. Therefore, drugs targeting novel targets are urgently required to overcome these challenges. The cholesterol side-chain cleavage enzyme (CYP11A1) is closely associated with steroidogenesis, and its downregulation is linked to adrenal dysfunction and several types of carcinoma. We previously found that overexpression of CYP11A1 inhibited epithelial-mesenchymal transition and induced G2/M arrest in the kidney cancer Caki-1 cell line. In this context, natural compounds that exhibit potent CYP11A1 stimulation activity can be promising therpaeutic agents for kidney cancer. METHODS: We screened a panel of 1374 natural compounds in a wound-healing assay using CYP11A1-transfected Caki-1 cells. Of these, 167 promising biologically active compounds that inhibited cancer cell migration by more than 75% were selected, and their half-maximal inhibitory concentrations (IC50) were determined. The IC50 of 159 compounds was determined and 38 compounds with IC50 values less than 50 µM were selected for further analysis. Steroid hormones (cholesterol and pregnenolone) levels in cells treated with the selected compounds were quantitated using LC-MS/MS to determine their effect on CYP11A1 activity. Western blotting for CYP11A1, autophagy signaling proteins, and ferroptosis regulators were performed to ivestigate the mechanisms underlying the action of the selected compounds. RESULTS: We screened five promising natural lead compounds that inhibited cancer cell proliferation after three screening steps. The IC50 of these compounds was determined to be between 5.9 and 14.6 µM. These candidate compounds increased the expression of CYP11A1 and suppressed cholesterol levels while increasing pregnenolone levels, which is consistent with the activation of CYP11A1. Our results showed that CYP11A1 activation inhibited the migration of cancer cells, promoted ferroptosis, and triggered autophagy signaling. CONCLUSIONS: This study indicates that the CYP11A1-overexpressing Caki-1 cell line is useful for screening drugs against kidney cancer. The two selected compounds could be utilized as lead compounds for anticancer drug discovery, and specifically for the development of antirenal cancer medication.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies.

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.