Primary Cultures Research Articles

Pleiotropy in FOXC1-attributable phenotypes involves altered ciliation and cilia-dependent signaling

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld–Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.

IRF3 regulates neuroinflammatory responses and the expression of genes associated with Alzheimer’s disease

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D: IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer’s disease, notably apolipoprotein-e. Using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 (ZBP1) as a target of IRF3 that is relevant across various neuroinflammatory disorders. Lastly, we show IRF3 phosphorylation and IRF3-dependent ZBP1 induction in response to Aβ in primary microglia cultures. Together, our results identify IRF3 as an important regulator of LPS and Aβ -mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

Correlation of Immunomodulatory Cytokines with Tumor Volume and Cerebrospinal Fluid in Vestibular Schwannoma Patients.

Sporadic vestibular schwannomas (VSs) often exhibit slow or negligible growth. Nevertheless, some VSs increase significantly in volume within a few months or grow continuously. Recent evidence indicates a role of inflammation in promoting VS growth. Therefore, our study aimed to identify cytokines, which are associated with larger VSs. The expression of different cytokines in VS tumor samples and VS primary cultures was investigated. Additionally, the concentration of cytokines in cell culture supernatants of VS primary cultures and cerebrospinal fluid (CSF) of VS patients and healthy controls were determined. Correlation analysis of cytokine levels with tumor volume, growth rate, Koos grade, age, and hearing was examined with Spearman's-rank test. The mRNA expression of CC-chemokine ligand (CCL) 18, growth differentiation factor (GDF) 15, and interferon regulatory factor 4 correlated positively with tumor volume. Moreover, the amount of GDF15 in the cell culture supernatant of primary cells correlated positively with tumor volume. The concentrations of the cytokines CCL2, CCL5, and CCL18 and transforming growth factor beta (TGFB) 1 in the CSF of the patients were significantly different from those in the CSF controls. Inhibition of immune cell infiltration could be a putative approach to prevent and control VS growth.

Characteristics of Organizational Culture on the Quality of Health Services in Meepago Region (Nabire Regency) Study in the Field of Public Administration

This study aims to emphasizes the characteristics of organizational culture and its impact on the quality of health services in public administration in Nabire Regency. The main argument in this research places Robbins' idea of ​​organizational culture into the characteristics of primary organizational culture. Method used in this research is descriptive qualitative, with the data source from observations, in-depth interviews, and documentation. The findings in this research showed that secondary organizational cultural characteristics also had a dominant influence in shaping organizational culture at the research location. The findings in this research show that primary cultural characteristics alone are not enough to achieve optimal quality of health services, especially in conditions in countries or regions that have not yet been settled and established, but on this occasion I dared to add new organizational cultural characteristics which I call cultural characteristics. Further research can develop the results of this research to investigate health services with a wider scope or in different cases in other places.

Apoptosis-Cell Cycle-Autophagy Molecular Mechanisms Network in Heterogeneous Aggressive Phenotype Prostate Hyperplasia Primary Cell Cultures Have a Prognostic Role.

Our study highlights the apoptosis, cell cycle, DNA ploidy, and autophagy molecular mechanisms network to identify prostate pathogenesis and its prognostic role. Caspase 3/7 expressions, cell cycle, adhesion glycoproteins, autophagy, nuclear shrinkage, and oxidative stress by flow-cytometry analysis are used to study the BPH microenvironment's heterogeneity. A high late apoptosis expression by caspases 3/7 activity represents an unfavorable prognostic biomarker, a dependent predictor factor for cell adhesion, growth inhibition by arrest in the G2/M phase, and oxidative stress processes network. The heterogeneous aggressive phenotype prostate adenoma primary cell cultures present a high S-phase category (>12%), with an increased risk of death or recurrence due to aneuploid status presence, representing an unfavorable prognostic biomarker, a dependent predictor factor for caspase 3/7 activity (late apoptosis and necrosis), and cell growth inhibition (G2/M arrest)-linked mechanisms. Increased integrin levels in heterogenous BPH cultures suggest epithelial-mesenchymal transition (EMT) that maintains an aggressive phenotype by escaping cell apoptosis, leading to the cell proliferation necessary in prostate cancer (PCa) development. As predictor biomarkers, the biological mechanisms network involved in apoptosis, the cell cycle, and autophagy help to establish patient prognostic survival or target cancer therapy development.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Dopaminergic neurons lacking Caspase-3 avoid apoptosis but undergo necrosis after MPTP treatment inducing a Galectin-3-dependent selective microglial phagocytic response

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc). Apoptosis is thought to play a critical role in the progression of PD, and thus understanding the effects of antiapoptotic strategies is crucial for developing potential therapies. In this study, we developed a unique genetic model to selectively delete Casp3, the gene encoding the apoptotic protein caspase-3, in dopaminergic neurons (TH-C3KO) and investigated its effects in response to a subacute regime of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, which is known to trigger apoptotic loss of SNpc dopaminergic neurons. We found that Casp3 deletion did not protect the dopaminergic system in the long term. Instead, we observed a switch in the cell death pathway from apoptosis in wild-type mice to necrosis in TH-C3KO mice. Notably, we did not find any evidence of necroptosis in our model or in in vitro experiments using primary dopaminergic cultures exposed to 1-methyl-4-phenylpyridinium in the presence of pan-caspase/caspase-8 inhibitors. Furthermore, we detected an exacerbated microglial response in the ventral mesencephalon of TH-C3KO mice in response to MPTP, which mimicked the microglia neurodegenerative phenotype (MGnD). Under these conditions, it was evident the presence of numerous microglial phagocytic cups wrapping around apparently viable dopaminergic cell bodies that were inherently associated with galectin-3 expression. We provide evidence that microglia exhibit phagocytic activity towards both dead and stressed viable dopaminergic neurons through a galectin-3-dependent mechanism. Overall, our findings suggest that inhibiting apoptosis is not a beneficial strategy for treating PD. Instead, targeting galectin-3 and modulating microglial response may be more promising approaches for slowing PD progression.

Lack of ceramide synthase 5 protects retinal ganglion cells from ocular hypertensive injury

Ceramides with varying acyl-chain lengths can have unique biological actions and hence, cellular responses to ceramides may depend not on their overall concentration but on that of individual ceramide species. The purpose of this study was to determine individual ceramide species impacting retinal ganglion cell (RGC) loss under the ocular hypertensive condition.Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human astrocytes were used to determine the effect of individual ceramide species on both RGC viability and astrocyte secretion of inflammatory cytokines in vitro. In in vivo experiments with wild-type (WT) and ceramide synthase 5 (CerS5) knockout mice, intraocular pressure was unilaterally elevated with microbead injection. Retinal function and morphology were evaluated using pattern electroretinography (pERG) and immunofluorescence, respectively. Ceramide levels were determined by LC-MS/MS analysis.Exposure to C16:0-, C18:0-, C18:1-, C20:0- and C24:0-ceramides significantly reduces RGC viability in vitro, with the very long chain C24:0-ceramide being the most neurotoxic; treatment with C18:0-, C18:1- and C24:0-ceramides stimulates an increase of TNF-α secretion by astrocytes. The retinas of CerS5 KO mice have significantly reduced levels of C16:0- and C18:1-ceramides compared to WT; ocular hypertensive eyes of these mice maintain higher pERG amplitudes and RGC numbers compared to WT.Individual ceramides with different chain lengths have different effects on RGCs and astrocytes. Our results demonstrate that suppressing C16:0- and C18:1-ceramide species effectively protects RGCs against ocular hypertensive injury. These results provide a basis for targeting specific ceramide species in the treatment of glaucoma.

Effects of crizotinib in strn-alk anaplastic thyroid cancer in primary culture in vitro

Effects of crizotinib in strn-alk anaplastic thyroid cancer in primary culture <i>in vitro</i>

Pathological characteristics of axons and alterations of proteomic and lipidomic profiles in midbrain dopaminergic neurodegeneration induced by WDR45-deficiency

BackgroundAlthough WD repeat domain 45 (WDR45) mutations have been linked to β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}-propeller protein-associated neurodegeneration (BPAN), the precise molecular and cellular mechanisms behind this disease remain elusive. This study aims to shed light on the impacts of WDR45-deficiency on neurodegeneration, specifically axonal degeneration, within the midbrain dopaminergic (DAergic) system. We hope to better understand the disease process by examining pathological and molecular alterations, especially within the DAergic system.MethodsTo investigate the impacts of WDR45 dysfunction on mouse behaviors and DAergic neurons, we developed a mouse model in which WDR45 was conditionally knocked out in the midbrain DAergic neurons (WDR45cKO). Through a longitudinal study, we assessed alterations in the mouse behaviors using open field, rotarod, Y-maze, and 3-chamber social approach tests. We utilized a combination of immunofluorescence staining and transmission electron microscopy to examine the pathological changes in DAergic neuron soma and axons. Additionally, we performed proteomic and lipidomic analyses of the striatum from young and aged mice to identify the molecules and processes potentially involved in the striatal pathology during aging. Further more, primary midbrain neuronal culture was employed to explore the molecular mechanisms leading to axonal degeneration.ResultsOur study of WDR45cKO mice revealed a range of deficits, including impaired motor function, emotional instability, and memory loss, coinciding with the profound reduction of midbrain DAergic neurons. The neuronal loss, we observed massive axonal enlargements in the dorsal and ventral striatum. These enlargements were characterized by the accumulation of extensively fragmented tubular endoplasmic reticulum (ER), a hallmark of axonal degeneration. Proteomic analysis of the striatum showed that the differentially expressed proteins were enriched in metabolic processes. The carbohydrate metabolic and protein catabolic processes appeared earlier, and amino acid, lipid, and tricarboxylic acid metabolisms were increased during aging. Of note, we observed a tremendous increase in the expression of lysophosphatidylcholine acyltransferase 1 (Lpcat1) that regulates phospholipid metabolism, specifically in the conversion of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC) in the presence of acyl-CoA. The lipidomic results consistently suggested that differential lipids were concentrated on PC and LPC. Axonal degeneration was effectively ameliorated by interfering Lpcat1 expression in primary cultured WDR45-deficient DAergic neurons, proving that Lpcat1 and its regulated lipid metabolism, especially PC and LPC metabolism, participate in controlling the axonal degeneration induced by WDR45 deficits.ConclusionsIn this study, we uncovered the molecular mechanisms underlying the contribution of WDR45 deficiency to axonal degeneration, which involves complex relationships between phospholipid metabolism, autophagy, and tubular ER. These findings greatly advance our understanding of the fundamental molecular mechanisms driving axonal degeneration and may provide a foundation for developing novel mechanistically based therapeutic interventions for BPAN and other neurodegenerative diseases.

Cant1 Affects Cartilage Proteoglycan Properties: Aggrecan and Decorin Characterization in a Mouse Model of Desbuquois Dysplasia Type 1.

Desbuquois dysplasia type 1 (DBQD1) is a recessive chondrodysplasia caused by mutations in the CANT1 gene, encoding for the Golgi Calcium-Activated Nucleotidase 1 (CANT1). The enzyme hydrolyzes UDP, the by-product of glycosyltransferase reactions, but it might play other roles in different cell types. Using a Cant1 knock-out mouse, we demonstrated that CANT1 is crucial for glycosaminoglycan (GAG) synthesis; however, its impact on the biochemical properties of cartilage proteoglycans remains unknown. Thus, in this work, we characterized decorin and aggrecan from primary chondrocyte cultures and cartilage biopsies of mutant mice at post-natal day 4 by Western blots and further investigated their distribution in the cartilage extracellular matrix (ECM) by immunohistochemistry. We demonstrated that the GAG synthesis defect caused by CANT1 impairment led to the synthesis and secretion of proteoglycans with shorter GAG chains compared with wild-type animals. However, this alteration did not result in the synthesis and secretion of decorin and aggrecan in the unglycanated form. Interestingly, the defect was not cartilage-specific since also skin decorin showed a reduced hydrodynamic size. Finally, immunohistochemical studies in epiphyseal sections of mutant mice demonstrated that the proteoglycan structural defect moderately affected decorin distribution in the ECM.

Visualization of Brain Tumors with Infrared-Labeled Aptamers for Fluorescence-Guided Surgery.

Gliomas remain challenging brain tumors to treat due to their infiltrative nature. Accurately identifying tumor boundaries during surgery is crucial for successful resection. This study introduces an innovative intraoperative visualization method utilizing surgical fluorescence microscopy to precisely locate tumor cell dissemination. Here, the focus is on the development of a novel contrasting agent (IR-Glint) for intraoperative visualization of human glial tumors comprising infrared-labeled Glint aptamers. The specificity of IR-Glint is assessed using flow cytometry and microscopy on primary cell cultures. In vivo effectiveness is studied on mouse and rabbit models, employing orthotopic xenotransplantation of human brain gliomas with various imaging techniques, including PET/CT, in vivo fluorescence visualization, confocal laser scanning, and surgical microscopy. The experiments validate the potential of IR-Glint for the intraoperative visualization of gliomas using infrared imaging. IR-Glint penetrates the blood-brain barrier and can be used for both intravenous and surface applications, allowing clear visualization of the tumor. The surface application directly to the brain reduces the dosage required and mitigates potential toxic effects on the patient. The research shows the potential of infrared dye-labeled aptamers for accurately visualizing glial tumors during brain surgery. This novel aptamer-assisted fluorescence-guided surgery (AptaFGS) may pave the way for future advancements in the field of neurosurgery.

Evaluation of the antineoplastic effect of cabozantinib in primary human cell cultures from anaplastic thyroid carcinoma

Evaluation of the antineoplastic effect of cabozantinib in primary human cell cultures from anaplastic thyroid carcinoma

PDE4B inhibition by nerandomilast: Effects on lung fibrosis and transcriptome in fibrotic rats and on biomarkers in human lung epithelial cells.

The PDE4 family is considered a prime target for therapeutic intervention in several fibro-inflammatory diseases. We have investigated the molecular mechanisms of nerandomilast (BI 1015550), a preferential PDE4B inhibitor. In addition to clinically relevant parameters of idiopathic pulmonary fibrosis (IPF; lung function measurement/high-resolution computed tomography scan/AI-Ashcroft score), whole-lung homogenates from a therapeutic male Wistar rat model of pulmonary fibrosis were analysed by next-generation sequencing (NGS). Data were matched with public domain data derived from human IPF samples to investigate how well the rat model reflected human IPF. We scored the top counter-regulated genes following treatment with nerandomilast in human single cells and validated disease markers discovered in the rat model using a human disease-relevant in vitro assay of IPF. Nerandomilast improved the decline of lung function parameters in bleomycin-treated animals. In the NGS study, most transcripts deregulated by bleomycin treatment were normalised by nerandomilast treatment. Most notably, a significant number of deregulated transcripts that were identified in human IPF disease were also found in the animal model and reversed by nerandomilast. Mapping to single-cell data revealed the strongest effects on mesenchymal, epithelial and endothelial cell populations. In a primary human epithelial cell culture system, several disease-related (bio)markers were inhibited by nerandomilast in a concentration-dependent manner. This study further supports the available knowledge about the anti-inflammatory/antifibrotic mechanisms of nerandomilast and provides novel insights into the mode of action and signalling pathways influenced by nerandomilast treatment of lung fibrosis.

STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.

Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.

An In Vitro Orbital Flow Model to Study Mechanical Loading Effects on Osteoblasts.

Flow induced by an orbital shaker is known to produce shear stress and oscillatory flow, but the utility of this model for studying mechanical loading effects in osteoblasts is not well defined. To test this, osteoblasts derived from the long bones of adult male C57BL/6J mice were plated on 6-well plates and subjected to orbital shaking at various frequencies (0.7, 1.4, and 3.3 Hz) for 30 and 60 min in serum-free differentiation media. The shear stress on cells produced by 0.7, 1.4, and 3.3 Hz shaking frequencies were 1.6, 4.5, and 11.8 dynes/cm2, respectively. ALP activity measured 72 h after shaking (orbital flow) showed a significant increase at 0.7 and 1.4 Hz, but not at 3.3 Hz, compared to static controls. Orbital flow-induced mechanical stress also significantly increased (25%) osteoblast proliferation at a 0.7 Hz flow compared to static controls. Additionally, expression levels of bone formation markers Osf2, Hif1a, Vegf, and Cox2 were significantly increased (1.5- to 3-fold, p < 0.05) in cells subjected to a 0.7 Hz flow compared to non-loaded control cells. We also evaluated the effect of orbital flow on key signaling pathways (mTOR, JNK, and WNT) known to mediate mechanical strain effects on osteoblasts. We found that blocking mTOR and WNT signaling with inhibitors significantly reduced (20-30%) orbital flow-induced ALP activity compared to cells treated using a vehicle. In contrast, inhibition of JNK signaling did not affect flow-induced osteoblast differentiation. In conclusion, our findings show that the flow produced by an orbital shaker at a lower frequency is an appropriate inexpensive model for studying the molecular pathways mediating mechanical strain effects on primary cultures of osteoblasts in vitro.

Hedgehog components are overexpressed in a series of liver cancer cases

Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC.

Performance of common primary and chromogenic culture media for MALDI-TOF MS identification of clinically relevant yeasts.

Timely and accurate identification of yeasts is essential for adequate treatment, considering the increase in antifungal resistance of some species, particularly for C. auris. Current matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) manufacturer's protocol for identification of yeasts requires 24- to 72-h cultivation on Sabouraud dextrose media (SAB), but not some of the mainstay primary culture media used in mycology such as inhibitory mold agar (IMA), Mycosel, CHROMagar Candida Plus, and CHROMagar Candida. As culture media can influence MALDI-TOF MS identification results, this study evaluated the accuracy and performance of identification of clinically relevant yeasts on these first-line media using the VITEK-MS MALDI-TOF MS system.IMPORTANCEIn this study, a panel of 140 strains (21 species) was used to assess the performance of the selected media. Although not in the manufacturer's list of accepted media, IMA and chromogenic media are suitable for the identification of yeasts on the VITEK-MS systems. CHROMagar Candida Plus allowed the identification of 135/140 isolates tested after 24-h incubation similar to SAB reference media (137/140). Yeast isolates that grew on Mycosel selective media were also reliably identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. VITEK-MS system with IVD database V3.2 correctly identified C. auris strains to the species level on CHROMagar Candida Plus alleviating the need for subcultivation and reduced turnaround time (24-72 h) to identification for patient screening.

Effects of chronic insecticide exposure on neuronal network development in vitro in rat cortical cultures.

Developmental exposure to carbamates, organophosphates, and pyrethroids has been associated with impaired neurodevelopmental outcomes. Sex-specific differences following chronic insecticide exposure are rather common in vivo. Therefore, we assessed the chronic effects of in vitro exposure to different carbamates (carbaryl, methomyl and aldicarb), organophosphates [chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), and 3,5,6,trichloropyridinol (TCP)], and pyrethroids [permethrin, alpha-cypermethrin and 3-phenoxy benzoic acid (3-PBA)] on neuronal network development in sex-separated rat primary cortical cultures using micro-electrode array (MEA) recordings. Our results indicate that exposure for 1week to carbaryl inhibited neurodevelopment in male cultures, while a hyperexcitation was observed in female cultures. Methomyl and aldicarb evoked a hyperexcitation after 2weeks of exposure, which was more pronounced in female cultures. In contrast to acute MEA results, exposure to ≥ 10µM CPF caused hyperexcitation in both sexes after 10days. Interestingly, exposure to 10µM CPO induced a clear hyperexcitation after 10days of exposure in male but not female cultures. Exposure to 100µM CPO strongly inhibited neuronal development. Exposure to the type I pyrethroid permethrin resulted in a hyperexcitation at 10µM and a decrease in neuronal development at 100µM. In comparison, exposure to ≥ 10µM of the type II pyrethroid alpha-cypermethrin decreased neuronal development. In female but not in male cultures, exposure to 1 and 10µM permethrin changed (network) burst patterns, with female cultures having shorter (network) bursts with fewer spikes per (network) burst. Together, these results show that MEA recordings are suitable for measuring sex-specific developmental neurotoxicity in vitro. Additionally, pyrethroid exposure induced effects on neuronal network development at human-relevant concentrations. Finally, chronic exposure has different effects on neuronal functioning compared to acute exposure, highlighting the value of both exposure paradigms.

Microchip construction for migration assays: investigating the impact of physical confinement on cell morphology and motility during vaccinia virus infection.

Vaccinia virus (VACV)-induced cell migration is thought to be closely related to the rapid transmission of viral infection in the body. The limited studies are mainly based on scratch assay using traditional cell culture techniques, which inevitably ignores the influences of extracellular microenvironment. Physical confinement, inherently presenting in vivo, has proven to be a critical extern cue in modulating migration behaviors of multiple cells, while its impacts on VACV-induced cell motility remain unclear. Herein, we developed a migration assay microchip featuring confined microchannel array to investigate the effect of physical confinement on infected cell morphology and motility during VACV infection. Results showed that different from the random cell migration observed in traditional scratch assay on planar substrate, VACV-infected cells exhibited accelerated directionally persistent migration under confinement microenvironment. Moreover, single-directed elongated dominant lamella appeared to contrast distinctly with multiple protrusions stretched in random directions under unconfined condition. Additionally, the Golgi complex tended to relocate behind the nucleus confined within the microchannel axis compared to the classical reorientation pattern. These differences in characteristic subcellular architecture and organelle reorientation of migrating cells revealed cell biological mechanisms underlying altered migration behavior. Collectively, our study demonstrates that physical confinement acting as a guidance cue has profound impacts on VACV-induced migration behaviors, which provides new insight into cell migration behavior and viral rapid spread during VACV infection.