Single Cerebral Organoid Mass Spectrometry of Cell-Specific Protein and Glycosphingolipid Traits.

Cerebral organoids are a prolific research topic and an emerging model system for neurological diseases in human neurobiology. However, the batch-to-batch reproducibility of current cultivation protocols is challenging and thus requires a high-throughput methodology to comprehensively characterize cerebral organoid cytoarchitecture and neural development. We report a mass spectrometry-based protocol to quantify neural tissue cell markers, cell surface lipids, and housekeeping proteins in a single organoid. Profiled traits probe the development of neural stem cells, radial glial cells, neurons, and astrocytes. We assessed the cell population heterogeneity in individually profiled organoids in the early and late neurogenesis stages. Here, we present a unifying view of cell-type specificity of profiled protein and lipid traits in neural tissue. Our workflow characterizes the cytoarchitecture, differentiation stage, and batch cultivation variation on an individual cerebral organoid level.

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs.

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa - spiders and birds - to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.

The evolution and ecology of multiple antipredator defences.

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such "defence portfolios" that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.

Spatiotemporal variation in the role of floral traits in shaping tropical plant-pollinator interactions.

The pollination syndrome hypothesis predicts that plants pollinated by the same pollinator group bear convergent combinations of specific floral functional traits. Nevertheless, some studies have shown that these combinations predict pollinators with relatively low accuracy. This discrepancy may be caused by changes in the importance of specific floral traits for different pollinator groups and under different environmental conditions. To explore this, we studied pollination systems and floral traits along an elevational gradient on Mount Cameroon during wet and dry seasons. Using Random Forest (Machine Learning) models, allowing the ranking of traits by their relative importance, we demonstrated that some floral traits are more important than others for pollinators. However, the distribution and importance of traits vary under different environmental conditions. Our results imply the need to improve our trait-based understanding of plant-pollinator interactions to better inform the debate surrounding the pollination syndrome hypothesis.

Endocrine-disrupting chemicals rapidly affect intercellular signaling in Leydig cells.

A decline in male fertility possibly caused by environmental contaminants, namely endocrine-disrupting chemicals (EDCs), is a topic of public concern and scientific interest. This study addresses a specific role of testicular gap junctional intercellular communication (GJIC) between adjacent prepubertal Leydig cells in endocrine disruption and male reproductive toxicity. Organochlorine pesticides (lindane, methoxychlor, DDT), industrial chemicals (PCB153, bisphenol A, nonylphenol and octylphenol) as well as personal care product components (triclosan, triclocarban) rapidly dysregulated GJIC in murine Leydig TM3 cells. The selected GJIC-inhibiting EDCs (methoxychlor, triclosan, triclocarban, lindane, DDT) caused the immediate GJIC disruption by the relocation of gap junctional protein connexin 43 (Cx43) from the plasma membrane and the alternation of Cx43 phosphorylation pattern (Ser368, Ser279, Ser282) of its full-length and two N-truncated isoforms. After more prolonged exposure (24 h), EDCs decreased steady-state levels of full-length Cx43 protein and its two N-truncated isoforms, and eventually (triclosan, triclocarban) also tight junction protein Tjp-1. The disturbance of GJIC was accompanied by altered activity of mitogen-activated protein kinases MAPK-Erk1/2 and MAPK-p38, and a decrease in stimulated progesterone production. Our results indicate that EDCs might disrupt testicular homeostasis and development via disruption of testicular GJIC, a dysregulation of junctional and non-junctional functions of Cx43, activation of MAPKs, and disruption of an early stage of steroidogenesis in prepubertal Leydig cells. These critical disturbances of Leydig cell development and functions during a prepubertal period might be contributing to impaired male reproduction health later on.

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Structure-Dependent Effects of Phthalates on Intercellular and Intracellular Communication in Liver Oval Cells.

Humans are exposed to phthalates released from plastics, cosmetics, or food on a daily basis. Phthalates have low acute liver toxicity, but their chronic exposures could induce molecular and cellular effects linked to adverse health outcomes, such as liver tumor promotion or chronic liver diseases. The alternation of gap junctional intercellular communication (GJIC) and MAPK-Erk1/2 pathways in liver progenitor or oval cells can disrupt liver tissue homeostatic mechanisms and affect the development and severity of these adverse outcomes. Our study with 20 different phthalates revealed their structurally dependent effects on liver GJIC and MAPK-Erk1/2 signaling in rat liver WB-F344 cell line with characteristics of liver oval cells. The phthalates with a medium-length side chain (3-6 C) were the most potent dysregulators of GJIC and activators of MAPK-Erk1/2. The effects occurred rapidly, suggesting the activation of non-genomic (non-transcriptional) mechanisms directly by the parental compounds. Short-chain phthalates (1-2 C) did not dysregulate GJIC even after longer exposures and did not activate MAPK-Erk1/2. Longer chain (≥7 C) phthalates, such as DEHP or DINP, moderately activated MAPK-Erk1/2, but inhibited GJIC only after prolonged exposures (>12 h), suggesting that GJIC dysregulation occurs via genomic mechanisms, or (bio)transformation. Overall, medium-chain phthalates rapidly affected the key tissue homeostatic mechanisms in the liver oval cell population via non-genomic pathways, which might contribute to the development of chronic liver toxicity and diseases.

Tumor-promoting cyanotoxin microcystin-LR does not induce procarcinogenic events in adult human liver stem cells.

HL1-hT1 cell line represents adult human liver stem cells (LSCs) immortalized with human telomerase reverse transcriptase. In this study, HL1-hT1 cells were found to express mesenchymal markers (vimentin, CD73, CD90/THY-1 and CD105) and an early hepatic endoderm marker FOXA2, while not expressing hepatic progenitor (HNF4A, LGR5, α-fetoprotein) or differentiated hepatocyte markers (albumin, transthyretin, connexin 32). In response to microcystin-LR (MC-LR), a time- and concentration-dependent formation of MC-positive protein bands in HL1-hT1 cells was observed. Cellular accumulation of MC-LR occurred most likely via mechanisms independent on organic anion transporting polypeptides (OATPs) or multidrug resistance (MDR) proteins, as indicated (a) by a gene expression analysis of 11 human OATP genes and 4 major MDR genes (MDR1/P-glycoprotein, MRP1, MRP2 and BCRP); (b) by non-significant effects of OATP or MDR1 inhibitors on MC-LR uptake. Accumulation of MC-positive protein bands in HL1-hT1 cells was associated neither with alterations of cell viability and growth, dysregulations of ERK1/2 and p38 kinases, reactive oxygen species formation, induction of double-stranded DNA breaks nor modulations of stress-inducible genes (ATF3, HSP5). It suggests that LSCs might have a selective, MDR1-independent, survival advantage and higher tolerance towards MC-induced cytotoxic, genotoxic or cancer-related events than differentiated adult hepatocytes, fetal hepatocyte or malignant liver cell lines. HL1-hT1 cells provide a valuable in vitro tool for studying effects of toxicants and pharmaceuticals on LSCs, whose important role in the development of chronic toxicities and liver diseases is being increasingly recognized.

Generation of the Human iPSC Line from Spontaneous Late-Onset Alzheimer's Disease Patient with ApoE3/3 Genotype and Sex-, Age-, and ApoE-Matched Healthy Control.

Human induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) isolated from a patient diagnosed with spontaneous late-onset Alzheimer's disease (AD) carrying ApoE3/3 gene and one age-, sex-, and ApoE-matched healthy control. Reprogramming was done using a commercially available Epi5 Reprogramming Kit containing OCT4, SOX2, KLF4, LIN28, and L-MYC as reprogramming factors. The pluripotency of the iPSC lines was verified by the expression of pluripotency markers and by their capacity to differentiate into all three embryonic germ layers in vitro. These newly established iPSC lines offer a valuable platform for in vitro modeling of AD.

Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

Cerebral organoids derived from patients with Alzheimer's disease with PSEN1/2 mutations have defective tissue patterning and altered development.

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study human neural development and disease. Especially in the field of Alzheimer's disease (AD), remarkable effort has been put into investigating molecular mechanisms behind this disease. Then, with the advent of 3D neuronal cultures and cerebral organoids (COs), several studies have demonstrated that this model can adequately mimic familial and sporadic AD. Therefore, we created an AD-CO model using iPSCs derived from patients with familial AD forms and explored early events and the progression of AD pathogenesis. Our study demonstrated that COs derived from three AD-iPSC lines with PSEN1(A246E) or PSEN2(N141I) mutations developed the AD-specific markers in vitro, yet they also uncover tissue patterning defects and altered development. These findings are complemented by single-cell sequencing data confirming this observation and uncovering that neurons in AD-COs likely differentiate prematurely.

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

An intergenerational approach to parasitoid fitness determined using clutch size.

Parasitoids, as important natural enemies, occur in high numbers and help maintain balance in natural ecosystems. Their fitness is traditionally studied as fertility based on the number of offspring in the F1 generation. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the clutch size-body size-fertility correlation among offspring. As a result of this correlation, when females adjust the number of offspring laid in a host, they determine not only the number of offspring produced but also the body size and reproductive potential of those offspring. Although parasitoid fertility has been determined several times from clutch size, here we use Anaphes flavipes to demonstrate the use of this relationship in an upgraded intergenerational approach to parasitoid fitness. We show that with a range of hosts simultaneously utilized by female parasitoids, identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can generate higher fertility in the F2 generation. Our approach provides an intergenerational perspective for determining individual fitness of gregarious parasitoids and new possibilities for the modelling of parasitoid population density.

Endocrine-disrupting chemicals affect Sertoli TM4 cell functionality through dysregulation of gap junctional intercellular communication in vitro.

The frequencies of adverse outcomes associated with male reproductive health, including infertility and testicular cancer, are increasing. These adverse trends are partially attributed to increased exposure to environmental agents such as endocrine-disrupting chemicals (EDCs). This study addresses effects on EDCs on adjacent prepubertal Sertoli TM4 cells, specifically on 1) testicular gap junctional intercellular communication (GJIC), one of the hallmarks of non-genotoxic carcinogenicity, 2) GJIC building blocks connexins (Cx), and 3) mitogen-activated protein kinases MAPKs. We selected eight representatives of EDCs: organochlorine chemicals such as pesticides dichlorodiphenyltrichloroethane, lindane, methoxychlor, and vinclozolin, industrial chemicals bisphenol A and 2,2',4,4',5,5'-hexachlorobiphenyl, and components of personal care products, triclocarban and triclosan. EDCs rapidly dysregulated GJIC in Sertoli TM4 cells mainly via MAPK p38 and/or Erk1/2 pathways by the intermediate hyper- or de-phosphorylation of Cx43 (Ser368, Ser282) and translocation of Cx43 from the plasma membrane, suggesting disturbed intracellular trafficking of Cx43 protein. Surprisingly, EDCs did not rapidly activate MAPK Erk1/2 or p38; on the contrary, TCC and TCS decreased their activity (phosphorylation). Our results indicate that EDCs might disrupt testicular homeostasis and development via testicular GJIC, junctional and non-junctional functions of Cx43 and MAPK-signaling pathways in Sertoli cells.

Linearization of Composite Material Damage Model Results and Its Impact on the Subsequent Stress-Strain Analysis.

To solve problems in the field of mechanical engineering efficiently, individual numerical procedures must be developed, and solvers must be adapted. This study applies the results of a carbon-fibre reinforced polymer (CFRP) analysis along with the nonlinear finite element damage (FE) method to the translation of a linear solver. The analyzed tensile test sample is modelled using the ply-by-ply method. To describe the nonlinear post-damage behavior of the material, the Hashin model is used. To validate the transformation, an analysis and comparison of the damage results of the linearized and nonlinear model is carried out. Job linearization was performed by collecting elements into groups based on their level of damage and pairing them with unique material cards. Potentially suitable mathematical functions are tested for the grouping and consolidation of the elements. The results show that the agreement of some presented methods depends on the damage level. The influence of the selected statistical functions on the result is shown here. The optimal solution is demonstrated, and the most efficient method of linearization is presented. The main motivation behind this work is that the problem has not been discussed in the literature and that there is currently no commercial software translator that provides the transference of models between solvers.

Strain-Field Modifications in the Surroundings of Impact Damage of Carbon/Epoxy Laminate.

The relationship between deformation and stress is crucial for any elasto-plastic body. This paper deals with the experimental identification of the basic parameters of the composite laminate model in relation to the finite element model. Standardized tensile, impact, and post-impact tests on a carbon fiber-reinforced epoxy laminate were used. The method by which the elasticity and failure parameters were obtained from the initial components is described. In the article, the modes of initiation and complete failure of samples in tensile tests, which are compared with the simulation, are presented. Furthermore, the article deals with the issue of the generation and detection of damage by low-speed impact, which can be caused by contact with moving objects, due to improper handling or maintenance. The results of impact analysis simulations are shown in the context of strain-field distribution changes obtained with the help of digital image correlation. The results showed high agreement between the calculations and the experiments. Based on this agreement, simulations of impact damage for various energies were performed. These simulations were used to determine the approximate sizes of the affected zones in relation to the impact energy. The results are finally discussed in the context of the possible use of structural health monitoring based on strain modifications.

Human iPSC-Derived Neural Models for Studying Alzheimer's Disease: from Neural Stem Cells to Cerebral Organoids.

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer's disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery.

Effect of host switching simulation on the fitness of the gregarious parasitoid Anaphes flavipes from a novel two-generation approach.

Herbivorous insects can escape the strong pressure of parasitoids by switching to feeding on new host plants. Parasitoids can adapt to this change but at the cost of changing their preferences and performance. For gregarious parasitoids, fitness changes are not always observable in the F1 generation but only in the F2 generation. Here, with the model species and gregarious parasitoid Anaphes flavipes, we examined fitness changes in the F1 generation under pressure from the simulation of host switching, and by a new two-generation approach, we determined the impact of these changes on fitness in the F2 generation. We showed that the parasitoid preference for host plants depends on hatched or oviposited learning in relation to the possibility of parasitoid decisions between different host plants. Interestingly, we showed that after simulation of parasitoids following host switching, in the new environment of a fictitious host plant, parasitoids reduced the fictitious host. At the same time, parasitoids also reduced fertility because in fictitious hosts, they are not able to complete larval development. However, from a two-generation approach, the distribution of parasitoid offspring into both native and fictitious hosts caused lower parasitoid clutch size in native hosts and higher individual offspring fertility in the F2 generation.

Investigation of Significant Parameters during Abrasive Waterjet Turning.

This paper presents an investigation of abrasive waterjet turning (AWJT). The purpose of the article was to investigate significant parameters of the turning process and to evaluate their impact on the turning product. The influence of the traverse speed, the rotational speed, and the relative position of the jet to the specimen (lateral jet shift) were investigated. Based on the previous research done in this field, the multi-pass tangential turning method was selected. Rotational speed does not seem to have a significant impact on the AWJ turning process. However, the relative position of the jet is a key parameter for improving the efficiency of the process. Increasing the lateral jet shift causes the volume of the material removed to increase until the optimal impact angle is reached. These findings need to be extended in order to adjust AWJT. Without these improvements, a comparison of jet to traditional technologies is inappropriate.

The Effects of Bilirubin and Lumirubin on the Differentiation of Human Pluripotent Cell-Derived Neural Stem Cells.

The 'gold standard' treatment of severe neonatal jaundice is phototherapy with blue-green light, which produces more polar photo-oxidation products that are easily excreted via the bile or urine. The aim of this study was to compare the effects of bilirubin (BR) and its major photo-oxidation product lumirubin (LR) on the proliferation, differentiation, morphology, and specific gene and protein expressions of self-renewing human pluripotent stem cell-derived neural stem cells (NSC). Neither BR nor LR in biologically relevant concentrations (12.5 and 25 µmol/L) affected cell proliferation or the cell cycle phases of NSC. Although none of these pigments affected terminal differentiation to neurons and astrocytes, when compared to LR, BR exerted a dose-dependent cytotoxicity on self-renewing NSC. In contrast, LR had a substantial effect on the morphology of the NSC, inducing them to form highly polar rosette-like structures associated with the redistribution of specific cellular proteins (ß-catenin/N-cadherin) responsible for membrane polarity. This observation was accompanied by lower expressions of NSC-specific proteins (such as SOX1, NR2F2, or PAX6) together with the upregulation of phospho-ERK. Collectively, the data indicated that both BR and LR affect early human neurodevelopment in vitro, which may have clinical relevance in phototherapy-treated hyperbilirubinemic neonates.