Single-cell Stage Research Articles

Effect of tributyltin exposure on the embryonic development and behavior of a molluscan model species, Lymnaea stagnalis

The presence of the organotin compound tributyltin (TBT) in aquatic ecosystems has been a serious environmental problem for decades. Although a number of studies described the negative impact of TBT on mollusks at different levels, investigations connected to its potential effects during embryogenesis have been neglected. For a better understanding of the impact of TBT on mollusks, in the present study, embryos of previously TBT-treated or not treated specimens of the great pond snail (Lymnaea stagnalis) were exposed to 100 ng L−1 TBT from egg-laying (single-cell stage) until hatching. According to our results, TBT significantly delayed hatching and caused shell malformation. TBT transiently decreased the locomotion (gliding) and also reduced the feeding activity, demonstrating for the first time that this compound can alter the behavioral patterns of molluscan embryos. The heart rate was also significantly reduced, providing further support that cardiac activity is an excellent indicator of metal pollution in molluscan species. At the histochemical level, tin was demonstrated for the first time in TBT-treated hatchlings with intensive reaction in the central nervous system, kidney, and hepatopancreas. Overall, the most notable effects were observed in treated embryos derived from TBT treated snails. Our findings indicate that TBT has detrimental effects on the development and physiological functions of Lymnaea embryos even at a sub-lethal concentration, potentially influencing their survival and fitness. Highlighting our observations, we have demonstrated previously unknown physiological changes (altered heart rate, locomotion, and feeding activity) caused by TBT, as well as visualized tin at the histochemical level in a molluscan species for the first time following TBT exposure. Further studies are in progress to reveal the cellular and molecular mechanisms underlying the physiological and behavioral changes described in the present study.

Novel MIP gene mutation causes autosomal-dominant congenital cataract.

To identify disease-causative mutations in families with congenital cataract. Two Chinese families with autosomal-dominant congenital cataract (ADCC) were recruited and underwent comprehensive eye examinations. Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family. Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis. The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis. Major intrinsic protein (MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos. Zebrafish embryonic lens phenotypes were screened using confocal microscopy. A novel heterozygous mutation (c.85G>A; p.G29R) in the MIP gene was identified in the proband of one family. A known heterozygous mutation (c.97C>T; p.R33C; rs864309693) in MIP was found in the proband of another family. In-silico prediction indicated that the novel mutation might affect the MIP protein function. Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP. Two missense mutations in the MIP gene in Chinese cataract families are identified, and one of which is novel. These findings expand the genetic spectrum of MIP mutations associated with cataracts. The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

First report of a morulated Ascaridoidea (Nematoda) egg in an avian coprolite from the Paleogene of the Paraíba Valley, State of São Paulo, Brazil.

Ascaridoidea (Nematoda) is a widespread superfamily of nematodes that comprises gastrointestinal parasites from all major groups of vertebrates. Although this taxon probably emerged in the Carboniferous, its Brazilian fossil record includes mostly eggs, found in ancient remains, collected in paleontological and archeological sites from the Mesozoic and Cenozoic Eras. The Tremembé Formation (Oligocene of the Taubaté Basin) has become an important source for paleoparasitological studies in avian coprolites during the third decade of the 21st century, with reports of eggs only at only a single cell stage, of embryonic development. Here we present the first egg of Ascaridoidea preserved containing morula, from a bird coprolite recovered from the shales of the Tremembé Formation. Three coprolites, from the outcrop of Aligra Comércio de Argila S/A, Taubaté municipality (State of São Paulo), were rehydrated and subjected to spontaneous sedimentation. Based on morphological and morphometric features and diet and zoopaleontological context, the trace fossils were assigned to piscivorous birds. The egg found showed morphological characteristics typical of Ascaridoidea: namely spherical form, ornamented, and somewhat thick shell. Moreover, this superfamily includes several taxa that infect piscivorous birds and fish in heteroxenous life cycles and produce eggs with similar features as the egg found in the present study. The paleoparasitological information associated with the paleofaunistic diversity of birds and fish from the Tremembé Formation, reveal that the ancient Brazilian paleoenvironments provided subsidies for the rise and success of nematodes infecting these animals during the Paleogene.

Immunomodulatory Effect of Arsenicum album on Zebrafish Embryos.

Background: Globally, 2.4 million neonates succumbed in 2019, constituting 47% of all under-5 mortalities[1]. India bore the highest neonatal mortality in the same year, attributed predominantly to infections[2]. Innate immunity, bestowed at birth, represents our primary defense against neonatal mortality. Exposure to crude forms of arsenic in zebrafish embryos has been shown to significantly down-regulate the innate immune responses[3-8]. Such studies reveal the realm of operation, susceptible cells, and mechanisms through which arsenic can engage with our system, aiding in the understanding of the Homoeopathic medicine Arsenicum album.Methods: Zebrafish were acclimatised for 7 days, under 28.5 ± 2 °C under a 12-hour light/dark cycle, then bred to collect embryos at the single-cell stage. Embryos were categorised into 8 triplicate groups: unexposed uninfected control, unexposed infected control, alcohol-exposed control, Arsenicum album exposed at 5 potencies (6x, 30c, 200c, 1M, LM/1). Embryos were exposed to medications in egg water medium for four days. From four days post-fertilization (dpf), they were transferred to four-litre tanks. On the seventh day post-exposure (dpe), they were infected with Edwardsiella tarda at 1 × 108 colony forming units (CFU)/ml for five hours. Fishes were collected at 5-hours and 30-hours post-infection (hpi) for assessing innate immunological responses. Survival rates, respiratory bursts and bacterial loads were statistically analysed.Results: The Arsenicum album LM/1 group had the highest survival rate, followed by the 1M group. Highest respiratory burst at 5 hpi and lowest bacterial load at 5 and 30 hpi were in the 1M group. LM/1 had highest respiratory burst at 30 hpi. The 6x group had the lowest survival rate, below the negative control. These findings highlight Arsenicum album's impact on innate immunity in zebrafish embryos.Conclusion: This study emphasizes Arsenicum album's innate immunomodulatory effect across potencies. Subsequent focused research is imperative to acquire a more nuanced comprehension.

Precision Lineage Deconvolution in Mixed Phenotype Acute Leukemia Using Cite-Seq Derived Hematopoietic Stages Identifies Lineage Dynamics Associated with Treatment Response

Introduction: Mixed phenotype Acute Leukemia (MPAL) is challenging due to ambiguous biology and lack of formal therapeutic guidelines. Although MPAL itself is rare, mixed phenotype lineage expression in secondary AML (sAML-MP) occurs with greater frequency and is associated with poor clinical outcomes. Multimodal single cell sequencing with cellular indexing of transcriptomes and epitopes (CITE-seq) offers more precise lineage characterization and assessment of lineage dynamics. Here we demonstrate a novel, unbiased approach to quantitative multistage hematopoietic lineage assessment in MPAL and sAML-MP using CITE-seq data. In addition, we demonstrate that this approach can be translated from single cell to bulk analysis. Methods: Sample preparation: Four MPAL and 6 sAML-MP samples displaying T-cell and myeloid subsets were identified and flow sorted into T and myeloid subgroups. RNA-seq was performed on each subset and analyzed using DESeq2. ComBat was used for batch correction when integrating with disparate data sources. Single cell lineage deconvolution: CITE-seq samples were processed using Seurat. A single cell deconvolution library was constructed using previously published hematopoietic stage clustering and labeling, and applied to CITE-seq data from five MPAL samples in the same dataset. Deconvolution algorithm parameters were optimized through a bootstrapping approach on in-silico sample mixtures. Bulk and translated lineage deconvolution: Bulk lineage deconvolution was performed using a published library of 13 stages of hematopoiesis derived from healthy donors.This was applied to bulk RNA-seq data from a cohort of published MPAL samples as well as the 6 sAML-MP and 4 MPAL samples described above. A pseudobulk was created for each CITE-seq lineage stage. Bulk lineage deconvolution was then re-run on the MPAL and sAML-MP samples using this library. Clinical Outcomes: A Wilcoxon signed-rank test was used to analyze differences among deconvolution-derived patient clusters, including response to induction therapy, specific gene mutations, and likelihood of transplant. Results: Lineage evolution with treatment: Deconvolution was applied on 17,848 MPAL cells and 35,038 control PBMC/BMMC cells. Antibody Derived Tag (ADT) features were consistent with lineage deconvolution stage assignments. CD38 was negatively correlated with HSC (-0.34, p < 0.001) and LMPP (-0.18, p < 0.001) while CD34 was positively correlated with both HSC (0.11, p < 0.001) and LMPP (0.27, p < 0.001) stages. MPAL 5 in particular was assessed at diagnosis and at two later time points. Unsupervised hierarchical clustering of all timepoints resulted in 10 clusters ( Figure A). The diagnostic time point was largely contained in clusters 4 (LMPP character, N = 1545 cells, 37.13%) and 9 (HSC character, N = 756 cells, 18.16%). The majority of both relapse timepoints were within cluster 4, with 76.77% (N=357 cells) of T1 and 73.92% (N=1100 cells) of T2 located in it. Bulk Unsupervised Analysis: Unsupervised analysis of sorted MPAL (N = 8), sorted AML-MP (N = 12), and unsorted MPAL (N = 24) bulk samples resulted in six distinct clusters, each with a distinct lineage signature ( Figure B). Some were enriched for more differentiated stages such as monocyte or GMP (clusters 6, 5), while others were enriched earlier stages such as LMPP (cluster 2). Secondary AML-MP samples were notably enriched in cluster 4. We found significant associations between lineage clusters and mutations in RUNX1 (p < 0.01), FLT3 (p = 0.004). Clinical Outcomes: Bulk RNA deconvolution cluster was significantly associated with complete remission (p = 0.0009). On average, individuals with an incomplete/no response to induction chemotherapy had a higher NK signature (p = 0.001), while individuals assigned to transplant had decreased LMPP character (p = 0.0495). Among the single-cell stages, decreased CLP 2 levels were associated with a poor response (p = 0.0224) while CMP / LMPP (p = 0.040) and CD4 N2 (p = 0.022) levels were decreased in patients who were able to undergo HSCT. Conclusions: Precise identification of lineage signatures in mixed phenotype leukemias shows promise in identifying clinically meaningful biological subsets of these diseases. Prospective analysis of lineage-derived biomarkers should be performed to undertake identification of formal risk stratification and treatment schemas.

Parallel evolution of the G protein-coupled receptor GrlG and the loss of fruiting body formation in the social amoeba Dictyostelium discoideum evolved under low relatedness.

Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines, we identified 38 single nucleotide polymorphisms in 29 genes. Each gene had 1 variant except for grlG (encoding a G protein-coupled receptor), which had 10 unique SNPs and 5 structural variants. Variants in the 5' half of grlG-the region encoding the signal peptide and the extracellular binding domain-were significantly associated with the loss of fruiting body formation; the association was not significant in the 3' half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5' half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage but rather by something-likely cheating-during the multicellular stage.

A model for the dissemination of circulating tumour cell clusters involving platelet recruitment and a plastic switch between cooperative and individual behaviours

BackgroundIn spite of extensive research, cancer remains a major health problem worldwide. As cancer progresses, cells acquire traits that allow them to disperse and disseminate to distant locations in the body – a process known as metastasis. While in the vasculature, these cells are referred to as circulating tumour cells (CTCs) and can manifest either as single cells or clusters of cells (i.e., CTC clusters), with the latter being the most aggressive. The increased metastatic potential of CTC clusters is generally associated with cooperative group benefits in terms of survival, including increased resistance to shear stress, anoikis, immune attacks and drugs. However, the adoption of a group phenotype poses a challenge when exiting the vasculature (extravasation) as the large size can hinder the passage through vessel walls. Despite their significant role in the metastatic process, the mechanisms through which CTC clusters extravasate remain largely unknown. Based on the observed in vivo association between CTC clusters and platelets, we hypothesized that cancer cells take advantage of the platelet-derived Transforming Growth Factor Beta 1 (TGF-β1) – a signalling factor that has been widely implicated in many aspects of cancer, to facilitate their own dissemination. To address this possibility, we evaluated the effect of exogenous TGF-β1 on an experimentally evolved non-small cell lung cancer cell line that we previously developed and used to investigate the biology of CTC clusters.ResultsWe found that exogenous TGF-β1 induced the dissociation of clusters in suspension into adherent single cells. Once adhered, cells released their own TGF-β1 and were able to individually migrate and invade in the absence of exogenous TGF-β1. Based on these findings we developed a model that involves a TGF-β1-mediated plastic switch between a cooperative phenotype and a single-celled stage that enables the extravasation of CTC clusters.ConclusionsThis model allows for the possibility that therapies can be developed against TGF-β1 signalling components and/or TGF-β1 target genes to suppress the metastatic potential of CTC clusters. Considering the negative impact that metastasis has on cancer prognosis and the lack of therapies against this process, interfering with the ability of CTC clusters to switch between cooperative and individual behaviours could provide new strategies to improve patient survival.

The developmental and structural uniqueness of the embryo of the extremophile viviparous nematode, Tokorhabditis tufae.

Viviparity, a reproductive form that supplies nutrients to the embryo during gestation, has repeatedly and independently occurred in multiple lineages of animals. During the convergent evolution of viviparity, various modifications of development, structure, and physiology emerged. A new species of nematode, Tokorhabditis tufae, was discovered in the alkaline, hypersaline, and arsenic-rich environment of Mono lake. Its reproductive form is viviparity because it is obligately live-bearing and the embryo increases in size during development. However, the magnitude of the increase in size and nutrient provisioning are unclear. We measured egg and embryo sizes at three developmental stages in T. tufae. Eggs and embryos of T. tufae at the threefold stage were respectively 2.6- and 3.6-fold larger than at the single-cell stage. We then obtained T. tufae embryos at the single-cell, lima bean, and threefold developmental stages and investigated the egg hatching frequency at three different concentrations of egg salt buffer. Removal of embryos from the uterus halted embryonic development at the single-cell and lima bean stages in T. tufae irrespective of the solution used for incubation, indicating the provision of nutrients within the uterus. Ultrastructural and permeability evaluation showed that the permeability barrier did not form during embryonic development, resulting in increased molecular permeability. This high permeability caused by the absence of the permeability barrier likely enables supply of nutrients from the mother. The structural and physiological modifications in T. tufae are like those in other viviparous animals. We conclude that T. tufae is a viviparous rather than an ovoviviparous nematode. T. tufae will facilitate investigation of the evolution of viviparity in animals.

Does symmetry preclude the evolution of senescence? A comment on Pen and Flatt 2021

Does symmetry preclude the evolution of senescence? A comment on Pen and Flatt 2021

Epiblast-like stem cells established by Wnt/β-catenin signaling manifest distinct features of formative pluripotency and germline competence

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility. We develop single-cell stage label transfer (scSTALT) to study the formative pluripotency continuum and reveal that EpiLSCs recapitulate a unique developmental period invivo, filling the gap of the formative pluripotency continuum between other published formative stem cells. WNT/β-catenin signaling activation counteracts differentiation effects of activin A and bFGF by preventing complete dissolution of naive pluripotency regulatory network. Moreover, EpiLSCs have direct competence toward germline specification, which is further matured by an FGF receptor inhibitor. Our EpiLSCs can serve as an invitro model for mimicking and studying early post-implantation development and pluripotency transition.

Autofluorescence prediction model for fluorescence unmixing and age determination.

Flow cytometry is a powerful tool for identifying and quantifying various cell markers, such as viability, vitality, and individual cell age, at single-cell stages. However, cell autofluorescence and marker fluorophore signals overlap at low fluorescence intensities. Thus, these signals must be unmixed before determining the age fraction. A comparison was made between principal component regression (PCR) and random forest (RF) to predict autofluorescence signals of Saccharomyces pastorianus var. carlsbergensis in a flow cytometer. RF provided better prediction results than the PCR and was therefore determined to be better suited for unmixing signals. In the subsequent application for unmixing the autofluorescence signal from the marker fluorophore signal, the Gaussian mixture analysis based on RF was in better agreement with the microscopy-determined replicative age distribution than the PCR-based method. The proposed approach of single-laser spectral unmixing and subsequent Gaussian mixture analysis showed that the microscopy data was consistent with the unmixed fluorescence spectra. The demonstrated approach enables fast and reliable unmixing of flow cytometric spectral data using a single-laser spectral unmixing method. This analysis method enables age determination of cells in industrial processes. This age determination allows for quantifying the yeast cell's age fractions, providing a detailed view of age-related changes. Additionally, the bud scar labeling technique can be used to determine age-related changes in Pichia pastoris yeast for biotechnological applications or recombinant protein expression.

96. Activating MAP2K1 Mutation in Zebrafish Endothelial Cells Causes Arteriovenous Shunts

Purpose: Arteriovenous malformation (AVM) is a sporadic vascular malformation defined by a nidus of irregular blood vessels connecting arteries to veins instead of a normal capillary bed. Somatic activating mutations in MAP2K1 cause extracranial AVM. The purpose of this study was to create an AVM animal model using zebrafish. Methods: Single-cell stage casper;Tg(gata1:dsred) zebrafish embryos were injected with 1 nL of transgenesis mixture: 100 ng/μL of either (1) Control (pTol2-Fli:GFP) or (2) Mutant (pTol2-Fli:GFP-kdrl:MAP2K1K57N) plasmid DNA + 40 ng/μL Tol2 transposase mRNA. Erythrocytes in these fish express the fluorescent protein dsRed. Embryos were anesthetized with 0.4 mg/mL Tricaine, embedded in 1% low-melt agarose and then imaged using a Zeiss LSM 800 Confocal Microscope or a Nikon SMZ18 Fluorescence Microscope. Confocal images were obtained using Zen Blue version 2.5. Embryos were imaged 72 hours post-fertilization. Blood flow was visualized using dsRed fluorescence. Results: Injection of MAP2K1K57N plasmid resulted in abnormal arteriovenous shunts (58/96 transgenic embryos, 60%). The phenotype consisted of either (1) a proximal shunt with blood flowing through a direct connection between proximal aortic vessels to the common cardinal vein and immediately back into the heart (39/58 embryos, 67%) or (2) a mid-trunk shunt between the dorsal artery and posterior cardinal vein (19/58 embryos, 33%). Shunts were not present in control zebrafish (n=65). Endothelial cells at the site of the shunt expressed high levels of the marker transgene confirming shunts contained mutant endothelial cells. Conclusion: Zebrafish endothelial cells expressing mutant MAP2K1 form abnormal arteriovenous shunts. The phenotype recapitulates extracranial human AVM. Mutant MAP2K1 zebrafish are a promising animal model for testing pharmacotherapy to treat AVM.

Insect egg morphology: evolution, development, and ecology.

The insect egg can be viewed through many lenses: it is the single-celled developmental stage, a resource investment in the next generation, an unusually large and complex cell type, and the protective vessel for embryonic development. In this review, I describe the morphological diversity of insect eggs and then identify recent advances in understanding the patterns of egg evolution, the cellular mechanisms underlying egg development, and notable aspects of egg ecology. I also suggest areas for particularly promising future research on insect egg morphology; these topics touch upon diverse areas such as tissue morphogenesis, life history evolution, organismal scaling, cellular secretion, and oviposition ecology.

Dynamic surface patterns on cells.

Dynamic pattern formations are commonly observed in multicellular systems, such as cardiac tissue and slime molds, and modeled using reaction-diffusion systems. Recent experiments have revealed dynamic patterns in the concentration profile of various cortical proteins at a much smaller scale, namely, embryos at their single-cell stage. Spiral waves of Rho and F-actin proteins have been reported in Xenopus frog and starfish oocytes [Bement et al., Nat. Cell Biol. 17, 1471 (2015)], while a pulsatile pattern of Rho and myosin proteins has been found in C. elegans embryo [Nishikawa et al., eLife 6, e30537 (2017)]. Here, we propose that these two seemingly distinct dynamic patterns are signatures of a single reaction-diffusion network involving active-Rho, inactive-Rho, actin, and myosin. We show that a small variation in the concentration of other ancillary proteins can give rise to different dynamical states from the same chemical network.

Random Integration Transgenesis in a Free-Living Regenerative Flatworm Macrostomum lignano.

Regeneration-capable flatworms are highly informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. Transgenesis is a powerful research tool for investigating gene function, but until recently, a transgenesis method was missing in flatworms, hampering their wider adoption in biomedical research. Here we describe a detailed protocol to create stable transgenic lines of the flatworm M. lignano using random integration of DNA constructs through microinjection into single-cell stage embryos.

AutoGGN: A gene graph network AutoML tool for multi-omics research

Omics data can be used to identify biological characteristics from genetic to phenotypic levels during the life span of a living being, while molecular interaction networks have a fundamental impact on life activities. Integrating omics data and molecular interaction networks will help researchers delve into comprehensive information hidden in the data. Here, we propose a new multimodal method — AutoGGN — to integrate multi-omics data with molecular interaction networks based on graph convolutional neural networks (GCNs). We evaluated AutoGGN using three classification tasks: single-cell embryonic developmental stage classification, pan-cancer type classification, and breast cancer subtyping. On all three tasks, AutoGGN showed better performance than other methods. This means AutoGGN has the potential to extract insights more effectively by means of integrating molecular interaction networks with multi-omics data. Additionally, in order to provide a better understanding of how our model makes predictions, we utilized the SHAP module and identified the key genes contributing to the classification, providing insight for the design of downstream biological experiments.

Exogenous Expression of pou5f3 Facilitates the Development of Somatic Cell Nuclear Transfer Embryos in Zebrafish at the Early Stage.

Enucleated oocytes can reprogram differentiated nuclei to totipotency after somatic cell nuclear transfer (SCNT), which is valuable in understanding nuclear reprogramming and generating genetically modified animals. To date, reprogramming efficiency is low and the development of SCNT embryos is not going as well as anticipated. To further disclose the reprogramming mechanisms during SCNT zebrafish embryo development, we examined the expression patterns of transcription regulation factors and regulated them by mRNA and morpholino microinjection. In this study, we show that stem cell-related transcription factors are downregulated in zebrafish SCNT embryos at the blastula stage. Exogenous expression of pou5f3 at the single-cell stage improves SCNT embryo development from the blastula to the gastrula stage. We also found that exogenous expression of klf4 or sox2 decreases SCNT embryo development from the blastula to the gastrula stage, while expression of nanog is necessary for the development of SCNT embryos. Our results conclude that zebrafish pou5f3 facilitates the development of SCNT embryos from the blastula to gastrula stage.

A liquid culture cancer spheroid model reveals low PI3K/Akt pathway activity and low adhesiveness to the extracellular matrix.

Three-dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer invitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage-independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical-basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK-2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single-cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb-like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel-cultured spheroids and 2D-cultured cells, explaining the susceptibility to the Akt-inhibiting anticancer drug. Subcutaneous xenografting and invitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel-cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage-independent spheroids and validates liquid cultures as a useful method in cancer spheroid research.

Son is necessary for proper vertebrate blood development.

The gene SON is on human chromosome 21 (21q22.11) and is thought to be associated with hematopoietic disorders that accompany Down syndrome. Additionally, SON is an RNA splicing factor that plays a role in the transcription of leukemia-associated genes. Previously, we showed that mutations in SON cause malformations in human and zebrafish spines and brains during early embryonic development. To examine the role of SON in normal hematopoiesis, we reduced expression of the zebrafish homolog of SON in zebrafish at the single-cell developmental stage with specific morpholinos. In addition to the brain and spinal malformations we also observed abnormal blood cell levels upon son knockdown. We then investigated how blood production was altered when levels of son were reduced. Decreased levels of son resulted in lower amounts of red blood cells when visualized with lcr:GFP transgenic fish. There were also reduced thrombocytes seen with cd41:GFP fish, and myeloid cells when mpx:GFP fish were examined. We also observed a significant decrease in the quantity of T cells, visualized with lck:GFP fish. However, when we examined their hematopoietic stem and progenitor cells (HSPCs), we saw no difference in colony-forming capability. These studies indicate that son is essential for the proper differentiation of the innate and adaptive immune system, and further investigation determining the molecular pathways involved during blood development should elucidate important information about vertebrate HSPC generation, proliferation, and differentiation.

Electrophysiological evaluation of human induced pluripotent stem cell-derived cardiomyocytes obtained by different methods.

The human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (CMs) (hiPSC-CMs) retain the same genetic information as the donor, and they have been shown to faithfully recapitulate the disease phenotypes of various genetic cardiac diseases. The hiPSC-CMs can be utilized in multiple types of studies and in most cases, the functionality of hiPSC-CMs is of interest. For the functional analyses, the hiPSC-CMs need to be manipulated after differentiation, e.g. enriched or dissociated into single-cell stage. For the functional assessments to be reliable and reproducible, the cell culture environment should support the cells in an optimal manner. The aim of the present study was to evaluate the effect of various differentiation methods, as well as coating materials used for the dissociated cells on the functionality of the differentiated hiPSC-CMs. The different protocols not only had different differentiation efficiencies, but they also yielded functionally different hiPSC-CMs. Additionally, the coating material had a major effect on the functionality of the hiPSC-CMs. The results of the present study emphasize that the cardiac differentiation method and the coating material have a major effect on hiPS-CMs' characteristics. Thus, when different hiPSC lines and results obtained in different labs are compared, extra care should be taken to check the conditions when results are compared.