Cell Cycle Kinetics and Sister Chromatid Exchange in Mosaic Turner Syndrome.

Turner syndrome (TS) is caused by a complete or partial absence of an X or Y chromosome, including chromosomal mosaicism, affecting 1 in 2500 female live births. Sister chromatid exchange (SCE) is used as a sensitive indicator of spontaneous chromosome instability. Cells from mosaic patients constitute useful material for SCE evaluations as they grow under the influence of the same genetic background and endogenous and exogenous factors. We evaluated the proliferation dynamics and SCE frequencies of 45,X and 46,XN cells of 17 mosaic TS patients. In two participants, the 45,X cells exhibited a proliferative disadvantage in relation to 46,XN cells after 72 h of cultivation. The analysis of the mean proliferation index (PI) showed a trend for a significant difference between the 45,X and 46,X+der(X)/der(Y) cell lineages; however, there were no intra-individual differences. On the other hand, mean SCE frequencies showed that 46,X+der(X) had the highest mean value and 46,XX the lowest, with 45,X occupying an intermediate position among the lineages found in at least three participants; moreover, there were intra-individual differences in five patients. Although 46,X+der(X)/der(Y) cell lineages, found in more than 70% of participants, were the most unstable, they had a slightly higher mean PI than the 45,X cell lineages in younger (≤17 years) mosaic TS participants. This suggests that cells with a karyotype distinct from 45,X may increase with time in mosaic TS children and adolescents.

Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.

Somatic epigenetic drift during shoot branching: a cell lineage-based model.

Plant architecture is shaped by the production of new organs, most of which emerge postembryonically. This process includes the formation of new lateral branches along existing shoots. Current evidence supports a detached-meristem model as the cellular basis of lateral shoot initiation. In this model, a small number of undifferentiated cells are sampled from the periphery of the shoot apical meristem (SAM) to act as precursors for axillary buds, which eventually develop into new shoots. Repeated branching thus creates cellular bottlenecks (i.e. somatic drift) that affect how de novo (epi)genetic mutations propagate through the plant body during development. Somatic drift could be particularly relevant for stochastic DNA methylation gains and losses (i.e. spontaneous epimutations), as they have been shown to arise rapidly with each cell division. Here, we formalize a special case of the detached-meristem model, where precursor cells are randomly sampled from the SAM periphery in a way that maximizes cell lineage independence. We show that somatic drift during repeated branching gives rise to a mixture of cellular phylogenies within the SAM over time. This process is dependent on the number of branch points, the strength of drift as well as the epimutation rate. Our model predicts that cell-to-cell DNA methylation heterogeneity in the SAM converges to nonzero states during development, suggesting that epigenetic variation is an inherent property of the SAM cell population. Our insights have direct implications for empirical studies of somatic (epi)genomic diversity in long-lived perennial and clonal species using bulk or single-cell sequencing approaches.

Classification and counting of cells in brightfield microscopy images: an application of convolutional neural networks.

Microscopy is integral to medical research, facilitating the exploration of various biological questions, notably cell quantification. However, this process's time-consuming and error-prone nature, attributed to human intervention or automated methods usually applied to fluorescent images, presents challenges. In response, machine learning algorithms have been integrated into microscopy, automating tasks and constructing predictive models from vast datasets. These models adeptly learn representations for object detection, image segmentation, and target classification. An advantageous strategy involves utilizing unstained images, preserving cell integrity and enabling morphology-based classification-something hindered when fluorescent markers are used. The aim is to introduce a model proficient in classifying distinct cell lineages in digital contrast microscopy images. Additionally, the goal is to create a predictive model identifying lineage and determining optimal quantification of cell numbers. Employing a CNN machine learning algorithm, a classification model predicting cellular lineage achieved a remarkable accuracy of 93%, with ROC curve results nearing 1.0, showcasing robust performance. However, some lineages, namely SH-SY5Y (78%), HUH7_mayv (85%), and A549 (88%), exhibited slightly lower accuracies. These outcomes not only underscore the model's quality but also emphasize CNNs' potential in addressing the inherent complexities of microscopic images.

Wiskott-Aldrich syndrome protein expression in female WAS carriers: A flow cytometry study from North India.

INTRODUCTION: Wiskott-Aldrich syndrome (WAS) is a rare X-linked inborn error of immunity characterized by microthrombocytopenia, infections, eczema, and increased predisposition to develop autoimmunity and malignancy. Flow cytometric assay for determining WAS protein (WASp) is a rapid and cost-effective tool for detecting patients. However, very few studies described WASp expression in female carriers. Most WAS carriers are clinically asymptomatic. Active screening of female family members helps identify female carriers, distinguish de novo mutations, and to select appropriate donor prior to curative stem cell transplantation. This study was undertaken to evaluate the diagnostic capability of flow cytometry-based WASp expression in peripheral blood cells to identify carriers and compare WASp expression in different blood cell lineages. PATIENTS AND METHODS: Female patients, heterozygous for WAS gene, were enrolled in this study conducted at Pediatric Allergy Immunology Unit, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Flow cytometric assessment of WASp expression in lymphocytes, monocytes, and neutrophils was carried out and compared with healthy control and affected patients. The results were expressed in delta (Δ) median fluorescence intensity (MFI) as well as stain index (SI), which is the ratio of ΔMFI of patient and ΔMFI of control. RESULTS: Thirteen mothers and two sisters of genetically confirmed WAS patients were enrolled in the study. All enrolled females were clinically asymptomatic and did not have microthrombocytopenia. Low WASp expression (SI < 1) was seen in lymphocytes and monocytes in 10 (66.6%) carriers. Females with variants in proximal exons (exons 1 and 2) were found to have lesser expression than those with distal (exons 3-12) variants. CONCLUSION: Flow cytometry is a rapid, easily available, cost-effective tool for WASp estimation. Lymphocytes followed by monocytes are the best cell lineages for WASp estimation in carrier females. However, genetic testing remains the gold standard, as carrier females with variants in distal exons may have normal WASp expression.

Epigenetically distinct synaptic architecture in clonal compartments in the teleostean dorsal pallium.

The dorsal telencephalon (i.e. the pallium) exhibits high anatomical diversity across vertebrate classes. The non-mammalian dorsal pallium accommodates various compartmentalized structures among species. The developmental, functional, and evolutional diversity of the dorsal pallium remain unillustrated. Here, we analyzed the structure and epigenetic landscapes of cell lineages in the telencephalon of medaka fish (Oryzias latipes) that possesses a clearly delineated dorsal pallium (Dd2). We found that pallial anatomical regions, including Dd2, are formed by mutually exclusive clonal units, and that each pallium compartment exhibits a distinct epigenetic landscape. In particular, Dd2 possesses a unique open chromatin pattern that preferentially targets synaptic genes. Indeed, Dd2 shows a high density of synapses. Finally, we identified several transcription factors as candidate regulators. Taken together, we suggest that cell lineages are the basic components for the functional regionalization in the pallial anatomical compartments and that their changes have been the driving force for evolutionary diversity.

The plant stem-cell niche and pluripotency: 15 years of an epigenetic perspective.

Pluripotent stem-cells are slowly dividing cells giving rise to daughter cells that can either differentiate to new tissues and organs, or remain stem-cells. In plants, stem-cells are located in specific niches of the shoot and root apical meristems (SAMs and RAMs). After ablation of stem-cell niches, pluripotent meristematic cells can establish new stem-cells, whereas the removal of the whole meristem destructs the regeneration process. In tissue cultures, after detached plant organs are transferred to rooting or callus induction medium (G5 or CIM), vasculature-associated pluripotent cells (VPCs) immediately start proliferation to form adventitious roots or callus, respectively, while other cell types of the organ explants basically play no part in the process. Hence, in contrast to the widely-held assumption that all plant cells have the ability to reproduce a complete organism, only few cell types are pluripotent in practice, raising the question how pluripotent stem-cells differ from differentiated cells. It is now clear that, in addition to gene regulatory networks of pluripotency factors and phytohormone signaling, epigenetics play a crucial role in initiation, maintenance and determination of plant stem-cells. Although, more and more epigenetic regulators have been shown to control plant stem-cell fate, only a few studies demonstrate how they are recruited and how they change the chromatin structure and transcriptional regulation of pluripotency factors. Here, we highlight recent breakthroughs but also revisited classical studies of epigenetic regulation and chromatin dynamics of plant stem-cells and their pluripotent precursor-cells, and point out open questions and future directions.

Overgrowth of Squamocolumnar Junction and Dysregulation of Stem Cell Lineages in the Stomach of Vitamin A-Deficient Mice.

Junctional epithelia are common sites for pathological transformations. In mice, the stratified epithelium of the forestomach joins the simple glandular epithelium of the cardia at the limiting ridge. We previously demonstrated the expression of vitamin A receptors in the gastric stem/progenitor cells and their progeny and found that excess retinoic acid enhances cellular dynamics of gastric epithelium. This study examines how deficiency of vitamin A would alter gastric epithelial stem cell lineages. Three-week-old mice of both genders were weaned and fed with a vitamin A deficient (VAD) diet for 4 or 8 months. Sex- and weight-matched littermate mice received a standard (control) diet. To label S-phase cells, all mice received a single intraperitoneal injection of 5-bromo-2-deoxyuridine before being euthanized. Stomach tissues were processed for microscopic examination and protein analysis to investigate stem cell lineages using different stains, lectins, or antibodies. The Student's t-test was used to compare quantified data showing differences between control and VAD groups. Eight-month-vitamin-A deficiency caused enlarged forestomach and overgrowth of the squamocolumnar junction with metaplastic and dysplastic cardiac glands, formation of intramucosal cysts, loss of surface mucosal integrity, increased amount of luminal surface mucus, and upregulation of trefoil factor 1 and H+,K+-ATPase. These changes were associated with decreased cell proliferation and upregulation of p63. In conclusion, vitamin A is necessary for maintaining gastric epithelial integrity and its deficiency predisposes the mouse stomach to precancerous lesions.

Early development of the breathing network.

Breathing (or respiration) is a complex motor behavior that originates in the brainstem. In minimalistic terms, breathing can be divided into two phases: inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). The neurons that discharge in synchrony with these phases are arranged in three major groups along the brainstem: (i) pontine, (ii) dorsal medullary, and (iii) ventral medullary. These groups are formed by diverse neuron types that coalesce into heterogeneous nuclei or complexes, among which the preBötzinger complex in the ventral medullary group contains cells that generate the respiratory rhythm (Chapter 1). The respiratory rhythm is not rigid, but instead highly adaptable to the physic demands of the organism. In order to generate the appropriate respiratory rhythm, the preBötzinger complex receives direct and indirect chemosensory information from other brainstem respiratory nuclei (Chapter 2) and peripheral organs (Chapter 3). Even though breathing is a hard-wired unconscious behavior, it can be temporarily altered at will by other higher-order brain structures (Chapter 6), and by emotional states (Chapter 7). In this chapter, we focus on the development of brainstem respiratory groups and highlight the cell lineages that contribute to central and peripheral chemoreflexes.

Prognostic Significance of Lineage Diversity in Bladder Cancer Revealed by Single-Cell Sequencing.

Bladder cancer is the most common malignant tumor of the urinary system. We investigated the clinical implications of cell lineages in bladder cancer by integrating single-cell and bulk transcriptome data. By investigating the single-cell transcriptional profiles of 12,424 cells from normal bladder, eleven cell types and five types of epithelial sub-population were identified. Based on the signature of cell types identified in single-cell profiles, deconvolution analysis was employed to estimate cell types and epithelial lineages in the bulk RNA sequencing bladder cancer cohort. Cancer subtypes with clinical implications were further identified based on the heterogeneity of the epithelial lineage across patients. This study suggests that the EMT-like subtype is robustly correlated with poor prognosis and the umbrella subtype is a positive factor for the patient survival. Our research has a high potential for accurate prognostic and therapeutic stratification of bladder cancer.

Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays.

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.

Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow.

Recent developments in clinical flow cytometry allow the simultaneous assessment of proliferative and anti-apoptotic activity in the different hematopoietic cell lineages and during their maturation process. This can further advance the flow cytometric diagnosis of myeloid malignancies. In this study we established indicative reference values for the Ki-67 proliferation index and Bcl-2 anti-apoptotic index in blast cells, as well as maturing erythroid, myeloid, and monocytic cells from normal bone marrow (BM). Furthermore, the cell fractions co-expressing both proliferation and anti-apoptotic markers were quantified. Fifty BM aspirates from femoral heads of patients undergoing hip replacement were included in this study. Ten-color/twelve-parameter flow cytometry in combination with a software-based maturation tool was used for immunophenotypic analysis of Ki-67 and Bcl-2 positive fractions during the erythro-, myelo-, and monopoiesis. Indicative reference values for the Ki-67 and Bcl-2 positive fractions were established for different relevant hematopoietic cell populations in healthy BM. Ki-67 and Bcl-2 were equally expressed in the total CD34 positive blast cell compartment and 30% of Ki-67 positive blast cells also showed Bcl-2 positivity. The Ki-67 and Bcl-2 positive fractions were highest in the more immature erythroid, myeloid and monocytic cells. Both fractions then gradually declined during the subsequent maturation phases of these cell lineages. We present a novel application of an earlier developed assay that allows the simultaneous determination of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices in maturing hematopoietic cell populations of the BM. Their differential expression levels during the maturation process were in accordance with the demand and lifespan of these cell populations. The indicative reference values established in this study can act as a baseline for further cell biological and biomedical studies involving hematological malignancies.

Corrigendum: Revisiting Stem Cell-Based Clinical Trials for Ischemic Stroke.

[This corrects the article DOI: 10.3389/fnagi.2020.575990.].

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Biochemical coordination of plasma cell genesis.

Antibody-secreting plasma cells are a central component of short- and long-term adaptive immunity. Yet, many fundamental questions about how activated B cells decide to yield functional plasma cells have yet to be answered. Likewise, the biochemical processes underpinning the ability of plasma cells to generate and secrete large numbers of antibodies, the capacity of some plasma cell to sustain antibody secretion, presumably without interruption, for decades, and the capacity of long-lived plasma cells to avoid apoptosis despite the high-energy demands associated with sustained robust antibody synthesis and secretion each remain mysterious processes. Our objective here is to review what is currently known about these processes with an emphasis on the earliest phases of plasma cell genesis. Along the way, we will work toward developing a model that ties the biochemistry of plasma cell function and survival. The chief idea imbedded in this model is that progress toward understanding plasma cell survival mechanisms may require increased focus on the unique cell autonomous processes inherent in plasma cell differentiation and function.

Ontogeny and heterogeneity of innate lymphoid cells and the noncoding genome.

Since their discovery a decade ago, it has become evident that innate lymphoid cells (ILCs) play critical roles in protective immune responses against intracellular and extracellular pathogens but are also central regulators of epithelial barrier integrity and tissue homeostasis. ILCs populate almost every tissue in mammalian organisms; therefore, not surprisingly, dysregulation of their functions contributes to the development and progression of multiple inflammatory and metabolic diseases. Our knowledge of the transcriptional programs governing the development, differentiation, and functions of the different groups of ILCs has increased dramatically in the last ten years. However, with the advent of new technologies, an unprecedented level of heterogeneity, plasticity, and developmental complexity has started to be revealed. In this review, we highlight recent advances in our understanding of ILC development and their biological functions. In particular, we aim to emphasize how our increasing knowledge of the chromatin landscape and the noncoding genome of these innate lymphocytes is allowing us to better understand their development and functions in different contexts during homeostasis and inflammation. Moreover, we propose that the design of more refined genetic tools to study tissue-specific ILCs and their functions can be accomplished by leveraging our understanding of how specific noncoding elements of the genome regulate gene expression in ILCs.

Not too fat to fight: The emerging role of macrophage fatty acid metabolism in immunity to Mycobacterium tuberculosis.

While the existence of a special relationship between Mycobacterium tuberculosis (Mtb) and host lipids has long been known, it remains a challenging enigma. It was clearly established that Mtb requires host fatty acids (FAs) and cholesterol to produce energy, build its distinctive lipid-rich cell wall, and produce lipid virulence factors. It was also observed that in infected hosts, Mtb constantly resides in a FA-rich environment that the pathogen contributes to generate by inducing a lipid-laden "foamy" phenotype in host macrophages. These observations and the proximity between lipid droplets and phagosomes containing bacteria within infected macrophages gave rise to the hypothesis that Mtb reprograms host cell lipid metabolism to ensure a continuous supply of essential nutrients and its long-term persistence in vivo. However, recent studies question this principle by indicating that in Mtb-infected macrophages, lipid droplet formation prevents bacterial acquisition of host FAs while supporting the production of FA-derived protective lipid mediators. Further, in vivo investigations reveal discrete macrophage phenotypes linking the FA metabolisms of host cell and intracellular pathogen. Notably, FA storage within lipid droplets characterizes both macrophages controlling Mtb infection and dormant intracellular Mtb. In this review, we integrate findings from immunological and microbiological studies illustrating the new concept that cytoplasmic accumulation of FAs is a metabolic adaptation of macrophages to Mtb infection, which potentiates their antimycobacterial responses and forces the intracellular pathogen to shift into fat-saving, survival mode.

A comparison of alternative mRNA splicing in the CD4 and CD8 T cell lineages.

T cells can be subdivided into a number of different subsets that are defined by their distinct functions. While the specialization of different T cell subsets is partly achieved by the expression of specific genes, the overall transcriptional profiles of all T cells appear very similar. Alternative mRNA splicing is a mechanism that facilitates greater transcript/protein diversity from a limited number of genes, which may contribute to the functional specialization of distinct T cell subsets. In this study we employ a combination of short-read and long-read sequencing technologies to compare alternative mRNA splicing between the CD4 and CD8 T cell lineages. While long-read technology was effective at assembling full-length alternatively spliced transcripts, the low sequencing depth did not facilitate accurate quantitation. On the other hand, short-read technology was ineffective at assembling full-length transcripts but was highly accurate for quantifying expression. We show that integrating long-read and short-read data together achieves a more complete view of transcriptomic diversity. We found that while the overall usage of transcript isoforms was very similar between the CD4 and CD8 lineages, there were numerous alternative spliced mRNA isoforms that were preferentially used by one lineage over the other. These alternative spliced isoforms included ones with different exon usage, exon exclusion or intron inclusion, all of which are expected to significantly alter the protein sequence.