Distribution Pattern Of Cells Research Articles

Estrogen receptor Esr1 induces early ovarian differentiation in Trachemys scripta

This study aims to explore the roles of three estrogen receptors (Esr1, Esr2, and Gper1) in early differentiation of embryonic gonads of Trachemys scripta. The expression characteristics of the receptor genes were studied first. The Esr1, Esr2, and Gper1 agonists PPT, WAY 200070, and G-1 were respectively injected into the embryos at the male-producing temperature (MPT) before initiation of gonadal differentiation. The sex reversal of the treated embryonic gonads was analyzed in terms of morphological structure of gonads, distribution pattern of germ cells, and expression of key genes and proteins involved in sex differentiation. The expression level of esr1 during the critical stage of sex differentiation was higher than those of esr2 and gper1 (very low expression) and was particularly high in the gonads at the female-producing temperature (FPT). After treatment with PPT, the MPT gonads presented obviously feminized morphology and structure, with the germ cells exhibiting a female distribution pattern. Furthermore, the mRNA expression levels of the key genes (dmrt1, amh, and sox9) for male differentiation were down-regulated significantly, while those of the key genes (foxl2 and cyp19a1) for female differentiation were up-regulated observably. The fluorescent signals of Amh and Sox9 expression almost disappeared, while Foxl2 and Arom were activated to express abundantly, which fully demonstrated the sex reversal of the gonads from male to female (sex reversal rate: 70.27%). However, the MPT gonads treated with WAY 200070 and G-1 still differentiated into testes, and the expression patterns of the key genes and proteins were similar to those in male gonads. The above results demonstrate that activation of Esr1 alone can fully initiate the early female differentiation process of gonads, suggesting that estrogen may induce early ovarian differentiation via Esr1 in Trachemys scripta. The findings provide a basis for further revealing the mechanisms of estrogen regulation in sex determination and differentiation of turtles.

P-200 Vitrification induces a deviation in cleavage morphokinetic patterns and transcriptomic signatures along mouse embryo development

Abstract Study question Are there cellular, molecular and/or epigenetic risks associated with cryopreservation that may affect embryo development and compromise the health of future newborns? Summary answer Cryopreservation significantly impacted mice pre-implantation embryo morphokinetics, altered transcriptomic profiles, and cell lineage distribution. However, global DNA methylation remained unaffected by cryopreservation. What is known already Cryopreservation is currently widely used in assisted reproduction. However, epidemiological studies suggest a negative impact in the long-term health of children born after frozen-embryo transfer (FET). Recent population-based register studies have reported: 1) an increased risk of infant mortality from birth up to 1 year of life in singletons conceived using cryopreserved embryos vs natural conceptions; 2) high birth weights for gestational age associated with FET; 3) an increased risk of obstetrical complications in singletons conceived using cryopreserved embryos; 4) a higher incidence of childhood cancer and imprinting disorders associated with FET. Study design, size, duration F1 hybrid (B6/CBA) mice zygotes were collected following uterus dissection (n = 200 embryos/collection), and half of those were vitrified for comparison. Each embryo’s morphokinetics was assessed through time-lapse imaging using GERI, at 37 °C, 6% CO2, 5% O2, up to 105 hours. Extended culture beyond blastocyst stage was also performed. Fresh and cryopreserved blastocysts at E5.0 were cultured up to E9.0-E10 in static conditions (IVC1 and IVC2 medium). Participants/materials, setting, methods Fresh and cryopreserved embryos at several developmental stages were used for: 1) immunofluorescence staining, 2) gene expression analysis through RT-qPCR and 3) single-cell RNAseq. Global methylation activity, cell lineage distribution and single-cell transcriptome were assessed using one of the previously described methodologies. Main results and the role of chance The morphokinetics of preimplantation embryos were examined by tracking the time required to reach 4-cell, 8-cell, compactation, morula, full blastocyst, and expanded blastocyst stage. A comprehensive analysis revealed notable delays in the developmental rhythm of cryopreserved embryos compared to fresh embryos (p < 0.01). Applying single-cell RNA sequencing (scRNAseq), transcriptomic profiles were generated for fresh and cryopreserved embryos, covering the zygote to blastocyst stages. Notably, distinct transcriptomic signatures for fresh and frozen embryos emerged within separate clusters at different developmental stages. These findings suggest a potential functional impact of cryopreservation on embryo development. To complement this, gene expression analysis of DNA methyltransferases and MAT enzymes was conducted through RT-qPCR at 8-cell and blastocyst stages. No significant differences were detected between fresh and cryopreserved conditions. Furthermore, immunofluorescence staining of nuclear 5mC and DNMT1 in 8-cell embryos and blastocysts demonstrated similar levels between fresh and cryopreserved embryos. Additionally, differential staining of embryonic and extraembryonic cells (OCT4, GATA6 and GATA3) in extended embryos (E9 and E10) revealed distinct cell distribution patterns between fresh and cryopreserved conditions. Limitations, reasons for caution Our research, conducted using a mouse experimental model, has limited applicability to humans due to considerable species differences in embryonic development. Additional research is needed to validate our findings and establish their relevance to human embryos, facilitating translational approaches. Wider implications of the findings Our study highlights a noticeable functional impact of vitrification on mouse embryos. Given the global incorporation of this method in fertility clinics and its potential association with increased disease risks, our findings emphasize the need for increased research to reduce uncertainties regarding the health of children born from cryopreservation methods. Trial registration number Not applicable

Anatomy of leaf blade, leaf sheath and pseudopetiole in Neotropical Bambusoideae (Poaceae): Insights into structure and taxonomic contributions

Anatomical investigations aiming to seek alternative characters for the taxonomy of bamboos are almost exclusively restricted to the leaf blade. Little is known about the anatomical structure of other parts of the leaf, such as the leaf sheath and pseudopetiole. The present paper analyzed the complete structure of foliage leaves, including the foliage leaf blade, foliage leaf sheath, and pseudopetiole of species belonging to the tribes Bambuseae (subtribes Guaduinae, Chusqueinae and Arthrostylidiinae) and Olyreae (subtribe Parianinae) to identify useful characters in the taxonomy of bamboos. We used scanning electron microscopy and light microscopy to describe micromorphological and anatomical features. All species sampled share the following features: foliage leaf blades with uniseriate epidermis; adaxial bulliform cells; mesophyll with arm cells, cavities, and collateral vascular bundles. Intercostal sclerenchyma associated to bulliform cells was observed only in Arthrostylidiinae. Micromorphologically, the shape and distribution of silica bodies, presence, type, and distribution of papillae on the long cells and subsidiary cells, and the presence and distribution of trichomes may be taxonomically informative in different taxonomic levels. We highlight the absence of papillae on the surfaces of foliage leaf blade of Guaduinae since the presence of papillae is considered a putative diagnosis feature supporting the sister relationship of this subtribe with Arthrostylidiinae. We describe the anatomical structure of foliage leaf sheath for the first time for Guaduinae, Chusqueinae and Parianinae. In Guaduinae, we found a unique distribution pattern of silica cells on the abaxial surface of the leaf sheath. Anatomical data about the pseudopetiole structure also were described for the first time in bamboos species; the arrangement of vascular bundles may be taxonomically informative, especially in Guaduinae.

Paneth cell ontogeny in term and preterm ovine models.

Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

Exploiting Geometric Features via Hierarchical Graph Pyramid Transformer for Cancer Diagnosis using Histopathological Images.

Cancer is widely recognized as the primary cause of mortality worldwide, and pathology analysis plays a pivotal role in achieving accurate cancer diagnosis. The intricate representation of features in histopathological images encompasses abundant information crucial for disease diagnosis, regarding cell appearance, tumor microenvironment, and geometric characteristics. However, recent deep learning methods have not adequately exploited geometric features for pathological image classification due to the absence of effective descriptors that can capture both cell distribution and gathering patterns, which often serve as potent indicators. In this paper, inspired by clinical practice, a Hierarchical Graph Pyramid Transformer (HGPT) is proposed to guide pathological image classification by effectively exploiting a geometric representation of tissue distribution which was ignored by existing state-of-the-art methods. First, a graph representation is constructed according to morphological feature of input pathological image and learn geometric representation through the proposed multi-head graph aggregator. Then, the image and its graph representation are feed into the transformer encoder layer to model long-range dependency. Finally, a locality feature enhancement block is designed to enhance the 2D local representation of feature embedding, which is not well explored in the existing vision transformers. An extensive experimental study is conducted on Kather-5K, MHIST, NCT-CRC-HE, and GasHisSDB for binary or multi-category classification of multiple cancer types. Results demonstrated that our method is capable of consistently reaching superior classification outcomes for histopathological images, which provide an effective diagnostic tool for malignant tumors in clinical practice.

SulA does not sequester FtsZ in Escherichia coli cells during the SOS response

In Escherichia coli, the SulA protein is synthesized during the SOS response to arrest cell division. Two possible models of SulA action were proposed: the sequestration and the capping. In current paper, to clarify which model better reflects the SulA effect on cell division upon the SOS response, the FtsZ/SulA ratio was estimated inside cells based on fusion of both FtsZ and SulA to fluorescent protein mNeonGreen. This allowed to quantify this ratio by fluorescence microscopy as well as western blotting; moreover, the effect of SulA on FtsZ distribution patterns in cells was analyzed based on fluorescence microscopy images. The SulA concentration in cells under the SOS response was shown to be several times (about 10) lower than that of FtsZ. The effect of SulA was unequal to corresponding decrease in FtsZ concentration. These results are supported by uneven FtsZ distribution in cells under the SOS response. Together the results of current work indicate that the division arrest by SulA protein in E. coli cells could not be explained by the sequestration model.

CCF-GNN: A Unified Model Aggregating Appearance, Microenvironment, and Topology for Pathology Image Classification.

Pathology images contain rich information of cell appearance, microenvironment, and topology features for cancer analysis and diagnosis. Among such features, topology becomes increasingly important in analysis for cancer immunotherapy. By analyzing geometric and hierarchically structured cell distribution topology, oncologists can identify densely-packed and cancer-relevant cell communities (CCs) for making decisions. Compared to commonly-used pixel-level Convolution Neural Network (CNN) features and cell-instance-level Graph Neural Network (GNN) features, CC topology features are at a higher level of granularity and geometry. However, topological features have not been well exploited by recent deep learning (DL) methods for pathology image classification due to lack of effective topological descriptors for cell distribution and gathering patterns. In this paper, inspired by clinical practice, we analyze and classify pathology images by comprehensively learning cell appearance, microenvironment, and topology in a fine-to-coarse manner. To describe and exploit topology, we design Cell Community Forest (CCF), a novel graph that represents the hierarchical formulation process of big-sparse CCs from small-dense CCs. Using CCF as a new geometric topological descriptor of tumor cells in pathology images, we propose CCF-GNN, a GNN model that successively aggregates heterogeneous features (e.g., appearance, microenvironment) from cell-instance-level, cell-community-level, into image-level for pathology image classification. Extensive cross-validation experiments show that our method significantly outperforms alternative methods on H&E-stained and immunofluorescence images for disease grading tasks with multiple cancer types. Our proposed CCF-GNN establishes a new topological data analysis (TDA) based method, which facilitates integrating multi-level heterogeneous features of point clouds (e.g., for cells) into a unified DL framework.

Visualizing intracellular membrane interactions and cell type-specific differentiation in ferroptosis and apoptosis with Boranil-Carbazole derivative

Intracellular lipid systems play essential roles in various physiological functions and cell growth processes. However, our understanding of the intricate interactions within this system, especially between mitochondria and lipid droplets, is limited, particularly in the context of cancer cells' altered lipid metabolism. To address this, our study introduces an N-B-O BODIPY-hexylcarbazole derivative, named Cz-Boranil, that sets a new benchmark in visualizing these critical interactions. Cz-Boranil's unique capability lies in its ability to display distinct intracellular distribution patterns in both normal and cancer cells, offering nuanced cell type-specific differentiation. More impressively, this probe tracks the coordinated interactions of lipid droplets and mitochondria during the critical processes of ferroptosis and apoptosis. We believe that the innovative capabilities of Cz-Boranil will revolutionize our understanding of intracellular lipid interactions and prove pivotal in identifying and studying cancerous cells.

Multiscale Imaging Techniques for Real‐Time, Noninvasive Diagnosis of Li‐Ion Battery Failures

With the increasing popularity of battery‐powered mobility, ensuring the safety and reliability of Li‐ion batteries (LIBs) has become critical for manufacturers. Despite advanced manufacturing processes for large‐scale Li‐ion cells, “latent defects” still unintentionally appear, due to imbalanced battery design, invisible faults, and extreme operating conditions. These defects cause performance degradation and can even lead to battery fires. Hence, early detection of latent defects, along with understanding the influence of cell parameters and operating conditions on battery failure scenarios, is crucial. For straightforward investigations and interpretations, noninvasive and in operando battery imaging techniques and methods have been proposed using X‐rays, neutrons, and ultrasound, as these can penetrate active and component materials and cell packaging. Moreover, magnetic‐field‐guided visualization of the current distribution pattern in cells under a current load has been proposed to identify invisible defects. This review thoroughly examines various imaging techniques for internal batteries, from the atomic and molecular levels in electrode materials and interfaces to macroscale battery systems. By assessing qualitative case studies and newly discovered phenomena, this review provides valuable insights into state‐of‐the‐art noninvasive battery imaging and its potential to improve the safety and reliability of LIB technology.

Gene expression pattern of K transporter GhHAK5 gene of potassium efficient and in-efficient cotton cultivars based on morphological physiognomies as affected by potassium nutrition and reduced irrigation

BackgroundUnder K deficiency the uptake and distribution pattern in plant cells is mediated through different transport proteins and channels which were controlled by specific gene family. Therefore, a hydroponic experiment was conducted under control condition for testing the gene expression pattern of the K transporter under adequate and low K supply levels. After that, a 2-year field experiment was conducted to evaluate five selected cotton cultivars (four K-efficient cultivars, viz., CIM-554, CYTO-124, FH-142, IUB-2013, and one K non-efficient, BH-212) screened from the initial hydroponics culture experiment and two levels of potassium (0 K2O kg·ha−1 and 50 K2O kg·ha−1) were tested under reduced irrigation (50% available water content; 50 AWC) and normal irrigation conditions (100% available water content; 100 AWC).ResultResults revealed that the transcript levels of GhHAK5aD in roots were significantly higher in K+ efficient cultivars than that in K+ non-efficient cultivars. The GhHAK5aD expression upon K+ deficiency was higher in roots but lower in shoots, indicating that GhHAK5aD could have a role in K+ uptake in roots, instead of transport of K+ from root to shoot. Similarly, under field conditions the cultivar FH-142 showed an increase of 22.3%, 4.9%, 2.4%, and 1.4% as compared with BH-212, IUB-2013, CYTO-124, and CIM-554, respectively, in seed cotton yield (SCY) with K application under reduced irrigation conditions. With applied K, the FH-142 showed an increase in net photosynthetic rate by 57.3% as compared with the rest of the cultivars under reduced irrigation over K control. However, the overall performance indicators of K-efficient cultivars like FH-142, CYTO-124, CIM-554, and IUB-2013 were better than BH-212 (K in-efficient) under reduced irrigation conditions with applied K at 50 kg·ha−1. Fiber quality trait improved significantly with K application under water deficit. The increase in micronaire was 3.6%, 4.7%, 7.8%, 3.4%, and 6.7% in BH-212, IUB-2013, CIM-554, CYTO-124, and FH-142, respectively, with K application at 50 kg·ha−1 over without K application under reduced irrigation conditions during the cotton growing season. Similarly, the cultivars FH-142 increased by 12% with K application under reduced irrigation as compared with other cultivars. The performance of K-efficient cultivars under reduced irrigation conditions was 30% better in SCY and quality traits with the application of K at 50 kg·ha−1 as compared with K-non-efficient cultivars. Similarly, water use efficiency (WUE) (40.1%) and potassium use efficiency (KUE) (20.2%) were also noted higher in case of FH-142 as compared with other cultivar with K application under reduced conditions.ConclusionHigher expression of GhHAK5aD gene was observed in K-efficient cultivars as compared with K-non-efficient cultivars in roots indicates that GhHAK5aD may be contributing to genotypic differences for K+ efficiency in cotton. K-efficient cotton cultivars can be used for the low-K environments and can also be recommended for general cultivars.

Novel Fabrication of 3-D Cell Laden Micro-Patterned Porous Structure on Cell Growth and Proliferation by Layered Manufacturing.

This study focuses on developing and characterizing a novel 3-dimensional cell-laden micro-patterned porous structure from a mechanical engineering perspective. Tissue engineering holds great promise for repairing damaged organs but faces challenges related to cell viability, biocompatibility, and mechanical strength. This research aims to overcome these limitations by utilizing gelatin methacrylate hydrogel as a scaffold material and employing a photolithography technique for precise patterned fabrication. The mechanical properties of the structure are of particular interest in this study. We evaluate its ability to withstand external forces through compression tests, which provide insights into its strength and stability. Additionally, structural integrity is assessed over time to determine its performance in in vitro and potential in vivo environments. We investigate cell viability and proliferation within the micro-patterned porous structure to evaluate the biological aspects. MTT assays and immunofluorescence staining are employed to analyze the metabolic activity and distribution pattern of cells, respectively. These assessments help us understand the effectiveness of the structure in supporting cell growth and tissue regeneration. The findings of this research contribute to the field of tissue engineering and provide valuable insights for mechanical engineers working on developing scaffolds and structures for regenerative medicine. By addressing challenges related to cell viability, biocompatibility, and mechanical strength, we move closer to realizing clinically viable tissue engineering solutions. The novel micro-patterned porous structure holds promise for applications in artificial organ development and lays the foundation for future advancements in large soft tissue construction.

PyCreas: a tool for quantification of localization and distribution of endocrine cell types in the islets of Langerhans.

Manifest diabetes, but also conditions of increased insulin resistance such as pregnancy or obesity can lead to islet architecture remodeling. The contributing mechanisms are as poorly understood as the consequences of altered cell arrangement. For the quantification of the different cell types but also the frequency of different cell-cell contacts within the islets, different approaches exist. However, few methods are available to characterize islet cell distribution in a statistically valid manner. Here we describe PyCreas, an open-source tool written in Python that allows semi-automated analysis of islet cell distribution based on images of pancreatic sections stained by immunohistochemistry or immunofluorescence. To ensure that the PyCreas tool is suitable for quantitative analysis of cell distribution in the islets at different metabolic states, we studied the localization and distribution of alpha, beta, and delta cells during gestation and prediabetes. We compared the islet cell distribution of pancreatic islets from metabolically healthy NMRI mice with that of New Zealand obese (NZO) mice, which exhibit impaired glucose tolerance (IGT) both preconceptionally and during gestation, and from C57BL/6N (B6) mice, which acquire this IGT only during gestation. Since substrain(s) of the NZO mice are known to show a variant in the Abcc8 gene, we additionally examined preconceptional SUR1 knock-out (SUR1-KO) mice. PyCreas provided quantitative evidence that alterations in the Abcc8 gene are associated with an altered distribution pattern of islet cells. Moreover, our data indicate that this cannot be a consequence of prolonged hyperglycemia, as islet architecture is already altered in the prediabetic state. Furthermore, the quantitative analysis suggests that states of transient IGT, such as during common gestational diabetes mellitus (GDM), are not associated with changes in islet architecture as observed during long-term IGT. PyCreas provides the ability to systematically analyze the localization and distribution of islet cells at different stages of metabolic disease to better understand the underlying pathophysiology.

Exercise prevents obesity by reducing gut-derived inflammatory signals to brown adipocytes in mice.

Gut dysbiosis impairs nonshivering thermogenesis (NST) in obesity. The antiobesogenic effects of exercise training might involve the modulation of gut microbiota and its inflammatory signals to the brown adipose tissue (BAT). This study evaluated whether high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) prevent overweight through reduced gut-derived inflammatory signals to BAT in high-fat-fed mice. Sixty male C57BL/6 mice (3 months old) comprised six experimental groups: control (C) diet group, C diet + HIIT (C-HIIT) group, C diet + MICT (C-MICT) group, high-fat (HF) diet group, HF diet + HIIT (HF-HIIT) group, and HF diet + MICT (HF-MICT) group. The protocols lasted for 10 weeks. HIIT and MICT restored body mass, mitigated glucose intolerance, and prevented hyperinsulinemia in HF-trained groups. A chronic HF diet caused dysbiosis, but HIIT and MICT prevented gut dysbiosis and preserved tight junction (TJ) gene expression. HF-HIIT and HF-MICT groups exhibited a similar pattern of goblet cell distribution, agreeing with the decreased plasma lipopolysaccharide concentrations and interscapular BAT (iBAT) Lbp-Cd14-Tlr4 expression. The lowered Nlrp3 and Il1β in the HF-HITT and HF-MICT groups complied with iBAT thermogenic capacity maintenance. This study shows reliable evidence that HIIT and MICT prevented overweight by restoring the diversity of the gut microbiota phyla and TJ gene expression, thereby reducing inflammatory signals to brown adipocytes with preserved thermogenic capacity. Both exercise modalities prevented overweight, but HIIT rescued Zo-1 and Jam-a gene expression, exerting more potent anti-inflammatory effects than MICT (reduced LPS concentrations), providing a sustained increase in thermogenesis with 78% less distance traveled.

Different patterns of mast cell distribution in B-cell non-Hodgkin lymphomas

Tumor growth, progression, and metastatic capability in non-Hodgkin lymphomas (NHLs) are influenced by different component of tumor microenvironment, including inflammatory cells. Among these latter, mast cells play a crucial role. The spatial distribution of mast cells inside the tumor stroma of different types of B-cell NHLs has not yet been investigated. The aim of this study is to analyze the pattern of distribution of mast cells in biopsy samples obtained from three different types of B-cell NHLs by utilizing an image analysis system and a mathematical model to allow a quantitative estimation to characterize their spatial distribution. As concerns the spatial distributions exhibited by mast cells in diffuse large B cell lymphoma (DLBCL), some clustering was detected in both activated B-like (ABC) and germinal center B-like (GBC) groups. In follicular lymphoma (FL), mast cell spatial distribution tends to uniformly fill the tissue space as far as the grade of the pathology increases. Finally, in marginal lymphoma tissue (MALT) lymphoma, mast cells maintain a significantly clustered spatial distribution, suggesting a lower tendency of the cells to fill the tissue space in this pathological condition. Overall, the data of this study confirm that the analysis of the spatial distribution of the tumor cells is of particular significance for the knowledge of the biological processes occurring in tumor stroma and for the development of parameters to characterize the morphologic organization of the cellular patterns in different types of tumors.

270-LB: Pancreatic Glial Cells Distribution Patterns Are Distinct between Human and Mouse

Glial cells are components of the central and peripheral nervous systems, which provide physical support for neuron and modulate synaptic transmission. We and others previously showed that in mice, pancreatic glial cells are located in close association with the extensive neuro-endocrine-vascular network that surrounds the islet perimeter. It is well known that differences exist in mouse and human islet architecture, in particular in vascular density and innervation. We therefore sought to determine whether density and/or organization of pancreatic glial cells also differs between mouse and human. Glial fibrillary acidic protein (GFAP) was used to identify pancreatic glial cells and was examined by confocal microscopy. In mouse pancreas, GFAP+ cells were frequently identified around the islet periphery and were less frequently seen in the core of the islet. In human pancreas, however, most of the GFAP+ cells were found in the exocrine tissue, resulting in fewer peri-islet glial cells relative to mice (table). High-fat diet feeding significantly increased %peri-islet GFAP+ area in mice while no difference was seen between obese and lean human donors (table). These findings indicate that pancreatic glial cell distribution patterns differ between mouse and human, raising the possibility that these cells may affect islet-cell function differently between the two species. Disclosure J. Niu: None. D. J. Hackney: None. P. Ahmadi: None. M. Hara: None. R. L. Hull-meichle: Employee; Veterans Administration, Research Support; Casma Therapeutics, Cystic Fibrosis Foundation, NIH - National Institutes of Health, Veterans Administration. J. Tong: None. Funding University of Washington

LAG3 in gastric cancer: it’s complicated

PurposeLymphocyte activation gene 3 (LAG3) is thought to contribute to T cell exhaustion within the tumor microenvironment of solid tumors. This study aimed to analyze the spatial distribution of LAG3 + cells in relation to clinicopathological and survival data in a large set of 580 primary resected and neoadjuvantly treated gastric cancers (GC).MethodsLAG3 expression was evaluated in tumor center and invasive margin using immunohistochemistry and whole-slide digital image analysis. Cases were divided into LAG3-low and LAG3-high expression groups based on (1) median LAG3 + cell density, (2) cut-off values adapted to cancer-specific survival using Cutoff Finder application.ResultsSignificant differences in spatial distribution of LAG3 + cells were observed in primarily resected GC, but not in neoadjuvantly treated GC. LAG3 + cell density showed evident prognostic value at following cut-offs: in primarily resected GC, 21.45 cells/mm2 in tumor center (17.9 vs. 10.1 months, p = 0.008) and 208.50 cells/mm2 in invasive margin (33.8 vs. 14.7 months, p = 0.006); and in neoadjuvantly treated GC, 12.62 cells/mm2 (27.3 vs. 13.2 months, p = 0.003) and 123.00 cells/mm2 (28.0 vs. 22.4 months, p = 0.136), respectively. Significant associations were found between LAG3 + cell distribution patterns and various clinicopathological factors in both cohorts. In neoadjuvantly treated GC, LAG3 + immune cell density was found to be an independent prognostic factor of survival (HR = 0.312, 95% CI 0.162–0.599, p < 0.001).ConclusionIn this study, a higher density of LAG3 + cells was associated with favorable prognosis. Current results support the need for extended analysis of LAG3. Differences in the distribution of LAG3 + cells should be considered, as they could influence clinical outcomes and treatment responses.Graphical abstract

Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is the most severe type that leads to 1% of male infertility. Wnt signaling governs normal sperm maturation. However, the role of Wnt signaling in spermatogonia in NOA has incompletely been uncovered, and upstream molecules regulating Wnt signaling remain unclear. Bulk RNA sequencing (RNA-seq) of NOA was used to identify the hub gene module in NOA utilizing weighted gene co-expression network analyses (WGCNAs). Single-cell RNA sequencing (scRNA-seq) of NOA was employed to explore dysfunctional signaling pathways in the specific cell type with gene sets of signaling pathways. Single-cell regulatory network inference and clustering (pySCENIC) for Python analysis was applied to speculate putative transcription factors in spermatogonia. Moreover, single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) determined the regulated genes of these transcription factors. Finally, spatial transcriptomic data were used to analyze cell type and Wnt signaling spatial distribution. The Wnt signaling pathway was demonstrated to be enriched in the hub gene module of NOA by bulk RNA-seq. Then, scRNA-seq data revealed the downregulated activity and dysfunction of Wnt signaling of spermatogonia in NOA samples. Conjoint analyses of the pySCENIC algorithm and scATAC-seq data indicated that three transcription factors (CTCF, AR, and ARNTL) were related to the activities of Wnt signaling in NOA. Eventually, spatial expression localization of Wnt signaling was identified to be in accordance with the distribution patterns of spermatogonia, Sertoli cells, and Leydig cells. In conclusion, we identified that downregulated Wnt signaling of spermatogonia in NOA and three transcription factors (CTCF, AR, and ARNTL) may be involved in this dysfunctional Wnt signaling. These findings provide new mechanisms for NOA and new therapeutic targets for NOA patients.

Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.

Axin2-CreERT2 mediated knockout of bone morphogenetic protein receptor type 1A caused abnormal secondary dentine and altered cell fate of Axin2-expressing odontogenic cells.

Inducing odontogenic differentiation and tubular dentine formation is extremely important in dentine repair and tooth regeneration. Bone morphogenic proteins (BMPs) signalling plays a critical role in dentine development and tertiary dentine formation, whilst how BMPR1A-mediated signalling affects odontoblastic differentiation of Axin2-expressing (Axin2+ ) odontogenic cells and tubular dentine formation remains largely unknown. This study aims to reveal the cellular and molecular mechanisms involved in the formation of secondary dentine. Axin2lacZ/+ mice harvested at post-natal 21 (P21) were used to map Axin2+ mesenchymal cells. Axin2CreERT2/+ ; R26RtdTomato/+ mice and Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice were generated to observe the tempo-spatial distribution pattern of Axin2-lineage cells and the effect of ablation of Axin2+ cells on dentinogenesis, respectively. A loss-of-function model was established with Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ (cKO) mice to study the role of BMP signalling in regulating Axin2+ cells. Micro-computed tomography, histologic and immunostainings, and other approaches were used to examine biological functions, including dentine formation, mineralization and cell differentiation in cKO mice. The results showed rich expression of Axin2 in odontoblasts at P21. Lineage tracing assay confirmed the wide distribution of Axin2 lineage cells in odontoblast layer and dental pulp during secondary dentine formation (P23 to P56), suggesting that Axin2+ cells are important cell source of primary odontoblasts. Ablation of Axin2+ cells (DTA mice) significantly impaired secondary dentine formation characterized with notably reduced dentine thickness (Mean of control: 54.11 μm, Mean of DTA: 27.79 μm, p = .0101). Furthermore, malformed osteo-dentine replaced the tubular secondary dentine in the absence of Bmpr1a with irregular cell morphology, abnormal cellular process formation and lack of cell-cell tight conjunction. Remarkably increased expression of osteogenic markers like Runx2 and DMP1 was detected, whilst DSP expression was observed in a dispersed manner, indicating an impaired odontogenic cell fate and failure in producing tubular dentine in cKO mice. Axin2+ cells are a critical population of primary odontoblasts which contribute to tubular secondary dentine formation, and BMP signalling pathway plays a vital role in maintaining the odontogenic fate of Axin2+ cells.

Investigating the spatial interaction of immune cells in colon cancer

The intricate network of interactions between cells and molecules in the tumor microenvironment creates a heterogeneous ecosystem. The proximity of the cells and molecules to their activators and inhibitors is essential in the progression of tumors. Here, we develop a system of partial differential equations coupled with linear elasticity to investigate the effects of spatial interactions on the tumor microenvironment. We observe interesting cell and cytokine distribution patterns, which are heavily affected by macrophages. We also see that cytotoxic Tcells get recruited and suppressed at the site of macrophages. Moreover, we observe that anti-tumor macrophages reorganize the patterns in favor of a more spatially restricted cancer and necrotic core. Furthermore, the adjoint-based sensitivity analysis indicates that the most sensitive model's parameters are directly related to macrophages. The results emphasize the widely acknowledged effect of macrophages in controlling cancer cells population and spatially arranging cells in the tumor microenvironment.