Biology subjects

Bunce, C.

Publications and source records attributed to Bunce, C..

6 recordsLinked to original sources

bioRxiv2024

Pyruvate from bone marrow mesenchymal stem cells supports myeloma redox homeostasis and anabolism

Multiple myeloma is an incurable cancer of plasma cells that depends on the bone marrow for its survival. Despite its prevalence, the molecular mechanisms underlying this malignancy remain poorly understood. In this study, we aim to bridge this knowledge gap by elucidating the metabolic interplay between myeloma cells and bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are crucial in supporting myeloma cell metabolism, contributing to their proliferation, survival, and resistance to chemotherapy. Through a combination of mathematical modelling and experimental co-cultures, we demonstrate that pyruvate - the end product of glycolysis - plays a key role in myeloma cell metabolism. Our findings reveal that myeloma cells predominantly rely on the uptake of pyruvate produced by neighbouring BMM-SCs via the plasma membrane proton-linked monocarboxylate transporters MCT-1 and MCT-2 encoded by the Slc16a1 and a2 genes, respectively. Furthermore, we show that pharmacological inhibition of the MCT-1/2, with AZD3965, triggers a cascade of compensatory metabolic responses, disrupting redox balance and significantly reducing the proliferation capacity of co-cultured myeloma cells.

cancer biology↗

bioRxiv2024

OoCount: A machine-learning based approach to mouse ovarian follicle counting and classification

The number and distribution of ovarian follicles in each growth stage provides a reliable readout of ovarian health and function. Leveraging techniques for three-dimensional (3D) imaging of ovaries in toto has the potential to uncover total, accurate ovarian follicle counts. However, because of the size and holistic nature of these images, counting oocytes is time consuming and difficult. The advent of deep-learning algorithms has allowed for the rapid development of ultra-fast, automated methods to analyze microscopy images. In recent years, these pipelines have become more user-friendly and accessible to non-specialists. We used these tools to create OoCount, a high-throughput, open-source method for automatic oocyte segmentation and classification from fluorescent 3D microscopy images of whole mouse ovaries using a deep-learning convolutional neural network (CNN) based approach. We developed a fast tissue-clearing and spinning disk confocal-based imaging protocol to obtain 3D images of whole mount perinatal and adult mouse ovaries. Fluorescently labeled oocytes from 3D images of ovaries were manually annotated in Napari to develop a machine learning training dataset. This dataset was used to retrain StarDist using a CNN within DL4MicEverywhere to automatically label all oocytes in the ovary. In a second phase, we utilize Accelerated Pixel and Object Classification, a Napari plugin, to classify labeled oocytes and sort them into growth stages. Here, we provide an end-to-end protocol for producing high-quality 3D images of the perinatal and adult mouse ovary, obtaining follicle counts and staging. We also demonstrate how to customize OoCount to fit images produced in any lab. Using OoCount, we can obtain accurate counts of oocytes in each growth stage in the perinatal and adult ovary, improving our ability to study ovarian function and fertility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/593993v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@8d7257org.highwire.dtl.DTLVardef@4dbe73org.highwire.dtl.DTLVardef@1283aaorg.highwire.dtl.DTLVardef@1822baf_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary sentenceThis protocol introduces OoCount, a high-throughput, open-source method for automatic oocyte segmentation and classification from fluorescent 3D microscopy images of whole mouse ovaries using a machine learning-based approach.

developmental biology↗

bioRxiv2023

Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1{beta} from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1{beta} secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-{kappa}B and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1{beta} in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases. Author SummaryAcute myeloid leukaemia (AML) and myelodysplastic syndromes MDS) are related blood cancers that are associated with frequent infections because the cancers suppress normal immunity. These infections are therefore generally considered as medical complications arising as a result of but separate to the cancer. However, we provide evidence here that infections may promote or drive cancer progression. We and others previously demonstrated that a human protein called NM23-H1 promotes the survival of AML cells by eliciting survival signals from other cells. NM23-H1 belongs to a highly conserved family of proteins that also occur in bacteria and fungi that cause infections in AML and MDS patients. Here we demonstrate that these bacterial and fungal proteins recapitulate the pro-survival effect of NM23-H1 on AML cells. We also determine that these effects are mediated via mechanisms already known to be important in the development and progression of AML and MDS. This study is the first to identify NM23-H1 like proteins from pathogenic microorganisms as novel activators of these pathways. These findings have important implications for how we understand infections in AML and MDS patients and suggest that in addition to being the consequence of these diseases, infections may also drive the cancer process.

microbiology↗

bioRxiv2021

Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLC) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially co-express Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a Sertoli- or granulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Finally, we found that WNT4 is a crucial regulator of the SLC lineage and is required for the formation of the rete testis. TeaserDescription of an uncharacterized multipotent gonadal cell lineage involved in testis and ovary development

developmental biology↗

bioRxiv2021

Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii.

Morphogenetic events during development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. Development of the rete ovarii correlated with events of ovary morphogenesis. Using genetic mouse models in which the oviduct and rete ovarii are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary.

developmental biology↗

bioRxiv2018

The RNA-Binding Protein DND1 Acts Sequentially as a Negative Regulator of Pluripotency and a Positive Regulator of Epigenetic Modifiers Required for Germ Cell Reprogramming

The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day 10.5 (E10.5). These cells undergo rapid mitotic proliferation, then enter a prolonged period of cell cycle arrest (G1/G0) during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein DND1 (DND1Ter/Ter), many germ cells fail to enter G1/G0, and give rise to teratomas, tumors in which many embryonic cell types are represented. To investigate the origin of these tumors, we sequenced the transcriptome of male germ cells in DND1Ter/Ter mutants at E12.5, E13.5, and E14.5, just prior to the formation of teratomas, and correlated this information with direct targets of DND1 identified by DO-RIP-Seq. Consistent with previous results, we found that DND1 controls the down regulation of many genes associated with pluripotency and active cell cycle, including elements of the mTor, Hippo and Bmp/Nodal signaling pathways. However, DND1 targets also include genes associated with male differentiation including a large group of chromatin regulators activated in wild type but not mutant germ cells during the transition between E13.5 and E14.5. These results suggest multiple functions of DND1, and link DND1 to the initiation of epigenetic modifications in male germ cells.

cell biology↗