Embryonic Cells Research Articles

Expanded detection and impact of BAP1 alterations in cancer.

Aberrant expression of the BAP1 (BRCA associated protein 1) tumor suppressor gene is a prominent risk factor for several tumor types and is important in tumor evolution and progression. Here we performed integrated multi-omics analyses using data from The Cancer Genome Atlas for 33 cancer types and over 10 000 individuals to identify alterations leading to BAP1 disruption. We combined existing variant calls and new calls derived from a de novo local realignment pipeline across multiple independent variant callers, increasing somatic variant detection by 41% from 182 to 257, including 11 indels ≥40 bp. The expanded detection of mutations highlights the power of new tools to uncover longer indels and impactful mutations. We developed an expression-based BAP1 activity score and identified a transcriptional profile associated with BAP1 disruption in cancer. BAP1 has been proposed to play a critical role in controlling tumor plasticity and normal cell fate. Leveraging human and mouse liver datasets, BAP1 loss in normal cells resulted in lower BAP1 activity scores and lower scores were associated with a less-differentiated phenotype in embryonic cells. Together, our expanded BAP1 mutant samples revealed a transcriptional signature in cancer cells, supporting BAP1's influences on cellular plasticity and cell identity maintenance.

Single-cell transcriptomics reveals evolutionary reconfiguration of embryonic cell fate specification in the sea urchin Heliocidaris erythrogramma.

Altered regulatory interactions during development likely underlie a large fraction of phenotypic diversity within and between species, yet identifying specific evolutionary changes remains challenging. Analysis of single-cell developmental transcriptomes from multiple species provides a powerful framework for unbiased identification of evolutionary changes in developmental mechanisms. Here, we leverage a "natural experiment" in developmental evolution in sea urchins, where a major life history switch recently evolved in the lineage leading to Heliocidaris erythrogramma, precipitating extensive changes in early development. Comparative analyses of scRNA-seq developmental time courses from H. erythrogramma and Lytechinus variegatus (representing the derived and ancestral states respectively) reveals numerous evolutionary changes in embryonic patterning. The earliest cell fate specification events and the primary signaling center are co-localized in the ancestral dGRN; remarkably, in H. erythrogramma they are spatially and temporally separate. Fate specification and differentiation are delayed in most embryonic cell lineages, although in some cases, these processes are conserved or even accelerated. Comparative analysis of regulator-target gene co-expression is consistent with many specific interactions being preserved but delayed in H. erythrogramma, while some otherwise widely conserved interactions have likely been lost. Finally, specific patterning events are directly correlated with evolutionary changes in larval morphology, suggesting that they are directly tied to the life history shift. Together, these findings demonstrate that comparative scRNA-seq developmental time courses can reveal a diverse set of evolutionary changes in embryonic patterning and provide an efficient way to identify likely candidate regulatory interactions for subsequent experimental validation.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

KBTBD2 controls bone development by regulating IGF-1 signaling during osteoblast differentiation.

Kelch repeat and BTB (POZ) domain-containing 2 (KBTBD2) is known for its pivotal role in metabolic regulation, particularly in adipocytes. However, its significance in skeletal development has remained elusive. Here, we uncover a previously unrecognized function of KBTBD2 in bone formation. Conditional knockout of Kbtbd2 in embryonic osteochondroprogenitor cells or osteoblasts results in impaired osteogenic differentiation, leading to reduced skeletal growth and mineralization. Mechanistically, the loss of KBTBD2 during osteogenesis leads to the accumulation of p85α, a regulatory subunit encoded by phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1), which exerts a potent inhibitory effect on insulin-like growth factor 1 (IGF-1)-induced activation of AKT. Moreover, our study extends the understanding of KBTBD2's relevance beyond bone biology to the context of SHORT syndrome, a rare genetic disorder marked by short stature and various physical abnormalities. We demonstrate that p85α harboring the p.(Arg649Trp) mutation, most frequently found in SHORT syndrome patients, exhibits reduced binding to KBTBD2, leading to impaired IGF-1-mediated activation of AKT. These findings reveal that KBTBD2 is essential in bone formation via regulating the IGF-1 signaling pathway and suggest loss of KBTBD2-mediated regulation of p85α as a potential mechanism for SHORT syndrome.

Aurora kinase as a putative target to tick control.

Aurora kinases (AURK) play a central role in controlling cell cycle in a wide range of organisms. They belong to the family of serine-threonine kinase proteins. Their role in the cell cycle includes, among others, the entry into mitosis, maturation of the centrosome and formation of the mitotic spindle. In mammals, 3 isoforms have been described: A, B and C, which are distinguished mainly by their function throughout the cell cycle. Two aurora kinase coding sequences have been identified in the transcriptome of the cattle tick Rhipicephalus microplus (Rm-AURKA and Rm-AURKB) containing the aurora kinase-specific domain. For both isoforms, the highest number of AURK coding transcripts is found in ovaries. Based on deduced amino acid sequences, it was possible to identify non-conserved threonine residues which are essential to AURK functions in vertebrates and which are not present in R. microplus sequences. A pan AURK inhibitor (CCT137690) caused cell viability decline in the BME26 tick embryonic cell line. In silico docking assay showed an interaction between Aurora kinase and CCT137690 with exclusive interaction sites in Rm-AURKA. The characterization of exclusive regions of the enzyme will enable new studies aimed at promoting species-specific enzymatic inhibition in ectoparasites.

Image Processing Application for Pluripotent Stem Cell Colony Migration Quantification

Human pluripotent stem cells (hPSCs) attract tremendous attention due to their unique properties. Manual extraction of trajectories of cell colonies in experimental image time series is labor intensive and subjective, thus the aim of the work was to develop a computer semi-automated protocol for colony tracking. The developed procedure consists of three major stages, namely, image registration, object detection and tracking. Registration using discrete Fourier transform and tracking based on the solution of a linear assignment problem was implemented as console programs in the Python 3 programming language using a variety of packages. Object detection was implemented as a multistep procedure in the ProStack in-house software package. The procedure consists of more than 40 elementary operations that include setting of several biologically relevant parameters, image segmentation and performing of quantitative measurements. The developed procedure was applied to the dataset containing bright-field images from time-lapse recording of the human embryonic cell line H9. The detection step took about 6 h for one image time series with a resolution of 2560 by 2160; about 1 min was required for image registration and trajectories extraction. The developed procedure was effective in detecting and analyzing the time series of images with “good” and “bad” phenotypes. The differences between phenotypes in the distance in pixels between the starting and finishing positions of trajectories, in the path length along the trajectory, and the mean instant speed and mean instant angle of the trajectories were identified as statistically significant by Mann–Whitney and Student’s tests. The measured area and perimeter of the detected colonies differed, on average, for different phenotypes throughout the entire time period under consideration. This result confirms previous findings obtained by analyzing static images.

Role of trace minerals in cow’s reproductive function and performance: a clinical theriogenology perspective*

Trace minerals (TM) have a crucial role in cattle reproduction. Although microelements are required in small amounts, their bioavailability is essential for the cow reproductive physiology, and for adequate fertility and productivity. They are particularly important for antioxidant protection against cellular damage (e.g. gametes and embryonic cells), hormone synthesis, and pregnancy maintenance. Oral TM supplementation is a common and highly recommended management practice in cattle operations. However, there is substantial variability in TM bioavailability in animals receiving oral TM supplementation. The strategic use of injectable TM supplementation (without replacing traditional oral TM supplements) before episodes of marked stress (e.g. parturition), higher metabolic demand with TM depletion (e.g. last trimester of pregnancy), active immune response (e.g. uterine involution or vaccination), and before breeding helps to maintain adequate TM and oxidative status during critical points of the reproductive program. This manuscript reviews the research-based evidence regarding the effects of TM supplementation on bovine reproduction and its impact on beef and dairy cattle reproductive performance.

Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

Bone Morphogenetic Protein (BMP) signaling is essential for craniofacial development, though little is known about the mechanisms that govern BMP secretion. We show that depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme. We show endogenous transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day 9.5 to 13.5 mice. Disruption of a potassium channel called Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Kcnj2 knockout mice have cleft palate and reduced BMP signaling. Our data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and intracellular calcium for mammalian craniofacial morphogenesis.

Abstract 4136861: Title: Protein interaction profiling of the Kv11.1 potassium channel reveals new therapeutic targets for Long QT Syndrome

Background: The voltage-gated potassium ion channel K V 11.1 plays a crucial role in cardiac repolarization. Genetic variants in K V 11.1 lead to Long QT Syndrome (LQTS), a condition associated with fatal arrhythmias. Approximately 90% of missense variants linked to LQTS cause intracellular protein transport (trafficking) dysfunction. Some K V 11.1 channel blockers act as chemical chaperones (e.g., E-4031) and increase K V 11.1 channel trafficking. However, these drugs cannot be used in LQTS patients. The underlying mechanisms, such as the protein networks involved in folding and quality control for K V 11.1 channel trafficking remain largely unexplored. Methods: We used affinity-purification (e.g., coimmunoprecipitation) coupled with mass spectrometry to quantify protein interaction changes in human embryonic kidney cells expressing wild-type K V 11.1 or two trafficking-deficient channel variants in the presence or absence of chemical chaperone E-4031. After co-immunoprecipitations and protein digestions, peptides were identified using multiplexed mass spectrometry. Results: We identified 572 protein interactions enriched in K V 11.1-expressing cells. We used bioinformatic analysis to make a cellular model showcasing likely K V 11.1 protein interactions localization from early biogenesis and transcription to plasma membrane expression (Figure 1). We revealed proteins responsible for protein folding, translation, proteasomal degradation, and others with most protein interactions significantly elevated in the genetic variants compared to wild-type (Figure 2). Upon 24-hour treatment with E-4031, some of these interactions were reduced towards wild-type and indicated potential molecular targets for therapeutic intervention (Figure 3). Conclusions: We report the discovery of novel K V 11.1 protein-protein interactions that could be therapeutically targeted to improve K V 11.1 trafficking and treat Long QT Syndrome.

Analyses of Off-Target Effects on Cardiac and Skeletal Muscles by Berberine, a Drug Used to Treat Cancers and Induce Weight Loss.

Previous reports from our laboratory describing the formation of myofibrils in cultured embryonic cardiac and skeletal muscle cells have proposed that myofibrillogenesis occurs in three steps of increasing protein organization: beginning with premyofibrils, followed by nascent myofibrils, and ending in mature myofibrils. Inhibitors of the ubiquitin proteasome system (UPS) prevented nascent myofibrils from progressing directly to mature myofibrils in cultured cardiac and skeletal muscle cells, supporting a three-step model of assembly in which some of the proteins in nascent myofibrils are proteolyzed to allow the assembly of mature myofibrils. Application of UPS inhibitors on cultured muscle cells suggests possible explanations for the off-target cardiac and skeletal muscle adverse effects of UPS drugs, which are used on cancer patients. Berberine, a plant derivative, has been used to treat various cancers, including multiple myelomas. In contrast to the use of UPS drugs, success was reported with Berberine in multiple myeloma patients with no off-target effects on their hearts. We have exposed cultured cardiac and skeletal muscle cells to Berberine, a ligase inhibitor of UHRF1 (ubiquitin-like with PHD and RING finger domains). Berberine inhibited myofibril assembly at the nascent myofibril stage in embryonic skeletal muscle cells but had no effect in the assembly of mature myofibrils in embryonic heart cells. RT-PCR experiments demonstrated Berberine inhibition of mRNA for muscle myosin II heavy chains but not for muscle actin mRNA in skeletal muscle cells. Berberine is also being used as a popular weight losing compound, because it is much cheaper and available without a prescription than the semaglutide containing weight losing drugs (Wegovy and Ozempic). In contrast to Berberine, semaglutide had no effects on myofibril assembly in culture assays for both cardiac and skeletal muscle cells. We postulate that analyses of cultured embryonic cardiac and skeletal muscle cells will provide a preclinical assay for the testing of novel cancer drugs with improved outcomes for patients, an important goal for cancer therapeutics.

The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability.

The spatial and temporal linear expression of Hox genes establishes a regional Hox code, which is crucial for the antero-posterior (A-P) patterning, segmentation, and neuronal circuit development of the hindbrain. RNF220, an E3 ubiquitin ligase, is widely involved in neural development via targeting of multiple substrates. Here, we found that the expression of Hox genes in the pons was markedly up-regulated at the late developmental stage (post-embryonic day E15.5) in Rnf220-/- and Rnf220+/- mouse embryos. Single-nucleus RNA sequencing (RNA-seq) analysis revealed different Hox de-repression profiles in different groups of neurons, including the pontine nuclei (PN). The Hox pattern was disrupted and the neural circuits were affected in the PN of Rnf220+/- mice. We showed that this phenomenon was mediated by WDR5, a key component of the TrxG complex, which can be polyubiquitinated and degraded by RNF220. Intrauterine injection of WDR5 inhibitor (WDR5-IN-4) and genetic ablation of Wdr5 in Rnf220+/- mice largely recovered the de-repressed Hox expression pattern in the hindbrain. In P19 embryonal carcinoma cells, the retinoic acid-induced Hox expression was further stimulated by Rnf220 knockdown, which can also be rescued by Wdr5 knockdown. In short, our data suggest a new role of RNF220/WDR5 in Hox pattern maintenance and pons development in mice.

CSIG-33. CERAMIDE-1-PHOSPHATE/CERK ARE REGULATORS OF ETMR GROWTH AND POTENTIAL THERAPEUTIC TARGETS

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm which occurs almost exclusively in infants and is associated with an extremely poor prognosis. To identify functional signaling lipids that contribute to the aggressive nature of ETMR and that can be targeted therapeutically, we carried out mass spectrometry imaging on human ETMR patient samples, the patient-derived cell line BT183, and normal neural stem cells. We identified an accumulation of ceramide-1-photphate (C1P) within the rapidly proliferating embryonal tumor cells in patient samples and the cell model. We also detected high accumulation of C1P within the aberrant vascular proliferations in patient samples. Ceramide-1-phosphate is a pleiotropic bioactive signaling lipid that controls cell fate. C1P and its synthesizing enzyme, ceramide kinase (CERK), are known mitogens that have been shown to regulate cell proliferation in several non-CNS cancers. C1P is known to mediate the upregulation of PI3K/Akt/mTOR, MAPK/MEK/ERK1/2, Rho kinase, JNK, to promote cell survival and migration. After spatially mapping C1P accumulation in patient samples and our in vitro 3D models, we next carried out IHC to correlate lipid accumulation with its synthesizing enzyme, CERK. CERK was found to be abundantly accumulated in both endothelial and tumor cells within the tumor microenvironment of patient samples. Western blot analysis of CERK demonstrated a significant increase in the ETMR 3D tumorspheres compared to the NSC neurospheres. Treatment of the ETMR 3D tumorspheres with the CERK inhibitor, NVP-231, potently reduced cell growth and viability. Western blot analysis of ETMR tumorspheres at 8 and 24 hours post-treatment demonstrated a time-dependent decrease in N-MYC and a subsequent increase in p53. Studies are ongoing to validate these findings in vivo and to elucidate the signaling pathways that C1P/CERK regulate in both the vascular and tumor cells in ETMR.

An Efficient and Stable Polarizing Agent for In-Cell Magic-Angle Spinning Dynamic Nuclear Polarization NMR Spectroscopy.

Nuclear Magnetic Resonance (NMR) spectroscopy would be a method of choice to follow biochemical events in cells because it can analyze molecules in complex environments. However, the intrinsically low sensitivity of NMR makes in-cell measurements challenging. Dynamic Nuclear Polarization (DNP) has emerged as a method to circumvent this limitation, but most polarizing agents developed for DNP are unstable in reducing cellular environments. Here, we introduce the use of Gd(III)-based DNP polarizing agents for in-cell NMR spectroscopy. Specifically, we show their persistent stability in cellular formulations, and we investigate the DNP performance of the Gd(III)-based complex [Gd(tpatcn)] in human embryonic kidney cell lysates and intact cells. For cell lysates, DNP enhancements up to -27 are obtained on the cellular signals, reproducible even after storage at room temperature for days. Mixing the [Gd(tpatcn)] solution with intact cells enables the observation of cellular signals with DNP, and DNP enhancement factors of about -40 are achieved.

The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

The spatial and temporal linear expression of Hox genes establishes a regional Hox code, which is crucial for the antero-posterior (A-P) patterning, segmentation, and neuronal circuit development of the hindbrain. RNF220, an E3 ubiquitin ligase, is widely involved in neural development via targeting of multiple substrates. Here, we found that the expression of Hox genes in the pons was markedly up-regulated at the late developmental stage (post-embryonic day E15.5) in Rnf220-/- and Rnf220+/- mouse embryos. Single-nucleus RNA sequencing (RNA-seq) analysis revealed different Hox de-repression profiles in different groups of neurons, including the pontine nuclei (PN). The Hox pattern was disrupted and the neural circuits were affected in the PN of Rnf220+/- mice. We showed that this phenomenon was mediated by WDR5, a key component of the TrxG complex, which can be polyubiquitinated and degraded by RNF220. Intrauterine injection of WDR5 inhibitor (WDR5-IN-4) and genetic ablation of Wdr5 in Rnf220+/- mice largely recovered the de-repressed Hox expression pattern in the hindbrain. In P19 embryonal carcinoma cells, the retinoic acid-induced Hox expression was further stimulated by Rnf220 knockdown, which can also be rescued by Wdr5 knockdown. In short, our data suggest a new role of RNF220/WDR5 in Hox pattern maintenance and pons development in mice.

Generation of a novel constitutive smooth muscle cell-specific Myh11 -driven Cre mouse model.

Dysfunction in either embryonic or postnatal vascular smooth muscle cells (SMCs) significantly contributes to the progression of various cardiovascular diseases. Therefore, elucidating the molecular mechanisms governing VSMC development and homeostasis is crucial. MYH11 is the most reliable lineage gene for SMCs and has been utilized to develop tamoxifen-inducible Cre driver lines for achieving SMC-specific gene manipulation by crossing with mice carrying the lox P -flanked gene, particularly in adult mice. For studies involving SMCs during embryogenesis, the commonly used constitutive Cre driver is controlled by the Tagln ( Sm22α ) promoter. However, this Cre driver exhibits activity in multiple non-SMC populations, including cardiomyocytes and skeletal muscle precursors, introducing confounding effects. Additionally, most existing SMC-specific Cre drivers are generated using a transgenic approach, raising concerns about random site integration and variable gene copy numbers. To address these limitations, we report a novel Cre mouse model generated by knock-in (KI) of a nuclear-localized Cre recombinase into the Myh11 gene locus using homologous recombination. We confirmed that the Cre activity precisely recapitulates endogenous Myh11 expression by crossing with Rosa26 mTmG or tdTomato reporter mice. Moreover, Myh11 -driven Cre can efficiently delete the floxed allele of the transcription factor Tead1 specifically in SMCs. The Tead1 SMC-specific knockout mice did not exhibit an overt phenotype, thereby circumventing the embryonic lethal phenotype mediated by Tagln -driven Cre, as we previously reported. These findings establish this novel Cre driver line as a robust tool for tracing the Myh11 -positive SMC lineage and manipulating gene function specifically in SMCs during embryonic development in mice.

Quantitative Analysis of Deer Bone Hydroethanolic Extract Using Label-Free Proteomics: Investigating Its Safety and Promoting Effect on Mouse Embryonic Osteoblastic Progenitor Cell Proliferation.

Deer bone is rich in proteins and free amino acids, offering high nutritional value and benefits such as strengthening bones and antioxidant properties. However, the development and utilization of deer bone resources are limited, and the safety evaluation of health foods is incomplete. We established a hydrogen ethanol extraction method for deer bone and analyzed the components of the deer bone hydroethanolic extract (DBHE) using liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography-mass spectrometry (GC-MS), and inductively coupled plasma mass spectrometry (ICP-MS). Using Label-free proteomics technology, we identified 69 proteins and 181 peptides. We also quantified 16 amino acids, 22 fatty acids, and 17 inorganic elements. Finally, we evaluated the safety of DBHE both in vitro and in vivo. The results indicated that DBHE did not exhibit any toxic effects at the doses we tested and can promote the proliferation of mouse embryonic osteoblastic progenitor cells (MC3T3-E1), demonstrating potential efficacy against osteoporosis and arthritis. This study provides a theoretical basis for the quality control, processing, and resource development of deer bone.

Sonodynamic Cancer Therapy by Mn(I)-tricarbonyl Complexes via Ultrasound-triggered CO Release and ROS Generation.

A novel ferrocene conjugated Mn(I)-tricarbonyl complex viz [Mn(Fc-tpy)(CO)3Br] (Mn2) where, Fc-tpy=4'-ferrocenyl-2,2':6',2''-terpyridine was synthesized and fully characterized along with its non-ferrocene analog [Mn(Ph-tpy)(CO)3Br] Ph-tpy=4'-phenyl-2,2':6',2''-terpyridine (Mn1) for ultrasound (US) activated anticancer applications. The X-ray structure of Mn2 confirmed its distorted octahedral geometry. Mn1 and Mn2, for the first time, showed US-triggered release of CO and ROS generation (1O2 and ⋅OH) in an aqueous solution from any Mn(I)-tricarbonyl complexes, indicating its potential for synergetic CO gas therapy and sonodynamic therapy. The above-mentioned in-solution chemistry was successfully translated into in vitro cellular models. These complexes showed unprecedented US-triggered toxicity against T-cell lymphoma and human breast cancer cells (IC50 for Mn2<1 μM) while were minimally toxic without US or against normal spleen and human embryonic kidney cells. Mn2 was ca. 12 fold more anticancer active than Mn1, indicating that the ferrocene conjugation augmented the US sensitivity. The apoptotic sonotoxicity of Mn2 was due to US-promoted mitochondrial depolarization via ROS generation and CO release. The apoptosis was triggered by caspase 3 activation. This is the first report of Mn(I)-tricarbonyl-based sonosensitizers for cancer SDT. Overall, this study, for the first time, establishes the effectiveness of 3d metal carbonyls in SDT.

Stem cell transplantation extends the reproductive life span of naturally aging cynomolgus monkeys

The ovary is crucial for female reproduction and health, as it generates oocytes and secretes sex hormones. Transplantation of mesenchymal stem cells (MSCs) has been shown to alleviate pathological ovarian aging. However, it is unclear whether MSCs could benefit the naturally aging ovary. In this study, we first examined the dynamics of ovarian reserve of Chinese women during perimenopause. Using a naturally aging cynomolgus monkey (Macaca fascicularis) model, we found that transplanting human embryonic stem cells-derived MSC-like cells, which we called M cells, into the aging ovaries significantly decreased ovarian fibrosis and DNA damage, enhanced secretion of sex hormones and improved fertility. Encouragingly, a healthy baby monkey was born after M-cell transplantation. Moreover, single-cell RNA sequencing analysis and in vitro functional validation suggested that apoptosis, oxidative damage, inflammation, and fibrosis were mitigated in granulosa cells and stromal cells following M-cell transplantation. Altogether, these findings demonstrate the beneficial effects of M-cell transplantation on aging ovaries and expand our understanding of the molecular mechanisms underlying ovarian aging and stem cell-based alleviation of this process.

Fish Cell Spheroids, a Promising In Vitro Model to Mimic In Vivo Research: A Review.

In vitro cell culture systems serve as instrumental platforms for probing biological phenomena and elucidating intricate cellular mechanisms. These systems afford researchers the opportunity to scrutinize cellular responses within a regulated environment, thereby circumventing the ethical and logistical challenges associated with in vivo experimentation. Three-dimensional (3D) cell cultures have emerged as a viable alternative to mimic in vivo environments. Within this context, spheroids are recognized as one of the most straightforward and efficacious models, presenting a promising substitute for conventional monolayer cultures. The application of 3D cultures of fish cells remains limited, focusing mainly on physiological and morphological characterization studies. However, given the capacity of spheroids to emulate in vivo conditions, researchers are exploring diverse applications of these 3D cultures. These include eco-toxicology, immunology, drug screening, endocrinology, and metabolism studies, employing a variety of cell types such as fibroblasts, hepatocytes, embryonic cells, gonadal cells, gastrointestinal cells, and pituitary cells. This review provides a succinct overview, concentrating on the most frequently employed methods for generating fish cell spheroids and their applications to date. The aim is to compile and highlight the significant contributions of these methods to the field and their potential for future research.

Female germline expression of OVO transcription factor bridges Drosophila generations.

OVO is required for female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1 allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo has several unannotated splice variations in the extended exon and a minor population of ovo-B transcripts have an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability and there is an early and continual developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.