Differentiation Marker Genes Research Articles

Pb inhibited C2C12 myoblast differentiation by regulating HDAC2

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Lead (Pb) exposure impaired the development and the health of bones, which slows the growth of children. However, it is far from clear what exactly the effects of Pb on skeletal muscle development are. In this study, C2C12 cells are commonly used as an in vitro model of muscle regeneration due to their ability to transition from a proliferative phase into differentiated myofibers. The dose of 1, 5, and 10 μM Pb were adopted to study the toxicity of Pb on C2C12 proliferation and differentiation. First, the effects of Pb on cell viability were detected and the results demonstrated that 5 μM and 10 μM Pb exposure decreased cell viability, while 1 μM Pb exposure has no obvious effects on cell viability. Then, 1–10 μM Pb exposure seriously reduced the C2C12 myoblasts differentiation, with the decrease of myogenic differentiation marker genes expression, including Muscle creatine kinase (MCK), Myosin Heavy Chain 4 (MYH4), Myogenin (MYOG), Myogenic Differentiation (MYOD). What’s more, it was found that the epigenetic modifier histone deacetylase-2 (HDAC2) was upregulated after Pb exposure on C2C12 myoblasts. Further studies conclusively showed knockdown of HDAC2 ameliorated Pb-damaged C2C12 myoblasts differentiation, indicating HDAC2 plays a vital role in the Pb-induced C2C12 myoblasts differentiation deficits. In summary, these results demonstrated that Pb exposure inhibited C2C12 myoblasts differentiation by regulating HDAC2.

Climate change and management intensity alter spatial distribution and abundance of P mineralizing bacteria and arbuscular mycorrhizal fungi in mountainous grassland soils

In mountainous grasslands management adaptations are required to maintain soil functions. We investigated climate change (CC) and management effects on the abundance and potential activity of microbiota catalyzing the major steps of P transformation which are still unknown in these grasslands.Soil samples were taken from intact plant-soil mesocosms managed extensively or intensively (two vs. five mowing and slurry applications, respectively). These mesocosms were previously translocated from high to lower altitudes to simulate two CC scenarios (CC1: +1 °C warming and mean annual precipitation (MAP) of 1347 mm and CC2: +3 °C warming and MAP of 956 mm), while control mesocosms (CC0) were relocated at their original site (6 °C and MAP of 1400 mm). Specific marker genes for P-solubilization (gcd), P-mineralization (phoN, phoD, phnX and appA), P-uptake (pitA and pstS), total bacteria and arbuscular mycorrhizal fungi (AMF) were quantified by quantitative real-time PCR. Spatial distributions of phosphatase activities were analyzed in situ by zymography analysis and total organic C, N and P contents were measured.Gene abundances and enzymatic activities were comparable for both managements under CC0, except for phytase-harboring (appA) microbiota which decreased under intensive management. The abundance of microbiota which catalyzes organic P (Po) mineralization (phoN and appA) and those harboring quinoprotein glucose dehydrogenase (gcd) for P solubilization significantly dropped by interacting effects of CC2 and extensive management. The same effect was found for microbes harboring specific P transporters (pitA and pstS). Under intensive management, microbiota catalyzing Po mineralization (phoN and appA), and alkaline phosphatase activities tended to increase in CC2. Noteworthy, the AMF abundance was reduced at 0–5 cm soil depth under CC. Our results suggest that CC and extensive management reduced microbial P solubilization, mineralization and uptake, while management intensification may increase P availability, which leads to shifts in nutrient stoichiometry and decreased AMF abundance.

Hidden risks: Simulated leakage of domestic sewage to secondary water supply systems poses serious microbiological risks

There are continuous reports about the pollution of the secondary water supply systems (SWSSs), among which domestic sewage leakage is the most serious. In this study, a pilot experiment lasting 70 days was conducted to explore the changes in physicochemical water quality and the microbial profiles in SWSSs polluted by different doses of domestic sewage through qPCR and high-throughput sequencing methods. The results showed that when domestic sewage entered the simulated water storage tank, a large amount of organic matter brought by domestic sewage quickly consumed chlorine disinfectants. High pollution levels (pollution index ≥ 1/1000) were accompanied by significant increases in turbidity and ammonia nitrogen concentration (p < 0.05) and by abnormal changes in sensory properties. Although different microbial community structures were found only at high pollution levels, qPCR results showed that the abundance of the bacterial 16S rRNA gene and some pathogenic gene markers in the polluted tank increased with the pollution level, and the specific gene marker of pathogens could be detected even at imperceptible pollution levels. In particular, the high detection frequency and abundance of Escherichia coli and Enterococcus faecails in polluted tank water samples demonstrated that they can be used for early warning. Moreover, it seems that the microorganisms that came with the domestic sewage lost their cultivability soon after entering SWSSs but could recover their activities during stagnation. In addition, the biofilm biomass in the polluted tank with high pollution levels increased faster at the initial stage, while after a longer contact time, it tended to remain at the same level as the control tank. This study emphasized the high microbial risk introduced by sewage water leakage even at imperceptible levels and could provide scientific suggestions for early warning and prevention of pollution to SWSSs.

Atp6i deficient mouse model uncovers transforming growth factor-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i−/−) arrested tooth root formation, indicated by truncated Hertwig’s epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i−/− mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i−/− mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-β (TGF-β) signaling in Atp6i−/− odontoblasts. Through treating pulp precursor cells with Atp6i−/− or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i−/− OC conditioned medium. Importantly, ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i−/− mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

FOXO1 regulates the formation of bovine fat by targeting CD36 and STEAP4

Intramuscular fat content is closely related to the quality of beef, where the forkhead box protein O1 (FOXO1) is involved in adipocyte differentiation and lipid metabolism, but the specific mechanism of its involvement is still unclear. In this study, interfering with FOXO1 promoted the G1/S transformation of bovine adipocytes by enhancing the expression of proliferation marker genes PCNA, CDK1, CDK2, CCNA2, CCNB1, and CCNE2, thereby positively regulating the proliferation of bovine adipocytes. Additionally, interfering with FOXO1 negatively regulated the expression of adipogenic differentiation marker genes PPARG and CEBPA, as well as lipid anabolism marker genes ACC, FASN, SCD1, SREBP1, FABP4, ACSL1, LPL, and DGAT1, thus reducing triglyceride (TG) content and inhibiting the generation of lipid droplets in bovine adipocytes. A combination of transcriptomic and metabolomics analyses revealed that FOXO1 could regulate the lipogenesis of cattle by influencing the AMPK and PI3K/AKT pathways. Importantly, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed that FOXO1 could regulate bovine lipogenesis by binding to the promoter regions of the CD36 and STEAP4 genes and affecting their transcriptional activities. These results provide a foundation for studying the role and molecular mechanism of FOXO1 in the bovine adipogenesis.

Fabrication of a Smart Fibrous Biomaterial That Harbors an Active TGF-β1 Peptide: A Promising Approach for Cartilage Regeneration.

The regeneration of articular cartilage remains a serious problem in various pathological conditions such as osteoarthritis, due to the tissue's low self-healing capacity. The latest therapeutic approaches focus on the construction of biomaterials that induce cartilage repair. This research describes the design, synthesis, and investigation of a safe, "smart", fibrous scaffold containing a genetically incorporated active peptide for chondrogenic induction. While possessing specific sequences and the respective mechanical properties from natural fibrous proteins, the fibers also incorporate a Transforming Growth Factor-β1 (TGF-β1)-derived peptide (YYVGRKPK) that can promote chondrogenesis. The scaffold formed stable porous networks with shear-thinning properties at 37 °C, as shown by SEM imaging and rheological characterization, and were proven to be non-toxic to human dental pulp stem cells (hDPSCs). Its chondrogenic capacity was evidenced by a strong increase in the expression of specific chondrogenesis gene markers SOX9, COL2, ACAN, TGFBR1A, and TGFBR2 in cells cultured on "scaffold-TGFβ1" for 21 days and by increased phosphorylation of intracellular signaling proteins Smad-2 and Erk-1/2. Additionally, intense staining of glycosaminoglycans was observed in these cells. According to our results, "scaffold-TGFβ1" is proposed for clinical studies as a safe, injectable treatment for cartilage degeneration.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/β-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

MiR-188-5p regulates the proliferation and differentiation of goat skeletal muscle satellite cells by targeting calcium/calmodulin dependent protein kinase II beta.

The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

P-506 Administration of low molecular weight heparin alleviate immune physiology in recurrent implantation failure modulating CCL2-CCR2 axis during frozen embryo transfer: effort from clinic to bench

Abstract Study question Does low molecular-weight heparin (LMWH) administration modulate CCL2-CCR2 axis in recurrent implantation failure (RIF) patients undergoing frozen embryo transfer (FET) to improve pregnancy salvage? Summary answer LMWH treatment attests beneficial immunological tolerance in vivo and in vitro through modulation of CCL2-CCR2 axis suggesting possible restoration of weak pro-inflammatory response in RIF. What is known already Immune imbalance in proportion of activated macrophages (M1) and alternatively activated macrophages (M2) impede endometrial receptivity and contribute to RIF. CCL2 (M2-asscociated) - CCR2 (t-helper-2 associated) axis orders macrophage polarization by influencing expression of functionally relevant and polarization associated genes and down-modulate pro-inflammatory cytokine production. Immunological effects of LMWH regulate pregnancy-promoting processes at foetal-maternal interface to provide an appropriate implantation process; outcomes being controversial. We aim to investigate the potential immunoregulatory properties of LMWH in patients with RIF undergoing FET, with particular regard to effects on macrophages and T-helper (Th) cells; two key players in promoting immune tolerance during pregnancy. Study design, size, duration This prospective cohort study constituted 121 women with RIF (≥3 failed embryo transfer) undergoing FET at Institute of Reproductive Medicine between January to October 2022. An incremental dose (4-12mg/day) of estradiol with (Group A; n = 62) or without (Group B; n = 59) LMWH (40 mg/day) from d2 to d12 with sequential ultrasound following intra-muscular progesterone (100mg/day) for 4 days was administered in all participants. Blood samples were collected to isolate plasma and peripheral-blood-mononuclear-cells (PBMCs). Participants/materials, setting, methods Monocytes were purified from PBMCs and cultured with GM-CSF or M-CSF to analyze factor/s which control CCL2-CCR2 expression along macrophage polarization by flow cytometry. Influence of CCL2 on GM-CSF-cultured macrophages (GM-MØ) and M-CSF-cultured macrophages (M-MØ) specific gene markers was analysed using quantitative RT-PCR (qRT-PCR). To assess immunological effects of LMWH in-vivo, Th-specific cytokine profile/s and respective chemokines were measured from plasma by Luminex xMAP technology. Statistical significance was set at p &amp;lt; 0.05. Main results and the role of chance The clinical pregnancy rate was higher in group A. (A vs B: 37.09% vs. 27.11%, p = 0.24). LMWH exposure significantly decreased the secretion of CCL2 (p &amp;lt; 0.01) and CCL22 (p &amp;lt; 0.001) in GM-CSF-cultured macrophages, while it increased CCL2, CCL20 production (p &amp;lt; 0.001) in both GM-MØ and M-MØ. However, expression of CCL7 was significantly lower (p &amp;lt; 0.01) in M-MØ and GM-MØ generated from CD14+ CD16+ monocytes and CD14 ++ CD16- monocytes respectively under LMWH exposure. qPCR analysis revealed a significant increase (p &amp;lt; 0.01) in the expression of various GM-MØ–specific genes including EGLN3, and SERPINE1 in macrophage supernatant post LMWH treatment. Expression of various M-MØ-specific marker/s such as IGF1, FOLR2, and HTR2B, is significantly up-regulated (p &amp;lt; 0.01) in Group B. Group A, although documented a decreased concentration/s (pg/ml) of Th17-type cytokine/s (IL-6 (1.88 ± 0.65 vs. 0.91 ± 0.43; p &amp;lt; 0.04 and IL-23 (17.94 ± 9.76 vs. 7.78 ± 3.43; p &amp;lt; 0.01)), at end of treatment, concentration of TGF-β was significantly higher (3909.05 ± 1248.35 vs. 2469.83 ± 1058.71; p &amp;lt; 0.01) which cue to probable maintenance in balance of inflammatory response at the time of implantation. No differences were observed in Th1-type cytokine level/s (IFN-γ and TNF-α). CXCL10, CXCL11, CXCL1, CXCL8, CCL17, CCL22 do not differ in vivo. Limitations, reasons for caution Beneficial effects of LMWH administration with a good sample size are clearly needed to confirm this hypothesis; hence result/s should be dealt with caution. Further to this, inclusion of other arm/s (corticosteroids, intralipid and/or intravenous immunoglobulin) will make the study much robust and conclusive. Wider implications of the findings The identification of atypical Th1-associated pro-inflammatory effects of LMWH drives to evaluate modulation of CCL2 expression to limit or potentiate macrophage activation in RIF; especially where anticoagulant effect is not the primary goal. Further, modification of cytokine expression/s might potentiate novel strategy derived from deregulated macrophage polarization. Trial registration number Not Applicable

#4362 A DISTINCT TRANSCRIPTOME SIGNATURE IN HUMAN RENAL SINUS ADIPOSE TISSUE – A POTENTIAL IMPORTANCE FOR RENAL DYSFUNCTION?

Abstract Background and Aims Previous studies have suggested that the anatomical location and function of adipose tissue (AT) is of relevance for normal as well as impaired renal function. Renal sinus AT (RSAT) is a perivascular fat compartment located around the renal arteries. The aim of this study was to perform a transcriptomic characterization of RS in comparison with omental (OAT) and subcutaneous (SAT) AT in healthy individuals. Method 30 kidney donors (15 men (50%), age 50 ± 11 years, BMI 25.8 ± 2.9 kg/m2, eGFR 83 ± 9 mL/min/1.73 m2, HOMA-IR 2.5 ± 1.1) were included in the study. RS, OAT and SAT biopsies were performed during laparoscopic unilateral nephrectomy. RNA-sequencing analyses and untargeted transcriptomics were performed. Analysis of differentially expressed genes (DEG) was employed to discover quantitative changes in expression levels between two tissue depots (RSAT) vs OAT and SAT, respectively. Gene Ontology (GO) term enrichment was used to summarize the function of genes that were differentially expressed. Results Paired samples of renal sinus fat (RSAT) to be compared with omental (OAT) or subcutaneous adipose tissue (SAT) were available in 26 and 22 subjects, respectively. Out of 12210 genes characterised in these AT depots, 463 genes (3,7%) were uniquely expressed in RS when compared to both OAT and SAT (co-expression Venn-diagram in figure 1). In addition, DEG analyses showed that 1639 genes had higher, whereas 384 had lower, expression in RS than in OAT. For RSAT versus SAT the corresponding numbers were 3789 and 2534, respectively. Among the gene that were differentially regulated, the following pathways were most significantly enhanced in RSAT compared to OAT or SAT (figure 2): chemotaxis and cell migration (e.g. cell and leukocyte chemotaxis and leukocyte migration), and immune response (e.g. T-cell activation, immune response-activating signal transduction, adaptive immune response). Conversely, ATP synthesis (e.g. oxidative phosphorylation, cellular respiration, mitochondrial complex) and extracellular matrix pathways were significantly attenuated in RS compared to SAT and OAT, respectively (not shown). A targeted analysis of specific gene markers that have previously been reported to be associated with chronic kidney disease (CKD) showed that AT expression of pro-inflammatory factors such as visfatin, IL-6, IL-11, IL-1β and IL-18 were higher in RSAT vs SAT or OAT (not shown). In addition, the expression of some genes, differently regulated in RSAT, and known to associated with progression of CKD, e.g. Fc Gamma Receptor IIa (FCGR2A), NF-kappa-B inhibitor beta (NFKBIB), Nuclear Factor, Erythroid 2 (NFE2) and Oncostatin M (OSM), were positively correlated with BMI (data not shown). Conclusion This study in healthy kidney donors indicates that the transcriptome signature of renal sinus AT seems to be distinct from subcutaneous and omental AT. Gene ontology analyses pointed towards an inflammatory environment and lower mitochondrial function in the renal sinus compared to other adipose tissue depots. These findings are likely to define depot-specific AT functions that may be of importance for renal function and disease.

Abstract A069: Microscale modeling of the human prostate bone metastatic niche

Abstract Difficulties in modeling the human bone metastasis microenvironment (BMME) niche contribute to limitations in our understanding of the cellular interactions that promote treatment resistance in prostate cancer. To address this, we have developed a multicellular, vascularized, and fully humanized 3D tissue chip model of bone metastases that will allow us to identify drivers of treatment resistance and test emerging cancer therapies in the context of multicellular networks. We utilized LumeNEXT, a closed microfluidics platform with a removable center rod allowing vessel formation, to create this bone microenvironment. To obtain major stromal and bone cell types found in the BMME, we differentiated primary bone marrow-derived mesenchymal stem cells (MSCs) into osteoblasts, fibroblasts, and adipocytes and differentiated patient-derived monocytes from peripheral blood into M2-polarized macrophages and osteoclasts. The primary stromal and bone cells were seeded into LumeNEXT devices and combined in co-cultured with primary prostate cancer tumor organoids in a collagen-based matrix. and deposited into LumeNEXT devices. After polymerization of the hydrogel, the center rod in each device was removed and the tunnels were seeded with endothelial cells to create functional microvasculature. We successfully differentiated the patient-derived cell types, optimized a novel multi-phenotype media (MPM) for co-culture, and maintained greater than 80% viability of all seven cell types in co-culture for at least 14 days in the LumeNEXT platform. qPCR analysis of the model confirmed that expression of differentiation marker genes was maintained after 14 days of co-culture for all cell types. Viability assays and time-lapse microscopy of cell tracker staining also showed viability and growth of prostate cancer organoids in co-culture. Integrated multi-analyte assay platforms were developed for cellular and molecular analysis of patient-derived primary tumor organoids including assessment of cellular phenotype, transcriptomic and epigenetic patterns in the context of multicellular networks. Additionally, we have aimed to develop assays to test emerging cancer-targeting therapies, such as antibody-drug conjugate (ADC). Functional testing of this model using ADC has demonstrated drug perfusion into the stroma through the microvessels, and ADC-specific tumor killing. In summary, we created a multicellular microfluidics model of human bone metastases. High viability of all seven cell types included in our model was maintained for 14 days and early ADC testing demonstrates functional microvasculature and tumor cell killing. The duration of viable co-culture multicellular cultures and promising functional testing demonstrates the suitability of this model for rapid drug testing. Future studies will focus on testing novel chemo-, hormone, and radiation therapies in this multicellular BMME model to determine mechanisms of microenvironment-mediated treatment resistance in prostate cancer. Citation Format: Adeline Ding, Cristina Sanchez de Diego, Sheena C. Kerr, Ravi Chandra Yada, Erika Heninger, Nan Sethakorn, Shannon Reese, Maria Virumbrales-Muñoz, Pete Geiger, Xavier Hazelberg, Joshua M. Lang, Dave Beebe. Microscale modeling of the human prostate bone metastatic niche [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A069.

Hmmr Acts as a Key Regulator in the ADSCs Proliferation and Mitosis.

Obesity is a common chronic health problem that requires lifelong efforts for the successful treatment. The proliferation of ADSCs is an essential step in the development of obesity. Identifying key regulators of ADSCs will be a novel strategy for adipogenesis inhibition and obesity prevention. In this study, transcriptomes of 15532 ADSCs were firstly profiled by single cell RNA-sequencing. On the basis of gene expression patterns, 15 cell subpopulations (six defined cell types) were distinguished. A subpopulation was identified as CD168+ ADSCs, and it was demonstrated to play a vital role in ADSCs proliferation. Furthermore, Hmmr, a specific marker gene of CD168+ ADSCs was found to be a critical gene associated with ADSCs proliferation and mitosis. Hmmr knockout resulted that ADSCs growth nearly arrested and aberrant nuclear division occurred. Finally, it was revealed that Hmmr promoted ADSCs proliferation through the extracellular signal-regulated kinase 1/2 signaling pathway. This study identified Hmmr as a key regulator in ADSCs proliferation and mitosis, and suggested that Hmmr may be a novel target for obesity prevention.

Gene expression variations and allele-specific expression of two rice and their hybrid in caryopses at single-nucleus resolution.

Seeds are an indispensable part of the flowering plant life cycle and a critical determinant of agricultural production. Distinct differences in the anatomy and morphology of seeds separate monocots and dicots. Although some progress has been made with respect to understanding seed development in Arabidopsis, the transcriptomic features of monocotyledon seeds at the cellular level are much less understood. Since most important cereal crops, such as rice, maize, and wheat, are monocots, it is essential to study transcriptional differentiation and heterogeneity during seed development at a finer scale. Here, we present single-nucleus RNA sequencing (snRNA-seq) results of over three thousand nuclei from caryopses of the rice cultivars Nipponbare and 9311 and their intersubspecies F1 hybrid. A transcriptomics atlas that covers most of the cell types present during the early developmental stage of rice caryopses was successfully constructed. Additionally, novel specific marker genes were identified for each nuclear cluster in the rice caryopsis. Moreover, with a focus on rice endosperm, the differentiation trajectory of endosperm subclusters was reconstructed to reveal the developmental process. Allele-specific expression (ASE) profiling in endosperm revealed 345 genes with ASE (ASEGs). Further pairwise comparisons of the differentially expressed genes (DEGs) in each endosperm cluster among the three rice samples demonstrated transcriptional divergence. Our research reveals differentiation in rice caryopsis from the single-nucleus perspective and provides valuable resources to facilitate clarification of the molecular mechanism underlying caryopsis development in rice and other monocots.

Risk prediction model construction for post myocardial infarction heart failure by blood immune B cells.

Myocardial infarction (MI) is a common cardiac condition with a high incidence of morbidity and mortality. Despite extensive medical treatment for MI, the development and outcomes of post-MI heart failure (HF) continue to be major factors contributing to poor post-MI prognosis. Currently, there are few predictors of post-MI heart failure. In this study, we re-examined single-cell RNA sequencing and bulk RNA sequencing datasets derived from the peripheral blood samples of patients with myocardial infarction, including patients who developed heart failure and those who did not develop heart failure after myocardial infarction. Using marker genes of the relevant cell subtypes, a signature was generated and validated using relevant bulk datasets and human blood samples. We identified a subtype of immune-activated B cells that distinguished post-MI HF patients from non-HF patients. Polymerase chain reaction was used to confirm these findings in independent cohorts. By combining the specific marker genes of B cell subtypes, we developed a prediction model of 13 markers that can predict the risk of HF in patients after myocardial infarction, providing new ideas and tools for clinical diagnosis and treatment. Sub-cluster B cells may play a significant role in post-MI HF. We found that the STING1, HSPB1, CCL5, ACTN1, and ITGB2 genes in patients with post-MI HF showed the same trend of increase as those without post-MI HF.

Cloning, identification and functional analysis of the goat transcription factor c-fos

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

Abstract 1693: Drug-tolerant persister cells contain heterogeneous subpopulations that utilize lung lineage pathways as drug resistance mechanism

Abstract Genomic discoveries of oncogenic driver alterations in lung cancer have led to development of effective target therapeutics; however, patients who initially respond to the therapy inevitably experience regrowth of the disease. Recent studies identified a reversible stage of cancer cells entering a quiescent state to ensure survival, called drug-tolerant persister (DTP) cells. The DTP stage is viewed as a major source of non-genetic drug resistance, while its epigenetic dynamic and regulatory mechanisms remain largely unknown. To determine the epigenetic activity reminiscent of a quiescent state during DTP development, we evaluated repressive epigenetic modifications during DTP progression and found that DTP cells had a markedly increased H3K27me3 level at the beginning of DTP stage. Treatment of EZH2 inhibitor together with target therapy synergistically inhibited cell growth, indicating the essential role of compressed chromatin structure for DTP survival. To further investigate the epigenomic heterogeneity of DTP cells, we performed single cell ATAC-seq on DTP cells. Unsupervised hierarchical clustering analysis revealed that the drug-induced DTP cells had two distinct populations compared with the untreated group with only one major population, suggesting an increased epigenomic heterogeneity. By analyzing the differentially accessible regions, motifs for TEADs and GATAs were specifically enriched in DTP cells, whose family members are well-established regulators of the lung development. This raised a possibility that the evolution of drug resistance shares similarities with organ regeneration, both require terminally differentiated cells regaining cell plasticity and serving as a progenitor cell to direct re-differentiation. Thus, we focused on alveolar lineage genes as a model to test our hypothesis that DTP cells hijack conserved developmental pathways for lung regeneration to ensure their survival and subsequent expansion. Our results showed that specific alveolar marker genes were transiently upregulated and exhibited translocation from cytoplasm to nuclei at early phase of DTP. Moreover, deletion of the alveolar lineage gene decreased cell re-growth under drug treatment. Our finding provides a new perspective of anti-relapse therapeutic strategies, that targeting lung lineage pathways to manipulate the activity of critical lineage factors during lung regeneration, for patients with minimal residual disease and relapse after target therapy. Citation Format: Yang Tian, Feng Jiang, Athina Jaffer, Benjamin Hopkins, Jian Jin, Hideo Watanabe. Drug-tolerant persister cells contain heterogeneous subpopulations that utilize lung lineage pathways as drug resistance mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1693.

LncRNA LINC00205 stimulates osteoporosis and contributes to spinal fracture through the regulation of the miR-26b-5p/KMT2C axis

BackgroundOsteoporosis (OP) is a common bone disease marked by decreased bone strength. Increasing evidence suggests that long non-coding RNA (lncRNAs) play important roles in the occurrence and progression of OP. This study aimed to investigate the role and mechanism of LINC00205 in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and OP.MethodsBone tissue samples were obtained from healthy controls and patients with osteoporosis with a spinal fracture (OP-Frx) or without a spinal fracture (OP-no-Frx). HMSCs were cultured and induced to undergo osteogenic differentiation. The expression of LINC00205, lysine (K)-specific methyltransferase 2C (KMT2C), and miR-26b-5p in bone tissues and cells was evaluated using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of LINC00205, miR-26b-5p, and KMT2C on calcium deposition, alkaline phosphatase (ALP) activity, and mRNA levels of the osteogenic differentiation marker genes [ALP, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2)] were investigated using alizarin red S staining, an ALP activity assay, and qRT-PCR, respectively. Dual-luciferase reporter assay was performed to ascertain the binding relationship between miR-26b-5p and LINC00205/KMT2C.ResultsLINC00205 and KMT2C were upregulated in patients with OP-Frx and OP-no-Frx, whereas miR-26b-5p was downregulated. Furthermore, LINC00205 and KMT2C expression decreased, whereas that of miR-26b-5p increased over time from day 7 to 21 of the osteogenic differentiation of hMSCs. The knockdown of LINC00205 and KMT2C significantly increased ALP activity, calcium deposition, and the expression of RUNX2, ALP, and OCN. In contrast, the inhibition of miR-26b-5p yielded the opposite result. These data suggest that LINC00205 inhibits the osteogenic differentiation of hMSCs by modulating the miR-26b-5p/KMT2C signaling axis.ConclusionLINC00205 promotes OP and is involved in spinal fractures. LINC00205 is also a potential negative regulator of the osteogenic differentiation of hMSCs.

Abstract 1219: Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines

Abstract Some types of thyroid cancers are known to arise from thyroid adenomas, as a malignant transition from benign adenoma, as proposed in the multi-step carcinogenesis model. Thyroid cancer studies using human thyroid cancer cell lines have been intensely performed by many researchers. However, mouse thyroid adenoma cell lines have not been well established and characterized. In order to obtain further insights into thyroid carcinogenesis process, we established five cell lines from thyroid adenomas developed in mice treated with goitrogen amitrole. Among them, two cell lines, CAT458/458s (CAT: cells from amitrole treated thyroids; CAT458s: a sub-line from CAT458) and CAT459 were found to be positive for E-cadherin and Claudin-1, representative epithelial markers, and negative for Vimentin, a mesenchymal marker, suggesting that they originated from thyroid epithelial cells. We found higher expression of DUOX1, a member of NADPH oxidase involved in thyroid hormone synthesis, in both CAT458s and 459 cells than normal mouse thyroids, presumably due to an unknown mechanism caused by amitrole treatment. NKX2-1, a master regulator of thyroid development, is not expressed in CAT458s cells while NKX2-1 is abundantly expressed in CAT459 cells. Consistent with this finding, NKX2-1-positive CAT459 cells showed higher mRNA expression of thyroid differentiation markers including Tg, Duox1, and Duox2. NKX2-1 overexpression in NKX2-1-negative CAT458s cells using a lentiviral infection system induced mRNA expressions of these thyroid differentiation marker genes, suggesting that partial thyroid differentiation of the adenoma cells is induced by NKX2-1. TSH and IGF-I are known to work synergistically for thyroid growth. However, Tshr mRNA expression is not detected in either CAT458s or 459 cells, suggesting that TSH signal is not responsible for their growth. On the other hand, IGF-IR protein is expressed and IGF-I induces AKT and S6 phosphorylation in both cells. We further tried to assess somatic gene mutations involved in adenoma development by whole-genome sequencing (WGS). WGS analysis revealed that somatic missense mutations are not found in genes known to be involved in thyroid carcinogenesis and cancer progression, such as Braf, Trp53, Ret, Tert, Hras, Kras, and Nras. This finding suggests that prominent oncogenic mutations are not required for thyroid adenoma development that may later progress to carcinoma. Currently we are validating several missense mutations, including mutations in tumor suppressor genes Atm and Tgfb1, found in these cell lines. In conclusion, we established mouse thyroid adenoma cell lines with different differentiation and molecular profiles. CAT458s and 459 cells would provide a tool to further clarify the process of thyroid adenoma development and thyroid differentiation. Citation Format: Yo-Taro Shirai, Nobuo Hoshi, Huaitian Liu, Maxwell P. Lee, Shioko Kimura. Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1219.

Predicting reprogramming-related gene expression from cell morphology in human induced pluripotent stem cells.

Purification is essential before differentiating human induced pluripotent stem cells (hiPSCs) into cells that fully express particular differentiation marker genes. High-quality iPSC clones are typically purified through gene expression profiling or visual inspection of the cell morphology; however, the relationship between the two methods remains unclear. We investigated the relationship between gene expression levels and morphology by analyzing live-cell, phase-contrast images and mRNA profiles collected during the purification process. We employed these data and an unsupervised image feature extraction method to build a model that predicts gene expression levels from morphology. As a benchmark, it was confirmed that the method can predict the gene expression levels from tissue images for cancer genes, performing as well as state-of-the-art methods. We then applied the method to iPSCs and identified two genes that are well predicted from cell morphology. Although strong batch (or possibly donor) effects resulting from the reprogramming process preclude the ability to use the same model to predict across batches, prediction within a reprogramming batch is sufficiently robust to provide a practical approach for estimating expression levels of a few genes and monitoring the purification process.

Medaka factor in the germline alpha (figα) is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor, figα, is one of the earliest marker genes of oocyte differentiation in vertebrates. In the present study, we made figα knockout medaka by CRISPR/Cas9, expecting aborted progress of oogenesis, to see if differentiation of somatic ovarian tissues is affected. Figα knockout male gonads differentiated normally into testes with functional sperm. The females, on the other hand, were sterile; there are oocytes only up to pachytene. No growing oocytes in diplotene were found. The phenotype was already apparent at 10 days after hatching, when diplotene oocytes start to develop in the control. Furthermore, several putative target genes of figα were not expressed in the mutant female gonads. Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred. Figα knockout female gonads differentiated morphologically into ovaries, suggesting that in medaka, figα knockout prevents oogenesis progress into diplotene stage, and ovarian differentiation do not need developed oocytes beyond pachytene stage. This ovarian phenotype reminded us of "pachytene checkpoint", which blocks meiotic progress into diplotene when chromosomal abnormalities are present. Therefore, we further analyzed phenotype of oocytes in the mutant ovaries. The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage. The number of apoptotic oocytes in the knockout ovaries was small. Finally, we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining. Taken together, figα knockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than "pachytene checkpoint" and arrested oocytes remained in the ovary. This is in contrast to figα knockout mice where all oocytes underwent apoptosis quickly after birth.