CBX2 is required to stabilize the testis pathway by repressing Wnt signaling.

XX and XY fetal gonads are initially bipotential, poised between the ovary and testis fate. Multiple lines of evidence suggest that commitment to testis fate requires the repression of genes associated with ovary fate. It was previously shown that loss of CBX2, the subunit of the Polycomb Repressive Complex 1 (PRC1) that binds H3K27me3 and mediates silencing, leads to ovary development in XY mice and humans. While it had been proposed that CBX2 is an activator of the testis-determining gene Sry, we investigated the alternative possibility that CBX2 has a direct role as a repressor of the antagonistic ovary-promoting pathway. To investigate this possibility, we developed a quantitative genome-wide profile of the repressive histone mark H3K27me3 and its active counterpart H3K4me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that testis and ovary sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue testis development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, an ovary-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development.

Numb regulates somatic cell lineage commitment during early gonadogenesis in mice.

During early gonadogenesis, proliferating cells in the coelomic epithelium (CE) give rise to most of the somatic cells in both XX and XY gonads. Previous dye-labeling experiments showed that a single CE cell could give rise to additional CE cells and to both supporting and interstitial cell lineages, implying that cells in the CE domain are multipotent progenitors, and suggesting that an asymmetric division is involved in the acquisition of gonadal cell fates. We found that NUMB is asymmetrically localized in CE cells, suggesting that it might be involved. To test this hypothesis, we conditionally deleted Numb on a Numbl mutant background just prior to gonadogenesis. Mutant gonads showed a loss of cell polarity in the surface epithelial layers, large interior cell patches expressing the undifferentiated cell marker LHX9, and a loss of differentiated cells in somatic cell lineages. These results indicate that NUMB is necessary for establishing polarity in CE cells, and that asymmetric divisions resulting from CE polarity are required for commitment to differentiated somatic cell fates. Surprisingly, germ cells, which do not arise from the CE, were also affected in mutants, which may be a direct or indirect effect of loss of Numb.

RASSF4 is required for skeletal muscle differentiation.

RASSF4, a member of the classical RASSF family of scaffold proteins, is associated with alveolar rhabdomyosarcoma, an aggressive pediatric cancer of muscle histogenesis. However, the role of RASSF4 in normal myogenesis is unknown. We demonstrate here that RASSF4 is necessary for early in vitro myogenesis. Using primary human myoblasts, we show that RASSF4 expression is dramatically increased during in vitro myogenic differentiation, and conversely that RASSF4-deficient myoblasts cannot differentiate, potentially because of a lack of upregulation of myogenin. In microscopy studies, we show that RASSF4 protein co-localizes with proteins of the myogenic microtubule-organizing center (MTOC) both before and after myogenic differentiation. RASSF4-deficient cells subject to differentiation conditions demonstrate a lack of shape change, suggesting that RASSF4 plays a role in promoting microtubule reorganization and myoblast elongation. In biochemical studies of myotubes, RASSF4 associates with MST1, suggesting that RASSF4 signals to MST1 in the myogenic differentiation process. Expression of MST1 in myoblasts partially reversed the effect of RASSF4 knockdown on differentiation, suggesting that RASSF4 and MST1 coordinately support myogenic differentiation. These data show that RASSF4 is critical for the early steps of myogenic differentiation.

Isolated Compounds from Turpinia formosana Nakai Induce Ossification.

Bone metabolism is a homeostatic process, imbalance in which leads to the onset of diseases such as osteoporosis and osteopenia. Although several drugs are currently available to treat such conditions, they are associated with severe side effects and do not enhance bone formation. Thus, identifying alternative treatment strategies that focus on enhancing bone formation is essential. Herein, we explored the osteogenic potential of Turpinia formosana Nakai using human osteoblast (HOb) cells. The plant extract was subjected to various chromatographic techniques to obtain six compounds, including one new compound: 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1). Compounds 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1), gentisic acid 5-O-ß-d-(6'-O-galloyl) glucopyranoside (2), strictinin (3), and (-)-epicatechin-3-O-ß-d-allopyranoside (6) displayed no significant cytotoxicity toward HOb cells, and thus their effects on various osteogenic markers were analyzed. Results showed that 1-3 and 6 significantly increased alkaline phosphatase (ALP) activity up to 120.0, 121.3, 116.4, and 125.1%, respectively. Furthermore, 1, 2, and 6 also markedly enhanced the mineralization process with respective values of up to 136.4, 118.9, and 134.6%. In addition, the new compound, 1, significantly increased expression levels of estrogen receptor-α (133.4%) and osteogenesis-related genes of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), bone morphogenetic protein (BMP)-2, bone sialoprotein (BSP), type I collagen (Col-1), and brain-derived neurotropic factor (BDNF) by at least 1.5-fold. Our results demonstrated that compounds isolated from T. formosana possess robust osteogenic potential, with the new compound, 1, also exhibiting the potential to enhance the bone formation process. We suggest that T. formosana and its isolated active compounds deserve further evaluation for development as anti-osteoporotic agents.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-ß-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

Coenzyme A protects against ferroptosis via CoAlation of thioredoxin reductase 2.

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Multiomic molecular characterization of the response to combination immunotherapy in MSS/pMMR metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a multiomic characterization was performed to identify predictive biomarkers associated with patient response to ICI combinations in MSS/pMMR CRC for the further development of ICI combinations. METHODS: Whole-exome sequencing, RNA sequencing, and multiplex fluorescence immunohistochemistry of tumors from patients with MSS/pMMR CRC, who received regorafenib plus nivolumab (REGONIVO) or TAS-116 plus nivolumab (TASNIVO) in clinical trials were conducted. Twenty-two and 23 patients without prior ICI from the REGONIVO and TASNIVO trials were included in this study. A biomarker analysis was performed using samples from each of these studies. RESULTS: The epithelial-mesenchymal transition pathway and genes related to cancer-associated fibroblasts were upregulated in the REGONIVO responder group, and the G2M checkpoint pathway was upregulated in the TASNIVO responder group. The MYC pathway was upregulated in the REGONIVO non-responder group. Consensus molecular subtype 4 was significantly associated with response (p=0.035) and longer progression-free survival (p=0.006) in the REGONIVO trial. CD8+ T cells, regulatory T cells, and M2 macrophages density was significantly higher in the REGONIVO trial responders than in non-responders. Mutations in the POLE gene and patient response were significantly associated in the TASNIVO trial; however, the frequencies of other mutations or tumor mutational burden were not significantly different between responders and non-responders in either trial. CONCLUSIONS: We identified molecular features associated with the response to the REGONIVO and TASNIVO, particularly those related to tumor microenvironmental factors. These findings are likely to contribute to the development of biomarkers to predict treatment efficacy for MSS/pMMR CRC and future immunotherapy combinations for treatment.

Testis development requires the repression of Wnt4 by Fgf signaling.

The bipotential gonad expresses genes associated with both the male and female pathways. Adoption of the male testicular fate is associated with the repression of many female genes including Wnt4. However, the importance of repression of Wnt4 to the establishment of male development was not previously determined. Deletion of either Fgf9 or Fgfr2 in an XY gonad resulted in up-regulation of Wnt4 and male-to-female sex reversal. We investigated whether the deletion if Wnt4 could rescue sex reversal in Fgf9 and Fgfr2 mutants. XY Fgf9/Wnt4 and Fgfr2/Wnt4 double mutants developed testes with male somatic and germ cells present, suggesting that the primary role of Fgf signaling is the repression of female-promoting genes. Thus, the decision to adopt the male fate is based not only on whether male genes, such as Sox9, are expressed, but also on the active repression of female genes, such as Wnt4. Because loss of Wnt4 results in the up-regulation of Fgf9, we also tested the possibility that derepression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we found that the relationship between these two signaling factors is not symmetric: loss of Fgf9 in XX Wnt4(-/-) gonads does not rescue their partial female-to-male sex-reversal.

Mitochondrial transplant after ischemia reperfusion promotes cellular salvage and improves lung function during ex-vivo lung perfusion.

BACKGROUND: In lung transplantation, ischemia-reperfusion injury associated with mitochondrial damage can lead to graft rejection. Intact, exogenous mitochondria provide a unique treatment option to salvage damaged cells within lung tissue. METHODS: We developed a novel method to freeze and store allogeneic mitochondria isolated from porcine heart tissue. Stored mitochondria were injected into a model of induced ischemia-reperfusion injury using porcine ex-vivo lung perfusion. Treatment benefits to immune modulation, antioxidant defense, and cellular salvage were evaluated. These findings were corroborated in human lungs undergoing ex-vivo lung perfusion. Lung tissue homogenate and primary lung endothelial cells were then used to address underlying mechanisms. RESULTS: Following cold ischemia, mitochondrial transplant reduced lung pulmonary vascular resistance and tissue pro-inflammatory signaling and cytokine secretion. Further, exogenous mitochondria reduced reactive oxygen species by-products and promoted glutathione synthesis, thereby salvaging cell viability. These results were confirmed in a human model of ex-vivo lung perfusion wherein transplanted mitochondria decreased tissue oxidative and inflammatory signaling, improving lung function. We demonstrate that transplanted mitochondria induce autophagy and suggest that bolstered autophagy may act upstream of the anti-inflammatory and antioxidant benefits. Importantly, chemical inhibitors of the MEK autophagy pathway blunted the favorable effects of mitochondrial transplant. CONCLUSIONS: These data provide direct evidence that mitochondrial transplant improves cellular health and lung function when administered during ex-vivo lung perfusion and suggest the mechanism of action may be through promotion of cellular autophagy. Data herein contribute new insights into the therapeutic potential of mitochondrial transplant to abate ischemia-reperfusion injury during lung transplant, and thus reduce graft rejection.

Synthesis, evaluation, and mechanism of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones as potent reversible SARS-CoV-2 entry inhibitors.

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001-0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to discovery that the synthesized compounds also inhibited Spike's receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced bodyweight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.

Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal.

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

Toll-like receptors and human disease: lessons from single nucleotide polymorphisms.

Toll-like receptors (TLRs), a large group of proteins which recognize various pathogen-associated molecular patterns, are critical for the normal function of the innate immune system. Following their discovery many single nucleotide polymorphisms within TLRs and components of their signaling machinery have been discovered and subsequently implicated in a wide range of human diseases including atherosclerosis, sepsis, asthma, and immunodeficiency. This review discusses the effect of genetic variation on TLR function and how they may precipitate disease.

Updated Efficacy Outcomes of Anti-PD-1 Antibodies plus Multikinase Inhibitors for Patients with Advanced Gastric Cancer with or without Liver Metastases in Clinical Trials.

PURPOSE: We previously reported preliminary activity of regorafenib plus nivolumab (REGONIVO) or lenvatinib plus pembrolizumab (LENPEM) in advanced gastric cancer (AGC). Meanwhile, several studies demonstrated liver metastases are less responsive to immunotherapy. PATIENTS AND METHODS: Combined efficacy outcomes with a longer follow-up in a phase Ib trial of REGONIVO and a phase II trial of LENPEM were examined in AGC with or without liver metastases (REGONIVO plus LENPEM cohort). We also investigated the efficacy of anti-PD-1 monotherapies (anti-PD-1 monotherapy cohort). A comparison of the immune microenvironment between gastric primary tumors and liver metastases was also conducted by multiplex IHC. RESULTS: In the REGONIVO plus LENPEM cohort, with a median follow-up of 14.0 months, objective response rate (ORR), median progression-free survival (mPFS), and median overall survival (mOS) were 46%, 7.8 months, and 15.6 months in patients with liver metastases, while 69%, 6.9 months, and 15.5 months in those without. In the anti-PD-1 monotherapy cohort, with a median follow-up of 27.6 months, ORR, mPFS, and mOS were 9%, 1.4 months, and 6.4 months in patients with liver metastases, while 22%, 2.3 months, and 9.0 months in those without. Multiplex IHC revealed liver metastases were associated with an abundance of immune-suppressive cells, such as tumor-associated macrophages and regulatory T cells, with fewer CD8+ T cells compared with gastric primary tumors. CONCLUSIONS: Anti-PD-1 antibodies plus regorafenib or lenvatinib for AGC showed promising antitumor activity with a longer follow-up, irrespective of liver metastases status, despite a more immune-suppressive tumor microenvironment in liver metastases.

BATF epigenetically and transcriptionally controls the activation program of regulatory T cells in human tumors.

Regulatory T (Treg) cells suppress effective antitumor immunity in tumor-bearing hosts, thereby becoming promising targets in cancer immunotherapy. Despite the importance of Treg cells in tumor immunity, little is known about their differentiation process and epigenetic profiles in the tumor microenvironment (TME). Here, we showed that Treg cells in the TME of human lung cancers harbored a completely different open chromatin profile compared with CD8+ T cells, conventional CD4+ T cells in the TME, and peripheral Treg cells. The integrative sequencing analyses including ATAC, single-cell RNA, and single-cell ATAC sequencing revealed that BATF, IRF4, NF-κB, and NR4A were important transcription factors for Treg cell differentiation in the TME. In particular, BATF was identified as a key regulator, which leveraged Treg cell differentiation through epigenetically controlling activation-associated gene expression, resulting in the robustness of Treg cells in the TME. The single-cell sequencing approaches also revealed that tissue-resident and tumor-infiltrating Treg cells followed a common pathway for differentiation and activation in a BATF-dependent manner heading toward Treg cells with the most differentiated and activated phenotypes in tissues and tumors. BATF deficiency in Treg cells remarkably inhibited tumor growth, and high BATF expression was associated with poor prognosis in lung cancer, kidney cancer, and melanoma. These findings indicate one of the specific chromatin remodeling and differentiation programs of Treg cells in the TME, which can be applied in the development of Treg cell-targeted therapies.

Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

Compounds Isolated from Wikstroemia taiwanensis Regulate Bone Remodeling by Modulating Osteoblast and Osteoclast Activities.

Bone remodeling, a dynamic process in which bone formation by osteoblast is preceded by bone resorption by osteoclast, is a vital physiological process for maintaining bone mass and strength, imbalances in which could precipitate osteoporosis. Due to the unilateral mechanism of the existing bone remodeling drugs, identifying compounds that could regulate the balance between osteoclast and osteoblast could improve the treatment of osteoporosis. Here, we show that compounds isolated from Wikstroemia taiwanensis modulate osteoclast and osteoblast activities. Specifically, astragalin (1) and kaempferol 3-O-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside (2), besides increasing mineral deposition, increased alkaline phosphatase activity (137.2% for 1 and 115.8% for 2) and ESR-α expression (112.8% for 1 and 122.5% for 2) in primary human osteoblasts. In contrast, compounds 1, 2, 3, and 5 inhibited tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB ligand-induced osteoclasts by 40.8, 17.1, 25.9, and 14.5% and also decreased the number of TRAP-positive cells by 51.6, 26.8, 20.5, and 18.6%, respectively. Our findings, therefore, showed that compounds isolated from W. taiwanensis could increase osteoblast activity while simultaneously decreasing osteoclast activity, and hence, warrant further evaluation for development as anti-osteoporosis agents.

In vivo and in vitro evaluation of the osteogenic potential of Davallia mariesii T. Moore ex Baker.

ETHNOPHARMACOLOGICAL RELEVANCE: Postmenopausal osteoporosis is a major bone health issue worldwide. There is an unmet medical need for osteoporosis treatments, a disease which disproportionately impacts women. Exploring botanicals to prevent or treat osteoporosis is currently an interest of investigations. Rhizomes of Davallia mariesii T. Moore ex Baker (Davalliacea) are used an indigenous herbal medicine in Asia for injuries due to fractures, contusions, and strains. AIM OF THE STUDY: In the present study, we investigated the osteogenic effect of the water extract of rhizomes of D. mariesii (DMH) on bone loss induced by an ovariectomy (OVX) in mice and also its impact on osteogenesis in primary human osteoblasts (HObs). Additionally, we performed a quantitative analysis of compounds in the DMH extract. MATERIALS AND METHODS: OVX C57BL/6J mice were orally administrated DMH extract for 12 weeks, and microarchitecture parameters were examined by microcomputed tomography. DMH extract was fractionated in a bio-guided manner, and fractions were isolated to obtain active compounds using HObs. Cell viability was evaluated by an MTT assay. Characteristics of early and late osteogenesis were analyzed by alkaline phosphatase activity and a mineralization assay. Molecular mechanisms were explored by a real-time quantitative PCR. Compounds in the DMH extract were identified and quantified using liquid chromatography tandem mass spectroscopy (LC-MS/MS). RESULTS: DMH improved bone mineral densities of vertebrae and the femur. Through microarchitectural observations, DMH significantly decreased the bone surface/volume ratio and trabecular separation, and also increased the connectivity density in the OVX group. Additionally, DMH inhibited osteoclast differentiation in receptor activator of nuclear factor-κB ligand-induced osteoclasts and increased bone formation in HObs. After bio-guided fractionation and isolation, we found that eriodictyol-7-O-ß-d-glucuronide (2) significantly increased alkaline phosphatase activity, and 5-O-ß-d-(6-O-vanilloylglucopyranosyl)gentisic acid (3) substantially enhanced mineral deposition. In HObs, compound 3 was more potent in upregulating expressions of bone morphogenetic protein-2, bone sialoprotein, osteopontin, osterix, and estrogen receptor-α. The amount of bioactive compound 3 in DMH was 5.68 ±â€¯0.64 mg/g of dry weight according to LC-MS/MS. CONCLUSION: For the first time we report that D. mariesii and its isolated compounds demonstrated potent osteogenic activities. Quantitative results of D. mariesii could be a reference for phytochemical analyses.

A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and Notch signaling.

The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue sarcoma of childhood, has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

DDR2 upregulation confers ferroptosis susceptibility of recurrent breast tumors through the Hippo pathway.

Recurrent breast cancer presents significant challenges with aggressive phenotypes and treatment resistance. Therefore, novel therapeutics are urgently needed. Here, we report that murine recurrent breast tumor cells, when compared with primary tumor cells, are highly sensitive to ferroptosis. Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), the receptor for collagen I, is highly expressed in ferroptosis-sensitive recurrent tumor cells and human mesenchymal breast cancer cells. EMT regulators, TWIST and SNAIL, significantly induce DDR2 expression and sensitize ferroptosis in a DDR2-dependent manner. Erastin treatment induces DDR2 upregulation and phosphorylation, independent of collagen I. Furthermore, DDR2 knockdown in recurrent tumor cells reduces clonogenic proliferation. Importantly, both the ferroptosis protection and reduced clonogenic growth may be compatible with the compromised YAP/TAZ upon DDR2 inhibition. Collectively, these findings identify the important role of EMT-driven DDR2 upregulation in recurrent tumors in maintaining growth advantage but activating YAP/TAZ-mediated ferroptosis susceptibility, providing potential strategies to eradicate recurrent breast cancer cells with mesenchymal features.