Bone marrow mesenchymal stem cells (BMSCs), which have multipotential differentiation and self-renewal ability, have been becoming an attractive source of seed cells for bone tissue engineering. Nonetheless, the precise underlying mechanisms of osteogenesis of BMSCs have not been fully understood. Retinoic acid-induced gene 3 (RAI3) has been found to play important roles in mesenchymal stem cells (MSCs) adipogenesis in our previous study. However, its function in the osteogenic differentiation of BMSCs remains unknown. In this study, we found that RAI3 was significantly reduced in osteogenically differentiated BMSCs; RAI3 knockdown promoted osteogenesis of BMSCs both in vitro and in vivo. Moreover, we found RAI3 knockdown significantly upregulated the expression level of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and AG-490 which can inhibit the STAT3 signaling reversed the enhancing effect of RAI3 knockdown on the osteogenic differentiation of BMSCs. These results suggest that RAI3 plays important roles in BMSCs osteogenesis with an involvement of the STAT3 signaling, which might open a new avenue to explore BMSCs osteogenesis for the application of BMSCs in bone regeneration. (C) 2020 Published by Elsevier Inc.
Embryonic stem cells (ESCs) provide an ideal model for investigating developmental processes and are great sources for developing regenerative medicine. Harnessing apoptosis facilitates accurate recapitulation of signalling events during embryogenesis and allows efficient expansion of the ESCs during differentiation. Bcl2, a key regulator of intrinsic anti-apoptotic pathway, encodes two splicing isoforms. However, the identification and functional comparison of Bcl2 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we provide the evidence that both Bcl2 splicing variants are expressed in mESCs. Despite the structural difference, they have similar subcellular localisation. Both Bcl2 alpha and Bcl2 beta enhance differentiation efficiency of the ESCs and effectively improve the survival and growth of ESCs under serum-free conditions. However, the functional effect of Bcl2 alpha was more potent than that of Bcl2 beta. Moreover, only Bcl2 alpha could maintain the long-term expansion and pluripotency of ESCs cultured in serum-free medium. Taken together, our results demonstrate previously unknown functional differences in Bcl2 alternative splicing isoforms in ESCs, and lay the foundation for future efforts to engineer ESCs for regenerative medicine. (C) 2020 The Authors. Published by Elsevier Inc.
3' uridylation is an essential modification associated with coding and noncoding RNA degradation in eukaryotes. In Arabidopsis, HESO1 was first identified as the major nucleotidyl transferase that uridylates most unmethylated miRNAs, and URT1 was later reported to play a redundant but important role in miRNA uridylation when HESO1 is absent. Two enzymes work sequentially and collaboratively to tail different forms of the same miRNAs in vivo. For mRNA, however, URT1 becomes the main enzyme to uridylate the majority of mRNA and repairs their deadenylated ends to restore the binding site for Poly(A) Binding Protein (PABP). HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails in fulfilling the same task. To understand the structural basis these two functional homologues possess for their different substrate preferences and catalytic behaviors, we first determined the crystal structures of URT1 in the absence and presence of UTP. Our structures, together with functional assay and sequence analysis, indicated that URT1 has a conserved UTP-recognition mechanism analogue to the terminal uridylyl transferases from other species whereas HESO1 may evolve separately to recognize UTP in a different way. Moreover, URT1 N552 may be an important residue in interacting with 30 nucleotide of RNA substrate. The URT1 structure we determined represents the first structure of uridylyl transferase from plants, shedding light on the mechanisms of URT1/HESO1-dependent RNA metabolism. (C) 2020 Elsevier Inc. All rights reserved.
Calorie restriction (CR) ameliorates various diseases including cardiovascular disease. However, its protection and underlying mechanisms against atherosclerosis remain un-fully elucidated. In this study, we fed apoE deficient (apoE(-/-)) mice in Control group a high-fat diet (HFD, 21% fat plus 0.5% cholesterol) or in CR group a CR diet (CRD, 2% fat plus 0.5% cholesterol, similar to 40% calorie restriction and same levels of cholesterol, vitamins, minerals and amino acids as in HFD). After 16 weeks feeding, compared with HFD, CRD substantially reduced atherosclerosis in mice. CRD increased SMC and collagen content but reduced macrophage content, necrotic core and vascular calcification in lesion areas. Mechanistically, CRD attenuated bodyweight gain, improved lipid profiles but had little effect on macrophage lipid metabolism. CRD also inhibited expression of inflammatory molecules in lesions. Taken together, our study demonstrates CRD effectively reduces atherosclerosis in apoE(-/-) mice, suggesting it as a potent and reproducible therapy for atherosclerosis management. (C) 2020 Elsevier Inc. All rights reserved.
The antimalarial drug Artemisinin has been reported to possess direct anti-tumor effects on various types of tumor cells. However, its anti-tumor potential has not been fully revealed, and its effects on tumor susceptibility to immune surveillance by the host are still unknown. Natural killer (NK) cells are the first line in tumor surveillance by the host, and have been recognized as a promising target for tumor immunotherapy. Here, we reported that Artemisinin sensitized tumor cells to NK cell cytolysis. Both human K562 and Raji tumor cells, and mouse YAC-1 tumor cells were more susceptible to human or mouse NK cell cytolysis in vitro after Artemisinin pretreatment. Conjugation formation between tumor cells and NK cells was increased after pretreatment with Artemisinin. Such effects on tumor cells by Artemisinin might not be the results of tumor recognition by NK cells, since major ligands of NK cell surface receptors were not affected. Mechanistically, although Artemisinin didn't induce tumor cell apoptosis, Artemisinin enriched apoptosis-related gene sets in these tumor cells, which might predispose tumor cells to apoptosis upon NK cell cytolysis. Moreover, NK cell numbers, percentages, maturation and functions were preserved in the presence of Artemisinin in vitro, suggesting that Artemisinin displays detrimental effects only on tumor cells but not on immune cells. These data reveal a novel anti-tumor mechanism of Artemisinin and demonstrate that Artemisinin could be a promising drug candidate for cancer treatment. (C) 2020 Elsevier Inc. All rights reserved.
As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type I PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian. (C) 2020 Elsevier Inc. All rights reserved.
Cotton Verticillium wilt caused by Verticillium dahliae (V. dahliae) is one of the most destructive fungal diseases and is difficult to control. However, resistant germplasm resources are scarce in cotton. Many studies have shown that host-induced gene silencing (HIGS) is a practical and effective technology in crop disease prevention by silencing virulence genes of pathogens. Acetolactate synthase (ALS) contains a catalytic subunit ILV2 and a regulatory subunit ILV6, which catalyzes the first common step reaction in branched-chain amino acid (BCAA) biosynthesis. We identified two acetolactate synthases, VdILV2 and VdILV6, which are homologs of ILV2 and ILV6, respectively, in Magnaporthe oryzae. To characterize the function of VdILV2 and VdILV6 in V. dahliae, we suppressed their expression in the strong pathogenic isolate Vd991 by using HIGS technology. VdILV2- or VdILV6-silenced V. dahliae had a dramatic reduction in pathogenicity. The results indicated that VdILV2 and VdILV6 are involved in the pathogenicity of V. dahliae. HIGS of VdILV2 or VdILV6 provides a novel fungicide target and an effective control to resist Verticillium wilt caused by V. dahliae. (C) 2020 Elsevier Inc. All rights reserved.
Diabetes mellitus is a metabolic disorder that can lead to blood-brain barrier (BBB) disruption and cognitive decline. However, the mechanisms of BBB breakdown in diabetes are still unclear. Soluble epoxide hydrolase (sEH) is an enzyme that degrades epoxyeicosatrienoic acids (EETs), which have multiple protective effects on vascular structure and functions. In the current study, we showed increased vascular permeability of the BBB, which was accompanied by upregulation of sEH and downregulation of 14,15-EET. Moreover, the sEH inhibitor t-AUCB restored diabetic BBB integrity in vivo, and 14,15-EET prevented ROS accumulation and MEC injury in vitro. t-AUCB or 14,15-EET treatment provoked AMPK/HO-1 activation under diabetic conditions in vivo and in vitro. Thus, we suggest that decreased EET degradation by sEH inhibition might be a potential therapeutic approach to attenuate the progression of BBB injury in diabetic mice via AMPK/HO-1 pathway activation. (C) 2020 Elsevier Inc. All rights reserved.
Recent developments in tissue clearing methods such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) have allowed for the three-dimensional analysis of biological structures in whole, intact tissue, providing greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, preventing the wide application of these techniques to fragile tissues such as developing embryos. Here, we present optimized passive clearing techniques, mPACT-A, that improve tissue rigidity without the expense of optical transparency. We also present a further modified mPACT-A protocol that is specifically optimized for handling mouse embryos, which are small and fragile, such that they easily dismantle when processed via established tissue clearing methods. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PRDM proteins, PRDM7 and PRDM12, in intact cleared mouse embryos at various stages of development. We observed strong PRDM7 and PRDM12 expression in the developing mouse nervous system, suggestive of potential roles in neural development that will be tested in future functional studies. (C) 2020 Elsevier Inc. All rights reserved.
Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that stimulates glucose-mediated insulin production by pancreatic beta cells. It is also associated with protective effects in multiple tissues. GLP-1 receptor is highly expressed in pulmonary tissue, hinting possible pulmonary delivery of GLP-1 drugs. However, little is known about the role of GLP-1 signaling in the lung, especially in mucus hypersecretory obstructive lung diseases. Here, we showed that treatment with exendin-4, a clinically available GLP-1 receptor agonist, up-regulates mucin expression in normal airway epithelial cells and in the lung of normal mice, indicating mucus stimulatory effect of GLP-1 under physiological condition. Exendin-4 also increased mucin expression in in vitro cellular and in vivo murine models of obstructive lung diseases via the activation of p38 MAP kinase. Notably, mucin induction in vivo exacerbated key pulmonary abnormalities including emphysematous phenotypes, implying that GLP-1 signaling in the lung is detrimental under pulmonary obstructive condition. Another GLP-1 receptor agonist liraglutide had similar induction of mucin. Together, our studies not only demonstrate novel physiological and pathological roles of GLP-1 in the lung but may also caution against the clinical use of inhaled GLP-1 receptor agonists in the patients with obstructive lung diseases. (C) 2020 Elsevier Inc. All rights reserved.
Integrin activation by Rap1-GTP is pivotal for lymphocyte trafficking. In this study, we show the phosphatidic acid (PA)-dependent membrane distribution of RA-GEF-1 and -2 (also known as Rapgef2 and 6), which are guanine nucleotide exchange factors for Rap1, plays important roles in lymphocyte migration. RA-GEF-1 associates with PA through 919-967 aa within CDC25 homology domain, and the deletion of this region of RA-GEF-1 inhibits chemokine-dependent migration. Chemokine stimulation induces temporal production of PA on the plasma membrane, which is not necessary for Rap1 activation, but the translocation of RA-GEFs. Thus, chemokine-dependent generation of PA is critical for lymphocyte migration through membrane localization of RA-GEFs. (C) 2020 Elsevier Inc. All rights reserved.
Extracellular Matrix (ECM) assembly and remodeling are critical physiological events in vivo, and abnormal ECM assembly or remodeling is related to pathological conditions such as osteoarthritis, fibrosis, cancers, and genetic diseases. ECM assembly/remodeling driven by cells represents more physiological processes. Collagen I (COL) is very abundant in tissues, which assembly/remodeling is mediated by biochemical and mechanical factors. How cells regulate COL assembly biomechanically still remains to be well understood. Here we used fluorescent COL in the medium to study how cells assembled ECM which represents more physiological structures. The results showed that MDCK cells actively recruited COL from the medium and helped assemble the fibers, which in turn facilitated cell branching morphogenesis, both displaying highly spatial associations and mutual dependency. Inhibition of cellular contraction force by ROCK and Myosin II inhibitors attenuated but did not block the COL fiber formation, while cell motion showed high consistency with the fiber assembly. Under ROCK or Myosin II inhibition, further analysis indicated high correlation between local cell movement and COL fiber strength as quantified from different regions of the same groups. Blocking cell motion by actin cytoskeleton disruption completely inhibited the fiber formation. These suggest that cell motion coordinated COL fiber assembly from the medium, possibly through generated strain on deposited COL to facilitate the fiber growth. (C) 2020 Published by Elsevier Inc.
Background: Atherosclerotic plaque rupture is the major trigger of acute cardiovascular risk events, and manipulation of M1/M2 macrophage homeostasis is an effective strategy for regulating atherosclerotic plaque stability. This study was aimed to illuminate the effects of oleoylethanolamide (OEA) on macrophage polarization and plaque stability. Methods: Macrophages derived from THP-1 were treated with OEA followed by LPS/IFN-gamma, and the markers of M1, M2 macrophages were monitored by western blot, real-time PCR and immunofluorescence staining. The effect of OEA on macrophage polarization in the arch of aortic arteries was tested by immunofluorescence staining and western blot, and the plaque stability was completed by Masson's trichrome and hematoxylin and eosin (HE) in apolipoprotein E (ApoE)(-/-) mice. Results: OEA treatment enhanced the expression of two classic M2 macrophage markers, macrophage mannose receptor (CD206) and transforming growth factor (TGF-beta), while the expression of iNOS (M1 macrophages) was decreased in THP-1-derived macrophages. Blocking of PPAR alpha using siRNA and inhibition of AMP-activated protein kinase (AMPK) by its inhibitor compound C attenuated the OEA-induced expression of M2 macrophage markers. In addition, OEA significantly suppressed M1, promoted M2 macrophage polarization, increased collagen content and decreased necrotic core size in atherosclerotic plaques in ApoE(-/-) mice, which were linked with the expression of PPAR alpha. Conclusions: OEA improved atherosclerotic plaque stability through regulating macrophage polarization via AMPK-PPAR alpha pathway. (C) 2020 Published by Elsevier Inc.
Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and beta 1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of beta-casein. However, the mechanism by which beta 1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with beta 1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of beta 1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that beta 1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of beta 1 integrin was not sufficient to increase beta-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of beta 1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of beta-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, beta 1 integrin, and beta-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking beta 1 integrin function, resulting in attenuated STAT5 activity and decreased beta-casein synthesis. These results indicated that beta 1 integrinwas a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for beta-casein expression in BMECs. (C) 2020 Elsevier Inc. All rights reserved.