The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

The Ethanolic Extract of Dictyopteris Divaricata Induces Apoptosis in Non-Small Cell Lung Cancer Cells by Inhibiting STAT3 Activity.

Dictyopteris divaricata (DD) has been reported to exert diverse pharmacological activities, including anti-inflammatory, antioxidant, and anticancer effects. In this study, we aimed to investigate the anticancer potential of the ethanolic extract of DD (EDD) in non-small cell lung cancer (NSCLC) cells and to explore the underlying mechanism. EDD significantly suppressed cell proliferation in H1299, PC9, and H1975 NSCLC cells. EDD treatment increased the proportion of Annexin V-positive cells and cells in sub-G1 phase, indicating the induction of apoptosis. This observation was further supported by the presence of fragmented nuclei and increased expression of cleaved PARP and cleaved caspase-3 in NSCLC cells following EDD treatment. Mechanistically, EDD decreased the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and Src. Transfection of constitutively activated STAT3 into H1975 cells partially attenuated EDD-induced apoptosis, highlighting the contribution of STAT3 inhibition to the anticancer activity of EDD. In addition, we identified fucosterol as a major constituent of EDD that exhibited similar anticancer potential in NSCLC cells. Taken together, our results demonstrate that EDD induces apoptosis in NSCLC cells by inhibiting STAT3 activity. We propose EDD as a potential candidate for the development of therapies targeting NSCLC.

Hexane Fraction of Adenophora triphylla var. japonica Root Extract Inhibits Angiogenesis and Endothelial Cell-Induced Erlotinib Resistance in Lung Cancer Cells.

The aim of this study was to investigate the anti-angiogenic effects of the hexane fraction of Adenophora triphylla var. japonica root extract (HAT) and its influence on the development of erlotinib resistance in human lung cancer cells. HAT significantly reduced the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream molecules were decreased via HAT, indicating its anti-angiogenic potential in endothelial cells (ECs). A docking analysis demonstrated that ß-sitosterol and lupeol, representative components of HAT, exhibit a high affinity for binding to VEGFR2. In addition, conditioned media from HAT-pretreated H1299 human lung cancer cells attenuated cancer-cell-induced chemotaxis of HUVECs, which was attributed to the decreased expression of angiogenic and chemotactic factors in H1299 cells. Interestingly, co-culture of erlotinib-sensitive PC9 human lung cancer cells with HUVECs induced erlotinib resistance in PC9 cells. However, co-culture with HAT-pretreated HUVECs partially restored the sensitivity of PC9 cells to erlotinib. HAT inhibited the development of erlotinib resistance by attenuating hepatocyte growth factor (HGF) production by ECs. Taken together, our results demonstrate that HAT exerts its anticancer effects by regulating the crosstalk between ECs and lung cancer cells.

Nuclear OsFKBP20-1b maintains SR34 stability and promotes the splicing of retained introns upon ABA exposure in rice.

Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome-associated protein OsFKBP20-1b during AS. We determined the AS landscape of wild-type and osfkbp20-1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron-containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20-1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays. We show that OsFKBP20-1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone-like protein in the spliceosome. OsFKBP20-1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress-responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20-1b after ABA treatment. Our findings reveal that spliceosome-associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.

The Ethanolic Extract of Trichosanthes Kirilowii Root Exerts anti-Cancer Effects in Human Non-Small Cell Lung Cancer Cells Resistant to EGFR TKI.

The aim of this study was to investigate whether the ethanol extract of the Trichosanthes kirilowii root (ETK), traditionally used to treat lung diseases, exhibits anticancer activity in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells. ETK treatment suppressed the growth of EGFR TKI-resistant NSCLC cells, including H1299, H1975, PC9/ER (erlotinib-resistant PC9) and PC9/GR (gefitinib-resistant PC9) cells, in a concentration- and time-dependent manner. Dose-dependent decline in anchorage-dependent and -independent colony formation was also detected following ETK treatment. We demonstrate that the growth-inhibitory effect of ETK was related to apoptosis induction, based on flow cytometry results showing ETK-induced increase in the percentage of cells with sub-G1 DNA and the population of annexin V-positive cells. Consistently, ETK induced chromatin condensation and cleavage of poly(ADP-ribose) polymerase (PARP). As a molecular mechanism, the phosphorylation level of signal transducer and activator of transcription 3 (STAT3) and Src was decreased by ETK. ETK-induced apoptosis was partially reversed by transfection of constitutively activated STAT3, indicating that STAT3 inactivation mediated ETK-induced apoptosis in EGFR TKI-resistant NSCLC cells. Our results provide basic evidence supporting the role of ETK as a novel therapeutic in EGFR TKI-resistant NSCLC.

Root Extract of Trichosanthes kirilowii Suppresses Metastatic Activity of EGFR TKI-Resistant Human Lung Cancer Cells by Inhibiting Src-Mediated EMT.

The roots of Trichosanthes kirilowii (TK) have been used in traditional oriental medicine for the treatment of respiratory diseases. In this study, we investigated whether an ethanolic root extract of TK (ETK) can regulate the metastatic potency of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant human lung cancer cells. The relative migration and invasion abilities of erlotinib-resistant PC9 (PC9/ER) and gefitinib-resistant PC9 (PC9/GR) cells were higher than those of parental PC9 cells. Mesenchymal markers were overexpressed, whereas epithelial markers were downregulated in resistant cells, suggesting that resistant cells acquired the EMT phenotype. ETK reduced migration and invasion of resistant cells. The expression levels of N-cadherin and Twist were downregulated, whereas Claudin-1 was upregulated by ETK, demonstrating that ETK suppresses EMT. As a molecular mechanism, Src was dephosphorylated by ETK. The anti-metastatic effect of ETK was reduced by transfecting PC9/ER cells with a constitutively active form of c-Src. Dasatinib downregulated N-cadherin, Twist, and vimentin, suggesting that Src regulates EMT in resistant cells. Notably, CuB played a key role in mediating the anti-metastatic activity of ETK. Collectively, our results demonstrate that ETK can attenuate the metastatic ability of EGFR-TKI-resistant lung cancer cells by inhibiting Src-mediated EMT.

Comparative computational RNA analysis of cardiac-derived progenitor cells and their extracellular vesicles.

Stem/progenitor cells, including cardiac-derived c-kit+ progenitor cells (CPCs), are under clinical evaluation for treatment of cardiac disease. Therapeutic efficacy of cardiac cell therapy can be attributed to paracrine signaling and the release of extracellular vesicles (EVs) carrying diverse cargo molecules. Despite some successes and demonstrated safety, large variation in cell populations and preclinical/clinical outcomes remains a problem. Here, we investigated this variability by sequencing coding and non-coding RNAs of CPCs and CPC-EVs from 30 congenital heart disease patients and used machine learning methods to determine potential mechanistic insights. CPCs retained RNAs related to extracellular matrix organization and exported RNAs related to various signaling pathways to CPC-EVs. CPC-EVs are enriched in miRNA clusters related to cell proliferation and angiogenesis. With network analyses, we identified differences in non-coding RNAs which give insight into age-dependent functionality of CPCs. By taking a quantitative computational approach, we aimed to uncover sources of CPC cell therapy variability.

An Ethanol Extract of Perilla frutescens Leaves Suppresses Adrenergic Agonist-Induced Metastatic Ability of Cancer Cells by Inhibiting Src-Mediated EMT.

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis.

Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Bidirectional relationship between cardiac extracellular matrix and cardiac cells in ischemic heart disease.

Ischemic heart diseases (IHDs), including myocardial infarction and cardiomyopathies, are a leading cause of mortality and morbidity worldwide. Cardiac-derived stem and progenitor cells have shown promise as a therapeutic for IHD but are limited by poor cell survival, limited retention, and rapid washout. One mechanism to address this is to encapsulate the cells in a matrix or three-dimensional construct, so as to provide structural support and better mimic the cells' physiological microenvironment during administration. More specifically, the extracellular matrix (ECM), the native cellular support network, has been a strong candidate for this purpose. Moreover, there is a strong consensus that the ECM and its residing cells, including cardiac stem cells, have a constant interplay in response to tissue development, aging, disease progression, and repair. When externally stimulated, the cells and ECM work together to mutually maintain the local homeostasis by initially altering the ECM composition and stiffness, which in turn alters the cellular response and behavior. Given this constant interplay, understanding the mechanism of bidirectional cell-ECM interaction is essential to develop better cell implantation matrices to enhance cell engraftment and cardiac tissue repair. This review summarizes current understanding in the field, elucidating the signaling mechanisms between cardiac ECM and residing cells in response to IHD onset. Furthermore, this review highlights recent advances in native ECM-mimicking cardiac matrices as a platform for modulating cardiac cell behavior and inducing cardiac repair.

Root Bark of Morus Alba L. Induced p53-Independent Apoptosis in Human Colorectal Cancer Cells by Suppression of STAT3 Activity.

The root bark of Morus alba L. (MA) used in traditional oriental medicine exerts various bioactivities including anticancer effects. In this study, we investigated the molecular mechanism underlying the methylene chloride extract of MA (MEMA)-induced apoptosis in colorectal cancer (CRC) cells. We observed that MEMA decreased cell viability and colony formation in both HCT116 p53+/+ cells and HCT116 p53-/- cells. In addition, MEMA increased the sub-G1 phase DNA content, the annexin V-positive cell population, and the expression of apoptosis marker proteins in both cell lines, indicating that MEMA induced apoptosis regardless of the p53 status. Interestingly, the phosphorylation level, transcriptional activity, and target genes expression of signal transducer and activator of transcription 3 (STAT3) were commonly decreased by MEMA. The overexpression of constitutively active STAT3 in HCT116 cells reversed MEMA-induced apoptosis, demonstrating that MEMA-triggered apoptosis was mediated by the inactivation of STAT3. Taken together, we suggest that MEMA can be applied not only to p53 wild-type CRC in the early stages but also to p53-mutant advanced CRC with hyperactivated STAT3. Even though a wide range of studies are required to validate the anticancer effects of MEMA, we propose MEMA as a novel material for the treatment of CRC.

The Root Extract of Peucedanum praeruptorum Dunn Exerts Anticancer Effects in Human Non-Small-Cell Lung Cancer Cells with Different EGFR Mutation Statuses by Suppressing MET Activity.

The aim of this study was to investigate the anticancer effects of the root extract of Peucedanum praeruptorum Dunn (EPP) in human non-small-cell lung cancer (NSCLC) cells and explore the mechanisms of action. We used four types of human lung cancer cell lines, including H1299 (epidermal growth factor receptor (EGFR) wild-type), PC9 (EGFR Glu746-Ala750 deletion mutation in exon 19; EGFR tyrosine kinase inhibitor (TKI)-sensitive), H1975 (EGFR L858R/T790M double-mutant; EGFR TKI-resistant), and PC9/ER (erlotinib-resistant) cells. EPP suppressed cell growth and the colony formation of NSCLC cells in a concentration-dependent manner. EPP stimulated chromatin condensation, increased the percentage of sub-G1 phase cells, and enhanced the proportion of annexin V-positive cells, demonstrating that EPP triggered apoptosis in NSCLC cells regardless of the EGFR mutation and EGFR TKI resistance status. The phosphorylation level of the signal transducer and activator of transcription 3 (STAT3) and AKT was decreased by EPP. The expression of STAT3 target genes was also downregulated by EPP. EPP reversed hepatocyte growth factor (HGF)-induced MET phosphorylation and gefitinib resistance. Taken together, our results demonstrate that EPP exerted anticancer effects not only in EGFR TKI-sensitive NSCLC cells, but also in EGFR TKI-resistant NSCLC cells, by suppressing MET activity.

ß2-Adrenergic Receptor Signaling Pathway Stimulates the Migration and Invasion of Cancer Cells via Src Activation.

Chronic stress has been reported to stimulate the release of catecholamines, including norepinephrine (NE) and epinephrine (E), which promote cancer progression by activating the adrenergic receptor (AR). Although previous studies showed that ß2-AR mediated chronic stress-induced tumor growth and metastasis, the underlying mechanism has not been fully explored. In this study, we aimed to investigate the molecular mechanism by which ß2-AR exerts a pro-metastatic function in hepatocarcinoma (HCC) cells and breast cancer (BC) cells. Our results showed that Hep3B human HCC cells and MDA-MB-231 human BC cells exhibited the highest ADRB2 expression among diverse HCC and BC cell lines. NE, E, and isoprenaline (ISO), adrenergic agonists commonly increased the migration and invasion of Hep3B cells and MDA-MB-231 cells. The phosphorylation level of Src was significantly increased by E/NE. Dasatinib, a Src kinase inhibitor, blocked E/NE-induced migration and invasion, indicating that AR agonists enhanced the mobility of cancer cells by activating Src. ADRB2 knockdown attenuated E/NE-induced Src phosphorylation, as well as the metastatic ability of cancer cells, suggesting the essential role of ß2-AR. Taken together, our results demonstrate that chronic stress-released catecholamines promoted the migration and invasion of HCC cells and BC cells via ß2-AR-mediated Src activation.

Blue laser-induced selective vasorelaxation by the activation of NOSs.

Phototherapy has been tried for treating cardiovascular diseases. In particular, ultraviolet and blue visible lights were suggested to be useful due to their nitric oxide (NO)-production ability in the skin. However, the effects of blue light on the arterial contractility are controversial. Here, we hypothesized that appropriate protocol of blue laser can induce selective vasorelaxation by activating vasodilating signaling molecules in arteries. Using organ chamber arterial mechanics, NO assay, Matrigel assay, and microarray, we showed that a 200-Hz, 300-µs, 445-nm pulsed-laser (total energy of 600 mJ; spot size 4 mm) induced selective vasorelaxation, without vasocontraction in rat mesenteric arteries. The laser stimulation increased NO production in the cord blood-endothelial progenitor cells (CB-EPCs). Both the laser-induced vasorelaxation and NO production were inhibited by a non-selective, pan-NO synthase inhibitor, L-NG-Nitro arginine methyl ester. Microarray study in CB-EPCs suggested up-regulation of cryptochrome (CRY)2 as well as NO synthase (NOS)1 and NOSTRIN (NOS trafficking) by the laser. In conclusion, this study suggests that the 445-nm blue puled-laser can induce vasorelaxation possibly via the CRY photoreceptors and NOSs activation. The blue laser-therapy would be useful for treating systemic hypertension as well as improving local blood flow depending on the area of irradiation.

The Root Extract of Scutellaria baicalensis Induces Apoptosis in EGFR TKI-Resistant Human Lung Cancer Cells by Inactivation of STAT3.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in managing lung cancer. The root of Scutellaria baicalensis (SB) traditionally used for fever clearance and detoxification possesses various bioactivities including anticancer effects. The purpose of this study was to investigate whether SB exhibited anticancer activity in EGFR TKI-resistant lung cancer cells and to explore the underlying mechanism. We used four types of human lung cancer cell lines, including H1299 (EGFR wildtype; EGFR TKI-resistant), H1975 (acquired TKI-resistant), PC9/ER (acquired erlotinib-resistant), and PC9/GR (acquired gefitinib-resistant) cells. The ethanol extract of SB (ESB) decreased cell viability and suppressed colony formation in the four cell lines. ESB stimulated nuclear fragmentation and the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3. Consistently, the proportion of sub-G1 phase cells and annexin V+ cells were significantly elevated by ESB, indicating that ESB induced apoptotic cell death in EGFR TKI-resistant cells. ESB dephosphorylated signal transducer and activator of transcription 3 (STAT3) and downregulated the target gene expression. The overexpression of constitutively active STAT3 reversed ESB-induced apoptosis, suggesting that ESB triggered apoptosis in EGFR TKI-resistant cells by inactivating STAT3. Taken together, we propose the potential use of SB as a novel therapeutic for lung cancer patients with EGFR TKI resistance.

SUMO Modification of OsFKBP20-1b Is Integral to Proper Pre-mRNA Splicing upon Heat Stress in Rice.

OsFKBP20-1b, a plant-specific cyclophilin protein, has been implicated to regulate pre-mRNA splicing under stress conditions in rice. Here, we demonstrated that OsFKBP20-1b is SUMOylated in a reconstituted SUMOylation system in E.coli and in planta, and that the SUMOylation-coupled regulation was associated with enhanced protein stability using a less SUMOylated OsFKBP20-1b mutant (5KR_OsFKBP20-1b). Furthermore, OsFKBP20-1b directly interacted with OsSUMO1 and OsSUMO2 in the nucleus and cytoplasm, whereas the less SUMOylated 5KR_OsFKBP20-1b mutant had an impaired interaction with OsSUMO1 and 2 in the cytoplasm but not in the nucleus. Under heat stress, the abundance of an OsFKBP20-1b-GFP fusion protein was substantially increased in the nuclear speckles and cytoplasmic foci, whereas the heat-responsiveness was remarkably diminished in the presence of the less SUMOylated 5KR_OsFKBP20-1b-GFP mutant. The accumulation of endogenous SUMOylated OsFKBP20-1b was enhanced by heat stress in planta. Moreover, 5KR_OsFKBP20-1b was not sufficiently associated with the U snRNAs in the nucleus as a spliceosome component. A protoplast transfection assay indicated that the low SUMOylation level of 5KR_OsFKBP20-1b led to inaccurate alternative splicing and transcription under heat stress. Thus, our results suggest that OsFKBP20-1b is post-translationally regulated by SUMOylation, and the modification is crucial for proper RNA processing in response to heat stress in rice.

Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs.

Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line "50", whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.

Golgi-localized cyclophilin 21 proteins negatively regulate ABA signalling via the peptidyl prolyl isomerase activity during early seedling development.

KEY MESSAGE: Plant possesses particular Golgi-resident cyclophilin 21 proteins (CYP21s) and the catalytic isomerase activities have a negative effect on ABA signalling gene expression during early seedling development. Cyclophilins (CYPs) are essential for diverse cellular process, as these catalyse a rate-limiting step in protein folding. Although Golgi proteomics in Arabidopsis thaliana suggests the existence of several CYPs in the Golgi apparatus, only one putative Golgi-resident CYP protein has been reported in rice (Oryza sativa L.; OsCYP21-4). Here, we identified the Golgi-resident CYP21 family genes and analysed their molecular characteristics in Arabidopsis and rice. The CYP family genes (CYP21-1, CYP21-2, CYP21-3, and CYP21-4) are plant-specific, and their appearance and copy numbers differ among plant species. CYP21-1 and CYP21-4 are common to all angiosperms, whereas CYP21-2 and CYP21-3 evolved in the Malvidae subclass. Furthermore, all CYP21 proteins localize to cis-Golgi, trans-Golgi or both cis- and trans-Golgi membranes in plant cells. Additionally, based on the structure, enzymatic function, and topological orientation in Golgi membranes, CYP21 proteins are divided into two groups. Genetic analysis revealed that Group I proteins (CYP21-1 and CYP21-2) exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity and regulate seed germination and seedling growth and development by affecting the expression levels of abscisic acid signalling genes. Thus, we identified the Golgi-resident CYPs and demonstrated that their PPIase activities are required for early seedling growth and development in higher plants.

Lupeol suppresses plasminogen activator inhibitor-1-mediated macrophage recruitment and attenuates M2 macrophage polarization.

Tumor-associated macrophages (TAMs) are closely related with poor prognosis of cancers. The current study investigated whether lupeol regulates TAMs by focusing on the recruitment and polarization of macrophages. We found that lupeol suppressed the recruitment of THP-1 macrophages (THP-1 cells differentiated into macrophages) towards H1299 lung carcinoma cells by inhibiting plasminogen activator inhibitor-1 (PAI-1) production from H1299 cells. The reduced migration of THP-1 macrophages by lupeol was recovered by adding recombinant human PAI-1 as a chemoattractant. Knockdown of PAI-1 or treatment of tiplaxtinin, a PAI-1 inhibitor, in H1299 cells abrogated the chemotaxis of macrophages. Furthermore, lupeol suppressed the interleukin (IL)-4- and IL-13-induced M2 macrophage polarization. The mRNA expression of M2 macrophage markers and the phosphorylation of signal transducer and activator of transcription 6 (STAT6) were commonly decreased by lupeol in RAW264.7 cells. In addition, lupeol-suppressed M2 macrophage polarization led to the reduced migration of Lewis lung carcinoma (LLC) cells. Taken together, our results suggest that lupeol attenuates PAI-1-mediated macrophage recruitment towards cancer cells and inhibits M2 macrophage polarization.

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.