Variation of sesquiterpene lactone contents in Lactuca altaica natural populations from Armenia

In recent years we initiated extensive studies on the characterization of the population structure of wild Lactuca relatives (WLRs) originating from their center of origin and diversity in Southwest Asia. A comparative phytochemical study of nine sesquiterpene lactones in natural populations of the wild lettuce L. altaica Fisch. & C.A. Mey. (Asteraceae) was performed, based on 22 plants, representing seven original individual seed samples derived from three localities representing three regions in Armenia. The compounds were profiled and quantified in leaves and roots of the plants, grown in a controlled glasshouse. The contents of major sesquiterpene lactones, that including the following eight guaianolides: cichorioside B, lactucin, 11β,13-dihydrolactucin, crepidiaside B, 8-deoxylactucin, jacquinelin, lactucopicrin/11β,13-dihydrolactucopicrin, as well as the germacranolide glucoside – lactuside A, were estimated by HPLC/PDA. The L. altaica plants could be characterized by the occurrence of lactuside A in their roots, and the mixture of lactucopicrin/11β,13-dihydrolactucopicrin in both their roots and leaves by relatively high amounts, similarly to results obtained for three commercial cultivars of L. sativa. The total content of sesquiterpene lactones in the roots was significantly higher than that in the leaves. This study is likely the first report of detailed screening of L. altaica natural populations and individuals, even by low sample size, for any trait. Species within the primary lettuce gene pool, should be considered as an attractive source of germplasm in further research and improvement of cultivated lettuce, Lactuca sativa L. While using interspecific hybridization in order to elevate the sesquiterpene levels in cultivated lettuces, the lactones quality (profile) and quantity, as well as the cross-ability level of the wild Lactuca spp. with the crop and fertility of the obtained offspring should be considered.     

Low level electricity increases the secretion of extracellular vesicles from cultured cells

Exosomes, a type of extracellular vesicles, can be collected from the conditioned medium of cultured cells, and are expected to be used in disease therapy and drug delivery systems. However, since the yield of exosomes from conditioned medium is generally low, investigations to develop new methods to increase exosome secretion and to elucidate the secretion mechanism have been performed. Our previous studies demonstrated that activation of intracellular signaling including Rho GTPase and subsequent endocytosis of extraneous molecules in cells could be induced by low level electricity (0.3–0.5 mA/cm²). Since exosomes are produced in the process of endocytosis and secreted by exocytosis via certain signaling pathways, we hypothesized that low level electric treatment (ET) would increase exosome secretion from cultured cells via intracellular signaling activation. In the present study, the influence of ET (0.34 mA/cm²) on extracellular vesicle (EV) secretion from cultured cells was examined by using murine melanoma and murine fibroblast cells. The results showed that the number of EV particles collected by ultracentrifugation was remarkably increased by ET in both cell lines without cellular toxicity or changes in the particle distribution. Also, protein amounts of the collected EVs were significantly increased in both cells by ET without alteration of expression of representative exosome marker proteins. Moreover, in both cells, the ratio of particle numbers to protein amount was not significantly changed by ET. Rho GTPase inhibition significantly suppressed ET-mediated increase of EV secretion in murine melanoma, indicating that Rho GTPase activation could be involved in ET-mediated EV secretion in the cell. Additionally, there were almost no differences in uptake of each EV into each donor cell regardless of whether the cells had been exposed to ET for EV collection. Taken together, these results suggest that ET could increase EV secretion from both cancer and normal cells without apparent changes in EV quality.     

血浆miR-29b与急性冠脉综合征患者胞外基质蛋白含量的关系及其诊断价值分析

目的:探究血浆微小RNA-29b(miR-29b)与急性冠脉综合征患者细胞外基质(ECM)蛋白含量的关系及其诊断价值。方法:选择2019年2月至2019年7月我院诊治的80例急性冠脉综合征患者作为观察组,另选择同期我院体检的80例健康志愿者作为对照组。检测两组受试者血浆miR-29b、透明质酸(HA)、层黏连蛋白(LN)和Ⅰ型前胶原蛋白(PCⅠ)水平,采用Pearson相关性分析血浆miR-29b与HA、LN和PCⅠ水平的相关性,采用受试者工作特征(ROC)曲线分析血浆miR-29b、HA、LN和PCⅠ对急性冠脉综合征的诊断价值。结果:与对照组相比,观察组的血浆miR-29b水平明显下降(P0.05),而血浆HA、LN和PCⅠ水平明显升高(P0.05)。血浆miR-29b水平与血浆HA、LN和PCⅠ水平均呈负相关关系(P0.05),ROC曲线分析结果显示,血浆miR-29b单独检测的敏感性和特异性分别为79.10%、71.60%,优于ECM三个指标的单独检测,且四项指标联合检测的敏感性和特异性高于单项检测。结论:急性冠脉综合征患者血浆miR-29b水平下调,且与ECM蛋白水平呈负相关,在急性冠脉综合征临床诊断中具有一定的价值。     

Oxidative modifications of the C-terminal domain of tropoelastin prevent cell binding

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin α(v)β(3) and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO(-)) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO(-) treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO(-) treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO(-) prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.     

The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.     

Fibromodulin suppresses nuclear factor-kappaB activity by inducing the delayed degradation of IKBA via a JNK-dependent pathway coupled to fibroblast apoptosis

Fibulin-5 (FBLN5) belongs to the Fibulin family of secreted extracellular matrix proteins, and our laboratory first established FBLN5 as a novel target for TGF-β in fibroblasts and endothelial cells. To better understand the pathophysiology of FBLN5, we carried out microarray analysis to identify fibroblast genes whose expressions were regulated by FBLN5 and TGF-β. In doing so, we identified fibromodulin (Fmod) as a novel target gene of FBLN5, and we validated the differential expression of Fmod and 12 other FBLN5-regulated genes by semi-quantitative real time PCR. Fmod belongs to the small leucine-rich family of proteoglycans, which are important constituents of mammalian extracellular matrices. Interestingly, parental 3T3-L1 fibroblasts displayed high levels of nuclear factor-κB (NF-κB) activity, although those engineered to express Fmod constitutively exhibited significantly reduced NF-κB activity, suggesting that Fmod functions to inhibit NF-κB signaling. By monitoring alterations in the activation of NF-κB and the degradation of its inhibitor, IκBα, we demonstrate for the first time that Fmod contributes to the constitutive degradation of IκBα protein in 3T3-L1 fibroblasts. Mechanistically, we observed Fmod to delay the degradation of IκBα by promoting the following: (i) activation of c-Jun N-terminal kinase; (ii) inhibition of calpain and casein kinase 2 activity; and (iii) induction of fibroblast apoptosis. Taken together, our study identified a novel function for Fmod in directing extracellular signaling, particularly the regulation of NF-κB activity and cell survival.     

Genetic analysis of the heparan modification network in Caenorhabditis elegans

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.     

The real-time detection of acupuncture-induced extracellular ATP mobilization in acupoints and exploration of its role in acupuncture analgesia

Our and in vitro studies had confirmed that mechanosensitive ATP release and accumulation in acupoints was elicited by acupuncture (AP), which might be a pivotal step for triggering AP analgesia. But to date, the dynamics of extracellular ATP (eATP) in the interstitial space during AP process was poorly known, mainly due to the low temporal resolution of the current detection approach. This study attempted to capture rapid eATP signals in vivo in the process of needling, and further explored the role of this eATP mobilization in initiating AP analgesic effect. Ipsilateral 20-min needling was applied on Zusanli acupoint (ST36) of complete Freund’s adjuvant (CFA)–induced ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in the interstitial space was microdialyzed and real-time quantified by luciferin-luciferase assay at 1-min interval with the aid of the microfluid chip. We revealed in behavioral tests that modulation of eATP levels in ST36 influenced AP analgesic effect on ankle arthritis. A transient eATP accumulation was induced by needling that started to mobilize at 4 min, climbed to the peak of 11.21 nM within 3.25 min and gradually recovered. Such AP-induced eATP mobilization was significantly impacted by ankle inflammation, needling depth, needle manipulation, and the presence of local ecto-nucleotidases. This work reveals that needling elicits a transient eATP mobilization in acupoints, which contributes to initiating AP analgesia. This study will help us better understand the peripheral mechanism of AP analgesia and guide clinicians to optimize the needle manipulations to improve AP efficacy.     

Redescription of Strombidium oculatum Gruber 1884 (Ciliophora,Oligotrichia)

The marine, tide pool-dwelling ciliate Stombidium oculatum was redescribed using live, stained, SEM, and TEM material prepared from samples collected from pools on the Isle of Man (Irish Sea) and Brittany (France). Also, we reviewed the older German and French works that reported on ciliates collected in the Mediterranean and Brittany, respectively. The Brittany and Isle of Man populations of the ciliate were considered identical. Some morphological and behavioural differences exist between the Brittany-Isle of Man populations and the Mediterranean populations, but they were insufficient to distinguish different taxa. Thus, taxa from all three locations were considered to be conspecific. Key features used to describe the ciliate were: morphology and ultrastructure of the free-swimming ciliate; cyst morphology; presence of mixotrophic-chloroplasts; presence of an eye spot composed of stigma obtained from chlorophyte prey; division, morphogenesis, and nuclear structure; live observations and behaviour, including the encystment-excystment cycle. Based on morphological and behavioural characteristics the taxon was distinguished from other similar species, and a neotype has been designated as no type material exists.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.