A Naturally Occurring Lgr4 Splice Variant Encodes a Soluble Antagonist Useful for Demonstrating the Gonadal Roles of Lgr4 in Mammals

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.     

Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.     

Loss of Lgr4 inhibits differentiation,migration and apoptosis,and promotes proliferation in bone mesenchymal stem cells

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4^+/– mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4^−/−) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4^−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.     

Tumor delivery of liposomal doxorubicin prepared with poly-L-glutamic acid as a drug-trapping agent

Context: Poly-l-glutamic acid (PGA) is an anionic polymer with a large number of carboxyl groups that can interact electrostatically with cationic drugs such as doxorubicin (DOX).

Objective: For stable encapsulation of DOX into liposomes, we prepared triethylamine (TEA)-PGA-liposomes using PGA as an internal trapping agent.

Methods: We prepared TEA-PGA-liposomes by remote loading of DOX with a TEA gradient into preformed liposomes prepared with 1, 2, or 4?mg/mL PGA (molecular weights 4800, 9800, and 20 500), and evaluated their biodistribution and antitumor effects on Lewis lung carcinoma (LLC) tumor-bearing mice.

Results: TEA-PGA-liposomes using the higher the molecular weight or concentration of PGA showed a slower release of DOX from the liposomes. TEA-PGA-liposomes prepared with a high concentration of PGA could enhance DOX accumulation in tumors and prolonged DOX circulation in the serum, indicating that DOX may be retained stably in the liposomal interior by interaction with PGA. Furthermore, injection of TEA-PGA-liposomes prepared with 4?mg/mL of PGA₉₈₀₀ or 2?mg/mL PGA₂₀₅₀₀ strongly inhibited tumor growth in LLC tumor-bearing mice.

Conclusions: PGA may be a potential trapping agent for liposomal DOX for tumor drug delivery.     

Bone marrow-derived mesenchymal stem cells promote cell proliferation of multiple myeloma through inhibiting T cell immune responses via PD-1/PD-L1 pathway

Objectives: This study aims to explore the effect of bone marrow mesenchymal stem cells (BMSCs) on multiple myeloma (MM) development and the underlying mechanism.

Materials and Methods: BMSCs from C57BL/6 J mice were isolated and the third passage was used for subsequent experiments. Additionally, a series of in vitro transwell coculture assays were performed to explore the effects of BMSCs on the proliferation of MM cells 5TGM1 and CD4⁺ T cells. Furthermore, a 5TGM1-induced MM mice model was established. Moreover, PD-L1 shRNA was transfected into BMSCs to investigate whether PD-1/PD-L1 pathway involved in BMSCs-mediated regulation of T cells and MM growth.

Results: Data revealed that BMSCs significantly promoted 5TGM1 proliferation in a dose-dependent manner. Furthermore, BMSCs administration exerted stimulatory effects on MM development in terms of shortening the mouse survival rate, promoting tumor growth, and enhancing inflammatory infiltration in the MM model mice. Moreover, BMSCs decreased the percentage of Th1 and Th17 cells, whereas increased that of Th2 and Treg cells. Their corresponding cytokines of these T cell subsets showed similar alteration in the presence of BMSCs. Additionally, BMSCs significantly suppressed CD4⁺ T cell proliferation. We also found that PD-L1 shRNA inhibited 5TGM1 proliferation likely through activation of CD4⁺ T cells. Further in vivo experiments confirmed that PD-L1 inhibition attenuated BMSCs-induced MM growth, inflammation infiltration and imbalance of Th1/Th2 and Th17/Treg.

Conclusion: In summary, our findings demonstrated that BMSCs promoted cell proliferation of MM through inhibiting T cell immune responses via PD-1/PD-L1 pathway.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Circulating fatty acids as biomarkers of dairy fat intake: data from the lifelines biobank and cohort study

Background: C14:0, C15:0, C17:0 and trans-C16:1(n-7) are often used as biomarkers for dairy fat intake. Trans-C18:1(n-7) and CLA, two fatty acids which are also present in dairy, have hardly been explored. We investigated whether trans-C18:1(n-7) and CLA can enrich the existing biomarker portfolio.

Methods: Data were obtained from Lifelines (n?=?769). Dairy fat intake was determined by FFQ. Fatty acids were measured in fasting plasma triglycerides (TG), phospholipids (PL) and cholesterol esters (CE).

Results: Median (25th–75th percentile) intakes of dairy and dairy fat were 322(209–447) and 12.3(8.4–17.4) g/d respectively. A pilot study showed that trans-C18:1(n-7) and CLA were only detectable in TG and PL. Of the established markers, TG C15:0 was most strongly associated with dairy fat intake (standardized β (std.β)?=?0.286, R²?=?0.111). Of the less established markers, TG trans-C18:1(n-7) was most strongly associated with dairy fat intake (Std.β?=?0.292, R²?=?0.115), followed by PL CLA (Std.β?=?0.272, R²?=?0.103) and PL trans-C18:1(n-7) (Std.β?=?0.269, R²?=?0.099). In TG, a combination of C15:0 and trans-C18:1(n-7) performed best (R²?=?0.128). In PL, a combination of C14:0, C15:0, trans-C18:1(n-7) and CLA performed best (R²?=?0.143).

Conclusion: Trans-C18:1(n-7) and CLA can be used as biomarkers of dairy fat intake. Additionally, combining established with less established markers allowed even stronger predictions for dairy fat intake.     

Dynamic variation of histone H3 trimethyl Lys4 (H3K4me3) and heterochromatin protein 1 (HP1) with employment length in nickel smelting workers

Objective: To investigate the dynamic variation in H3K4me3 and HP1 with employment length in nickel smelting workers.

Methods: Blood samples were collected from 140 nickel smelting workers and 140 age-matched office workers to test for H3K4me3, and HP1 levels.

Results: H3K4me3 was statistically significantly different (p?<?0.05) between the two groups and positively correlated with employment length (r_s?=_?0.267). HP1 was not correlated with employment length (p?=?0.066) but was significantly different between the two groups.

Conclusions: Chronic exposure to nickel can induce oxidative damage, and increase H3K4me3 expression and inhibit HP1 expression.     

Leucine-rich repeat-containing G-protein coupled receptor 5/GPR49 activates G12/13-Rho GTPase pathway

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5/GPR49) is highly expressed in adult stem cells of various tissues, such as intestine, hair follicles, and stomach. LGR5 is also overexpressed in some colon and ovarian tumors. Recent reports show that R-spondin (RSPO) family ligands bind to and activate LGR5, enhancing canonical Wnt signaling via the interaction with LRP5/6 and Frizzled. The identity of heterotrimeric G-proteins coupled to LGR5, however, remains unclear. Here, we show that Rho GTPase is a downstream target of LGR5. Overexpression of LGR5 induced SRF-RE luciferase activity, a reporter of Rho signaling. RSPOs, ligands for LGR4, LGR5, and LGR6, however, did not induce SRF-RE reporter activity in the presence of LGR5. Consistently, LGR5-induced activity of the SRF-RE reporter was inhibited by Rho inhibitor C3 transferase and RhoA N19 mutant, and knockdown of Gα_12/13 genes blocked the reporter activity induced by LGR5. In addition, focal adhesion kinase, NF-κB and c-fos, targets of Rho GTPase, were shown to be regulated by LGR5. Here, we have demonstrated, for the first time, that LGR5 is coupled to the Rho pathway through G_12/13 signaling.     

C-Phycocyanin elicited antitumor efficacy via cell-cycle arrest,apoptosis induction,and invasion inhibition in esophageal squamous cell carcinoma

Objectives: Mounting evidence has demonstrated that C-Phycocyanin (C-PC) exhibits marked antitumor activity in a wide type of tumors, such as pancreas cancer, breast carcinoma, lung cancer, and colon cancer. The current study aimed to confirm the antitumor efficacy of C-PC in esophageal squamous cell carcinoma (ESCC).

Methods: The efficacy of C-PC was evaluated against the proliferation of ESCC cell lines EC9706 and EC1 by CCK-8 kit and in a mice model of ESCC EC9706. Cell cycle and apoptosis were investigated by flow cytometry, and cell invasion was determined via transwell chamber. Protein expression was examined by Western blots.

Results: We found that C-PC exhibited anti-proliferation ability in a time-dependent manner and a dose-dependent manner in ESCC EC9706 and EC1 cells. Besides, C-PC markedly arrested cell cycle in the G0/G1 phase, induced cell apoptosis and suppressed cell invasion ability in both EC9706 and EC1 cells (p?<?.01). Notably, C-PC evoked the elevations of Bax, PARP, and cleaved-caspase-3 protein, but reduced cyclin D1, CDK4, Bcl-2, MMP-2, and MMP-9 expression levels. Further investigation from in vivo experiment revealed that C-PC displayed significant antitumor efficacy in the xenografted EC9706 model.

Conclusions: Our data presented herein suggest C-PC exerts antitumor efficacy in ESCC.     

Longitudinal analysis of tear cathepsin S activity levels in male non-obese diabetic mice suggests its potential as an early stage biomarker of Sjögren’s Syndrome

Context: Cathepsin S (CTSS) activity is elevated in Sjögren’s Syndrome (SS) patient tears.

Objective: To evaluate longitudinal expression of tear and tissue CTSS activity relative to other disease indicators in Non-Obese Diabetic (NOD) mice.

Methods: CTSS activity was measured in tears and lacrimal glands (LG) from male 1–6?month (M) NOD and 1 and 6?M BALB/c mice. Lymphocytic infiltration was quantified by histopathology, while disease-related proteins (Rab3D, CTSS, collagen 1) were quantified using q-PCR and immunofluorescence.

Results: In NOD LG, lymphocytic infiltration was noted by 2?M and established by 3?M (p?<?0.01). IFN-?, TNF-α, and MHC II expression were increased by 2?M (p?<?0.01). Tear CTSS activity was significantly elevated at 2?M (p?<?0.001) to a maximum of 10.1-fold by 6?M (p?<?0.001). CTSS activity in LG lysates was significantly elevated by 2?M (p?<?0.001) to a maximum of 14-fold by 3?M (p?<?0.001). CTSS and Rab3D immunofluorescence were significantly increased and decreased maximally in LG acini by 3?M and 2?M, respectively. Comparable changes were not detected between 1 and 6?M BALB/c mouse LG, although Collagen 1 was decreased by 6?M in LG of both strains.

Conclusion: Tear CTSS activity is elevated with other early disease indicators, suggesting potential as an early stage biomarker for SS.     

Taxonomical notes on Chinese camaenids with description of three new species (Gastropoda: Pulmonata)

The present study deals with four Chinese camaenid species based on museum collections and newly obtained materials. Pseudiberus liuae Wu, n. sp., diagnosed by two long mucous glands and the smallest shell size in the genus and inhabiting bare rock like other congeneric members, is described from southern Gansu. Aegista (Plectotropis) wardi (Preston, 1912) is conchologically re-described and moved out of Aegista Alber, 1860 to Pseudiberus Ancey, 1887 based on the keeled periphery and the absence of hairs, scales or their scars on the teleoconch, which are present in Aegista but partially absent in Pseudiberus. The first Chinese fluorescent snail Bradybaena qixiaensis Wu & Asami n. sp. is reported from Nanjing, Jiangsu. The species shares many characters with the Japanese fluorescent snail Bradybaena pellucida Kuroda & Habe, 1953 but is distinct in the pattern of microsculpture on the internal surface of the penis. Nesiohelix yeni Wu & Asami n. sp., sympatric with N. moreletiana (Heude, 1882), is distinguished from its congener by possession of a bubble-shaped penial caecum.

Pseudiberus liuae Wu in Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:3C9299AA-5089-4E43-9B26-85A0D6C23B66

Bradybaena qixiaensis Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:7991C4D5-5E0B-46DF-8B19-BE26511806CD

Nesiohelix yeni Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:68CFF173-AACC-4DA8-B347-9ABB5CA569A3     

LGR4 is required for sequential molar development

Tooth development requires proliferation, differentiation, and specific migration of dental epithelial cells, through well-organized signaling interactions with mesenchymal cells. Recently, it has been reported that leucine-rich repeat-containing G protein coupled receptor 4 (LGR4), the receptor of R-spondins, is expressed in many epithelial cells in various organs and tissues and is essential for organ development and stem cell maintenance. Here, we report that LGR4 contributes to the sequential development of molars in mice. LGR4 expression in dental epithelium was detected in SOX2⁺ cells in the posterior end of the second molar (M2) and the early tooth germ of the third molar (M3). In keratinocyte-specific Lgr4-deficient mice (Lgr4^{K5 KO}), the developmental defect became obvious by postnatal day 14 (P14) in M3. Lgr4^{K5 KO} adult mice showed complete absence or the dwarfed form of M3. In M3 development in Lgr4^{K5 KO} mice, at Wnt/β-catenin signal activity was down-regulated in the dental epithelium at P3, as indicated by lymphoid enhancer-binding factor-1 (LEF1) expression. We also confirmed the decrease, in dental epithelium of Lgr4^{K5 KO} mice, of the number of SOX2⁺ cells and the arrest of cell proliferation at P7, and observed abnormal differentiation at P14. Our data demonstrated that LGR4 controls the sequential development of molars by maintaining SOX2⁺ cells in the dental epithelium, which have the ability to form normal molars.     

A lyophilized sterically stabilized liposome-containing docetaxel: in vitro and in vivo evaluation

Objectives: In this study, an improved lyophilized PEGylated liposomal formulation of docetaxel (DOC) has been developed.

Methods: PEGylated docetaxel liposome (PL-DOC) was prepared by thin-film evaporation method and lyophilization. The effect of various components of the lipids and their compatibility with DOC on the entrapment efficiency (EE) of liposome was investigated. The lyophilized PL-DOC was characterized by morphology, particle size, zeta potential, EE, release in vitro and stability. Pharmacokinetics and biodistribution in vivo of lyophilized PL-DOC were also investigated.

Results: The optimal liposome formulation was egg phosphatidylcholine (EPC):cholesterol (CH):DSPE-PEG2000:DOC?=?56:40:4:4 (molar ratio). Sucrose and mannitol were chosen as cryoprotectant in the lyophilization (cryoprotectant-to-lipid (C/L) mass ratio = 8:1). The size of lyophilized PL-DOC was 152.3?±?1.0?nm with negative charge and the EE was 89.75?±?1.79%. Compared with nonlyophilized PL-DOC, the lyophilized PL-DOC was more stable at 4?°C for six months. The lyophilized PL-DOC also showed the good stability after reconstituted by 5% glucose injection. In vitro release study of PL-DOC showed that PL-DOC had a sustained release effect. After i.v. administration at the dose of 10?mg/kg in rats, a significant increase in the AUC_0-∞, MRT_0-∞ and t_1/2 was observed in PL-DOC group compared with conventional docetaxel liposome (CL-DOC) and DOC injection (DOC-I) group. Biodistribution studies in mice showed that PL-DOC significantly decreased the uptake by the organs of mononuclear phagocytic system (MPS), such as liver and spleen, while prolonging the retention time of DOC in the plasma.

Conclusion: Our PEGylated liposome formulation reported in this study could potentially produce viable clinical strategies for improved delivery of DOC for the treatment of human cancer.     

Hesperetin mitigates acrolein-induced apoptosis in lung cells in vitro and in vivo

Objectives: A number of studies have suggested that acrolein-induced lung injury and pulmonary diseases are associated with the depletion of antioxidants and the production of reactive oxygen species. Therefore, compounds that scavenge reactive oxygen species may exert protective effects against acrolein-induced apoptosis. Because hesperetin, a natural flavonoid, has been reported to have an antioxidant activity, we investigated the effect of hesperitin against acrolein-induced apoptosis of lung cells.

Methods: We evaluated the protective role of hesperetin in acrolein-induced lung injury using Lewis lung carcinoma (LLC) cells and mice.

Results: Upon exposure of LLC cells and mice to acrolein, hesperetin ameliorated the lung inbjury through attenuation of oxidative stress.

Conclusion: In the present report, we demonstrate that hesperetin exhibits a protective effect against acrolein-induced apoptosis of lung cells in both in vitro and in vivo models. Our study provides a useful model to investigate the potential application of hesperetin for the prevention of lung diseases associated with acrolein toxicity.     

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster

Objectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na₂MoO₄).

Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food.

Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration.

Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.     

A new nitrobenzoxadiazole-based GSTP1-1 inhibitor with a previously unheard of mechanism of action and high stability

Context: The nitrobezoxadiazole derivative NBDHEX is a potent inhibitor of glutathione transferase P1-1 (GSTP1-1) endowed with outstanding anticancer activity in different tumor models.

Objective: To characterize by in vitro biochemical and in silico studies the NBDHEX analogues named MC2752 and MC2753.

Materials and methods: Synthesis of MC2752 and MC2753, biochemical assays and in silico docking and normal-mode analyses.

Results: The presence of a hydrophobic moiety in the side chain of MC2753 confers unique features to this molecule. Unlike its parent drug NBDHEX, MC2753 does not require GSH to trigger the dissociation of the complex between GSTP1-1 and TRAF2, and displays high stability towards the nucleophilic attack of the tripeptide under physiological conditions.

Discussion and conclusion: MC2753 may represent a lead compound for the development of novel GSTP1-1 inhibitors not affected in their anticancer action by fluctuations of cellular GSH levels, and characterized by an increased half-life in vivo.