Estradiol‐Induced Anorexia Is Independent of Leptin and Melanin‐Concentrating Hormone

Objective: Treatment of male rodents with estradiol (E₂) is associated with anorexia and weight loss by poorly understood mechanisms. We examined the role of the orexigenic hypothalamic peptide melanin‐concentrating hormone (MCH) and the appetite‐inhibiting, fat‐derived hormone leptin in mediating E₂‐induced anorexia. Research Methods and Procedures: We studied the effect of E₂ treatment (implantation of either E₂ pellet or matching placebo) in male C57Bl/6J mice, as well as in a lean mouse model (MCH knockout mice) and an obese model (leptin‐deficient ob/ob mice). We also studied the effect of E₂ treatment in the context of high‐fat diet. Results: We confirmed E₂ dose‐dependent anorexia in male wild type mice fed a normal chow diet. E₂ treatment was associated with a significant decrease in body fat, serum leptin levels, and arcuate hypothalamic proopiomelanocortin expression. E₂‐implanted mice also showed increased hypothalamic neuropeptide Y and MCH expression. As MCH has been implicated in E₂‐induced hypophagia, we performed E₂ pellet implantation in MCH knockout mice and observed hypophagia and weight loss, indicating that MCH is not an essential mediator of E₂‐induced anorexia. E₂‐implanted ob/ob mice also had hypophagia and weight loss, indicating that leptin is not essential for E₂‐induced anorexia. High‐fat diet significantly exacerbated the effect of E₂ treatment, leading to a 99.6% decrease in food intake at 48 hours and a 30% loss of body weight within 1 week. Discussion: The anorectic effects of E₂ were independent of MCH and leptin. Our results suggested that E₂ may have effects on nutrient preferences.     

17β‐estradiol supplementation attenuates ovariectomy‐induced increases in ATGL signaling and reduced perilipin expression in visceral adipose tissue

Adipocytes from post‐menopausal females have higher basal lipolytic rates than pre‐menopausal females, which contributes to increased risk of developing dyslipidemia following menopause. The purpose of this study was to delineate cellular mechanisms affecting adipose tissue function in the ovariectomized (OVX) mouse and also determine if physical activity or estrogen supplementation alter any detected changes. Female C57/Bl6 mice were placed into SHAM, OVX sedentary (OVX), OVX exercise (OVX‐Ex), and OVX sedentary + 17β‐estradiol (OVX + E₂) groups. Visceral fat mass, glycerol, and NEFA levels were significantly higher in OVX mice compared to SHAM animals, but were not elevated in the E₂‐treated animals. Voluntary running failed to change circulating levels of glycerol or NEFA in OVX mice, but did partially attenuate the increase in visceral fat mass. Adipose triglyceride lipase (ATGL) protein content was significantly elevated in visceral fat from OVX and OVX‐Ex groups compared to SHAM, while ATGL–CGI‐58 interaction was significantly higher in OVX than SHAM and OVX + E₂ mice. No significant differences in HSL phosphorylation were detected between groups, however, ERK1/2 phosphorylation was significantly elevated in the OVX mice. To determine if ERK1/2 function was critical for the increased glycerol levels, visceral fat was treated with MEK inhibitor PD98059, with no differences in glycerol release detected. Perilipin protein content was decreased significantly in OVX and OVX‐Ex mice compared to SHAM. Thus, these data suggest that increased ATGL signaling and reduced perilipin protein content may contribute to increased NEFA and glycerol levels in OVX mice, which are attenuated with E₂ treatment, but not by exercise. J. Cell. Biochem. 110: 420–427, 2010. © 2010 Wiley‐Liss, Inc.     

Modulating the structure of phenylalanine‐incorporated ascidiacyclamide through fluorination

We designed four fluorinated Phe‐incorporated ascidiacyclamide ([Phe]ASC) analogs, (cyclo(?Xxx1‐oxazoline2‐d ‐Val3‐thiazole4‐Ile5‐oxazoline6‐d ‐Val7‐thiazole8‐)), [(4‐F)Phe]ASC (Xxx1: 4‐fluorophenylalanine), [(3,5‐F₂)Phe]ASC (Xxx1: 3,5‐difluorophenylalanine), [(3,4,5‐F₃)Phe]ASC (Xxx1: 3,4,5‐trifluorophenylalanine) and [(F₅)Phe]ASC (Xxx1: pentafluorophenylalanine), to modulate the π‐electron density of the aromatic ring of the Phe residue. X‐ray diffraction analysis, ¹H NMR and CD spectra all suggested that the interactions between the benzene ring of the Xxx1 residue and the alkyl groups of oxazoline2 contribute to the stability of the folded structure of these analogs. Substituting fluorines for the hydrogens progressively weakened those interactions through reducing the π‐electron density, thereby mediating transformation from the folded to square structure. As a result, [(F₅)Phe]ASC preferred the square form more than the other analogs did. Also contributing to the preference for the square form may be the hindrance of the rotation around the Cα–Cβ bond by the two ortho‐fluoro substituents of [(F₅)Phe]ASC. These findings demonstrate that the structure of ASC can be modulated by using fluorine as an electron‐withdrawing group. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The effects of peptides with estrogen‐like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells

Hormone replacement therapy (HRT) for post‐menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post‐menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen‐mimetic peptide 1 (EMP‐1) (VSWFFE) in comparison to estrogen (E₂) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP‐1‐like E₂ inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP‐1 did not affect E₂ stimulation of DNA, but inhibited E₂ inhibition of cell proliferation at high concentration. All effects by the combination of EMP‐1 and E₂ were abolished at hg. A44‐stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co‐incubated with E₂ at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E₂ stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45‐stimulated CK‐specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP‐1. EMP‐1 and A44 similar to E₂‐stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP‐1 provided it acts similarly in vivo can replace E₂ for treatment of post‐menopausal women in hyperglycemia due to diabetes. J. Cell. Biochem. 110: 1142–1146, 2010. Published 2010 Wiley‐Liss, Inc.     

Medium‐Chain Fatty Acids Attenuate Agonist‐Stimulated Lipolysis,Mimicking the Effects of Starvation

Objective: To test the hypothesis that incorporation of medium‐chain fatty acids (FAs) into adipocyte triglycerides alters intracellular lipolysis. Research Methods and Procedures: 3T3‐L1 adipocytes were pretreated with octanoate for various incubation periods. After the removal of exogenous FAs, cells were incubated with different lipolytic agonists. To determine the effects on lipolysis, we measured the following: the release of glycerol and FAs, lipase activity, protein levels of hormone‐sensitive lipase (HSL), and perilipin A; translocation of HSL; phosphorylation of perilipin A; and levels of cellular adenosine triphosphate, cyclic adenosine monophosphate, and H₂O₂. To compare the effects of starvation with those caused by octanoate pretreatment, we measured glycerol release and H₂O₂ generation in rat adipocytes of starved donors. Results: Pretreatment of adipocytes with octanoate in vitro increased basal lipolysis but decreased the cellular response for agonists. The same effects were seen in starvation in vivo. Preincubation with octanoate for 48 hours did not affect basal lipase activity, HSL, and perilipin protein levels, but it reduced agonist‐stimulated perilipin phosphorylation and HSL translocation toward fat droplets. This was associated with a reduction in basal cellular adenosine triphosphate levels and agonist‐stimulated cyclic adenosine monophosphate generation. Starvation and octanoate pretreatment both increased intracellular H₂O₂ concentrations, which might also contribute to the inhibition on agonist‐stimulated lipolysis. Discussion: Pretreatment with octanoate seems to induce changes in adipocyte lipolysis in a pattern mimicking the effects of starvation. Such changes could contribute, in part, to weight loss in animals and humans associated with dietary medium‐chain FAs.     

Low‐intensity pulsed ultrasound induced enhanced adipogenesis of adipose‐derived stem cells

Objective

The aim of this study was to investigate effects of low‐intensity pulsed ultrasound (LIPUS) on differentiation of adipose‐derived stem cells (ASCs), in vitro.

Materials and methods

Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR‐γ1, and APN, was examined by real‐time PCR. Immunofluorescence (IF) staining was performed to test for PPAR‐γ at the protein level.

Results

Our data revealed that specific patterns of LIPUS up‐regulated levels of both PPAR‐γ1 and APN mRNA, and PPAR‐γ protein.

Conclusions

In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

     

Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of post‐menopausal women with severe pelvic organ prolapse

This study investigates the effect of local oestrogen therapy (LET) on the expression of proteins participating in collagen/elastin biogenesis and immune markers in vaginal tissues of post‐menopausal women with severe pelvic organ prolapse (POP). Vaginal biopsies were collected from the anterior vaginal wall of informed and consented 52 post‐menopausal women with severe POP undergoing total hysterectomy. Twenty‐nine of the 52 women were treated with LET (in the form of vaginal oestrogen cream or tablet), while the remaining 23 untreated patients served as the controls. This study was approved by Sinai Health System REB. Vaginal tissue specimens were analysed for gene and protein expression using real‐time RT‐PCR and Luminex assays, protein localization and immune cell infiltration were assessed by immunohistochemistry. Forty‐four cytokines were detected. We found that LET application: (a) significantly increased (P < 0.05) gene and protein expression levels of extracellular matrix (ECM) structural proteins, collagen and elastin, as well as the expression of ECM maturation enzyme BMP1; (b) decreased protein expression level of ECM degradation enzymes MMP1, MMP2 and MMP3 accompanied by an increase in their tissue inhibitors, TIMP1 and TIMP4; (c) significantly increased (P < 0.05) the gene and protein expression levels of 14 vaginal cytokines involved in leucocyte infiltration, which was confirmed by immunohistochemistry. Our results indicate that LET plays an important role in the activation of immune system within the local vaginal environment, limiting the undesirable ECM degradation, which supports the strengthening of vaginal ECM in post‐menopausal women, therefore resisting menopause/age‐related changes and inducing urogenital tract tissue regeneration.     

Evaluation of Ki‐67 and Bcl‐2 antigen expression in breast carcinomas of women treated with raloxifene

Objectives: To evaluate the effect of raloxifene on Ki‐67 and Bcl‐2 antigen expression in operable, stage II, oestrogen‐receptor‐positive invasive ductal breast carcinomas. Materials and methods: Twenty post‐menopausal women who had taken 60 mg of raloxifene daily for 28 days prior to definitive surgery were enrolled in the investigation. Two tumour samples were obtained by incisional biopsy during the study, one at the time of confirmation of diagnosis of invasive ductal carcinoma and evaluation of oestrogen receptor status, and the other 29 days later, at the time of definitive surgery. Immunohistochemistry was performed on tumour samples, prior to and after raloxifene treatment, to evaluate Ki‐67 and Bcl‐2 expression. Friedman and McNemar tests were used for statistical analysis of the data, significance being established at 5%. Results: Mean percentage of Ki‐67‐stained nuclei was 24.86 ± 2.95 prior to raloxifene treatment and 13.33 ± 1.52 after treatment (P < 0.001). Prior to raloxifene treatment, only 9/20 cases (45%) were classified as Bcl‐2‐positive, whereas after treatment, 17/20 (85%) were classified as Bcl‐2‐positive (P < 0.013). Conclusions: Raloxifene treatment significantly reduced Ki‐67 antigen expression and increased Bcl‐2 expression in breast carcinomas of post‐menopausal women.     

Antiproliferative Evaluation of Some 2‐[2‐(2‐Phenylethenyl)‐cyclopent‐3‐en‐1‐yl]‐1,3‐benzothiazoles: DFT and Molecular Docking Study

The 2‐[2‐(2‐phenylethenyl)cyclopent‐3‐en‐1‐yl]‐1,3‐benzothiazoles were synthesized from the reactions of 7‐benzylidenebicyclo[3.2.0]hept‐2‐en‐6‐ones with 2‐aminobenzenethiol. The antiproliferative activities of 2‐[2‐(2‐phenylethenyl)cyclopent‐3‐en‐1‐yl]‐1,3‐benzothiazoles were determined against C6 (rat brain tumor) and HeLa (human cervical carcinoma cells) cell lines using BrdU cell proliferation ELISA assay. Cisplatin and 5‐fluorouracil (5‐FU) were used as standards. The most active compound was 2‐{(1S,2S)‐2‐[(E)‐2‐(4‐methylphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole against C6 cell lines with IC₅₀=5.89 μm value (cisplatin, IC₅₀=14.46 μm and 5‐FU, IC₅₀=76.74 μm ). Furthermore, the most active compound was 2‐{(1S,2S)‐2‐[(E)‐2‐(2‐methoxyphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole against HeLa cell lines with IC₅₀=3.98 μm (cisplatin, IC₅₀=37.95 μm and 5‐FU, IC₅₀=46.32 μm ). Additionally, computational studies of related molecules were performed by using B3LYP/6‐31G+(d,p) level in the gas phase. Experimental IR and NMR data were compared with the calculated results and were found to be compatible with each other. Molecular electrostatic potential (MEP) maps of the most active 2‐{(1S,2S)‐2‐[(E)‐2‐(2‐methoxyphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole against HeLa and the most active 2‐{(1S,2S)‐2‐[(E)‐2‐(4‐methylphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole against C6 were investigated, aiming to determine the region that the molecule is biologically active. Biological activities of mentioned molecules were investigated with molecular docking analyses. The appropriate target protein (PDB codes: 1 M17 for the HeLa cells and 1JQH for the C6 cells) was used for 2‐{(1S,2S)‐2‐[(E)‐2‐(2‐methoxyphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole and 2‐{(1S,2S)‐2‐[(E)‐2‐(4‐methylphenyl)ethenyl]cyclopent‐3‐en‐1‐yl}‐1,3‐benzothiazole molecules exhibiting the highest biological activity against HeLa and C6 cells in the docking studies. As a result, it was determined that these molecules are the best candidates for the anticancer drug.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.     

Machine learning reveals sex‐specific 17β‐estradiol‐responsive expression patterns in white perch (Morone americana) plasma proteins

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17β‐estradiol (E₂) induction. Semiquantitative nanoLC‐MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two‐way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E₂‐induced plasma samples using the protein expression data. E₂‐induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain‐containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E₂‐responsive proteins in fishes and therefore may be useful indicators of estrogen induction.     

Analysis of cell growth and gene expression of porcine adipose tissue‐derived mesenchymal stem cells as nuclear donor cell

In several laboratory animals and humans, adipose tissue‐derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming‐related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4‐year‐old female miniature pig. The ASC expressed cell‐surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation‐inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P < 0.05), and SOX2 showed significantly higher expression in ASC than in the other two cell types (P < 0.05). After somatic cell nuclear transfer (SCNT), the development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

Oestrogen induces epithelial‐mesenchymal transition in endometriosis via circ_0004712/miR‐148a‐3p sponge function

Endometriosis is a common, chronic gynaecologic disease affecting up to 10% of women in their reproductive age and leading to pain and infertility. Oestrogen (E₂)‐induced epithelial‐mesenchymal transition (EMT) process has been considered as a key factor of endometriosis development. Recently, the dysregulated circular RNAs (circRNAs) have been discovered in endometriosis tissues. However, the molecular mechanism of circRNAs on the E₂‐induced EMT process in endometriosis is still unknown. Here, we demonstrated that circ_0004712 up‐regulated by E₂ treatment in endometrial epithelial cells. Knock‐down the expression of circ_0004712 significantly suppressed E₂‐induced cell migration activity. Meanwhile, we identified miR‐148a‐3p as a potential target miRNA of circ_0004712. Inhibited the expression of miR‐148a‐3p could recovered the effect of circ_0004712 knock‐down in E₂‐treated endometrial epithelial. Furthermore, Western blot assay showed that E₂ treatment could increase the expression and activity of β‐catenin, snail and N‐cadherin and reduce the expression of E‐cadherin. The expression and activity of β‐catenin pathway were recovered by circ_0004712 knock‐down or miR‐148a‐3p overexpression. Altogether, the results demonstrate that circ_0004712/miR‐148a‐3p plays an important role in E₂‐induced EMT process in the development of endometriosis, and the molecular mechanism may be associated with the β‐catenin pathway. This work highlighted the importance of circRNAs in the development of endometriosis and provide a new biomarker for diagnosis and therapies.     

Investigating a possible role for the bacterial signal molecules N‐acylhomoserine lactones in Balanus improvisus cyprid settlement

Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N‐acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL‐producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL‐degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3‐oxo‐C6‐HSL), rhlI from Pseudomonas aeruginosa (C4‐HSL/C6‐HSL), vanI from V. anguillarum (3‐oxo‐C10‐HSL) and sulI from Sulfitobacter sp. BR1 (C4‐HSL, 3‐hydroxy‐C6‐HSL, C8‐HSL and 3‐hydroxy‐C10‐HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8‐HSL, 3‐oxo‐C10‐HSL and C12‐HSL) at concentrations similar to those found within natural biofilms (5 μm ) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL‐signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.