Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

INTRODUCTION: Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. OBJECTIVES: To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. METHODS: A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. RESULTS: Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. CONCLUSION: The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway.

Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10-7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Suppressing chlorophyll degradation by silencing OsNYC3 improves rice resistance to Rhizoctonia solani, the causal agent of sheath blight.

Necrotrophic fungus Rhizoctonia solani Kühn (R. solani) causes serious diseases in many crops worldwide, including rice and maize sheath blight (ShB). Crop resistance to the fungus is a quantitative trait and resistance mechanism remains largely unknown, severely hindering the progress on developing resistant varieties. In this study, we found that resistant variety YSBR1 has apparently stronger ability to suppress the expansion of R. solani than susceptible Lemont in both field and growth chamber conditions. Comparison of transcriptomic profiles shows that the photosynthetic system including chlorophyll biosynthesis is highly suppressed by R. solani in Lemont but weakly in YSBR1. YSBR1 shows higher chlorophyll content than that of Lemont, and inducing chlorophyll degradation by dark treatment significantly reduces its resistance. Furthermore, three rice mutants and one maize mutant that carry impaired chlorophyll biosynthesis all display enhanced susceptibility to R. solani. Overexpression of OsNYC3, a chlorophyll degradation gene apparently induced expression by R. solani infection, significantly enhanced ShB susceptibility in a high-yield ShB-susceptible variety '9522'. However, silencing its transcription apparently improves ShB resistance without compromising agronomic traits or yield in field tests. Interestingly, altering chlorophyll content does not affect rice resistance to blight and blast diseases, caused by biotrophic and hemi-biotrophic pathogens, respectively. Our study reveals that chlorophyll plays an important role in ShB resistance and suppressing chlorophyll degradation induced by R. solani infection apparently improves rice ShB resistance. This discovery provides a novel target for developing resistant crop to necrotrophic fungus R. solani.

Uterine-specific SIRT1 deficiency confers premature uterine aging and impairs invasion and spacing of blastocyst, and stromal cell decidualization, in mice.

A distinct age-related alteration in the uterine environment has recently been identified as a prevalent cause of the reproductive decline in older female mice. However, the molecular mechanisms that underlie age-associated uterine adaptability to pregnancy are not known. Sirtuin 1 (SIRT1), a multifunctional NAD+-dependent deacetylase that regulates cell viability, senescence and inflammation during aging, is reduced in aged decidua. Thus, we hypothesize that SIRT1 plays a critical role in uterine adaptability to pregnancy and that uterine-specific ablation of Sirt1 gene accelerates premature uterine aging. Female mice with uterine ablation of Sirt1 gene using progesterone receptor Cre (PgrCre) exhibit subfertility and signs of premature uterine aging. These Sirt1-deficient mothers showed decreases in litter size from their 1st pregnancy and became sterile (25.1 ± 2.5 weeks of age) after giving birth to the third litter. We report that uterine-specific Sirt1 deficiency impairs invasion and spacing of blastocysts, and stromal cell decidualization, leading to abnormal placentation. We found that these problems traced back to the very early stages of hormonal priming of the uterus. During the window of receptivity, Sirt1 deficiency compromises uterine epithelial-stromal crosstalk, whereby estrogen, progesterone and Indian hedgehog signaling pathways are dysregulated, hampering stromal cell priming for decidualization. Uterine transcriptomic analyses also link these causes to perturbations of histone proteins and epigenetic modifiers, as well as adrenomedullin signaling, hyaluronic acid metabolism, and cell senescence. Strikingly, our results also identified genes with significant overlaps with the transcriptome of uteri from aged mice and transcriptomes related to master regulators of decidualization (e.g. Foxo1, Wnt4, Sox17, Bmp2, Egfr and Nr2f2). Our results also implicate accelerated deposition of aging-related fibrillar Type I and III collagens in Sirt1-deficient uteri. Collectively, SIRT1 is an important age-related regulator of invasion and spacing of blastocysts, as well as decidualization of stromal cells.

Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics analysis.

INTRODUCTION: Data-dependent acquisition (DDA) is the most commonly used MS/MS scan method for lipidomics analysis on orbitrap-based instrument. However, MS instrument associated software decide the top N precursors for fragmentation, resulting in stochasticity of precursor selection and compromised consistency and reproducibility. We introduce a novel workflow using biologically relevant lipids to construct inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow. OBJECTIVES: To ensure consistent coverage of biologically relevant lipids in LC-MS/MS-based lipidomics analysis. METHODS: Biologically relevant ion list was constructed based on LIPID MAPS and lipidome atlas in MS-DIAL 4. Lipids were extracted from mouse tissues and used to assess different MS/MS scan workflow (DDA, BRI-DIA, and hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer. RESULTS: DDA resulted in more MS/MS events, but the total number of unique lipids identified by three methods (DDA, BRI-DIA, and hybrid MS/MS scan mode) is comparable (580 unique lipids across 44 lipid subclasses in mouse liver). Major cardiolipin molecular species were identified by data generated using BRI-DIA and hybrid methods and allowed calculation of cardiolipin compositions, while identification of the most abundant cardiolipin CL72:8 was missing in data generated using DDA method, leading to wrong calculation of cardiolipin composition. CONCLUSION: The method of using inclusion list comprised of biologically relevant lipids in DIA MS/MS scan is as efficient as traditional DDA method in profiling lipids, but offers better consistency of lipid identification, compared to DDA method. This study was performed using Orbitrap Exploris 480, and we will further evaluate this workflow on other platforms, and if verified by future work, this biologically relevant ion fragmentation workflow could be routinely used in many studies to improve MS/MS identification capacities.

Activated microglia promote invasion and barrier dysfunction of brain endothelial cells via regulating the CXCL13/CXCR5 axis.

The blood brain barrier (BBB) is a protective border that prevents noxious substances from gaining access to the central nervous system (CNS). CXCL13 is a chemokine from the CXC chemokine family, which has been shown to destroy the barrier function of umbilical vein endothelial cells with its receptor CXCR5. Here, we aimed to investigate the role of CXCL13/CXCR5 signaling axis in BBB. The invasive ability of bEnd.3 cells was determined by the Transwell invasion assay. The barrier integrity of bEnd.3 cells was assessed by detecting trans-endothelial electrical resistance, the permeability to fluorescein isothiocyanate-dextran, and the expression levels of the tight junction protein E-cadherin. Lipopolysaccharide (LPS)-activated microglia promoted invasion and barrier dysfunction, and upregulated CXCR5 and p-p38 expression levels in cocultured bEnd.3 cells. However, the effects of activated microglia were alleviated by knocking down CXCR5 in cocultured bEnd.3 cells. Furthermore, recombinant CXCL13 promoted invasion and barrier dysfunction, and upregulated the expression levels of p-p38 in bEnd.3 cells; however, its effects were abolished by treating bEnd.3 cells with the p38 inhibitor SB203580. Our data tentatively demonstrated that LPS-activated microglial cells may promote invasion and barrier dysfunction in bEnd.3 cells by regulating the CXCL13/CXCR5 axis and p38 signaling.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†.

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the ß3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Magnolol suppressed cell migration and invasion and induced cell apoptosis via inhibition of the NF-κB signaling pathway by upregulating microRNA-129 in multiple myeloma.

Multiple myeloma (MM) is incurable cancer in the blood system. Magnolol is an effective component against various cancers. This study tried to investigate the effect and mechanism of magnolol on MM via regulating miR-129. Human normal plasma cells (nPCs) and MM cells U266 and LP1 were used in this study, accompanied by treatment of magnolol. The miR-129 inhibitor was transfected into U266 and LP1 cells during experiments. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were tested by wound healing assay and Transwell assay. And Annexin-V-FITC/PI assay was utilized to assess cell apoptosis. miR-129, miR-1271-5p, miR-342-3p, and miR-124-3p expressions were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and western blot was adopted to evaluate Cyclin D1, matrix metalloprotein (MMP)-7, MMP-9, phosphorylation (p)-IκBα, p-p65, and p65 protein levels. In U266 and LP1 cells, with magnolol concentration increasing, cell viability, migration, and invasion rates, Cyclin D1, MMP-7, and MMP-9 expressions decreased, while cell apoptosis rose. And magnolol increased the miR-129 expression in MM cells. Besides, miR-129 inhibitor antagonized the above-mentioned effect of magnolol and partly offset the magnolol-induced decrease of p-IκBα and p-p65 expression, as well as the ratio of p-p65 to p65 in U266 and LP1 cells. Magnolol suppressed cell migration and invasion and induced cell apoptosis via inhibiting NF-κB pathway activation, by upregulating miR-129 in MM.

Amino Acids in Cell Signaling: Regulation and Function.

Amino acids are the main building blocks for life. Aside from their roles in composing proteins, functional amino acids and their metabolites play regulatory roles in key metabolic cascades, gene expressions, and cell-to-cell communication via a variety of cell signaling pathways. These metabolic networks are necessary for maintenance, growth, reproduction, and immunity in humans and animals. These amino acids include, but are not limited to, arginine, glutamine, glutamate, glycine, leucine, proline, and tryptophan. We will discuss these functional amino acids in cell signaling pathways in mammals with a particular emphasis on mTORC1, AMPK, and MAPK pathways for protein synthesis, nutrient sensing, and anti-inflammatory responses, as well as cell survival, growth, and development.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Distal acupoint stimulation versus peri-incisional stimulation for postoperative pain in open abdominal surgery: a systematic review and implications for clinical practice.

BACKGROUND: Acute postoperative pain remains a major clinical problem that affects patient recovery. Distal acupoint and peri-incisional stimulation are both used for relieving acute postoperative pain in hospital. Our objective was to assess and compare the effects of distal and peri-incisional stimulation on postoperative pain in open abdominal surgery. METHODS: MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and Chinese databases CNKI and Wanfangdata were searched to identify eligible randomized controlled trials. Intensity of postoperative pain, opioid consumption and related data were extracted and analyzed using a random effects model. Risk of bias was assessed. Subgroup analyses were conducted when data were enough. RESULTS: Thirty-five trials were included, in which 17 trials studied distal stimulation, another 17 trials studied peri-incisional stimulation and one studied the combination of the two approaches. No studies that directly compared the two approaches were identified. Subgroup analysis showed that both distal and peri-incisional stimulation significantly alleviated postoperative resting and movement pain from 4 h to 48 h after surgery by 6 to 25 mm on a 100 mm visual analogue scale. Peri-incisional stimulation showed a better reduction in postoperative opioid consumption. No studies compared the effects of the combined peri-incisional and distal stimulation with either mode alone. Overall the quality of evidence was moderate due to a lack of blinding in some studies, and unclear risk of allocation concealment. CONCLUSION: Both distal and peri-incisional modes of stimulation were effective in reducing postoperative pain. Whether a combined peri-incisional stimulation and distal acupuncture has superior results requires further studies.

Innate differences and colostrum-induced alterations of jejunal mucosal proteins in piglets with intra-uterine growth restriction.

Mammalian neonates undergo rapid transitions from a sterile uterine environment with a continuous intravenous supply of nutrients to a microbe-rich environment with intermittent ingesting of colostrum/milk via the gut. Currently, little is known about the colostrum-induced alterations of intestinal mucosal proteins in piglets with intra-uterine growth restriction (IUGR). In this study, we sought to investigate the innate differences and effects of colostrum on alterations in small-intestinal proteomes of IUGR piglets. Two IUGR (approximately 0·9 kg) and two normal-birth weight (NBW; approximately 1·3 kg) piglets were obtained from each of six sows at birth. One half (n 12; 6 IUGR v. 6 NBW) of the selected newborn piglets were killed to obtain jejunum samples, and the other half (n 12; 6 IUGR v. 6 NBW) of the newborn piglets were allowed to suckle colostrum from their own mothers for 24 h before jejunum sample collection. On the basis of proteomic analysis, we identified thirty-one differentially expressed proteins in the jejunal mucosa between IUGR and normal neonates before or after colostrum consumption. The intestinal proteins altered by colostrum feeding play important roles in the following: (1) increasing intestinal integrity, transport of nutrients, energy metabolism, protein synthesis, immune response and, therefore, cell proliferation; and (2) decreasing oxidative stress, and therefore cell apoptosis, in IUGR neonates. However, colostrum only partially ameliorated the inferior status of the jejunal mucosa in IUGR neonates. These findings provide the first evidence in intestinal protein alterations of IUGR neonates in response to colostrum ingestion, and thus render new insights into the mechanisms responsible for impaired growth in IUGR neonates and into new nutritional intervention strategies.

RIG-I Signaling via MAVS Is Dispensable for Survival in Lethal Influenza Infection In Vivo.

Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines. It requires interaction with an adaptor molecule, mitochondrial antiviral-signaling protein (MAVS), to activate downstream signaling pathways. To elucidate the mechanism(s) by which RIG-I-dependent recognition of IAV infection in vivo triggers innate immune responses, we infected mutant mice lacking RIG-I or MAVS with influenza A virus (IAV) and measured their innate immune responses. As has previously been demonstrated with isolated deletion of the virus recognition receptors TLR3, TLR7, and NOD2, RIG-I or MAVS knockout (KO) did not result in higher mortality and did not reduce IAV-induced cytokine responses in mice. Infected RIG-I KO animals displayed similar lung inflammation profiles as did WT mice, in terms of the protein concentration, total cell count, and inflammatory cell composition in the bronchoalveolar lavage fluid. RNA-Seq results demonstrated that all types of mice exhibited equivalent antiviral and inflammatory gene responses following IAV infection. Together, the results indicated that although RIG-I is important in innate cytokine responses in vitro, individual deletion of the genes encoding RIG-I or MAVS did not change survival or innate responses in vivo after IAV infection in mice.

Decreased epithelial progesterone receptor A at the window of receptivity is required for preparation of the endometrium for embryo attachment.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

Synergistic effect of regulatory T cells and proinflammatory cytokines in angiogenesis in the endometriotic milieu.

STUDY QUESTION: Do regulatory T cells (Tregs) contribute to angiogenesis in endometriosis? SUMMARY ANSWER: High levels of CCL17 and CCL22 cause the recruitment of Tregs, upregulate the immunosuppression of Tregs and, in turn, may promote angiogenesis in endometrial cells in synergy with proinflammatory cytokines. WHAT IS ALREADY KNOWN: The peritoneal fluid of patients with endometriosis has a higher percentage of Tregs than that of normal individuals; however, the regulatory role of Tregs in the disease remains unclear. STUDY DESIGN, SIZE, DURATION: This study used primary human endometrial stromal cells (ESCs), monocytes (Mo), Tregs and human umbilical vein endothelial cells (HUVECs). All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: The migration of Tregs was evaluated by the transwell migration assay. The activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase and p38 signaling pathways was examined using the In-Cell WesternTM (LI-COR®) western blot analysis system, as well as by traditional western blot analysis. Changes in the expression of CCL22, CCL17, transforming growth factor-beta 1 (TGF-ß1), Interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), IL-8 and vascular endothelial growth factor (VEGF) in cell-culture supernatant were detected by ELISA. We analyzed the Tregs by multicolor flow cytometry to directly test the expression of CCR4, CD4, CD25, Foxp3, CTLA-4, CD39 and CD73. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed that ESCs-Mo co-culture produced significantly higher levels of CCL22 and CCL17 than ESCs or Mo cultured alone, and that estradiol (E2) or progesterone (P) further promoted this upregulation, demonstrating stronger chemotaxis on Tregs. The co-culture of ESCs with Mo stimulated TGF-ß1 secretion by Tregs, which could be inhibited by anti-CCL22 or/and anti-CCL17 neutralizing antibodies (Abs). The expression of CCR4 by Tregs was upregulated in ESCs-Mo co-culture, especially by treatment with E2 and/or P, and this effect could be abolished by anti-CCL22 and/or anti-CCL17-neutralizing Abs. The Treg-ESCs-Mo co-culture treated with E2 (10-8 mol/l) and P (10-8 mol/l) could enhance the immunosuppression of Tregs, as proved by the elevated expression of Foxp3, CTLA-4, CD39 and CD73 on Tregs. ESCs-Mo co-culture could significantly promote the secretion of IL-1ß and TNF-α. TGF-ß1 from Tregs could activate p38/ERK1/2 signaling pathways in ESCs, and IL-1ß and TNF-α produced by ESCs-Mo co-culture had synergistic roles with TGF-ß1. TGF-ß1 and the proinflammatory cytokines IL-1ß or TNF-α could synergistically promote IL-8 and VEGF expression in ESCs via the p38/ERK1/2 signaling pathways. The high levels of IL-8 and VEGF in the supernatant of ESCs stimulated the angiogenesis of HUVECs. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This study was only performed in vitro using eutopic ESCs, instead of ectopic cells, from endometriosis patients. Therefore, it is necessary to do further experiments to determine whether Tregs promote angiogenesis in the endometriotic milieu in synergy with proinflammatory cytokines in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Co-targeting Tregs and proinflammatory cytokines may be an effective treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Ministry of Science and Technology of China 2015CB943300 to L.D.-J.; National Natural Science Foundation of China, item number 81200425 to  W.X.-Q.; National Natural Science Foundation of China, item number 81471548 to L.D.-J.; and The Research Fund for the Doctoral Program of Higher Education of China to W.X.-Q. (20110071120093). The authors have no conflicts of interest to declare.

RIG-I overexpression decreases mortality of cigarette smoke exposed mice during influenza A virus infection.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines which participate in clearing viral infections. Cigarette smoke (CS) exposure increases the frequency and severity of respiratory tract infections. METHODS: We generated a RIG-I transgenic (TG) mouse strain that expresses the RIG-I gene product under the control of the human lung specific surfactant protein C promoter. We compared the mortality and host immune responses of RIG-I TG mice and their litter-matched wild type (WT) mice following challenge with influenza A virus (IAV). RESULTS: RIG-I overexpression increased survival of IAV-infected mice. CS exposure increased mortality in WT mice infected with IAV. Remarkably, the effect of RIG-I overexpression on survival during IAV infection was enhanced in CS-exposed animals. CS-exposed IAV-infected WT mice had a suppressed innate response profile in the lung compared to sham-exposed IAV-infected WT mice in terms of the protein concentration, total cell count and inflammatory cell composition in the bronchoalveolar lavage fluid. RIG-I overexpression restored the innate immune response in CS-exposed mice to that seen in sham-exposed WT mice during IAV infection, and is likely responsible for enhanced survival in RIG-I TG mice as restoration preceded death of the animals. CONCLUSIONS: Our results demonstrate that RIG-I overexpression in mice is protective for CS enhanced susceptibility of smokers to influenza infection, and that CS mediated RIG-I suppression may be partially responsible for the increased morbidity and mortality of the mice exposed to IAV. Thus, optimizing the RIG-I response may be an important treatment strategy for CS-enhanced lung infections, particularly those due to IAV.

Functional Roles of Fructose: Crosstalk between O-Linked Glycosylation and Phosphorylation of Akt-TSC2-MTOR Cell Signaling Cascade in Ovine Trophectoderm Cells.

During pregnancy, the placentae of ungulate mammals (e.g., cows, sheep, and pigs) convert glucose into fructose which is the most abundant hexose sugar in fetal fluids and blood. However, the role of fructose, the most enigmatic component of carbohydrate metabolism in fetal-placental tissues, is largely ignored since it is not metabolized via the glycolytic pathway or the Krebs cycle as an energy source. Here we provided evidence for biological functions of fructose that affect proliferative behavior of the conceptus trophectoderm/chorion via activation of the Akt-TSC2-MTOR signaling cascade. The phosphorylation for activation of this cascade is mediated by O-GlcNAcylation from UDP-N-acetylglucosamine (UDP-GlcNAc), a primary product of the hexosamine biosynthesis pathway. These results reveal novel functional roles of fructose in promoting embryonic/fetal growth and development during pregnancy, and also provide new insight into understanding the relationship between excessive fructose intake and metabolic disorders.

Uterine Histotroph and Conceptus Development. II. Arginine and Secreted Phosphoprotein 1 Cooperatively Stimulate Migration and Adhesion of Ovine Trophectoderm Cells via Focal Adhesion-MTORC2 Mediated Cytoskeleton Reorganization.

In all mammalian species, critical events, including uterine receptivity and development of the conceptus (embryo/fetus and its associated extraembryonic membranes), must be intricately orchestrated and carefully timed during the window of implantation. Otherwise, failure of conceptuses to implant is inevitable, which accounts for 50%-75% of failures to establish pregnancy. Unlike human and rodent blastocysts, the blastocysts of pigs and ruminants undergo rapid transitions from spherical to tubular and filamentous conceptuses in response to histotroph during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1; also known as osteopontin) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy; however, little is known about their relationship and synergistic effects on conceptus development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their migratory and adhesive responses to individual and combined effects of arginine and recombinant SPP1 (rSPP1) that contains an Arg-Gly-Asp (RGD) binding sequence. Migration and adhesion of oTr1 cells were significantly stimulated by rSPP1, whereas arginine alone only induced a significant increase in cell migration. However, the combination of arginine and rSPP1 had an additive effect on migration, and a synergistic effect on adhesion of oTr1 cells. Those cooperative effects of arginine and SPP1 were mediated by focal adhesion assembly-MTORC2-cytoskeletal reorganization and MAPK pathways. Collectively, results suggest that arginine and SPP1 in histotroph affect cellular events required for rapid elongation of ovine conceptuses during the peri-implantation period of pregnancy.