Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

<p>Abstract</p> <p>Background</p> <p>Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW) proteins. We have optimised a procedure to overcome this limitation.</p> <p>Results</p> <p>Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics.</p> <p>Conclusions</p> <p>This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.</p

A genome-wide function of THSC/TREX-2 at active genes prevents transcription–replication collisions

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3 end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription– replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.Spanish Ministry of Economy and Competitiveness [BFU2010-16372]; Junta de Andalucía [CVI4567 and P12/BIO-1238]; European Union (FEDER); and a JAE predoctoral training grant from the Spanish Research Council (CSIC) [to J.M.S-P.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16372].Peer reviewe

Interleukin-11 alters placentation and causes preeclampsia features in mice

Preeclampsia is an insidious disease, unique to humans, affecting ∼8% of pregnancies. There are no early detection tests or pharmacological treatments. Impaired placentation is widely accepted to contribute to the pathogenesis. However, the mechanisms remain elusive, given the complications of studying first-trimester placental development in women. A major limitation for the study of new treatments is the lack of available animal models that recapitulate the full spectrum of preeclampsia features. We have developed a mouse model characterized by elevated levels of the cytokine Interleukin-11 (IL11). This study provides evidence of a novel pathway causative of preeclampsia features in vivo. It also provides a novel in vivo mouse model that is useful for preclinical studies to test potential therapeutics

Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states

Evaluation of DNA vaccine targeting strategies and expression library immunisation against lethal erythrocytic stage Malaria

Abstract not availabl

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression.

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications

Evaluation of DNA vaccine targeting strategies and expression library immunisation against lethal erythrocytic stage Malaria

Abstract not availabl

Therapeutically blocking Interleukin-11 Receptor-a enhances doxorubicin cytotoxicity in high grade type I endometrioid tumours

High grade type I endometrial cancers have poor prognosis. Interleukin (IL)11 is elevated in tumours and uterine lavage with increasing tumour grade in women. IL11 regulates cell cycle, invasion and migration and we recently demonstrated that IL11 receptor (R)α inhibition impaired low and moderate grade endometrial tumourigenesis in vivo. In this report, we hypothesized that micro-RNA(miR)-1 regulates IL11 and that IL11 promotes high grade endometrial tumour growth. We aimed to determine whether combination treatment using an anti-human IL11Rα blocking antibody (Ab) and doxorubicin chemotherapeutic impairs high grade tumour growth. MiR-1 was absent in human endometrial tumours versus human benign endometrium (n = 10/group). Transfection with miR-1 mimic restored miR-1 expression, down-regulated IL11 mRNA and impaired cell viability in grade 3-derived AN3CA human endometrial epithelial cancer cells. AN3CA cell proliferation was reduced in response to Ab and doxorubicin combination treatment versus Ab, IgG control, or doxorubicin alone. Subcutaneous xenograft tumours were established in female Balb/c athymic nude mice using AN3CA cells expressing IL11 and IL11Rα. Administration of recombinant human IL11 to mice (n = 4/group) activated IL11 downstream target, signal transducers and activators of transcription (STAT3) and significantly increased tumour growth (p < 0.05), suggesting that IL11 promotes high grade tumour growth. IL11Rα blocking Ab reduced STAT3 phosphorylation and combination treatment with doxorubicin resulted in a significant reduction in tumour growth (p < 0.05) compared to Ab, doxorubicin, or IgG control. Our data suggest that therapeutically targeting IL11Rα in combination with doxorubicin chemotherapy could inhibit high grade type I endometrioid cancer growth

miR-29c overexpression and COL4A1 downregulation in infertile human endometrium reduces endometrial epithelial cell adhesive capacity in vitro implying roles in receptivity

The endometrium is a highly complex tissue that is vulnerable to subtle gene expression changes and is the first point of contact for an implanting blastocyst. Successful blastocyst implantation can only occur when the endometrium is receptive during a short window with each menstrual cycle. microRNAs are small, non-coding RNAs that negatively regulate their gene targets. miR-29c has previously been identified to be differentially regulated across the fertile menstrual cycle, however it has not been investigated in association with infertility. We hypothesised that miR-29c dysregulation in the infertile endometrium would negatively influence endometrial adhesion and blastocyst implantation outcomes during the mid-secretory, receptive phase. miR-29c expression was elevated in early and mid-secretory phase infertile endometrium and localised to the epithelial compartments of endometrial tissue. Overexpression of miR-29c in vitro impaired endometrial epithelial adhesion, and reduced collagen type IV alpha 1 (COL4A1) mRNA expression. COL4A1 was immunolocalised to the luminal and glandular epithelial basement membranes in early and mid-secretory phase fertile and infertile endometrium for the first time. Knockdown of COL4A1 impaired endometrial epithelial adhesion suggesting a role in endometrial receptivity and implantation. Our data suggests miR-29c overexpression with infertility may impair the adhesive capacity of the endometrium, potentially contributing to implantation failure and infertility