BackgroundPreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo.MethodsDetermine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy.ResultsPIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control).ConclusionsPIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients (n=14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14-3.55, chi-squared=12.74, P=0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11-0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared=4.0, P=0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored.

Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo development in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25ng/ml (37% and 42%) compared with PIFscr (18%; P<0.001), regardless of the proportion of peripheral-blood NKCD56+ cells to lymphocytes. Pre-incubation of blood from infertile patients with sPIF for 24h decreased NKCD69+ expression versus incubatino with PIFscr (P<0.05). In conclusion, sPIF inhibits NK cell cytotoxicity by reducing NKCD69 expression, suggesting a significant role in RPL patients. There is a continuous search to identify safe and effective agents to counteract recurrent pregnancy loss (RPL). Preimplantation factor (PIF) secreted by the embryo at the 2-cell stage is present throughout viable pregnancy but absent in nonviable pregnancy. Its immunomodulatory (not suppressive) effects promote embryo acceptance and maintenance by mother/host, control inflammation, facilitate uterine environment and placental embedding. Synthetic PIF (sPIF) was used to complete PIF's role as a targeted, safe treatment for immune-based RPL. Previous reports showed sPIF's significant protective systemic effect against maternal factors present in RPL serum. Herein is examined sPIF's ability to inhibit the local protective toxicity induced by natural killer (NK) immune cells in a representative number of RPL patients. When elevated in blood, NK cells are associated with RPL. Low-dose physiological sPIF was highly effective to inhibit NK cell toxicity. Side-by-side comparison showed that sPIF is equally effective at a lower dose than intravenous gamma immunoglobulin or Intralipid treatment currently used. The sPIF effect on NK cells was targeted, indicating specific action. Overall, sPIF may represent a safe, effective and nontoxic immune-based therapy against RPL.

BackgroundEarly identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome.MethodsArtificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC.ResultsPIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer.ConclusionData herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI.

Edited by Roger J. ColbranTransient receptor potential canonical type 5 (TRPC5) is a Ca 2؉ -permeable cation channel that is highly expressed in the brain and is implicated in motor coordination, innate fear behavior, and seizure genesis. The channel is activated by a signal downstream of the G-protein-coupled receptor (GPCR)-G q/11 -phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report that Arg-593, a residue located in the E4 loop near the TRPC5 extracellular Gd 3؉ binding site, is critical for conferring the sensitivity to GPCR-G q/11 -PLC-dependent gating on TRPC5. Indeed, guanosine 5-O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5 R593A mutant, whereas the addition of Gd 3؉ rescues the mutant's sensitivity to GPCR-G q/11 -PLC-dependent gating. Computer modeling suggests that Arg-593 may cross-bridge the E3 and E4 loops, forming the "molecular fulcrum." While validating the model using site-directed mutagenesis, we found that the Tyr-542 residue is critical for establishing a functional Gd 3؉ binding site, the Tyr-541 residue participates in fine-tuning Gd 3؉ -sensitivity, and that the Asn-584 residue determines Ca 2؉ permeability of the TRPC5 channel. This is the first report providing molecular insights into the molecular mechanisms regulating the sensitivity to GPCR-G q/11 -PLC-dependent gating of a receptor-operated channel.Transient receptor potential canonical type 5 (TRPC5) 2 channels are predominantly expressed in the nervous system. The highest levels of TRPC5 expression are found in mammalian brain regions such as the amygdala, hippocampus, cerebellum, cerebral cortex, and substantia nigra (1, 2). TRPC5 activation is implicated in regulating neurite outgrowth, growth cone morphology, and dendritic morphogenesis (3-5). In humans, TRPC5 gene mutations are associated with male mental retardation (2). TRPC5 knock-out mice exhibit deficits in motor coordination and innate fear behavior (6, 7). Genetic ablation of TRPC5 also reduces pilocarpine-induced seizures, long term potentiation, and seizure-induced neuronal cell death in the mouse hippocampus (8, 9). Plasma membrane insertion of TRPC5 in pyramidal hippocampal neurons is associated with the generation of prolonged cholinergic depolarization and bursting during epileptiform seizure discharges, suggesting that elevated TRPC5 activity may lead to an imbalance in hippocampal neuronal networks (10).Although TRPC5 channels have critical physiological roles, the molecular mechanisms involved in regulating TRPC5 activity remain not fully elucidated. TRPC5 proteins are localized to the plasma membrane and function as Na ϩ -and Ca 2ϩ -permeable channels. A TRPC5 channel consists of four subunits, each subunit containing six ␣-helical transmembrane domains (S1-S6, Fig. 1A) and a pore loop located between the S5 and S6 transmembrane domains (1, 11). The conduction pathway of the channel is formed by the S6 transmembrane domain and pore loop residues. Act...

In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein–protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation.

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1subunit (CaVα1) that is engaged in protein–protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3protein–protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3protein–protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1. Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein–protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.

Regulation of uterine contractility is an important aspect of women’s health. Phenylephrine, a selective agonist of the α1-adrenoceptor and a potent smooth muscle constrictor, is widely used in women even during pregnancy to relieve cold-related symptoms, to treat postpartum haemorrhoid, and during routine eye exams. We performed isometric tension recordings to investigate the effect of phenylephrine on mouse uterine contractility. Phenylephrine decreased spontaneous and oxytocin-induced contractions in non-pregnant mouse uterine rings and strips with an IC50 of ~1 μM. Prazosin, an inhibitor of α1-adrenoceptor, did not prevent phenylephrine-mediated relaxations. Conversely, ICI118551, an antagonist of β2-adrenoceptors, inhibited phenylephrine relaxation. In the presence of ICI118551, high concentrations (>30 μM) of phenylephrine caused mouse uterine contractions, suggesting that β-adrenoceptor-mediated inhibition interferes with the phenylephrine contractile potential. Phenylephrine-dependent relaxation was reduced in the uterus of pregnant mice. We used primary mouse and human uterine smooth muscle cells (M/HUSMC) to establish the underlying mechanisms. Phenylephrine stimulated large increases in intracellular cAMP in M/HUSMCs. These cAMP transients were decreased when HUSMCs were cultured in the presence of oestrogen and progesterone to mimic the pregnancy milieu. Thus, phenylephrine is a strong relaxant in the non-pregnant mouse uterus, but exhibits diminished effect in the pregnant uterus.