Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Selective inhibition of BTK prevents murine lupus and antibody-mediated glomerulonephritis.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Effect of interferon-tau administration on endometrium of nonpregnant ewes: a comparison with pregnant ewes.

In ruminants, conceptus interferon-tau (IFNT) alters maternal physiology to accommodate a pregnancy. We hypothesized that the effectiveness of IFNT on extending corpus luteum (CL) life span in nonpregnant ewes would depend upon the dose and manner of administration and would be correlated with the response in gene expression in endometrium. We anticipated that IFNT, whether administered im or by uterine infusion, would mimic changes observed in pregnancy. Ewes were assigned to five treatments: 1) uterine infusion of saline; 2) uterine infusion of ovine IFNT4 (200 microg/d); 3) saline im injection; 4) im injection of IFNT4 at low dose (200 microg/d); and 5) high dose (2 mg/d). CL life span was increased in groups 2 and 5, but not in 1, 3, and 4. Endometrial RNA extracted from groups 1-5 on d 14 and from d 14 pregnant and nonbred (cyclic) ewes was used to assess expression of 70 genes on microarrays. When pregnant and cyclic ewes were compared, 30 genes were up-regulated and nine down-regulated during pregnancy. Responses were slightly less in groups 2 and 5 but were much lower in group 4. The majority of the highly up-regulated genes were associated with antiviral responses. Those down-regulated included ones for IGF-II, hypoxia-inducible factor 1alpha, oxytocin receptor, prostaglandin F synthase, and cyclooxygenase-2. Quantitative PCR for selected genes confirmed these data and revealed that similar gene expression changes occurred in the CL of pregnant and group 2 ewes. IFNT treatment mimics pregnancy, but relatively high doses of im-injected IFNT are required to elicit a full endometrial response.

Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers.

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.

Crizotinib reduces the rate of dark adaptation in the rat retina independent of ALK inhibition.

Crizotinib (Xalkori) is a tyrosine kinase inhibitor of both anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor (c-Met). Though not predicted from standard nonclinical toxicological evaluation, visual disturbance became a frequently observed adverse event in humans. To understand the possible mechanism of this vision effect, an in vivo electroretinogram (ERG) study was conducted to assess retinal functional changes following oral administration of crizotinib. Immunohistochemical (IHC) staining of ALK and c-Met in the neural retinas of human, non-human primate, dog, rat, and mouse was used to aid in the animal model selection. ALK IHC staining was identified predominantly in the ganglion cell and inner nuclear layers of most species evaluated, in the inner plexiform layer in human and rodent, and in the nerve fiber layer in human and rat only. There was no apparent staining of any layer of the neural retina for c-Met in any of the species evaluated. ERG measurements identified a significant reduction in b-wave amplitude during the initial phase of dark adaptation in the crizotinib-treated rats. ERGs were also taken following oral administration of PF-06463922 (an ALK-selective inhibitor), for an understanding of potential kinase involvement. ERG effects were not observed in PF-06463922-treated animals when comparable exposures in the vitreous humor were achieved. Collectively, our results suggest that the ERG b-wave amplitude decreases during dark adaption following crizotinib administration may be related to signaling changes within the retina in rats, likely independent of ALK inhibition.

Expression of proto-oncogene cFMS protein in lung, breast, and ovarian cancers.

We performed immunohistochemistry for macrophage colony-stimulating factor 1 receptor (also known as c-fms proto-oncogene product) on tissue microarrays of human nontumor lung, pulmonary squamous cell carcinomas (SCC) and adenocarcinomas (ADC), and breast and ovarian carcinomas using a commercially available anti-cFMS antibody. The specificity of the antibody was validated by Western blot and mass spectrometry analysis. Staining of cFMS was restricted to stromal fibroblasts in pulmonary SCC and ADC specimens and was not identified in tumor epithelium or epithelium and stromal cells of nontumor lung. Evaluation of pulmonary SCC (n=63) and ADC (n=71) specimens revealed stromal fibroblast cFMS staining in 60% (38 of 63) and 35% (25 of 71) of the tumor samples, respectively. A similar pattern of stromal fibroblast cFMS staining was observed in breast (n=21) and ovarian (n=50) carcinomas. It was reported that glucocorticoids induced cFMS expression in breast carcinomas and choriocarcinomas. To investigate whether stromal cFMS expression in lung cancers was associated with glucocorticoid signaling, glucocorticoid receptor protein distribution was evaluated in lung tissue microarrays by immunohistochemistry. Stromal fibroblast glucocorticoid receptor staining was only observed in 18% (2 of 11) of pulmonary SCC and 6% (1 of 17) of ADC specimens, suggesting that cFMS expression may not be directly mediated by glucocorticoids in stromal fibroblasts of lung cancers. The tumor stromal cell expression of cFMS in certain tumor types (lung, ovarian, and breast) suggests the potential for more diverse tumor therapeutic options and presents an attractive target for drug development.

Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

Osteopontin improves in vitro development of porcine embryos and decreases apoptosis.

An optimal environment for fertilization and early embryonic development is provided by the mammalian oviduct and uterus. The secretory cells lining the lumen of the oviduct and uterus synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. Western blotting in this study demonstrated that a 50-kDa secreted phosphoprotein 1 (SPP1) form was present in the uterus on Days 0, 3, and 5 in pregnant and nonbred gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5 in pregnant gilts, but in nonbred gilts the concentration of SPP1 on Day 0 was higher than Day 3, but not Day 5. In addition, we show that addition of 0.1 microg/ml SPP1 to the culture medium after fertilization increased the percent cleaved (24 hr: 23.6 +/- 1.29(a) vs. 18.7 +/- 0.65(b) (2-cell %)), and the percent blastocyst (37.2 +/- 1.12(a) vs. 30.9 +/- 0.56(b)) derived from IVF (P < 0.05). In parthenogenetic-derived embryos the percent cleaved was increased due to SPP1 at 24 hr (24.0 +/- 1.59(a) vs. 19.7 +/- 1.59(b) (>2-cell %)), and at 48 hr (72.9+/- 2.99(a) vs. 63.3 +/- 2.99(b)), but not the percent blastocyst. By TUNEL assay, SPP1 decreased both apoptosis (7.9 +/- 0.04(a) vs. 13.1 +/- 0.02(b)) and the percent fragmentation (45.2 +/- 0.07(a) vs. 58.8 +/- 0.03(b)). We conclude that SPP1 can improve development in vitro possibly by reducing the rate of apoptosis.

Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes.

This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 microg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 microg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 microg/ml SPP1). The ZP of 0.1 microg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermatozoa bound to the ZP during IVF increased after 4 h of 1.0 microg/ml SPP1 treatment compared to 0 or 0.1 microg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.

Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression.

The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function.

Large-scale generation and analysis of expressed sequence tags from porcine ovary.

One method to identify the factors that control ovarian function is to characterize the genes that are expressed in ovary. In the present study, cDNA libraries from fetal, neonatal, and prepubertal porcine ovaries, pubertal ovaries on different days of the estrous cycle (Days 0 [follicle], 5, and 12 [follicle and corpus luteum]), and follicles isolated from weaned sows (diameter, 2, 4, 6, and 8 mm) were constructed and sequenced. A total of 22 176 cDNAs were sequenced, of which 15 613 were of sufficient quality for clustering. Clustering of cDNAs resulted in 8507 contigs, 6294 (74%) of which were comprised of a single sequence. Sixty-eight percent of the contigs had consensus sequences that were homologous to existing Tentative Consensus (TC) sequences or mature transcripts (ET) in The Institute for Genomic Research Porcine Gene Index. The consensus sequences were classified according to the Gene Ontology Index. Most cDNA-encoded proteins were components of the nucleus, ribosome, or mitochondrion. The proteins primarily functioned in binding, catalysis, and transport. Nearly 75% of the proteins were involved in metabolism and cell growth and/or maintenance. Analysis of the cDNA frequency across different libraries demonstrated differential gene expression within different-size follicles, between follicles and corpora lutea, and across developmental time-points. The expression of selected genes (analyzed by ribonuclease protection assay and Northern blotting) was consistent with the frequency of their respective cDNA in the individual libraries. This porcine ovary unigene set will be useful for identifying factors and mechanisms controlling ovarian follicular development in a variety of species.

Developmental expression of 2489 gene clusters during pig embryogenesis: an expressed sequence tag project.

Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3' end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5' end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.