Autophagy and SARS-CoV-2-Old Players in New Games.

At present it is well-defined that autophagy is a fundamental process essential for cell life but its pro-viral and anti-viral role has been stated out with the COVID pandemic. However, viruses in turn have evolved diverse adaptive strategies to cope with autophagy driven host defense, either by blocking or hijacking the autophagy machinery for their own benefit. The mechanisms underlying autophagy modulation are presented in the current review which summarizes the accumulated knowledge on the crosstalk between autophagy and viral infections, with a particular emphasizes on SARS-CoV-2. The different types of autophagy related to infections and their molecular mechanisms are focused in the context of inflammation. In particular, SARS-CoV-2 entry, replication and disease pathogenesis are discussed. Models to study autophagy and to formulate novel treatment approaches and pharmacological modulation to fight COVID-19 are debated. The SARS-CoV-2-autophagy interplay is presented, revealing the complex dynamics and the molecular machinery of autophagy. The new molecular targets and strategies to treat COVID-19 effectively are envisaged. In conclusion, our finding underline the importance of development new treatment strategies and pharmacological modulation of autophagy to fight COVID-19.

Machine Learning Approach for Muscovy Duck (Cairina moschata) Semen Quality Assessment.

This study aimed to develop a comprehensive approach for assessing fresh ejaculate from Muscovy duck (Cairina moschata) drakes to fulfil the requirements of artificial insemination in farm practices. The approach combines sperm kinetics (CASA) with non-kinetic parameters, such as vitality, enzyme activities (alkaline phosphatase (AP), creatine kinase (CK), lactate dehydrogenase (LDH), and γ-glutamyl-transferase (GGT)), and total DNA methylation as training features for a set of machine learning (ML) models designed to enhance the predictive capacity of sperm parameters. Samples were classified based on their progressive motility and DNA methylation features, exhibiting significant differences in total and progressive motility, curvilinear velocity (VCL), velocity of the average path (VAP), linear velocity (VSL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and live normal sperm cells in favour of fast motility ones. Additionally, there were significant differences in enzyme activities for AP and CK, with correlations to LDH and GGT levels. Although motility showed no correlation with total DNA methylation, ALH, wobble of the curvilinear trajectory (WOB), and VCL were significantly different in the newly introduced classification for "suggested good quality", where both motility and methylation were high. The performance differences observed while training various ML classifiers using different feature subsets highlight the importance of DNA methylation for achieving more accurate sample quality classification, even though there is no correlation between motility and DNA methylation. The parameters ALH, VCL, triton extracted LDH, and VAP were top-ranking for "suggested good quality" predictions by the neural network and gradient boosting models. In conclusion, integrating non-kinetic parameters into machine-learning-based sample classification offers a promising approach for selecting kinetically and morphologically superior duck sperm samples that might otherwise be hindered by a predominance of lowly methylated cells.

Endothelial inflammation and dysfunction in COVID-19.

The biggest challenge in the COVID-19 pandemic besides the spread of the SARS-CoV-2 virus is to reduce mortality rates. As the number of cases continues to rise and new variants, some with at least partial resistance to vaccines, emerge, the need for better understanding of the underlying pathology of the disease and for improved therapeutic strategies grows urgently. The endothelium is a main target of most viral infections in the body. The dysregulation of the normal functions of endothelial cells (ECs) contributes greatly to the thrombo-inflammatory storm and subsequent blood clot associated deaths in COVID-19 patients. Therefore, in this review we emphasize on the importance of ECs in healthy resting state and in inflammation. We summarize the current understanding of SARS-CoV-2 pathogenicity and the key contributions of in vitro cell culture models some of which have established the ACE2 (angiotensin-converting enzyme 2) receptors as the main gates for viral entry in the cell. Lastly, we focus on 3D biofabrication methods for the design of better in vitro models that mimic the host environment including interactions of multiple cell types, simulation of blood flow and real-time viral infections. The development and implementation of such experimental platforms are critical to elucidate host-pathogen interactions and to test new antiviral drugs and vaccines in a controlled, safe, and highly reproducible and predictive manner.

Alternative RNA Splicing-The Trojan Horse of Cancer Cells in Chemotherapy.

Almost all transcribed human genes undergo alternative RNA splicing, which increases the diversity of the coding and non-coding cellular landscape. The resultant gene products might have distinctly different and, in some cases, even opposite functions. Therefore, the abnormal regulation of alternative splicing plays a crucial role in malignant transformation, development, and progression, a fact supported by the distinct splicing profiles identified in both healthy and tumor cells. Drug resistance, resulting in treatment failure, still remains a major challenge for current cancer therapy. Furthermore, tumor cells often take advantage of aberrant RNA splicing to overcome the toxicity of the administered chemotherapeutic agents. Thus, deciphering the alternative RNA splicing variants in tumor cells would provide opportunities for designing novel therapeutics combating cancer more efficiently. In the present review, we provide a comprehensive outline of the recent findings in alternative splicing in the most common neoplasms, including lung, breast, prostate, head and neck, glioma, colon, and blood malignancies. Molecular mechanisms developed by cancer cells to promote oncogenesis as well as to evade anticancer drug treatment and the subsequent chemotherapy failure are also discussed. Taken together, these findings offer novel opportunities for future studies and the development of targeted therapy for cancer-specific splicing variants.

Synthetic PreImplantation Factor (sPIF) induces posttranslational protein modification and reverses paralysis in EAE mice.

An autoimmune response against myelin protein is considered one of the key pathogenic processes that initiates multiple sclerosis (MS). The currently available MS disease modifying therapies have demonstrated to reduce the frequency of inflammatory attacks. However, they appear limited in preventing disease progression and neurodegeneration. Hence, novel therapeutic approaches targeting both inflammation and neuroregeneration are urgently needed. A new pregnancy derived synthetic peptide, synthetic PreImplantation Factor (sPIF), crosses the blood-brain barrier and prevents neuro-inflammation. We report that sPIF reduces paralysis and de-myelination of the brain in a clinically-relevant experimental autoimmune encephalomyelitis mice model. These effects, at least in part, are due to post-translational modifications, which involve cyclic AMP dependent protein kinase (PKA), calcium-dependent protein kinase (PKC), and immune regulation. In terms of potential MS treatment, sPIF was successfully tested in neurodegenerative animal models of perinatal brain injury and experimental autoimmune encephalitis. Importantly, sPIF received a FDA Fast Track Approval for first in human trial in autommuninty (completed).

The core sequence of PIF competes for insulin/amyloid ß in insulin degrading enzyme: potential treatment for Alzheimer's disease.

The central pathological feature of Alzheimer's disease (AD) is the sequential proteolytic processing of amyloid precursor protein (APP) to amyloid-ß peptides (Aß) agglomeration. The clearance of Aß may be induced by the large zinc-binding protease insulin degrading enzyme (IDE). IDE is the common link between AD and Type II diabetes as insulin is an IDE target as well. Not surprisingly, the search for safe and effective drugs modulating IDE is ongoing. A new pregnancy derived peptide, PreImplantation Factor (PIF), inhibits neuro-inflammation and crosses the blood-brain-barrier. Importantly, we report that the (R3I4K5P6) core sequence of the PIF peptide modulates IDE function and results in decreased Aß agglomeration in neuronal cells. Using bioinformatics we show that PIF binds to the IDE complex and sterically competes for the same place as insulin or Aß. The predicted RIKP sequence and especially the specific I4 and P6 amino acids are essential for hydrophobic interactions with the IDE complex. In terms of potential AD treatment, PIF was successfully tested in neurodegenerative animal models of perinatal brain injury and experimental autoimmune encephalitis. Importantly, sPIF received a FDA Fast Track Approval and orphan drug designation for first-in-human trial in autoimmunity.

Correction: PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

[This corrects the article DOI: 10.18632/oncotarget.16028.].

HLA-DR Genotyping and Mitochondrial DNA Analysis Reveal the Presence of Family Burials in a Fourth Century Romano-British Christian Cemetery.

In Colchester, Britain's oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the "vault complex" and demonstrate a continental European origin of the individuals investigated.

Preimplantation Factor (PIF) Promotes HLA-G, -E, -F, -C Expression in JEG-3 Choriocarcinoma Cells and Endogenous Progesterone Activity.

BACKGROUND/AIMS: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and -C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells. METHODS: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. RESULTS: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1ß, IL-8, GM-CSF and TGF-ß1), resulting in improved maternal signalling. CONCLUSION: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders.

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos' development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by <3 kDa RPL serum fraction likely by reducing reactive oxygen species (ROS). We reveal that protein disulfide isomerase/thioredoxin (PDI/TRX) is a prime PIF target in the embryo, rendering it an important ROS scavenger. The 16F16-PDI/TRX inhibitor drastically reduced blastocyst development while exogenous PIF increased >2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor's pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

Micro-RNA-204 Participates in TMPRSS2/ERG Regulation and Androgen Receptor Reprogramming in Prostate Cancer.

Cancer progression is driven by genome instability incurred rearrangements such as transmembrane protease, serine 2 (TMPRSS2)/v-ets erythroblastosis virus E26 oncogene (ERG) that could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. Previously, we found dualistic miR-204 effects, acting either as a tumor suppressor or as an oncomiR in ERG fusion-dependent manner. Here, we provided further evidence for an important role of miR-204 for TMPRSS2/ERG and androgen receptor (AR) signaling modulation and fine tuning that prevents TMPRSS2/ERG overexpression in prostate cancer. Based on proximity-based ligation assay, we designed a novel method for detection of TMPRSS2/ERG protein products. We found that miR-204 is TMPRSS2/ERG oncofusion negative regulator, and this was mediated by DNA methylation of TMPRSS2 promoter. Transcriptional factors runt-related transcription factor 2 (RUNX2) and ETS proto-oncogene 1 (ETS1) were positive regulators of TMPRSS2/ERG expression and promoter hypo-methylation. Clustering of patients' sera for fusion protein, transcript expression, and wild-type ERG transcript isoforms, demonstrated not all patients harboring fusion transcripts had fusion protein products, and only few fusion positive ones exhibited increased wild-type ERG transcripts. miR-204 upregulated AR through direct promoter hypo-methylation, potentiated by the presence of ERG fusion and RUNX2 and ETS1. Proteomics studies provided evidence that miR-204 has dualistic role in AR cancer-related reprogramming, promoting prostate cancer-related androgen-responsive genes and AR target genes, as well as AR co-regulatory molecules. miR-204 methylation regulation was supported by changes in molecules responsible for chromatin remodeling, DNA methylation, and its regulation. In summary, miR-204 is a mild regulator of the AR function during the phase of preserved AR sensitivity as the latter one is required for ERG-fusion translocation.

Toll-Like Receptor 2 Mediates In Vivo Pro- and Anti-inflammatory Effects of Mycobacterium Tuberculosis and Modulates Autoimmune Encephalomyelitis.

Mycobacteria display pro- and anti-inflammatory effects in human and experimental pathology. We show here that both effects are mediated by Toll-like receptor 2 (Tlr2), by exploiting a previously characterized Tlr2 variant (Met82Ile). Tlr2 82ile promoted self-specific proinflammatory polarization as well as expansion of ag-specific FoxP3(+) Tregs, while Tlr2 82met impairs the expansion of Tregs and reduces the production of IFN-γ and IL-17 proinflammatory cytokines. Preferential dimerization with Tlr1 or Tlr6 could not explain these differences. In silico, we showed that Tlr2 variant Met82Ile modified the binding pocket for peptidoglycans and participated directly to a putative binding pocket for sugars and cadherins. The distinct pro- and anti-inflammatory actions impacted severity, extent of remission, and distribution of the lesions within the central nervous system of experimental autoimmune encephalomyelitis. Thus, Tlr2 has a janus function in vivo as mediator of the role of bacterial products in balancing pro- and anti-inflammatory immune responses.

PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders.

PreImplantation factor prevents atherosclerosis via its immunomodulatory effects without affecting serum lipids.

PreImplantation factor (PIF) is a 15-amino acid peptide endogenously secreted by viable embryos, regulating/enabling maternal (host) acceptance/tolerance to the "invading" embryo (allograft) all-while preserving maternal immunity to fight infections. Such attributes make PIF a potential therapeutic agent for chronic inflammatory diseases. We investigated whether PIF's immunomodulatory properties prevent progression of atherosclerosis in the hyper-cholesterolaemic ApoE-deficient murine model. Male, high-fat diet fed, ApoE-deficient (ApoE-/-) mice were administered either PBS, scrambled PIF (0.3-3 mg/kg) or PIF (0.3-3 mg/kg) for seven weeks. After treatment, PIF (3 mg/kg)-treated ApoE-/- mice displayed significantly reduced atherosclerosis lesion burden in the aortic sinus and aortic arch, without any effect on lipid profile. PIF also caused a significant reduction in infiltration of macrophages, decreased expression of pro-inflammatory adhesion molecules, cytokines and chemokines in the plaque, and reduced circulating IFN-γ levels. PIF preferentially binds to monocytes/neutrophils. In vitro, PIF attenuated monocyte migration (MCP-1-induced chemotaxis assay) and in vivo in LPS peritonitis model. Also PIF prevented leukocyte extravasation (peritonitis thioglycollate-induced model), demonstrating that PIF exerts its effect in part by modulation of monocyte function. Inhibition of the potassium channel KCNAB3 (Kv1.3) and of the insulin degrading enzyme (IDE) was demonstrated as potential mechanism of PIF's immunomodulatory effects. In conclusion, PIF regulates/lowers inflammation and prevents atherosclerosis development without affecting circulating lipids. Overall our findings establish PIF as a strong immunomodulatory drug candidate for atherosclerosis therapy.

Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production.

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1ß secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1ß restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1ß secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1ß expression, while NOD2 inversely promoted IL-1ß. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction.

miR-204 is dysregulated in metastatic prostate cancer in vitro.

During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. We aimed to investigate miR-204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR-204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c-MYB, ETS1 and RUNX2 transcript and protein expression and the miR-204 affected global proteome. We further investigated if these transcription factors exert an effect on miR-204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR-204 effects, either acting as a tumor suppressor on c-MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR-204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR-204 also differentially affected mRNA splicing and protein stability. miR-204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR-204, c-MYB, RUNX2 and ETS1, the c-MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro-RNA loss, but also significant genome rearrangement-driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro-survival and metastatic potential.

NLRs Challenge Impacts Tight Junction Claudins In Sertoli Cells.

AIM: The present study aims to investigate the NALP3 system and its effect on claudins in Sertoli cells using a mouse adult Sertoli cell line as a model. We focus on the Sertoli cell biology looking for the possible implications for male reproductive functions. MATERIALS AND METHODS: Adult Sertoli cells were transfected with NAPL3 siRNA and treated with NOD1 (ie-DAP) and NOD2 (MDP) receptor ligands. Two dimensional gel electrophoresis was performed on lysates of non-challenged and MDP-treated Sertoli cells. RESULTS: There were positive claudin-5 and claudin-11 expression levels on transcript (RT-qPCR) levels. Specific protein spots in 2D gels were detected after bioinformatics analysis. This study demonstrates direct induction of tight-junction proteins probably favouring junction stability. CONCLUSIONS: The innate immunity and tight-junction pathway integration probably have a protective role for both blood-testis immune barrier and spermatogenesis compartmentalisation maintained by the very same barrier. This integration also points the way for mechanistic research of the disturbances inflicted during an inflammatory response in the testis niche.

PIF direct immune regulation: Blocks mitogen-activated PBMCs proliferation, promotes TH2/TH1 bias, independent of Ca(2+).

PreImplantation Factor (PIF(9&15)) secreted by viable embryos exerts an essential transplant acceptance and immune regulatory role in pregnancy. Synthetic PIF replicates endogenous PIF's effect in pregnant and non-pregnant immune disorder models. PIF binds macrophages to regulate CD3/CD28-induced T-cell response. We present evidence that PIF regulates the co-stimulatory T-cell receptor, CD2, which binds to and is activated by phytohemagglutinin (PHA), a potent mitogen, confirming PIF's ability to systemically respond to diverse immune stimulants. PIF's effect on PHA-activated PBMC (male and non-pregnant females) proliferation and cytokine secretion was tested, showing that both PIF(9&15) block PHA-induced PBMC proliferation and promote anti-inflammatory IL10 secretion, while reducing pro-inflammatory IFNγ secretion. Thus favoring a T(H)2 cytokine bias. Surface plasmon resonance spectroscopy, immunocytochemistry and Flex station experiments reveal that PIF effect is direct. PIF targets intracellular targets but does not affect early Ca(2+) mobilization. By promoting the CD2 receptor in activated T-cells and through inhibition of co-ligand CD58 expression, PIF regulates antigen-presenting cell (APC)-T-cell interactions required for PHA action. Structure-based design demonstrated that PIF15 offers improved target specificity as compared to PIF9. Collectively, PIF directly regulates mitogen-induced PBMC activation. Results support PIF translation for therapy of immune disorders.

Insight into PreImplantation Factor (PIF*) mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP) through essential RIKP [corrected] binding site.

BACKGROUND: Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. METHODS: FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. RESULTS: PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase ß-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. CONCLUSION: Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.

Quantified colocalization reveals heterotypic histocompatibility class I antigen associations on trophoblast cell membranes: relevance for human pregnancy.

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.