Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.

STUDY QUESTION: Are uterine fluid-derived extracellular vesicles (UF-EVs) a 'liquid biopsy' reservoir of biomarkers for real-time monitoring of endometrial status? SUMMARY ANSWER: The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. WHAT IS KNOWN ALREADY: EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. STUDY DESIGN, SIZE, DURATION: Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH + 2) and receptive (LH + 7) phase of proven fertile women and from the receptive (LH + 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: For paired UF-endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n = 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n = 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. MAIN RESULTS AND THE ROLE OF CHANCE: A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson's r = 0.70 P < 0.0001; Spearman's ρ = 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH + 7 receptive versus the LH + 2 non-receptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n = 238 genes included in the commercially available endometrial receptivity array and the LH + 7 versus LH + 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)=9.38 (P < 0.001) for transcripts up-regulated in LH + 7 in the commercial array and enriched in LH + 7 UF-EVs, and a NES = -5.40 (P < 0.001) for transcripts down-regulated in LH + 7 in the commercial array and depleted in LH + 7 UF-EVs. When analyzing LH + 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional profile between the commercially available endometrial receptivity array genes and the comparison of LH + 7 UF-EVs of women with successful versus failed implantation, demonstrated a significant enrichment with a NES = 2.14 (P = 0.001) for transcripts up-regulated in the commercial array in the receptive phase and enriched in UF-EVs of women who conceived, and a not significant NES = -1.18 (P = 0.3) for transcripts down-regulated in the commercial array and depleted in UF-EVs. In terms of physical features, UF-EVs showed a homogeneity among the different groups analyzed except for a slight but significant difference in EV size, being smaller in women with a successful implantation compared to patients who failed to conceive after euploid blastocyst transfer (mean diameter ± SD 205.5± 22.97 nm vs 221.5 ± 20.57 nm, respectively, P = 0.014). LARGE SCALE DATA: Transcriptomic data were deposited in NCBI Gene Expression Omnibus (GEO) and can be retrieved using GEO series accession number: GSE158958. LIMITATIONS, REASONS FOR CAUTION: Separation of RNA species associated with EV membranes might have been incomplete, and membrane-bound RNA species-rather than the internal RNA content of EVs-might have contributed to our RNA-seq results. Also, we cannot definitely distinguish the relative contribution of exosomes, microvesicles and apoptotic bodies to our findings. When considering patients undergoing ART, we did not collect UFs in the same cycle of the euploid embryo transfer but in the one immediately preceding. We considered this approach as the most appropriate in relation to the novel, explorative nature of our study. Based on our results, a validation of UF-EV RNA-seq analyses in the same cycle in which embryo transfer is performed could be hypothesized. WIDER IMPLICATIONS OF THE FINDINGS: On the largest sample size of human EVs ever analyzed with RNA-seq, this study establishes a gene signature to use for less-invasive endometrial receptivity tests. This report is indeed the first to show that the transcriptome of UF-EVs correlates with the endometrial tissue transcriptome, that RNA signatures in UF-EVs change with endometrial status, and that UF-EVs could serve as a reservoir for potential less-invasive collection of receptivity markers. This article thus represents a step forward in the design of less-invasive approaches for real-time monitoring of endometrial status, necessary for advancing the field of reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a competitive grant from European Society of Human Reproduction and Embryology (ESHRE Research Grant 2016-1). The authors have no financial or non-financial competing interests to disclose. TRIAL REGISTRATION NUMBER: NA.

Ultra-performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions.

RATIONALE: Because of the large molecular weight, the structural complexity and the similarity with endogenous immunoglobulins present in high concentrations, in vivo quantitative studies with therapeutic monoclonal antibodies are particularly challenging. In this work, an UPLC/MRM MS-based methodology is described for the quantification of trastuzumab in human serum by monitoring a novel specific peptide marker located within its heavy chain Complementarity-Determining Region (CDR). METHODS: For maximum sensitivity and selectivity, specific transitions of this diagnostic proteotypic peptide were optimized and monitored at m/z 364.1 â†’ 437.3 (quantitation ion) and m/z 364.1 â†’ 358.0 (confirmation ion). As a proof-of-concept, the methodology was applied to the determination of trastuzumab in human serum over a clinically relevant range from 0.02 to 200 µg/mL. The methodology has been evaluated in terms of specificity, linearity, accuracy, precision, detection and quantitation limits. RESULTS: An excellent linear response has been obtained in the range from 0.036 to 3.6 fmol/µL for the standard peptide and from 0.03 to 285 fmol/µL for the trastuzumab in human serum with typical R2 values of 0.99. The limit of detection (LOD) and limit of quantification (LOQ) are 0.005 fmol/µL and 0.05 fmol/µL, respectively, with mean bias and RSD values of 18% and 1%, respectively, for quality control samples. CONCLUSIONS: The strategy used to set up the UPLC/MRM MS methodology based on monitoring specific peptide markers within CDRs can be potentially applied to the detection and quantification of other humanized or human mAbs in biological fluids. Copyright © 2017 John Wiley & Sons, Ltd.

Ecology of duckweed ponds used for nutrient recovery from wastewater.

The microorganism community that grows under duckweed shelter can play an important role on treatment processes. Therefore, the present study aimed to assess the zooplankton dynamic and microbial community in duckweed ponds (DPs) applied for domestic wastewater treatment under open field conditions. A pilot system comprised of two DPs in series (DP1 and DP2), with 10 m2 each, received domestic wastewater through a flow rate of 200 L·day-1. Thus, the system was monitored during 314 days through samples collected and analysed weekly. Also, the zooplankton organisms were identified and quantified. DNA sequencing was performed in order to identify the bacterial populations. The findings showed a high efficiency of nutrient removal with 93% and 91% of total phosphorus and total nitrogen, respectively. A high density of microcrustaceans was observed in DP1 reaching 4,700 org.100 mL-1 and rotifers (over than 32,000 org.100 mL-1) in DP2, that could be related to the low suspended solids concentration (<30 mg·L-1) and turbidity (<10 NTU). The bacterial community showed a strong heterogeneity between samples collected along the seasons. Through these findings, it is possible to realise that the understanding of ecology could help to enhance the operation and designs of DPs.

Biochar reduces short-term nitrate leaching from a horizon in an apple orchard.

Nitrogen leaching in croplands is a worldwide problem with implications both on human health and on the environment. Efforts should be taken to increase nutrient use efficiency and minimize N losses from terrestrial to water ecosystems. Soil-applied biochar has been reported to increase soil fertility and decrease nutrient leaching in tropical soils and under laboratory conditions. Our objective was to evaluate the effect of biochar addition on short-term N leaching from A soil horizon in a mature apple orchard growing on subalkaline soils located in the Po Valley (Italy). In spring 2009, 10 Mg of biochar per hectare was incorporated into the surface 20-cm soil layer by soil plowing. Cumulative nitrate (NO) and ammonium (NH) leaching was measured in treated and control plots 4 mo after the addition of biochar and the following year by using ion-exchange resin lysimeters installed below the plowed soil layer. Cumulative NO leaching was not affected by biochar after 4 mo, whereas in the following year it was significantly ( < 0.05) reduced by 75% over the control (from 5.5 to 1.4 kg ha). Conversely, NH leaching was very low and unaffected by soil biochar treatment. The present study shows that soil biochar addition can significantly decrease short-term nitrate leaching from the surface layer of a subalkaline soil under temperate climatic conditions.

The WNT receptor ROR2 drives the interaction of multiple myeloma cells with the microenvironment through AKT activation.

Multiple myeloma is the second most frequent hematological cancer after lymphoma and remains an incurable disease. The pervasive support provided by the bone marrow microenvironment to myeloma cells is crucial for their survival. Here, an unbiased assessment of receptor tyrosine kinases overexpressed in myeloma identified ROR2, a receptor for the WNT noncanonical pathway, as highly expressed in myeloma cells. Its ligand, WNT5A is the most abundant growth factor in the bone marrow of myeloma patients. ROR2 mediates myeloma cells interactions with the surrounding bone marrow and its depletion resulted in detachment of myeloma cells from their niche in an in vivo model, triggering apoptosis and thus markedly delaying disease progression. Using in vitro and ex vivo 3D-culture systems, ROR2 was shown to exert a pivotal role in the adhesion of cancer cells to the microenvironment. Genomic studies revealed that the pathways mostly deregulated by ROR2 overexpression were PI3K/AKT and mTOR. Treatment of cells with specific PI3K inhibitors already used in the clinic reduced myeloma cell adhesion to the bone marrow. Together, our findings support the view that ROR2 and its downstream targets represent a novel therapeutic strategy for the large subgroup of MM patients whose cancer cells show ROR2 overexpression.

K-ras activation generates an inflammatory response in lung tumors.

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

The AP-1 transcription factor JunB is essential for multiple myeloma cell proliferation and drug resistance in the bone marrow microenvironment.

Despite therapeutic advances, multiple myeloma (MM) remains an incurable disease, predominantly because of the development of drug resistance. The activator protein-1 (AP-1) transcription factor family has been implicated in a multitude of physiologic processes and tumorigenesis; however, its role in MM is largely unknown. Here we demonstrate specific and rapid induction of the AP-1 family member JunB in MM cells when co-cultured with bone marrow stromal cells. Supporting a functional key role of JunB in MM pathogenesis, knockdown of JUNB significantly inhibited in vitro MM cell proliferation and survival. Consistently, induced silencing of JUNB markedly decreased tumor growth in a murine MM model of the microenvironment. Subsequent gene expression profiling revealed a role for genes associated with apoptosis, DNA replication and metabolism in driving the JunB-mediated phenotype in MM cells. Importantly, knockdown of JUNB restored the response to dexamethasone in dexamethasone-resistant MM cells. Moreover, 4-hydroxytamoxifen-induced activation of a JunB-ER fusion protein protected dexamethasone-sensitive MM cells against dexamethasone- and bortezomib-induced cytotoxicity. In summary, our results demonstrate for the first time a specific role for AP-1/JunB in MM cell proliferation, survival and drug resistance, thereby strongly supporting that this transcription factor is a promising new therapeutic target in MM.

Multiple forms of protein kinase from normal human brain and glioblastoma.

The biochemical characteristics of the protein kinase (PK; adenosine triphosphate-protein phosphotransferase, EC 2.7.1.37) isozymes in subcellular preparations from normal human brain cortex and glioblastoma were investigated after chromatography on diethylaminoethyl cellulose, and the following results have been obtained. Two major isozyme forms, eluted by 50 and 200 mM phosphate buffer, are present in both cytosol and membrane-derived preparations from cerebral cortex. Furthermore, these isozyme forms have properties similar to those referred to as type I and type II cyclic adenosine 3':5'-monophosphate-dependent PK. In these chromatographic isozymes, cyclic adenosine 3';5'-monophosphate is more active in stimulating the basal PK enzyme than is cyclic guanosine 3':5'-monophosphate. In glioblastoma, the PK activity from cytosol and particulate preparations is resolved by diethylaminoethyl cellulose in four peaks. In cytosol, the major portion of the enzyme is eluted with a 300 mM buffer (about 50% of the total basal PK activity) and is cyclic nucleotide dependent. On the contrary, in glioblastoma particulate, the PK enzyme is mainly eluted at 50 and 100 mM buffer; neither of these isozymes is cyclic nucleotide dependent. As for cytosol, only the particulate isozyme eluted at 300 mM buffer is strongly activated by cyclic nucleotides. Finally, in both glioblastoma subcellular preparations, only a type II cyclic adenosine 3':5'-monophosphate-dependent PK is present.

Interaction of sulfpride and ergot derivatives on rat brain DOPAC concentration and prolactin secretion in vivo.

Sulpiride, which differs from classical neuroleptics by not producing major extrapyramidal side effects, is a potent antiemetic agent and stimulates prolactin secretion in both laboratory animals and man. In parallel it increases dopamine synthesis in both striatum and nucleus accumbens. Bromocriptine and metergoline are two effective agents in suppressing prolactin release and postulated to stimulate dopamine receptors. The interactions of these two ergot derivatives with sulpiride have been investigated on prolactin release and on striatal and limbic DOPAC accumulation. Bromocriptine at all doses tested was able to suppress the increased in vivo prolactin secretion observed after sulpiride administration. Metergoline antagonized the sulpiride-induced prolactin increase only at low doses; on the contrary higher doses potentiated it. High concentrations of bromocriptine suppressed the sulpiride-induced increased of DOPAC levels in striatum and n. accumbens, while metergoline potentiated the sulpiride-induced accumulation of brain DOPAC.

Bromocriptine and lisuride stimulate the accumulation of cyclic AMP in intact slices but not in homogenates of rat neostriatum.

The effects of bromocriptine and lisuride on cyclic AMP concentrations in homogenates and in intact slices of rat neostriatum were investigated. Significant increases in cyclic AMP concentration were found after a 10-min exposure to bromocriptine and lisuride in striatal intact slices. On the contrary, as previously found, the two dopaminergic ergot derivatives did not stimulate dopamine-senstiive adenylate cyclase present in striatal homogenates. The stimulatory effects observed only in intact tissues were blocked by the specific dopamine receptor blocking agent fluphenazine. It is tempting to conclude that dopaminergic ergot derivatives have a site of action different from that stimulated by classic dopamine agonists in tissue homogenates.

Temperature effect on subclasses of muscarinic receptors in rat colon, heart and cerebral cortex.

The high-affinity muscarinic antagonist /3H/-Quinuclidinyl benzilate (/3H/-QNB) has been used to label muscarinic receptors in a crude membrane fraction of rat cerebral cortex, colon and heart. The inhibition of /3H/-QNB binding by Atropine, Oxotremorine and Pirenzepine was investigated at three temperatures: 37 degrees C, 22 degrees C and 10 degrees C. The IC50 values and the proportion of high (Rt1) and low (Rt2) affinity binding sites were determined for the three compounds. When the temperature were lowered from 37 degrees C to 10 degrees C, in the agonist and antagonist dissociation constants decreased in all tissues. Changes in temperature did not modify Rt1 or Rt2 values for Oxotremorine and Pirenzepine. The results show marked temperature-dependent modifications of IC50 values for muscarinic receptors of high- and low-affinity sites in rat cerebral cortex, colon or heart.

Determination of nuvenzepine in human plasma by a sensitive [3H]pirenzepine radioreceptor binding assay.

A sensitive method for the quantitation of small amounts of nuvenzepine, a new M1-selective antimuscarinic drug, in plasma is described. The analytical method involves the use of a radioreceptor binding assay based on [3H]pirenzepine displacement in rat cerebral cortex homogenates; no previous extraction is required. The method is reliable, with an interassay CV ranging from 5 to 10%, and allows the analysis of greater than 100 samples/experiment. The limit of detection is approximately 0.1 ng/assay. Using this method we have determined the plasma levels of nuvenzepine in eight healthy volunteers treated PO with 15 or 25 mg of nuvenzepine.HCl. The pharmacokinetic parameters obtained were (for 15 and 25 mg): Cmax, 64 and 131 ng/mL; AUC0-infinity, 851 and 1379 ng.h/mL; t1/2, 8.6 and 7.2 h. These values are in good agreement with those obtained using an HPLC method. Therefore, this radioreceptor binding assay proved to be simple, rapid, and specific for the determination of low levels of nuvenzepine in human plasma.

Changes in polyamines, auxins and peroxidase activity during in vitro rooting of Fraxinus angustifolia shoots: an auxin-independent rooting model.

Among shoots of Fraxinus angustifolia Vahl raised in vitro, 76% rooted after culture on root induction medium for 5 days in darkness followed by culture on root expression medium for 15 days in light. The addition of 20.7 microM indole-butyric acid (IBA) to the root induction medium did not significantly increase the rooting percentage (88%). Putrescine, spermidine, cyclohexylamine (CHA) and aminoguanidine (AG) enhanced rooting up to 100% (98.66% for AG), when applied during root induction in the absence of IBA, otherwise these compounds inhibited rooting, as did spermine and difluoromethylornithine (DFMO) + difluoromethylarginine (DFMA). The root induction phase was characterized by a temporary increase in endogenous free indole-acetic acid (IAA) and putrescine concentrations during root induction, whereas the root expression phase was characterized by increased peroxidase activity and low concentrations of polyamines. These changes were specifically associated with the rooting process and did not depend on the presence of exogenous IBA, because application of exogenous IBA enhanced the amount of IAA in the cuttings but did not affect rooting or the pattern of changes in polyamines and peroxidase. The effects of CHA, AG and DFMO + DFMA on endogenous concentrations of auxins and polyamines highlight the close relationship between the effects of IAA and putrescine in root induction and suggest that polyamine catabolism has an important role in root formation and elongation.

Effectiveness and tolerability of ketoprofen lysine, once a day, in patients with rheumatic disorders.

Ketoprofen lysine (KL) at a dose of 160 mg b.i.d. has been shown to have favourable anti-inflammatory and analgesic properties in a large number of patients. In the present paper investigators describe their experience with a new therapeutic schedule of KL, 320 mg given once a day to patients with various rheumatic disorders. With this new schedule there was satisfactory clinical improvement of most of the clinical parameters and good tolerability, with a low incidence of gastric troubles. The favourable clinical effects and the patients' good compliance with once-a-day KL make it a useful drug for successful treatment of rheumatic disorders.

Effects of suprofen on renal function in patients with rheumatoid arthritis.

Suprofen is a new potent analgesic with antiinflammatory properties that appears to inhibit prostaglandin synthetase in a tissue-selective manner, having relatively little effect on the kidneys of experimental animals. The effects were studied of one week of treatment of rheumatoid arthritis patients with suprofen or ibuprofen on Na+ and K+ excretion, creatinine clearance, urinary enzymes that are markers for tubular damage, and urinary prostaglandins such as PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin). Neither compound caused changes in renal function related to the week of treatment, but significant decreases in prostaglandins were observed: this change was fully reversible after discontinuation of the drug.

Monoclonal antibody against human growth hormone receptor.

GHR shows a high degree of homology with the prolactin receptor and with the other receptors that belong to the hemopoietic receptor superfamily. This paper describes a monoclonal antibody (MAb) (2B4B6) specific for both the extracellular domain of human GHR and human growth hormone (GH) binding protein. Mice were immunized against a seven-aminoacid peptide sequence screened by FASTA (sequence similarity search served by Genome-Net) from the European Bioinformatics Institute to exclude the existence of human membrane proteins with significant sequence homology. MAbs were screened against the peptide sequence and 2B4B6 was selected for its capability to recognize the full-length hGHBP. As evaluated by both enzyme-linked immunoadsorbent assay (ELISA) and FACS analysis, this MAb seems to recognize and bind to a hGHR positive cell line, IM-9, as well as a murine cell line, BaF3 (8/6), transfected with a chimeric construct, hGHR/hG-CSFR and expressing hGHR on the cell membrane. Studies investigating the biological effects of this MAb showed that anti-hGHR mediated inhibition of cell proliferation was not due to competition with GH binding but rather to prevention of receptor dimerization. Because of its specificity, this MAb may be usefully applied in situations in which GHR and receptors with a high degree of homology, such as PRLR (prolactin receptor), are expressed simultaneously, as occurs in the immune system.

Recombinant bacterial cells as efficient biocatalysts for the production of nucleosides.

The preparation of nucleosides as well as their base-modified analogues using purified nucleoside phosphorylase enzymes or, more conveniently, using whole bacterial cells is described. The development of genetically modified strains of Escherichia coli, able to over-produce Uridine-phosphorylase and Purine-nucleoside-phosphorylase in the same cells, improves the specific biocatalytic activity and the consequent industrial scale approach.