Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling.

Urinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

Next-generation sequencing reveals novel rare fusion events with functional implication in prostate cancer.

Gene fusions, mainly between TMPRSS2 and ERG, are frequent early genomic rearrangements in prostate cancer (PCa). In order to discover novel genomic fusion events, we applied whole-genome paired-end sequencing to identify structural alterations present in a primary PCa patient (G089) and in a PCa cell line (PC346C). Overall, we identified over 3800 genomic rearrangements in each of the two samples as compared with the reference genome. Correcting these structural variations for polymorphisms using whole-genome sequences of 46 normal samples, the numbers of cancer-related rearrangements were 674 and 387 for G089 and PC346C, respectively. From these, 192 in G089 and 106 in PC346C affected gene structures. Exclusion of small intronic deletions left 33 intergenic breaks in G089 and 14 in PC346C. Out of these, 12 and 9 reassembled genes with the same orientation, capable of generating a feasible fusion transcript. Using PCR we validated all the reliable predicted gene fusions. Two gene fusions were in-frame: MPP5-FAM71D in PC346C and ARHGEF3-C8ORF38 in G089. Downregulation of FAM71D and MPP5-FAM71D transcripts in PC346C cells decreased proliferation; however, no effect was observed in the RWPE-1-immortalized normal prostate epithelial cells. Together, our data showed that gene rearrangements frequently occur in PCa genomes but result in a limited number of fusion transcripts. Most of these fusion transcripts do not encode in-frame fusion proteins. The unique in-frame MPP5-FAM71D fusion product is important for proliferation of PC346C cells.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

Dose-response effects of a new growth hormone receptor antagonist (B2036-PEG) on circulating, hepatic and renal expression of the growth hormone/insulin-like growth factor system in adult mice.

The effects of growth hormone (GH) in regulating the expression of the hepatic and renal GH and insulin-like growth factor (IGF) system were studied by administering a novel GH receptor antagonist (GHRA) (B2036-PEG) at different doses (0, 1.25, 2.5, 5 and 10 mg/kg/day) to mice for 7 days. No differences were observed in the groups with respect to body weight, food consumption or blood glucose. However, a dose-dependent decrease was observed in circulating IGF-I levels and in hepatic and renal IGF-I levels at the highest doses. In contrast, in the 5 and 10 mg/kg/day GHRA groups, circulating and hepatic transcriptional IGF binding protein-3 (IGFBP-3) levels were not modified, likely resulting in a significantly decreased IGF-I/IGFBP-3 ratio. Hepatic GH receptor (GHR) and GH binding protein (GHBP) mRNA levels increased significantly in all GHRA dosage groups. Endogenous circulatory GH levels increased significantly in the 2.5 and 5 mg/kg/day GHRA groups. Remarkably, increased circulating IGFBP-4 and hepatic IGFBP-4 mRNA levels were observed in all GHRA administration groups. Renal GHR and GHBP mRNA levels were not modified by GHRA administration at the highest doses. Also, renal IGFBP-3 mRNA levels remained unchanged in most GHRA administration groups, whereas IGFBP-1, -4 and -5 mRNA levels were significantly increased in the 5 and 10 mg/kg/day GHRA administration groups. In conclusion, the effects of a specific GHR blockade on circulating, hepatic and renal GH/IGF axis reported here, may prove useful in the future clinical use of GHRAs.

The effect of epidermal growth factor and IGF-I infusion on hepatic and renal expression of the IGF-system in adult female rats.

Systemic administration of epidermal growth factor (EGF) in neonatal rats results in reduced body weight gain and decreased circulating levels of IGF-I, suggesting its involvement in EGF-induced growth retardation. We investigated the effect of EGF and/or IGF-I administration for 7 days on circulating IGF-I and IGFBP levels and hepatic and renal IGF-system mRNA expression profiles in adult female rats. EGF administration (30 microg/rat/day) did not influence body weight, liver or kidney weight. In contrast, IGF-I (400 microg/rat/day) and EGF/IGF-I administration increased both body weight and kidney weight. Also, serum IGF-I and the 30 kDa IGFBPs (IGFBP-1 and -2) were significantly increased in these groups. Serum IGFBP-3 levels increased in the IGF-I group along with increased hepatic IGFBP-1 and -3 mRNA levels. In contrast, in the EGF administration group serum IGFBP-3 levels were significantly decreased; however, the mRNA levels remained unchanged. In the EGF/IGF-I administration group, serum IGF-I and IGFBP-3 levels were significantly lowered when compared with the IGF-I administration group. This was in contrast to the effect on kidney weight increase that was identical for the IGF-I and EGF/IGF-I groups. The decrease in serum IGFBP-3 was not reflected at the hepatic IGFBP-3 mRNA level. IGFBP-3 expression might be regulated at a post-transcriptional level although EGF induced IGFBP-3 proteolysis could not be demonstrated in vitro. We conclude that EGF administration reduced serum IGFBP-3 whereas IGF-I administration increased the level of IGFBP-3 and IGF-I and resulted in an increased body and kidney weight in adult female rats.

Repression of androgen-regulated gene expression by dominant negative androgen receptors.

The androgen receptor (AR) is a ligand-dependent transcription activator responsible for male sexual development. In order to specifically inhibit the AR pathway, dominant negative ARs were constructed by inactivation of the major transactivation domains of the wild type AR and fusing this mutant (AR122) to the Krüppel-associated box (KRAB) repressor domain and/or histone deacetylase (HDAC1). The HDAC1-KRAB-AR122 protein was the most successful dominant negative AR, capable of repressing the wild type AR ninefold when co-expressed at a 1:1 plasmid ratio. A maximal repression of 41-fold was achieved when HDAC1-KRAB-AR122 was cotransfected with the wild type AR at a 4:1 plasmid ratio. HDAC1-KRAB-AR122 repressed transcription in a ligand-dependent manner since it inhibited a constitutively active AR mutant (AR5) only in the presence of agonists. High concentrations of partial agonists such as RU486, cyproterone acetate, and estradiol were also capable of triggering repression by HDAC1-KRAB-AR122. The potent dominant negative AR proteins might prove useful tools to inhibit AR function in vitro and in vivo.

The IGF system during fetal-placental development of the mouse.

Insulin-like growth factors (IGF-I and -II) promote cellular mitosis and differentiation and have been implicated in fetal and placental growth. Together with the IGF receptors and IGF binding proteins (IGFBPs) they form a complex network, with tissue specific activity. This review will discuss the data generated to elucidate the functions of the IGF system during mouse development.

Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer.

Prostate cancer (PCa) is the most frequent male malignancy and the second most common cause of cancer-related death in Western countries. Current clinical and pathological methods are limited in the prediction of postoperative outcome. It is becoming increasingly evident that small non-coding RNA (ncRNA) species are associated with the development and progression of this malignancy. To assess the diversity and abundance of small ncRNAs in PCa, we analyzed the composition of the entire small transcriptome by Illumina/Solexa deep sequencing. We further analyzed the microRNA (miRNA) expression signatures of 102 fresh-frozen patient samples during PCa progression by miRNA microarrays. Both platforms were cross-validated by quantitative reverse transcriptase-PCR. Besides the altered expression of several miRNAs, our deep sequencing analyses revealed strong differential expression of small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). From microarray analysis, we derived a miRNA diagnostic classifier that accurately distinguishes normal from cancer samples. Furthermore, we were able to construct a PCa prognostic predictor that independently forecasts postoperative outcome. Importantly, the majority of miRNAs included in the predictor also exhibit high sequence counts and concordant differential expression in Illumina PCa samples, supported by quantitative reverse transcriptase-PCR. Our findings provide miRNA expression signatures that may serve as an accurate tool for the diagnosis and prognosis of PCa.