The Two Stem Cell MicroRNA Gene Clusters C19MC and miR-371-3 Are Activated by Specific Chromosomal Rearrangements in a Subgroup of Thyroid Adenomas

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias

Symptom Remission and Brain Cortical Networks at First Clinical Presentation of Psychosis: The OPTiMiSE Study

Individuals with psychoses have brain alterations, particularly in frontal and temporal cortices, that may be particularly prominent, already at illness onset, in those more likely to have poorer symptom remission following treatment with the first antipsychotic. The identification of strong neuroanatomical markers of symptom remission could thus facilitate stratification and individualized treatment of patients with schizophrenia. We used magnetic resonance imaging at baseline to examine brain regional and network correlates of subsequent symptomatic remission in 167 medication-naïve or minimally treated patients with first-episode schizophrenia, schizophreniform disorder, or schizoaffective disorder entering a three-phase trial, at seven sites. Patients in remission at the end of each phase were randomized to treatment as usual, with or without an adjunctive psycho-social intervention for medication adherence. The final follow-up visit was at 74 weeks. A total of 108 patients (70%) were in remission at Week 4, 85 (55%) at Week 22, and 97 (63%) at Week 74. We found no baseline regional differences in volumes, cortical thickness, surface area, or local gyrification between patients who did or did not achieved remission at any time point. However, patients not in remission at Week 74, at baseline showed reduced structural connectivity across frontal, anterior cingulate, and insular cortices. A similar pattern was evident in patients not in remission at Week 4 and Week 22, although not significantly. Lack of symptom remission in first-episode psychosis is not associated with regional brain alterations at illness onset. Instead, when the illness becomes a stable entity, its association with the altered organization of cortical gyrification becomes more defined

PET/CT imaging of pancreatic carcinoma targeting the “cancer integrin” αvβ6

Purpose!#!Fluorine-18 fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG PET/CT) is the standard for staging aggressive non-Hodgkin lymphoma (NHL). Limited data from prospective studies is available to determine whether initial staging by FDG PET/CT provides treatment-relevant information of bone marrow (BM) involvement (BMI) and thus could spare BM biopsy (BMB).!##!Methods!#!Patients from PETAL (NCT00554164) and OPTIMAL>60 (NCT01478542) with aggressive B-cell NHL initially staged by FDG PET/CT and BMB were included in this pooled analysis. The reference standard to confirm BMI included a positive BMB and/or FDG PET/CT confirmed by targeted biopsy, complementary imaging (CT or magnetic resonance imaging), or concurrent disappearance of focal FDG-avid BM lesions with other lymphoma manifestations during immunochemotherapy.!##!Results!#!Among 930 patients, BMI was detected by BMB in 85 (prevalence 9%) and by FDG PET/CT in 185 (20%) cases, for a total of 221 cases (24%). All 185 PET-positive cases were true positive, and 709 of 745 PET-negative cases were true negative. For BMB and FDG PET/CT, sensitivity was 38% (95% confidence interval [CI]: 32-45%) and 84% (CI: 78-88%), specificity 100% (CI: 99-100%) and 100% (CI: 99-100%), positive predictive value 100% (CI: 96-100%) and 100% (CI: 98-100%), and negative predictive value 84% (CI: 81-86%) and 95% (CI: 93-97%), respectively. In all of the 36 PET-negative cases with confirmed BMI patients had other adverse factors according to IPI that precluded a change of standard treatment. Thus, the BMB would not have influenced the patient management.!##!Conclusion!#!In patients with aggressive B-cell NHL, routine BMB provides no critical staging information compared to FDG PET/CT and could therefore be omitted.!##!Trial registration!#!NCT00554164 and NCT01478542

Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions

Background: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. Results: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. Conclusion: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.Dentistry, Faculty ofOral Biological and Medical Sciences (OBMS), Department ofNon UBCReviewedFacult

Genomic organization of the fusion gene on the derivative chromosome 1 resulting from a translocation t(1;19)(p35.2;13.4) in cell line S40.2.

<p>Detailed schematic overview illustrating the origin of the fusion transcripts <i>PUM1-FUS-19q-I</i> (Genbank Accession number GQ334687) and PUM1-FUS-19q-II (Genbank Accession number GQ334688) identified in cell line S40.2. The genomic region of PUM1 in 1p35.2 (horizontal gray bar) fuses after exon 10 of <i>PUM1</i> (exons: vertical light gray bars) to the genomic region of <i>C19MC</i> in 19q13.4 (horizontal red bar). The two vertical yellow bars indicate 3′-sequences located after exon 1–10 of <i>PUM1</i> in <i>PUM1-FUS-19q-I</i> and <i>PUM1-FUS-19q-II</i>, respectively, both originating from alternative splicing. The fusion transcripts were detected either by 3′-RACE-PCR (<i>PUM1-FUS-19q-I</i>) or RT-PCR (<i>PUM1-FUS-19q-II</i>) experiments. The quantified miRNAs have been highlighted by their names.</p

Expression analysis of miR-517a by RT-PCR.

<p>PCR reactions were performed and then analyzed in 4% small DNA Agarose. The expected DNA-fragment has a size of 62 bp, Ultra low range Ladder (Fermentas) was used as Marker (M). Lane 1: S40.2, 2: S40.2 without reverse transcriptase (–RT), 3: S121, 4: S121–RT, 5: thyroid (normal), 6: thyroid–RT, 7: placenta, 8: placenta-RT, 9: S270.2, 10: S270.2–RT, 11: S290.1, 12: S290.1–RT, 13: S141.2, 14: S325, 15: S211, 16: S211–RT, 17: fetal RNA, 18: adult testis, 19: fetal RNA-RT, 20: S141.2-RT, 21: adult testis-RT, 22: S325-RT (for details of the cell lines and tumor samples see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009485#pone-0009485-t001" target="_blank">Table 1</a>).</p

Delineation of <i>PUM1</i> breakpoint by metaphase FISH.

<p>Part of metaphase of cell line S40.2 after FISH with two overlapping BAC clones RP11-201O14 (green) and RP11-1136E4 (red) both spanning the whole genomic sequence of <i>PUM1</i> in 1p35.2. The breakpoint in 1p35.2 is located within <i>PUM1</i> indicated by a separation of RP11-201O14 and RP11-1136E4. Because of weak signals of RP11-1136E4 remaining on the der(1) the breakpoint is located within RP11-1136E4 distal to RP11-201O14.</p

Scheme of the chromosomal region 19q13.4 with the two miRNA clusters C19MC and miR-371-3.

<p>Protein coding genes are represented by gray bars whereas genes of miRNA clusters are given as blue (C19MC cluster) and green (miR-371-3 cluster) lines, respectively. The common breakpoint cluster (BPC) of benign thyroid tumors of about 150 kb is indicated by a vertical arrow. miR-512-1 (pre-miR) is coding for mature-miR-512-5p, miR-371 (pre-miR) is coding for mature-miR371-3p. Gene symbols refer to the following protein coding genes: <i>ZNF331</i>  =  <i>zinc finger protein 331</i>, <i>DPRX</i>  =  <i>divergent-paired related homeobox</i>, <i>NLRP12</i>  =  <i>NLR family</i>, <i>pyrin domain containing 12.</i></p