Christopher I. Jones

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

Background:Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).Methods:MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.Results:Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.Conclusion:We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

The 5' → 3' exoribonuclease XRN1/Pacman and its functions in cellular processes and development.

XRN1 is a 5′ → 3′ processive exoribonuclease that degrades mRNAs after they have been decapped. It is highly conserved in all eukaryotes, including homologs in Drosophila melanogaster (Pacman), Caenorhabditis elegans (XRN1), and Saccharomyces cerevisiae (Xrn1p). As well as being a key enzyme in RNA turnover, XRN1 is involved in nonsense‐mediated mRNA decay and degradation of mRNAs after they have been targeted by small interfering RNAs or microRNAs. The crystal structure of XRN1 can explain its processivity and also the selectivity of the enzyme for 5′ monophosphorylated RNA. In eukaryotic cells, XRN1 is often found in particles known as processing bodies (P bodies) together with other proteins involved in the 5′ → 3′ degradation pathway, such as DCP2 and the helicase DHH1 (Me31B). Although XRN1 shows little specificity to particular 5′ monophosphorylated RNAs in vitro, mutations in XRN1 in vivo have specific phenotypes suggesting that it specifically degrades a subset of RNAs. In Drosophila, mutations in the gene encoding the XRN1 homolog pacman result in defects in wound healing, epithelial closure and stem cell renewal in testes. We propose a model where specific mRNAs are targeted to XRN1 via specific binding of miRNAs and/or RNA‐binding proteins to instability elements within the RNA. These guide the RNA to the 5′ core degradation apparatus for controlled degradation. WIREs RNA 2012, 3:455–468. doi: 10.1002/wrna.1109

Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper.

Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. In this paper, we show a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, we demonstrate that a null mutation in pacman results in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. Our results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms.

The 5'-3' exoribonuclease Pacman (Xrn1) regulates expression of the heat shock protein Hsp67Bc and the microRNA miR-277-3p in Drosophila wing imaginal discs

Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm5) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, we show that eight mRNAs were increased in level by > 2-fold in the pcm5 mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm5 mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes.

Functions of microRNAs in Drosophila development.

Control of mRNA translation and degradation has been shown to be key in the development of complex organisms. The core mRNA degradation machinery is highly conserved in eukaryotes and relies on processive degradation enzymes gaining access to the mRNA. Control of mRNA stability in eukaryotes is also intimately linked to the regulation of translation. A key question in the control of mRNA turnover concerns the mechanisms whereby particular mRNAs are specifically degraded in response to cellular factors. Recently, microRNAs have been shown to bind specifically to mRNAs and regulate their expression via repression of translation and/or degradation. To understand the molecular mechanisms during microRNA repression of mRNAs, it is necessary to identify their biologically relevant targets. However, computational methods have so far proved unreliable, therefore verification of biologically important targets at present requires experimental analysis. The present review aims to outline the mechanisms of mRNA degradation and then focus on the role of microRNAs as factors affecting particular Drosophila developmental processes via their post-transcriptional effects on mRNA degradation and translation. Examples of experimentally verified targets of microRNAs in Drosophila are summarized.

RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1

Ribonucleases are critically important in many cellular and developmental processes and defects in their expression are associated with human disease. Pacman/XRN1 is a highly conserved cytoplasmic exoribonuclease which degrades RNAs in a 5′-3′ direction. In Drosophila, null mutations in pacman result in small imaginal discs, a delay in onset of pupariation and lethality during the early pupal stage. In this paper, we have used RNA-seq in a genome-wide search for mRNAs misregulated in pacman null wing imaginal discs. Only 4.2% of genes are misregulated ±>2-fold in pacman null mutants compared to controls, in line with previous work showing that Pacman has specificity for particular mRNAs. Further analysis of the most upregulated mRNAs showed that Pacman post-transcriptionally regulates the expression of the secreted insulin-like peptide Dilp8. Dilp8 is related to human IGF-1, and has been shown to coordinate tissue growth with developmental timing in Drosophila. The increased expression of Dilp8 is consistent with the developmental delay seen in pacman null mutants. Our analysis, together with our previous results, show that the normal role of this exoribonuclease in imaginal discs is to suppress the expression of transcripts that are crucial in apoptosis and growth control during normal development.

The roles of the exoribonucleases DIS3L2 and XRN1 in human disease

RNA degradation is a vital post-transcriptional process which ensures that transcripts are maintained at the correct level within the cell. DIS3L2 and XRN1 are conserved exoribonucleases that are critical for the degradation of cytoplasmic RNAs. Although the molecular mechanisms of RNA degradation by DIS3L2 and XRN1 have been well studied, less is known about their specific roles in the development of multicellular organisms or human disease. This review focusses on the roles of DIS3L2 and XRN1 in the pathogenesis of human disease, particularly in relation to phenotypes seen in model organisms. The known diseases associated with loss of activity of DIS3L2 and XRN1 are discussed, together with possible mechanisms and cellular pathways leading to these disease conditions.

Effectiveness of antipsychotics used in first-episode psychosis: a naturalistic cohort study.

Background One year of antipsychotic treatment from symptom remission is recommended following a first episode of psychosis (FEP). Aims To investigate the effectiveness of commonly used antipsychotic medications in FEP. Method A retrospective cohort study of naturalistic treatment of patients (N=460) accepted by FEP services across seven UK sites. Treatment initiation to all-cause discontinuation determined from case files. Results Risk of treatment discontinuation is greatest within 3 months of treatment initiation. Risperidone had longest median survival time. No significant differences were observed in time to discontinuation between commonly used antipsychotics on multivariable Cox regression analysis. Poor adherence and efficacy failure were the most common reasons for discontinuation. Conclusions Effectiveness differences appear not to be a current reason for antipsychotic choice in FEP. Adherence strategies and weighing up likely adverse effects should be the clinical focus. Declaration of interest R.W., A.T. and S.M. have received research grant, speaker honoraria and conference attendance funding from all companies marketing antipsychotics. Copyright and usage

Scabies outbreaks in ten care homes for elderly people: a prospective study of clinical features, epidemiology, and treatment outcomes

Summary Background Scabies outbreaks in residential and nursing care homes for elderly people are common, subject to diagnostic delay, and hard to control. We studied clinical features, epidemiology, and outcomes of outbreaks in the UK between 2014 and 2015. Methods We did a prospective observational study in residential care homes for elderly people in southeast England that reported scabies outbreaks to Public Health England health protection teams. An outbreak was defined as two or more cases of scabies (in either residents or staff) at a single care home. All patients who provided informed consent were included; patients with dementia were included if a personal or nominated consultee (ie, a family member or nominated staff member) endorsed participation. Dermatology-trained physicians examined residents at initial clinical visits, which were followed by two mass treatments with topical scabicide as per local health protection team guidance. Follow-up clinical visits were held 6 weeks after initial visits. Scabies was diagnosed through pre-defined case definitions as definite, probable, or possible with dermatoscopy and microscopy as appropriate. Findings 230 residents were examined in ten outbreaks between Jan 23, 2014, and April 13, 2015. Median age was 86·9 years (IQR 81·5–92·3), 174 (76%) were female, and 157 (68%) had dementia. 61 (27%) residents were diagnosed with definite, probable, or possible scabies, of whom three had crusted scabies. Physical signs differed substantially from classic presentations. 31 (51%) of the 61 people diagnosed with scabies were asymptomatic, and only 25 (41%) had burrows. Mites were visualised with dermatoscopy in seven (11%) patients, and further confirmed by microscopy in three (5%). 35 (57%) cases had signs of scabies only on areas of the body that would normally be covered. Dementia was the only risk factor for a scabies diagnosis that we identified (odds ratio 2·37 [95% CI 1·38–4·07]). At clinical follow-up, 50 people who were initially diagnosed with scabies were examined. No new cases of scabies were detected, but infestation persisted in ten people. Interpretation Clinical presentation of scabies in elderly residents of care homes differs from classic descriptions familiar to clinicians. This difference probably contributes to delayed recognition and suboptimal management in this vulnerable group. Dermatoscopy and microscopy were of little value. Health-care workers should be aware of the different presentation of scabies in elderly people, and should do thorough examinations, particularly in people with dementia. Funding Public Health England and British Skin Foundation.

A Novel Role for the 3'-5' Exoribonuclease Dis3L2 in Controlling Cell Proliferation and Tissue Growth.

ABSTRACT In a complex organism, cell proliferation and apoptosis need to be precisely controlled in order for tissues to develop correctly. Excessive cell proliferation can lead to diseases such as cancer. We have shown that the exoribonuclease Dis3L2 is required for the correct regulation of proliferation in a natural tissue within the model organism Drosophila melanogaster. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3′-5′ direction. We show that knockdown of dis3L2 in the Drosophila wing imaginal discs results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq we identified a small set of mRNAs that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, we identified 2 that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). We also demonstrate a compensatory effect between Dis3L2 and the 5′-3′ exoribonuclease Pacman demonstrating that these 2 exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome.