Whole-genome-scale identification of novel non-protein-coding RNAs controlling cell proliferation and survival through a functional forward genetics strategy.

Identification of cell fate-controlling lncRNAs is essential to our understanding of molecular cell biology. Here we present a human genome-scale forward-genetics approach for the identification of lncRNAs based on gene function. This approach can identify genes that play a causal role, and immediately distinguish them from those that are differentially expressed but do not affect cell function. Our genome-scale library plus next-generation-sequencing and bioinformatic approach, radically upscales the breadth and rate of functional ncRNA discovery. Human gDNA was digested to produce a lentiviral expression library containing inserts in both sense and anti-sense orientation. The library was used to transduce human Jurkat T-leukaemic cells. Cell populations were selected using continuous culture ± anti-FAS IgM, and sequencing used to identify sequences controlling cell proliferation. This strategy resulted in the identification of thousands of new sequences based solely on their function including many ncRNAs previously identified as being able to modulate cell survival or to act as key cancer regulators such as AC084816.1*, AC097103.2, AC087473.1, CASC15*, DLEU1*, ENTPD1-AS1*, HULC*, MIRLET7BHG*, PCAT-1, SChLAP1, and TP53TG1. Independent validation confirmed 4 out of 5 sequences that were identified by this strategy, conferred a striking resistance to anti-FAS IgM-induced apoptosis.

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma.

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of female adults with poorly controlled atopic asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 13, 17 A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFα, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, we utilised a previously published assessment of the circulating "blood microbiome" performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in just the asthma group or just in the control group, and many more were observed to be expressed at significantly different levels in the asthma group compared to the control group. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthmatic state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

What is normal? Next generation sequencing-driven analysis of the human circulating miRNAOme.

BACKGROUND: MicroRNAs (miRNAs) are short non-protein-coding RNA species that have a regulatory function in modulating protein translation and degradation of specific mRNAs. MicroRNAs are estimated to target approximately 60% of all human mRNAs and are associated with the regulation of all physiological processes. Similar to many messenger RNAs (mRNA), miRNAs exhibit marked tissue specificity, and appear to be dysregulated in response to specific pathological conditions. Perhaps, one of the most significant findings is that miRNAs are detectable in various biological fluids and are stable during routine clinical processing, paving the way for their use as novel biomarkers. Despite an increasing number of publications reporting individual miRNAs or miRNA signatures to be diagnostic of disease or indicative of response to therapy, there is still a paucity of baseline data necessary for their validation. To this end, we utilised state of the art sequencing technologies to determine the global expression of all circulating miRNAs within the plasma of 18 disease-free human subjects. RESULTS: In excess of 500 miRNAs were detected in our study population with expression levels across several orders of magnitude. Ten highly expressed miRNAs accounted for 90% of the total reads that mapped showing that despite the range of miRNAs present, the total miRNA load of the plasma was predominated by just these few species (50% of which are blood cell associated). Ranges of expression were determined for all miRNA detected (>500) and a set of highly stable miRNAs identified. Finally, the effects of gender, smoking status and body mass index on miRNA expression were determined. CONCLUSIONS: The data contained within will be of particular use to researchers performing miRNA-based biomarker screening in plasma and allow shortlisting of candidates a priori to expedite discovery or reduce costs as required.

The hallmarks of osteoarthritis and the potential to develop personalised disease-modifying pharmacological therapeutics.

Osteoarthritis (OA) is an age-related condition and the leading cause of pain, disability and shortening of adult working life in the UK. The incidence of OA increases with age, with 25% of the over 50s population having OA of the knee. Despite promising preclinical data covering various molecule classes, there is regrettably at present no approved disease-modifying OA drugs (DMOADs). With the advent of next generation sequencing technologies, other therapeutic areas, in particular oncology, have experienced a paradigm shift towards defining disease by its molecular composition. This paradigm shift has enabled high resolution patient stratification and supported the emergence of personalised or precision medicines. In this review we evaluate the potential for the development of OA therapeutics to undergo a similar paradigm shift given that OA is increasingly being recognised as a heterogeneous disease affecting multiple joint tissues. We highlight the evidence for the role of these tissues in OA pathology as different "hallmarks" of OA biology and review the opportunities to identify and develop targeted disease-modifying pharmacological therapeutics. Finally, we consider whether it is feasible to expect the emergence of personalised disease-modifying medicines for patients with OA and how this might be achieved.

Beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle does not modulate disease severity in the rodent meniscectomy model of osteoarthritis.

OBJECTIVE: To examine whether beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model. METHODS: Male Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n=15) or saline vehicle (n=20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n=10; saline pre-treated n=10) or a sham control surgical procedure (saline pre-treated n=5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint. RESULTS: Clenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery. CONCLUSIONS: Our data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to "slow twitch" in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability.