Roles and dynamics of 3-methylcytidine in cellular RNAs.

Modified nucleotides within cellular RNAs significantly influence their biogenesis, stability, and function. As reviewed here, 3-methylcytidine (m3C) has recently come to the fore through the identification of the methyltransferases responsible for installing m3C32 in human tRNAs. Mechanistic details of how m3C32 methyltransferases recognize their substrate tRNAs have been uncovered and the biogenetic and functional relevance of interconnections between m3C32 and modified adenosines at position 37 highlighted. Functional insights into the role of m3C32 modifications indicate that they influence tRNA structure and, consistently, lack of m3C32 modifications impairs translation. Development of quantitative, transcriptome-wide m3C mapping approaches and the discovery of an m3C demethylase reveal m3C to be dynamic, raising the possibility that it contributes to fine-tuning gene expression in different conditions.

Differentially expressed messenger RNA/proteins can distinguish teratoma from necrosis in postchemotherapy retroperitoneal lymph node dissection tissue.

BACKGROUND: Before postchemotherapy retroperitoneal lymph node dissection (pcRPLND), in patients with metastasized germ cell tumors (GCTs), those harboring necrosis (NEC) cannot be distinguished from those who have teratoma (TER), resulting in relevant overtreatment, whereas microRNA-371a-3p may be predictive for viable GCT. The purpose of this study was to explore messenger RNA (mRNA) and proteins to distinguish TER from NEC in pcRPLND tissue. METHODS: The discovery cohort consisted in total of 48 patients, including 16 each with TER, viable GCT, and NEC. Representative areas were microdissected. A NanoString panel and proteomics were used to analyze 770 genes and >5000 proteins. The most significantly and differentially expressed combination of both parameters, mRNA and its associated protein, between TER and NEC was validated using immunohistochemistry (IHC) in an independent validation cohort comprising 66 patients who were not part of the discovery cohort. RESULTS: The authors observed that anterior gradient protein 2 homolog (AGR2) and keratin, type I cytoskeletal 19 (KRT19) were significantly differentially expressed in TER versus NEC in mRNA and protein analyses (proteomics). The technical validation using IHC was successful in the same patients. These proteins were further validated by IHC in the independent patient cohort and exhibited significantly higher levels in TER versus NEC (p < .0001; area under the curve, 1.0; sensitivity and specificity, 100% each). CONCLUSIONS: The current study demonstrated that KRT19 and AGR2 mRNA and protein are overexpressed in TER versus NEC in pcRPLND tissue and might serve as a future diagnostic target to detect TER, for instance, by functional imaging, to avoid overtreatment. PLAIN LANGUAGE SUMMARY: The proteins and the corresponding genes called AGR2 and KRT19 can differentiate between teratoma and necrosis in remaining tumor masses after chemotherapy in patients who have metastasized testicular cancer. This may be a way to improve presurgical diagnostics and to reduce the current overtreatment of patients with necrosis only, who could be treated sufficiently by surveillance.

Integrated Analysis of miRNA and mRNA Regulation Network in Hypertension.

Hypertension is the most common chronic disease. Early diagnosis is helpful for early medical intervention. The miRNAs and the messenger RNAs (mRNAs) network may be valuable disease diagnosis markers. We aimed to explore the diagnostic value of the miRNA-mRNA network for hypertension patients. Data of miRNAs and mRNAs expression were obtained from the Gene Expression Omnibus database. The weighted gene co-expression network analysis was performed to screen hypertension-related gene modules, and these genes undergone functional enrichment analysis using "clusterProfiler" package. Differential expression analysis was applied on miRNAs expression profiles using "limma" package. TargetScanHuman and miRDB databases were used to select target mRNAs. Cytoscape software was used to visualize the miRNA-mRNA regulation network. P value < 0.05 was considered statistically significant after t test. There were 123 screened mRNAs which were enriched in 161 Gene ontology (GO) terms and 14 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Thirty-five differentially expressed miRNAs (DEMs) are found in the GSE75670. Totally 36 miRNA-mRNA pairs were obtained after the integrated analysis, and three mRNAs and the hsa-miRNA-5589-5p were identified as key joints. Hub genes, KIAA0513, ARID3A and LRPAP1, and key hsa-miRNA-5589-5p are potential diagnostic biomarkers for hypertension. Our findings are promising in the clinical application, conducive to early detection and prompt intervention of hypertension.

Screening Biomarkers of Sudden Coronary Death Based on mRNA Expression Profile of Rat Myocardial Tissues.

OBJECTIVES: To explore the differential expression of messenger RNA (mRNA) in myocardial tissues of rats with sudden coronary death (SCD), and to provide ideas for the forensic identification of SCD. METHODS: The rat SCD model was established, and the transcriptome sequencing was performed by next-generation sequencing technology. Differentially expressed genes (DEGs) in myocardial tissues of SCD rats were screened by using the R package limma. A protein-protein interaction (PPI) network was constructed by using the STRING database and Cytoscape 3.8.2 on DEG, and hub genes were screened based on cytoHubba plug-in. Finally, the R package clusterProfiler was used to analyze the biological function and signal pathway enrichment of the selected DEG. RESULTS: A total of 177 DEGs were associated with SCD and were mainly involved in the renin-angiotensin system and PI3K-Akt signaling pathway. The genes including angiotensinogen (AGT), complement component 4a (C4a), Fos proto-oncogene (FOS) and others played key roles in the development of SCD. CONCLUSIONS: Genes such as AGT, C4a, FOS and other genes are expected to be potential biomarkers for forensic identification of SCD. The study based on mRNA expression profile can provide a reference for forensic identification of SCD.

Signatures of altered long noncoding RNAs and messenger RNAs expression in the early acute phase of spinal cord injury.

Spinal cord injury (SCI) is a highly severe disease and it can lead to the destruction of the motor and sensory function resulting in temporary or permanent disability. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt that play a critical role in central nervous system (CNS) injury. However, the exact roles of lncRNAs and messenger RNAs (mRNAs) in the early acute phase of SCI remain to be elucidated. We examined the expression of mRNAs and lncRNAs in a rat model at 2 days after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. Subsequently, a comprehensive bioinformatics analysis was also performed to clarify the interaction between DE mRNAs. A total of 3,193 DE lncRNAs and 4,308 DE mRNAs were identified between the injured group and control group. Classification, length distribution, and chromosomal distribution of the dysregulated lncRNAs were also performed. The gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. A protein-protein interaction (PPI) network indicated that IL6, TOP2A, CDK1, POLE, CCNB1, TNF, CCNA2, CDC20, ITGAM, and MYC were the top 10 core genes. The subnetworks from the PPI network were identified to further elucidate the most significant functional modules of the DE mRNAs. These data may provide novel insights into the molecular mechanism of the early acute phase of SCI. The identification of lncRNAs and mRNAs may offer potential diagnostic and therapeutic targets for SCI.

Different expression profile of mRNA and long noncoding RNA in autoimmune thyroid diseases patients.

Increasing evidence has found that long noncoding RNAs (lncRNAs) and message RNAs (mRNAs) play an important role in the progress of autoimmune thyroid diseases (AITD). So, in this study, the different expressed of lncRNA and mRNA was screened by microarray analysis and quantitative real-time polymerase chain reaction (PCR). To further investigate the relationship among the differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene ontology (GO) were used for biofunctions and signaling pathways analysis, respectively. Finally, the interaction relationship between lncRNA and mRNAs was analysis with lncRNA-mRNA coexpression network. The result found that the abnormal expression of lncRNAs and mRNAs were 1615 and 1913, respectively. The altered genes included CD40LG, IFNG, CTLA4, FAS, STAT1, STAT3, and STAT4. These were enriched in presentation and antigen processing, Th1 and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, B cell receptor signaling pathway, Th17 cell differentiation, and cell adhesion molecules (CAMs), all of which had been suggested to be associated with immunopathogenic mechanisms and AITD-induced pathophysiologic changes. A coexpression network profile was contained with 126 network nodes and 477 connections which were based on seven mRNAs and 119 interacted lncRNAs. The outcomes of differentially expressed lncRNAs and their coreralated mRNAs in our study revealed that lncRNAs involved in immunopathogenic mechanisms may play a crital role in the pathogenesis of AITD.

Identification of long noncoding RNAs biomarkers for diagnosis and prognosis in patients with colon adenocarcinoma.

BACKGROUND: In the study, we aimed to find key long noncoding RNAs (lncRNAs) significantly associated with the diagnosis of colon adenocarcinoma (COAD) and lncRNA signatures to provide survival risk prognosis for patients with COAD. METHODS: The lncRNA and messenger RNA (mRNA) expression profiles and clinical information of patients with COAD were obtained from the Cancer Genome Atlas database. The differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between COAD and normal tissues were identified. The optimal diagnostic and prognostic lncRNA biomarkers for COAD were identified by using feature selection procedure and classification model. Functional enrichment analysis revealed the possible roles of mRNAs coexpressed with lncRNAs in some cancer-related biological processes and pathways. Receiver operating characteristic curve of these lncRNA biomarkers were obtained by using 10-fold cross-validation. Univariate and multivariate Cox regression analysis for these DElncRNAs were performed to obtain the lncRNAs related to overall survival time. The expression levels of selected DElncRNAs were validated by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: A total of 169 DElncRNAs (60 downregulated and 109 upregulated) and 1236 DEmRNAs (708 downregulated and 528 upregulated) between COAD and normal tissues were identified. Eight lncRNAs of XXbac-B476C20.9, PP7080, CDKN2B-AS1, LINC00092, CA3-AS1, HAND2-AS1, CTD-2269F5.1, and LINC01082 were selected as optimal diagnostic lncRNA biomarkers for COAD. The expression of eight optimal diagnostic lncRNA biomarkers in qRT-PCR validation was consistent with our integrated analysis. Among them, XXbac-B476C20.9 was not only one of the eight optimal diagnostic lncRNA biomarkers, but also related to the prognosis of COAD. CONCLUSION: Our study demonstrated the promising potential of XXbac-B476C20.9 as an independent biomarker for diagnosis and prognosis of patients with COAD.

Interactome analysis of the human Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (hMTr1) protein.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.

Identification specific miRNA in t(4;14) multiple myeloma based on miRNA-mRNA expressing profile correlation analysis.

BACKGROUND: Multiple myeloma (MM) is a common malignancy belonging to the hematological system. The translocation t(4;14)(p16.3;q32.3) is a critical cytogenetic change of MM, which is presenting a poor prognosis. The specific microRNAs (miRNAs) that are involved in t(4;14) myeloma are still unknown. Thus, the main purpose of this research was to identify specific miRNAs in t(4;14) positive myeloma. METHODS: The expression profiles of miRNA and messenger RNA (mRNA) in t(4; 14) positive and negative samples were obtained from the gene expression omnibus data series. The miRNA-mRNA regulatory network was constructed based on two self-defined regulation models. Subsequently, we performed the topology analysis for mining the hub genes, and Pearson's correlation coefficient analysis was used to calculate the relevance of the hub genes and specific miRNAs. RESULTS: Thirteen differentially expressed miRNAs and 206 differential mRNAs were extracted between t(4;14) positive group and negative group. The network consisted of 8 miRNAs and 154 mRNAs in 2 reverse regulated models, which showed a total of 485 interactions, including 376 cis-regulated and 109 trans-regulated relationships. The miR-125a-3p, miR-125a-5p, miR-99b-5p, and let-7e were powerful miRNAs correlating with the FGFR3, MAP1B, MYRIP, and CDC42BPA under the relevance analysis in the subnetwork. CONCLUSION: In our study, a distinctive correlation analysis of miRNA-mRNA was established to excavate specific miRNAs and hub target mRNAs in patients with t(4;14), but it was only a matter of theoretical principles. The further experimental explorations are needed to confirm valuable diagnostic and therapeutic symbols specific associated with t(4;14) in the future.

Oral testing for high-risk human papillomavirus DNA and E6/E7 messenger RNA in healthy individuals at risk for oral infection.

BACKGROUND: Testing for oral high-risk human papillomavirus (HPV) DNA may be useful for identifying individuals at increased risk for HPV-driven oropharyngeal cancer (OPC). However, positivity for HPV DNA provides no information on the transforming potential of the infection. In contrast, the detection of high-risk HPV E6/E7 messenger RNA (mRNA) may help to identify clinically significant infections because of the indispensable role of E6/E7 viral oncoproteins in the carcinogenic process. METHODS: Oral rinses were collected with a mouthwash from cancer-free individuals at increased risk for oral HPV infection. High-risk HPV DNA and mRNA were evaluated via the testing of the oral rinses with the Linear Array HPV genotyping test and the Aptima HPV assay, respectively. RESULTS: Overall, 310 subjects with no clinical evidence of lesions of the oral cavity and oropharynx were included in the study. Thirty-three (10.6%) harbored high-risk HPV DNA in their oral rinse. These cases, together with 10 random samples negative for high-risk HPV DNA, were tested with the Aptima assay. A valid result was obtained for 41 of the 43 specimens (95.3%). Among the 31 cases that were positive for high-risk HPV DNA and had a valid Aptima result, 4 (12.9%) were positive for HPV mRNA. HPV mRNA was not detected in any of the samples negative for high-risk HPV DNA. CONCLUSIONS: HPV mRNA is detectable in oral rinses of cancer-free subjects. Oral HPV mRNA testing may be useful in the screening and/or early detection of HPV-driven OPC by possibly identifying active and transforming oral infections. The testing of individuals at increased risk for HPV-related OPC via simply and noninvasively collected oral specimens is an attractive option for future screening strategies.

"The acid test"-validation of the ParaDNA® Body Fluid ID Test for routine forensic casework.

The identification of the cellular origin and composition of crime scene-related traces can provide crucial insight into a crime scene reconstruction. In the last decade, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined. Besides capillary electrophoretic (CE) and real-time quantitative PCR (RT-qPCR)-based approaches for mRNA detection, melt curve analysis bears potential as a simple-to-use method for BFI. The ParaDNA® Body Fluid ID Test relies on HyBeacon® probes and was developed as a rapid test for mRNA-based BFI of six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual, and peripheral blood. The herein presented work was performed as an "acid test" of the system and should clarify whether the approach matches the requirements of forensic routine casework in German police departments. Tested samples consisted of single source as well as of mixed samples.

A novel 3' untranslated region mutation in the SLC29A3 gene associated with pigmentary hypertrichosis and non-autoimmune insulin-dependent diabetes mellitus syndrome.

BACKGROUND: Pigmentary hypertrichosis and non-autoimmune insulin-dependent diabetes mellitus (PHID) is one of the rare H syndrome diseases mainly characterized by hyperpigmentation, hypertrichosis, sensorineural hearing loss, cardiac complications, developmental delay, and diabetes mellitus (DM). Mutations in the coding regions of the SLC29A3 gene that encodes for an equilibrative nucleoside transporter (ENT3) have been reported to cause the phenotypic spectrum of the H syndrome. Disease-causing mutations in the untranslated regions (UTRs) of the SLC29A3 gene have not been previously described in the literature. The aim of the study is to describe and assess the pathogenicity of a novel 3'UTR mutation in the SLC29A3 gene associated with the PHID phenotype in two Turkish patients. METHODS: The mutation was identified by a targeted gene approach. To understand the pathogenicity of this 3'UTR mutation, RNA and protein expression studies were performed by using the quantitative real-time polymerase chain reaction method and western blotting, respectively, using fibroblasts cultured from the patients' skin biopsies. RESULTS: SLC29A3 and ENT3 expression levels were both decreased in the patients compared to controls matched for passage numbers, RNA, and protein extraction methods. CONCLUSIONS: A novel 3'UTR mutation in the SLC29A3 gene is associated with the PHID syndrome, highlighting a potentially new pathological mechanism for this disease. The involvement of the 3'UTR has not been previously established in any of the H syndrome disease cluster or in any complex syndrome of DM.

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.

Altered Long Noncoding RNA and Messenger RNA Expression in Experimental Intracerebral Hemorrhage - a Preliminary Study.

BACKGROUND/AIMS: Functional recovery in the chronic phase is a difficult problem in intracerebral hemorrhage (ICH) treatment. Long noncoding RNAs (lncRNAs) are demonstrated to be involved in central nervous system (CNS) disorders. However, the roles of lncRNAs in post-ICH injury and repair are poorly understood, especially those that may be attributed to long-term neurological deficit. The present study depicted the lncRNA and messenger RNA (mRNA) profile by microarray at late stage after an experimental ICH. METHODS: LncRNA and mRNA microarray was used to first identify differentially expressed genes. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine bio-functions and signaling pathways, with which differentially expressed genes are most closely related. Quantitative real-time polymerase chain reaction (PCR) was used to validate the results of microarray. Finally, the lncRNA-mRNA co-expression network was constructed to find the interaction of genes. RESULTS: A total of 625 differentially expressed lncRNAs and 826 expressed mRNAs were identified. Altered genes were enriched in mitochon-drial matrix, G-protein coupled receptor signaling pathway, and olfactory transduction, which may be associated with ICH-induced pathophysiologic changes in the long term. A co-expression network profile based on 5 validated differentially expressed lncRNAs and 205 interacted mRNAs was composed of 210 nodes and 298 connections. CONCLUSION: Mitochondrial matrix, reduced G-protein coupled receptor activity, and impaired olfactory transduction may be involved in the sequelae following ICH. Further, these dysregulated lncRNAs and mRNAs may be the promising therapeutic targets to overcome obstacles in functional recovery following ICH.

An Intersectional Study of LncRNAs and mRNAs Reveals the Potential Therapeutic Targets of Buyang Huanwu Decoction in Experimental Intracerebral Hemorrhage.

BACKGROUND/AIMS: Both experimental and clinical studies have revealed satisfactory effects of the traditional Chinese formula Buyang Huanwu decoction (BYHWD) in improving post-intracerebral hemorrhage (ICH) neurological deficiencies. However, the multifaceted mechanisms of BYHWD in ICH treatment are not comprehensively understood. The present study explored various therapeutic targets of BYHWD by using lncRNA and mRNA transcriptomics. METHODS: LncRNA and mRNA microarrays were used to identify differentially expressed genes. ICH-induced upregulated genes (ICH vs sham) and BYHWD-induced downregulated genes (BYHWD vs ICH) were first identified. The intersection between these 2 sets was determined to identify ICH-induced highly expressed genes that were reversed by BYHWD. Then, the genes downregulated after ICH and the genes upregulated after BYHWD treatment were used to generate another set of intersections. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were subsequently performed to determine relative biological functions and signaling transduction pathways according to genes within the intersections. Quantitative real-time PCR was used to validate changes in gene expression observed using the microarray. Finally, a lncRNA-mRNA co-expression network was established to identify links among the genes within the intersections. RESULTS: A total of 18 differentially expressed lncRNAs and 33 differentially expressed mRNAs were identified using 2 lncRNA arrays (ICH vs sham and BYHWD vs ICH). The altered genes were enriched in the hemoglobin complex, oxygen transport and oxygen transporter and were closely associated with pyruvate metabolism. The co-expression network consisted of 53 nodes and 595 connections (308 positive interactions and 287 negative interactions). CONCLUSION: The hemoglobin complex, oxygen transport, oxygen transporter activity and pyruvate metabolism are possible therapeutic targets of BYHWD in ICH treatment. The present study provides the basis and direction for future investigations to explore the mechanisms by which BYHWD protects against long-term neurological deficiencies after ICH.

RNA splicing in human disease and in the clinic.

Defects at the level of the pre-mRNA splicing process represent a major cause of human disease. Approximately 15-50% of all human disease mutations have been shown to alter functioning of basic and auxiliary splicing elements. These elements are required to ensure proper processing of pre-mRNA splicing molecules, with their disruption leading to misprocessing of the pre-mRNA molecule and disease. The splicing process is a complex process, with much still to be uncovered before we are able to accurately predict whether a reported genomic sequence variant (GV) represents a splicing-associated disease mutation or a harmless polymorphism. Furthermore, even when a mutation is correctly identified as affecting the splicing process, there still remains the difficulty of providing an exact evaluation of the potential impact on disease onset, severity and duration. In this review, we provide a brief overview of splicing diagnostic methodologies, from in silico bioinformatics approaches to wet lab in vitro and in vivo systems to evaluate splicing efficiencies. In particular, we provide an overview of how the latest developments in high-throughput sequencing can be applied to the clinic, and are already changing clinical approaches.

Efficacy and safety of dovitinib in pretreated patients with advanced squamous non-small cell lung cancer with FGFR1 amplification: A single-arm, phase 2 study.

BACKGROUND: Fibroblast growth factor receptor 1 (FGFR1) amplification is a potential driving oncogene in squamous cell cancer (SCC) of the lung. The current phase 2 study evaluated the efficacy and tolerability of dovitinib, an FGFR inhibitor, in patients with advanced SCC of the lung. METHODS: Patients with pretreated advanced SCC of the lung whose tumors demonstrated FGFR1 amplification of > 5 copies by fluorescence in situ hybridization were enrolled. Dovitinib at a dose of 500 mg was administered orally, once daily, on days 1 to 5 of every week, followed by 2 days off. The primary endpoint was overall response. Secondary endpoints were progression-free survival, overall survival, and toxicity. RESULTS: All 26 patients were men with a median age of 68 years (range, 52-80 years). The majority of patients were ever-smokers. The median duration of dovitinib administration (28 days per cycle) was 2.5 months (range, 0.7-8.6 months). The overall response rate was 11.5% (95% confidence interval [95% CI], 0.8%-23.8%) and the disease control rate was 50% (95% CI, 30.8%-69.2%), with 3 patients achieving partial responses. Response durations for the patients with partial responses were ≥4.5 months, ≥ 5.1 months, and 6.1 months, respectively. After a median follow-up of 15.7 months (range, 1.2-25.6 months), the median overall survival was 5.0 months (95% CI, 3.6-6.4 months) and the median progression-free survival was 2.9 months (95% CI, 1.5-4.3 months). The most common grade 3 or higher adverse events were fatigue (19.2%), anorexia (11.5%), and hyponatremia (11.5%) (event severity was graded based on National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). CONCLUSIONS: Treatment with dovitinib demonstrated modest efficacy in patients with advanced SCC with FGFR1 amplification. Further studies to evaluate other biomarkers correlated with the efficacy of dovitinib in patients with SCC are warranted. Cancer 2016;122:3024-3031. © 2016 American Cancer Society.

Nuclear cyclophilins affect spliceosome assembly and function in vitro.

Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing.

Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA.

Messenger RNA (mRNA) is rapidly growing as a therapeutic modality for vaccination and the treatment of a wide range of diseases. As a result, there is an increased demand for mRNA-based analytical methods capable of assessing purity and stability, which are considered critical quality attributes (CQAs). In recent decades capillary electrophoresis (CE) has emerged alongside liquid chromatography (LC) as an important tool for the assessment of purity and stability of mRNA therapeutics. CE offers a variety of advantages over conventional LC or gel-based analytical methods, including reduced injection volume, increased resolution, and increased separation efficiency. In this study we compared CE-based analytical methods: the Agilent RNA 6000 Nano Kit, the Revvity RNA Reagent Kit, the Sciex RNA 9000 Purity and Integrity Kit, and the Agilent HS RNA Kit. These methods were evaluated on their vendor-recommended instruments: the Bioanalyzer, LabChip GXII, PA800 Plus, and Fragment Analyzer, respectively. We assessed the ability of these methods to measure mRNA integrity, purity, and stability. Furthermore, several parameters for each method were also assessed: selectivity, precision, resolution, analysis time, and ease of use. Based on our results, all four methods are suitable for use in the characterization of in vitro transcribed (IVT) mRNA, depending on the intended application. The Sciex RNA 9000 Purity and Integrity kit method achieved the highest selectivity and resolving power compared with the other methods, making it the most suitable for high-resolution, in-depth sample characterization. In comparison, the Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit achieved lower selectivity and resolution, but their faster analysis times make them more suitable for high-throughput and screening applications.

Replacing poly(ethylene glycol) with RAFT lipopolymers in mRNA lipid nanoparticle systems for effective gene delivery.

Lipid nanoparticles (LNPs) have emerged as promising carriers to efficiently transport mRNA into cells for protein translation, as seen with the mRNA vaccines used against COVID-19. However, they contain a widely used polymer - poly(ethylene glycol) (PEG) - which lacks the functionality to be easily modified (which could effectively control the physicochemical properties of the LNPs such as its charge), and is also known to be immunogenic. Thus, it is desirable to explore alternative polymers which can replace the PEG component in mRNA LNP vaccines and therapeutics, while still maintaining their efficacy. Herein, we employed reversible addition-fragmentation chain transfer (RAFT) polymerisation to synthesise five PEG-lipid alternatives that could stabilise LNPs encapsulating mRNA or pDNA molecules. Importantly, the resultant RAFT lipopolymer LNPs exhibit analogous or higher in vivo gene expression and antigen-specific antibody production compared to traditional PEG-based formulations. Our synthesis strategy which allows the introduction of positive charges along the lipopolymer backbone also significantly improved the in vivo gene expression. This work expands the potential of RAFT polymer-conjugated LNPs as promising mRNA carriers and offers an innovative strategy for the development of PEG-free mRNA vaccines and therapeutics.