A novel saliva-based miRNA profile to diagnose and predict oral cancer.

Oral cancer (OC) is the most common form of head and neck cancer. Despite the high incidence and unfavourable patient outcomes, currently, there are no biomarkers for the early detection of OC. This study aims to discover, develop, and validate a novel saliva-based microRNA signature for early diagnosis and prediction of OC risk in oral potentially malignant disorders (OPMD). The Cancer Genome Atlas (TCGA) miRNA sequencing data and small RNA sequencing data of saliva samples were used to discover differentially expressed miRNAs. Identified miRNAs were validated in saliva samples of OC (n = 50), OPMD (n = 52), and controls (n = 60) using quantitative real-time PCR. Eight differentially expressed miRNAs (miR-7-5p, miR-10b-5p, miR-182-5p, miR-215-5p, miR-431-5p, miR-486-3p, miR-3614-5p, and miR-4707-3p) were identified in the discovery phase and were validated. The efficiency of our eight-miRNA signature to discriminate OC and controls was: area under curve (AUC): 0.954, sensitivity: 86%, specificity: 90%, positive predictive value (PPV): 87.8% and negative predictive value (NPV): 88.5% whereas between OC and OPMD was: AUC: 0.911, sensitivity: 90%, specificity: 82.7%, PPV: 74.2% and NPV: 89.6%. We have developed a risk probability score to predict the presence or risk of OC in OPMD patients. We established a salivary miRNA signature that can aid in diagnosing and predicting OC, revolutionising the management of patients with OPMD. Together, our results shed new light on the management of OC by salivary miRNAs to the clinical utility of using miRNAs derived from saliva samples.

Does the registry speak your language? A case study of the Global Angelman Syndrome Registry.

Global disease registries are critical to capturing common patient related information on rare illnesses, allowing patients and their families to provide information about their condition in a safe, accessible, and engaging manner that enables researchers to undertake critical research aimed at improving outcomes. Typically, English is the default language of choice for these global digital health platforms. Unfortunately, language barriers can significantly inhibit participation from non-English speaking participants. In addition, there is potential for compromises in data quality and completeness. In contrast, multinational commercial entities provide access to their websites in the local language of the country they are operating in, and often provide multiple options reflecting ethnic diversity. This paper presents a case study of how the Global Angelman Syndrome Registry (GASR) has used a novel approach to enable multiple language translations for its website. Using a "semi-automated language translation" approach, the GASR, which was originally launched in English in September 2016, is now available in several other languages. In 2020, the GASR adopted a novel approach using crowd-sourcing and machine translation tools leading to the availability of the GASR in Spanish, Traditional Chinese, Italian, and Hindi. As a result, enrolments increased by 124% percent for Spain, 67% percent for Latin America, 46% percent for Asia, 24% for Italy, and 43% for India. We describe our approach here, which we believe presents an opportunity for cost-effective and timely translations responsive to changes to the registry and helps build and maintain engagement with global disease communities.

First detection and complete genome sequence of a new potexvirus naturally infecting Adenium obesum.

Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named "Adenium obesum virus X" (AobVX), isolated from Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.

Salivary micro RNAs as biomarkers for oropharyngeal cancer.

BACKGROUND: Despite the rising incidence, particularly of the human papillomavirus (HPV)-associated fraction of oropharyngeal cancer (OPC), there are no early detection methods for OPC. Considering the close association between saliva and head and neck cancers, this study was designed to investigate salivary micro RNA (miRNAs) associated with OPC, especially focusing on HPV-positive OPC. METHODS: Saliva was collected from OPC patients at diagnosis and patients were clinically followed up ≤5 years. Salivary small RNA isolated from HPV-positive OPC patients (N = 6), and HPV-positive (N = 4) and negative controls (N = 6) were analysed by next-generation sequencing to identify dysregulated miRNAs. Discovered miRNAs were validated by quantitative PCR using two different assays in a separate cohort of patients (OPC = 91, controls = 92). The relative expression was calculated considering SNORD-96A as the normalizer. Candidate miRNAs with diagnostic and prognostic potential were evaluated by generalized logistic regression. RESULTS: A panel consisting of nine miRNAs was identified to have the best diagnostic performance to discriminate HPV-positive OPC from HPV-positive controls (AUC- validation-1 = 94.8%, validation-2 = 98%). Further, a panel consisting of six miRNAs were identified to discriminate OPC from controls regardless of the HPV status (AUC- validation-1 = 77.2%, validation-2 = 86.7%). In addition, the downregulation of hsa-miR-7-5p was significantly associated with poor overall survival of OPC patients (HR = 0.638). A panel consisting of nine miRNAs were identified for the prediction of the overall survival of the OPC patients (log-rank test-p = 0.0008). CONCLUSION: This study highlights that salivary miRNAs can play an essential role in the detection and prognostication of OPC.

Automated Proteomics Workflows for High-Throughput Library Generation and Biomarker Detection Using Data-Independent Acquisition.

Sequential window acquisition of all theoretical mass spectra-mass spectrometry underpinned by advanced bioinformatics offers a framework for comprehensive analysis of proteomes and the discovery of robust biomarkers. However, the lack of a generic sample preparation platform to tackle the heterogeneity of material collected from different sources may be a limiting factor to the broad application of this technique. We have developed universal and fully automated workflows using a robotic sample preparation platform, which enabled in-depth and reproducible proteome coverage and characterization of bovine and ovine specimens representing healthy animals and a model of myocardial infarction. High correlation (R2 = 0.85) between sheep proteomics and transcriptomics datasets validated the developments. The findings suggest that automated workflows can be employed for various clinical applications across different animal species and animal models of health and disease.

Genome architecture and diverged selection shaping pattern of genomic differentiation in wild barley.

Divergent selection of populations in contrasting environments leads to functional genomic divergence. However, the genomic architecture underlying heterogeneous genomic differentiation remains poorly understood. Here, we de novo assembled two high-quality wild barley (Hordeum spontaneum K. Koch) genomes and examined genomic differentiation and gene expression patterns under abiotic stress in two populations. These two populations had a shared ancestry and originated in close geographic proximity but experienced different selective pressures due to their contrasting micro-environments. We identified structural variants that may have played significant roles in affecting genes potentially associated with well-differentiated phenotypes such as flowering time and drought response between two wild barley genomes. Among them, a 29-bp insertion into the promoter region formed a cis-regulatory element in the HvWRKY45 gene, which may contribute to enhanced tolerance to drought. A single SNP mutation in the promoter region may influence HvCO5 expression and be putatively linked to local flowering time adaptation. We also revealed significant genomic differentiation between the two populations with ongoing gene flow. Our results indicate that SNPs and small SVs link to genetic differentiation at the gene level through local adaptation and are maintained through divergent selection. In contrast, large chromosome inversions may have shaped the heterogeneous pattern of genomic differentiation along the chromosomes by suppressing chromosome recombination and gene flow. Our research offers novel insights into the genomic basis underlying local adaptation and provides valuable resources for the genetic improvement of cultivated barley.

Identification of Candidate mRNA Isoforms for Prostate Cancer-Risk SNPs Utilizing Iso-eQTL and sQTL Methods.

Single nucleotide polymorphisms (SNPs) impacting the alternative splicing (AS) process (sQTLs) or isoform expression (iso-eQTL) are implicated as important cancer regulatory elements. To find the sQTL and iso-eQTL, we retrieved prostate cancer (PrCa) tissue RNA-seq and genotype data originating from 385 PrCa European patients from The Cancer Genome Atlas. We conducted RNA-seq analysis with isoform-based and splice event-based approaches. The MatrixEQTL was used to identify PrCa-associated sQTLs and iso-eQTLs. The overlap between sQTL and iso-eQTL with GWAS loci and those that are differentially expressed between cancer and normal tissue were identified. The cis-acting associations (FDR < 0.05) for PrCa-risk SNPs identified 42, 123, and 90 PrCa-associated cassette exons, intron retention, and mRNA isoforms belonging to 25, 95, and 83 genes, respectively; while assessment of trans-acting association (FDR < 0.05) yielded 59, 65, and 196 PrCa-associated cassette exons, intron retention and mRNA isoforms belonging to 35, 55, and 181 genes, respectively. The results suggest that functional PrCa-associated SNPs can play a role in PrCa genesis by making an important contribution to the dysregulation of AS and, consequently, impacting the expression of the mRNA isoforms.

IsomiR-eQTL: A Cancer-Specific Expression Quantitative Trait Loci Database of miRNAs and Their Isoforms.

The identification of expression quantitative trait loci (eQTL) is an important component in efforts to understand how genetic variants influence disease risk. MicroRNAs (miRNAs) are short noncoding RNA molecules capable of regulating the expression of several genes simultaneously. Recently, several novel isomers of miRNAs (isomiRs) that differ slightly in length and sequence composition compared to their canonical miRNAs have been reported. Here we present isomiR-eQTL, a user-friendly database designed to help researchers find single nucleotide polymorphisms (SNPs) that can impact miRNA (miR-eQTL) and isomiR expression (isomiR-eQTL) in 30 cancer types. The isomiR-eQTL includes a total of 152,671 miR-eQTLs and 2,390,805 isomiR-eQTLs at a false discovery rate (FDR) of 0.05. It also includes 65,733 miR-eQTLs overlapping known cancer-associated loci identified through genome-wide association studies (GWAS). To the best of our knowledge, this is the first study investigating the impact of SNPs on isomiR expression at the genome-wide level. This database may pave the way for researchers toward finding a model for personalised medicine in which miRNAs, isomiRs, and genotypes are utilised.

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants.

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21-22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Global Evolutionary History and Dynamics of Dengue Viruses Inferred from Whole Genome Sequences.

Dengue is an arboviral disease caused by dengue virus (DENV), leading to approximately 25,000 deaths/year and with over 40% of the world's population at risk. Increased international travel and trade, poorly regulated urban expansion, and warming global temperatures have expanded the geographic range and incidence of the virus in recent decades. This study used phylogenetic and selection pressure analyses to investigate trends in DENV evolution, using whole genome coding sequences from publicly available databases alongside newly sequenced isolates collected between 1963-1997 from Southeast Asia and the Pacific. Results revealed very similar phylogenetic relationships when using the envelope gene and the whole genome coding sequences. Although DENV evolution is predominantly driven by negative selection, a number of amino acid sites undergoing positive selection were found across the genome, with the majority located in the envelope and NS5 genes. Some genotypes appear to be diversifying faster than others within each serotype. The results from this research improve our understanding of DENV evolution, with implications for disease control efforts such as Wolbachia-based biocontrol and vaccine design.

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis.

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

The role of bronchoscopy in patients with SARS-CoV-2 pneumonia.

BACKGROUND: The role of bronchoscopy in coronavirus disease 2019 (COVID-19) is a matter of debate. PATIENTS AND METHODS: This observational multicentre study aimed to analyse the prognostic impact of bronchoscopic findings in a consecutive cohort of patients with suspected or confirmed COVID-19. Patients were enrolled at 17 hospitals from February to June 2020. Predictors of in-hospital mortality were assessed by multivariate logistic regression. RESULTS: A total of 1027 bronchoscopies were performed in 515 patients (age 61.5±11.2 years; 73% men), stratified into a clinical suspicion cohort (n=30) and a COVID-19 confirmed cohort (n=485). In the clinical suspicion cohort, the diagnostic yield was 36.7%. In the COVID-19 confirmed cohort, bronchoscopies were predominantly performed in the intensive care unit (n=961; 96.4%) and major indications were: difficult mechanical ventilation (43.7%), mucus plugs (39%) and persistence of radiological infiltrates (23.4%). 147 bronchoscopies were performed to rule out superinfection, and diagnostic yield was 42.9%. There were abnormalities in 91.6% of bronchoscopies, the most frequent being mucus secretions (82.4%), haematic secretions (17.7%), mucus plugs (17.6%), and diffuse mucosal hyperaemia (11.4%). The independent predictors of in-hospital mortality were: older age (OR 1.06; p<0.001), mucus plugs as indication for bronchoscopy (OR 1.60; p=0.041), absence of mucosal hyperaemia (OR 0.49; p=0.041) and the presence of haematic secretions (OR 1.79; p=0.032). CONCLUSION: Bronchoscopy may be indicated in carefully selected patients with COVID-19 to rule out superinfection and solve complications related to mechanical ventilation. The presence of haematic secretions in the distal bronchial tract may be considered a poor prognostic feature in COVID-19.

Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when 'exosome-depleted serum' is utilised.

Extracellular vesicles (EVs) are important intercellular communication messengers. Half of the published studies in the field are in vitro cell culture based in which bovine serum in various concentrations and forms is used to facilitate the production of extracellular vesicles. 'Exosome depleted serum' is the type of bovine serum most widely used in the production of human EVs. Herein, we demonstrate that, despite the initial caution raised in 2014 about the persistence of bovine EVs, 'exosome depleted serum' was still used in 46% of publications on human or rodent EVs between 2015 and 2019. Using nanoparticle tracking analysis combined with detergent lysis of vesicles as well as bovine CD9 ELISA, we show that there were approximately 5.33 x 107/mL of bovine EVs remaining in the 'exosome depleted serum'. Importantly, the 'exosome depleted serum' was relatively enriched in small EVs by approximately 2.7-fold relative to the large EVs compared to that in the original serum. Specifically, the percentage of small EVs in total vesicles had increased from the original 48% in the serum before ultracentrifugation to 92% in the 'exosome depleted serum'. Furthermore, the pervasive bovine EVs carried over by the 'exosome depleted serum', even when the lowest concentration (0.5%) was used in cell culture, resulted in a significant contamination of human EVs in cell culture conditioned medium. Our findings indicate that the use 'exosome depleted serum' in cell culture-based studies may introduce artefacts into research examining the function of human and rodent EVs, in particular those involving EV miRNA. Thus, we appeal to the researchers in the EV field to seriously reconsider the practice of using 'exosome depleted serum' in the production of human and other mammalian EVs in vitro.

DNA damage contributes to transcriptional and immunological dysregulation of testicular cells during Chlamydia infection.

Chlamydia is the most commonly reported sexually transmitted bacterial infection, with 127 million notifications worldwide each year. Both males and females are susceptible to the pathological impacts on fertility that Chlamydia infections can induce. However, male chlamydial infections, particularly within the upper reproductive tract, including the testis, are not well characterized. In this study, using mouse testicular cell lines, we examined the impact of infection on testicular cell lineage transcriptomes and potential mechanisms for this impact. The somatic cell lineages exhibited significantly fragmented genomes during infection. Likely resulting from this, each of the Leydig, Sertoli and germ cell lineages experienced extensive transcriptional dysregulation, leading to significant changes in cellular biological pathways, including interferon and germ-Sertoli cell signalling. The cell lineages, as well as isolated spermatozoa from infected mice, also contained globally hypomethylated DNA. Cumulatively, the DNA damage and epigenetic-mediated transcriptional dysregulation observed within testicular cells during chlamydial infection could result in the production of spermatozoa with abnormal epigenomes, resulting in previously observed subfertility in infected animals and congenital defects in their offspring.

Temperature modulates immune gene expression in mosquitoes during arbovirus infection.

The principal vector of dengue, Zika and chikungunya viruses is the mosquito Aedes aegypti, with its ability to transmit pathogens influenced by ambient temperature. We use chikungunya virus (CHIKV) to understand how the mosquito transcriptome responds to arbovirus infection at different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18, 28 and 32°C, and found that higher temperature correlated with higher virus levels, particularly at 3 days post infection, but lower temperature resulted in reduced virus levels. RNAseq analysis indicated significantly altered gene expression levels in CHIKV infection. The highest number of significantly differentially expressed genes was observed at 28°C, with a more muted effect at the other temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2, was not substantially altered in response to CHIKV. The upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28°C. Functional annotations suggested that genes in immune response and metabolic pathways related to energy supply and DNA replication were involved in temperature-dependent changes. Time post infection also led to substantially different gene expression profiles, and this varied with temperature. In conclusion, temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher temperatures.

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.