BackgroundSugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.MethodsWe generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.ResultsA total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.DiscussionThe new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

Background: Hepatitis B is a liver infection disease caused by the Hepatitis B Virus (HBV) that can become chronic and develop into hepatocellular carcinoma. HBV was classified as a double-stranded DNA virus. Currently, there is a report showing that HBV virus-encoded miRNA called HBV-miR-3 controls the replication of HBV. However, the regulation of HBV-miR-3 in host cells remains unclear. Objective: This study aimed to investigate the regulation of HBV-miR-3 in host gene target which is related to chronic HBV infection and HCC process. Methods: In this study, we analyzed the read count of HBV-miR-3 from next-generation sequencing of chronic hepatitis patients in Pegylated interferon alpha-2a (PEG-IFN-α-2a) treatment. To understand the regulation of HBV-miR-3 in host cells, the HBV-miR-3 recognition sites were predicted in host target genes using miRDB. The effect of HBV-miR-3 in host cells was examined using qPCR and 3′ UTR dual luciferase assay. Results: The read count of HBV-miR-3 was found in chronic hepatitis patients before treatment. Moreover, the decrease of HBV-miR-3 was correlated with response group of chronic hepatitis patients after treatment. On the other hand, the abundance of HBV-miR-3 showed no difference in nonresponse group of chronic patients after PEG-IFN-α-2a treatment. To study the role of HBV-miR-3 in patients, four HBV-miR-3 target regions from Protein phosphatase 1A (PPM1A) and DIX domain containing 1 (DIXDC1) were identified in the human genome using miRDB. Interestingly, we found that HBV-miR-3 hybridized with PPM1A mRNA. The mRNA expression from RT-qPCR showed no difference between HepG2 transfected with pSilencer_scramble or pSilencer_HBV-miR-3. However, the reporter assay showed that PPM1A mRNA was suppressed by HBV-miR-3. The protein expression of PPM1A showed a decrease in cells overexpressing HBV-miR-3. Finally, the HBV-miR-3 can promote cell proliferation in cells overexpressing HBV-miR-3. Conclusion: This study is the first report showed the HBV encoded miRNA can regulate host gene expression. HBV-miR-3 silenced PPM1A by inhibiting the translation process of PPM1A. The downregulation of PPM1A promotes cell proliferation related to HCC development.

Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili.

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7−/ − MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7−/ − MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7−/ − MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7−/ − MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7−/ − MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7−/ − MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7−/ − MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.