A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of theAFGPgenomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essentialcispromoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadidAFGPoriginated from entirely nongenic DNA. The gadidAFGPevolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.

Myocardial injury is one of the main symptoms of sepsis. However, the mechanisms underlying sepsis-induced myocardial dysfunction remain unclear. In the present study, the concentration of cardiac troponin T (CTnT) in serum was measured using an enzyme-linked immunosorbent assay kit. The levels of interleukin (IL)-1β and IL-18 were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and the level of malondialdehyde (MDA) was determined using a corresponding kit. Myocardial pathology was analyzed via hematoxylin and eosin staining. RT-qPCR analysis and western blotting and/or immunohistochemistry were used to quantify the expression levels of thioredoxin-interacting protein (TNXIP), NOD-like receptor pyrin domain containing 3 (NLRP3), cleaved caspase-1, caspase-1, catalase and manganese-superoxide dismutase (MnSOD). The viability of cells was determined using a cell counting kit-8. Apoptosis and reactive oxygen species (ROS) were examined using flow cytometry. Models of sepsis-induced myocardial injury were successfully established; evidence included increases in the levels of CTnT, IL-1β, IL-18 and MDA and myocardial tissue damage in vivo , and decreased cell viability and improvements in IL-1β and IL-18 in vitro . The levels of TXNIP, NLRP3 and cleaved caspase-1 were upregulated in the sepsis models. Small interfering RNA targeting TNXNIP (siTXNIP) increased cell viability, reduced the apoptotic rate and attenuated the release of IL-1β and IL-18. The levels of TXNIP, NLRP3 and cleaved caspase-1 and production of ROS were suppressed by siTXNIP, accompanied by increases in catalase and MnSOD. TXNIP/NLRP3 serves an important role in the development of sepsis-induced myocardial damage.

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant ( Camellia sinensis ), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762 bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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