Animal influenza viruses continue to pose a threat to human public health. The Eurasian avian-like H1N1 (EA H1N1) viruses are widespread in pigs throughout Europe and China and have caused human infections in several countries, indicating their pandemic potential. To carefully monitor the evolution of the EA H1N1 viruses in nature, we collected nasal swabs from 103,110 pigs in 22 provinces in China between October 2013 and December 2019, and isolated 855 EA H1N1 viruses. Genomic analysis of 319 representative viruses revealed that these EA H1N1 viruses formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, and two of these genotypes (G4 and G5) were widely distributed in pigs. Animal studies indicated that some strains have become highly pathogenic in mice and highly transmissible in ferrets via respiratory droplets. Moreover, two-thirds of the EA H1N1 viruses reacted poorly with ferret serum antibodies induced by the currently used H1N1 human influenza vaccine, suggesting that existing immunity may not prevent the transmission of the EA H1N1 viruses in humans. Our study reveals the evolution and pandemic potential of EA H1N1 viruses and provides important insights for future pandemic preparedness.

2'-O-methylation (Nm) is one of the most abundant RNA epigenetic modifications and plays a vital role in the post-transcriptional regulation of gene expression. Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in mRNAs or relatively rare non-coding RNAs (ncRNAs). Here, we developed a new method for enriching Nm sites by using RNA exoribonuclease and periodate oxidation reactivity to eliminate 2'-hydroxylated (2'-OH) nucleosides, coupled with sequencing (Nm-REP-seq). We revealed several novel classes of Nm-containing ncRNAs as well as mRNAs in humans, mice, and drosophila. We found that some novel Nm sites are present at fixed positions in different tRNAs and are potential substrates of fibrillarin (FBL) methyltransferase mediated by snoRNAs. Importantly, we discovered, for the first time, that Nm located at the 3'-end of various types of ncRNAs and fragments derived from them. Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods. Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution. Using this quantitative method, we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format. The strengths of identified promoters covered a 37-fold range by flow cytometry. PzipA and PsltA were identified as the strongest promoters, which were 2.9- and 1.5-fold higher than that of the commonly used constitutive promoter PgpdA. Thus, we applied PzipA and PsltA to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A. nidulans. The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives, namely, fumiganins A and B. Our method provides an innovative strategy for natural product discovery in fungi.

DNMT1 is a DNA methyltransferase that catalyzes and maintains methylation in CpG dinucleotides. It blocks the entrance of DNA into the catalytic pocket via the replication foci targeting sequence (RFTS) domain. Recent studies have shown that an H3-tail-conjugated two-mono-ubiquitin mark (H3Ub2) activates DNMT1 by binding to the RFTS domain. However, the activation mechanism of DNMT1 remains unclear. In this work, we combine various sampling methods of extensive simulations, including conventional molecular dynamics, Gaussian-accelerated molecular dynamics, and coarse-grained molecular dynamics, to elucidate the activation mechanism of DNMT1. Geometric and energy analyses show that binding of H3Ub2 to the RFTS domain of DNMT1 results in the bending of the α4-helix in the RFTS domain at approximately 30°-35°, and the RFTS domain rotates ∼20° anti-clockwise and moves ∼3 Å away from the target recognition domain (TRD). The hydrogen-bonding network at the RFTS-TRD interface is significantly disrupted, implying that the RFTS domain is dissociated from the catalytic core, which contributes to activating the auto-inhibited conformation of DNMT1. These results provide structural and dynamic evidence for the role of H3Ub2 in regulating the catalytic activity of DNMT1.

The effects and regulation of Beclin-1-an autophagy-related protein-have not been fully defined, however, a negative correlation has been reported between Beclin-1 expression and carcinogenesis. Meanwhile, no compound has been shown to directly inhibit its activity. Here, we evaluate piceatannol, a naturally occurring polyphenolic compound, as a potential targeting agonist of Beclin-1, to assess its efficacy as an antitumor agent against gastric cancer. More specifically, we determine the effects of piceatannol treatment on cell viability using a monitoring system and colony forming assay. Piceatannol was found to efficiently inhibit the proliferation of several human gastric cancer cell lines. Autophagic flux is increased by piceatannol treatment, and correlates with inhibition of cell proliferation and colony formation. Additionally, microscale thermophoresis and surface plasmon resonance results show a direct interaction between piceatannol and Beclin-1, which reduces the phosphorylation activity of Beclin-1 at the Ser-295 site. Notably, piceatannol impairs the binding of Beclin-1 to Bcl-2 and enhances the recruitment of binding of UV radiation resistance-associated gene protein, which further triggers Beclin-1-dependent autophagy signaling. An increase in autophagic activity via treatment with the mTOR inhibitor, everolimus, effectively sensitizes piceatannol-induced antitumor effects. Xenograft models confirmed that piceatannol inhibits tumor development and elicits a potent synergistic effect with everolimus in vivo. Taken together, the findings of this study strongly support the application of combinatorial piceatannol and everolimus therapy in future clinical trials for gastric cancer patients.

CD19 chimeric antigen receptor (CAR) T cells have shown robust efficacy in relapsed and refractory acute lymphoblastic leukemia (R/R ALL), but compromising result in chronic lymphoblastic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). CD19 relapse and the lack of CAR-T cell persistence which result in treatment failure are considerable obstacles to overcome. CAR-T targeting CD20 is an option for salvaging CD19 CAR-T failure. Previous studies have established variant structures of bispecific CAR-T which could avoid antigen-loss and immune escape. Here, we constructed tandem and loop CAR structures targeting both CD19 and CD20 antigen. Bispecific CAR-T cells could eliminate either CD19 or CD20 negative lymphoma cells, suggesting they exhibited dual antigen targeting of CD19 and CD20. By comparing the efficiency of four bispecific CAR modified T cells, it was found that loop2019 CAR was the best structure among them to eradicate lymphoma cell lines and patients' primary lymphoma or CLL cells in a very low dose in vitro and prolong the survival time dramatically in lymphoma xenograft mice model. These data highlighted the potential of loop2019 CAR-T in clinical treatment.

Phased small interfering RNAs (phasiRNAs) are abundantly expressed in anthers and linked to environment-related male fertility in grasses, yet how they function under different environmental conditions remains unclear. Here, we identified a rice (Oryza sativa) low temperature-induced Argonaute (AGO) protein, OsAGO1d, that is responsible for generating phasiRNAs and preserving male fertility at low temperature. Loss of OsAGO1d function causes low-temperature male sterility associated with delayed programmed cell death of tapetal cells during anther development. OsAGO1d binds miR2118 and miR2275 family members and triggers phasiRNA biogenesis; it also binds 21-nt phasiRNAs with a 5' terminal U. In total, phasiRNAs from 972 loci are OsAGO1d-dependent. OsAGO1d protein moves from anther wall cells into meiocytes, where it loads miR2275 to produce 24-nt phasiRNAs. Together, our results show that OsAGO1d acts as a mobile signal to fine-tune phasiRNA production and this function is important for male fertility at low temperature.