circMORC3-encoded novel protein negatively regulates antiviral immunity through synergizing with host gene MORC3.

The protein-coding ability of circRNAs has recently been a hot topic, but the role of protein-coding circRNAs in antiviral innate immunity of teleost fish has rarely been reported. Here, we identified a novel circRNA, termed circMORC3, derived from Microrchidia 3 (MORC3) gene in Miichthys miiuy. circMORC3 can inhibit the expression of antiviral cytokines. In addition, circMORC3 encodes a novel peptide with a length of 84 amino acids termed MORC3-84aa. MORC3-84aa not only significantly inhibited TRIF-mediated activation of IRF3 and NF-κB signaling pathways, but also effectively suppressed the expression of antiviral cytokines triggered by RNA virus Siniperca chuatsi rhabdovirus (SCRV). We found that MORC3-84aa directly interacted with TRIF and negatively regulated TRIF protein expression. In addition, host gene MORC3 attenuates SCRV-induced IFN and ISG expression. Mechanistically, MORC3-84aa promotes autophagic degradation of TRIF by enhancing K6-linked ubiquitination and inhibits TRIF-mediated activation of the type I interferon signaling pathway. And the host gene MORC3 not only repressed IRF3 protein expression but also inhibited IRF3 phosphorylation levels. Our study shows that circMORC3 and host gene MORC3 played a synergistic role in viral immune escape.

CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape.

It is known that about 10 circular RNAs (circRNAs) can encode functional polypeptides in higher mammals. However, it is not clear whether the functional polypeptides that can be translated by circRNAs are only the products of the evolution of higher animals, or also widely exist in other lower organisms. In addition, it is also unclear whether the two ways of translating polypeptides using IRES and m6A in the one circRNA are exclusive or coexistent. Here, we discovered a novel circRNA derived from the 3'-5' RNA helicase Ythdc2 (Ythdc2) gene in lower vertebrate fish, namely circYthdc2, which can translate into a 170 amino acid polypeptide (Ythdc2-170aa) through IRES sequence or m6A modification, and is involved in antiviral immune of fish. Moreover, SCRV infection can promote circYthdc2 translate Ythdc2-170aa. Then, we found that both Ythdc2-170aa and Ythdc2 can promote the degradation of STING by promoting the ubiquitination modification of K11 and K48 link of STING, and weaken the host's antiviral innate immunity. Notably, when circYthdc2 is abundant, Ythdc2 preferentially degrades circYthdc2 and no longer promotes the degradation of STING. Further studies have shown that circYthdc2 is highly conserved from lower vertebrates to higher mammals, and human circYthdc2 can also encode the same polypeptide and play a similar function to that of fish circYthdc2. This discovery confirms for the first time that the ability of circRNA to encode functional proteins is evolutionarily conserved, and finds that the ways of polypeptide translation by the same circRNA were diverse, which is of great significance for further elucidating the function and evolution of circRNAs in vertebrates.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

IMPORTANCE: The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

The comparative studies of complete chloroplast genomes in Actinidia (Actinidiaceae): novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships.

The genus Actinidia, also called kiwifruit, is characterized with abundant balanced nutritional metabolites, including exceptionally high vitamin C content. However, the traditional classification could not fully reflect the actual Actinidia species' relationships, which need further revision through more accurate approaches. Compared to the nuclear genome, the chloroplast genome has simple heredity characteristics, conserved genome structure and small size, suitable for deciphering complicated species' phylogenetic relationships. Here, the genome-wide comprehensive comparative analyses were performed over 29 independent chloroplast genomes' sequences derived from 25 Actinidia taxa. The average genome size is 156,673.38 bp, with an average 37.20% GC content. The long repeat sequences rather than SSRs (simple sequence repeats) in Actinidia were revealed to be the causal agent leading to the chloroplast genome size expansion. The clpP gene sequences with exon merge and intron deletion were annotated in all the 29 chloroplast genomes tested, which has been previously reported to be lost in Actinidia species. Comprehensive sequence analyses indicated the distinct variation at the clpP gene locus was Actinidiaceae-specific, emerging after the Actinidiaceae-other Ericales species divergence. Four highly divergent sequences (i.e., rps16 ~ trnQ-UUG, rps4 ~ trnT-UGU, petA ~ psbJ, and rps12 ~ psbB) evolved in the LSC (large single-copy) and SSC (small single-copy) regions embodying rps12 ~ psbB (including clpP gene and its up/downstream noncoding sequence) were identified as variation hot spots in Actinidia species. Based on either LSC region alone, combined sequences of LSC and SSC or the whole chloroplast genome sequences, three identical phylogenetic trees of the 25 Actinidia taxa with relatively improved resolution were reconstructed, consistently supporting the reticulate evolutionary lineage in Actinidia. Our findings could help to better understand the evolution characteristics of chloroplast genomes and phylogenetic relationships among Actinidia species.

Circular RNA circRara promote the innate immune responses in miiuy croaker, Miichthys miiuy.

With the in-depth study of circRNA, more and more biological studies have shown that circRNAs play an important role in mammals, such as cell proliferation, apoptosis, invasion, development and disease state. However, the regulatory mechanism of circRNA in lower vertebrates remains unclear. Here, we found a new circular RNA and named it circRara. We carried out the experimental study on its antiviral and antibacterial response, cell proliferation and activity. The results showed that circRara had a positive regulatory effect on the antiviral and antibacterial response, cell proliferation and activity in miiuy croaker. First, we found that the expression of circRara could be up-regulated under the stimulation of LPS and poly (I: C), but not the expression of linear Rara. In addition, the increase of circRara can increase the production of inflammatory factors and antiviral genes, which was confirmed by double luciferase reporter gene experiment and qPCR. These results will help to further understand the immunomodulatory mechanism of circRNA in teleost fish.

First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China.

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 µm in size (average diameter = 18.22 ± 1.00 µm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, ß-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 µl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 µl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days' inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

Enhancing anti-tumor therapy with agmatine-cholesterol conjugate liposomes: in vitro and in vivo evidence.

In this study, we synthesized a novel compound, agmatine-cholesterol conjugate (AG-Chol), to enhance the anti-tumor activity of drug-loaded liposomes. We replaced cholesterol with AG-Chol in preparing doxorubicin hydrochloride (DOX) liposomes by using an active loading method for DOX. We assessed the physical and chemical properties of the resulting AG-Liposomes and evaluated their efficacy in vitro and in vivo. The results showed that AG-Liposomes were stable with high encapsulation efficiency. Compared with the control liposomes, AG-Liposomes exhibited a slower drug release rate in the release medium at pH 6.8. The in vitro cell experiments demonstrated that AG-Liposomes had higher tumor cell uptake rate, stronger migration inhibition rate, higher apoptosis rate, better anti-clonogenic ability, and higher lysosome escape ability than the control liposomes. In vivo distribution results demonstrate that liposomes prepared with AG-Chol instead of cholesterol can significantly enhance their tumor targeting abilities and reduce their distribution to non-targeted sites. In vivo tumor suppression experiments showed that AG-Liposomes had a higher tumor suppression rate than the control liposomes without causing apparent toxicity to normal tissues, as evidenced by histological staining. Therefore, substituting cholesterol with AG-Chol in the preparation of liposomes can result in enhanced lysosome escape, improved tumor targeting, and increased efficacy of anti-tumor drugs.

CircMIB2 therapy can effectively treat pathogenic infection by encoding a novel protein.

The mRNA therapy is widely used in the treatment of diseases due to its efficient characteristics, and the COVID-19 vaccine is the application of mRNA therapy. However, due to the instability of mRNA, mRNA vaccines often need lots of modifications to ensure its stability. Recent research shows that circRNA with stable RNA structure can encode protein, which provides a new direction for mRNA therapy. Here, we discovered a novel circRNA (circMIB2) derived from E3 ubiquitin-protein ligase MIB2 (MIB2) gene in lower vertebrate fish, which can translate into a 134 amino acid protein (MIB2-134aa) through m6A modification, and is involved in innate immunity. MIB2-134aa is completely consistent with the amino acid sequence of the two domains of host gene MIB2 protein; host gene MIB2 can target TRAF6 through the two domains and inhibit the innate immune response by promoting the ubiquitination degradation of the K11-link of TRAF6, MIB2-134aa also targets TRAF6 through these same domains. Interestingly, MIB2-134aa greatly reduced the degradation of TRAF6 by its host gene MIB2. More importantly, we found that circRNA therapy of circMIB2 can significantly inhibit the colonization of Vibrio anguillarum in zebrafish, and it provides a new direction for the treatment of pathogenic diseases of fish.

Magnolol-loaded cholesteryl biguanide conjugate hydrochloride nanoparticles for triple-negative breast cancer therapy.

The potential of combination therapy using nanoparticle delivery systems in improving triple-negative breast cancer treatment efficacy remains to be explored. Here, we report a novel nanoparticle system using a cholesterol biguanide conjugate hydrochloride (CBH) as both a drug and carrier to load magnolol (MAG). Poly(ethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) and aminoethyl anisamide-poly(ethylene glycol)-poly(lactic-co-glycolic acid) (AEAA-PEG-PLGA) were added to form nanoparticles. Nanoparticles accumulated most in tumor tissues when the weight ratio of AEAA-PEG-PLGA to mPEG-PLGA was 4:1. MAG and CBH exerted a synergistic inhibitory effect on 4 T1 cells. An in vitro study showed that nanoparticles displayed the highest tumor cell uptake rate, highest apoptosis rate, and strongest inhibitory effect on tumor cell migration and monoclonal formation. CBH might promote nanoparticle uptake by cells and lysosomal escape. After intravenous administration to mice with 4 T1 breast tumors in situ, the nanoparticles inhibited tumor growth without obvious toxicity. Western blot results showed that nanoparticles altered the levels of p53, p-AKT, and p-AMPK in the tumor tissue. Moreover, cell apoptosis was found in the same area of H&E-stained and TUNEL-stained tumors treated with the nanoparticles. Collectively, this nanoparticle system provides a novel combination drug delivery strategy for treating triple-negative breast cancer.

[The influence of occupational exposure and smoking on lung ventilation of coke oven workers].

OBJECTIVE: To study the influence of occupational exposure and smoking on lung ventilation function of coke oven workers. METHODS: Environmental monitoring was performed on the top, side and bottom of some coke ovens. Lung ventilation function test was performed in 234 coke oven workers. RESULTS: The poison concentration in environment had such tendency as it was the highest on the top, then the side, and the lowest at the bottom. The standardized forced expiratory volume in the 1st second (FEV(1), 90.8% +/- 8.6%) and forced expiratory rate (FEV(1)%, 95.4% +/- 12.4%) of those coke oven workers who smoked were significantly lower than those non-smoking workers (100.9% +/- 14.3%, 108.9% +/- 17.6%); among those smoking workers, the partial correlation coefficients between forced vital capacity (FVC) and benzene soluble, benzopyrene, and smoking index were -0.249, -0.187 and -0.368 respectively; and the coefficients between FEV(1) and the three aspects were -0.255, -0.191 and -0.388; and the coefficients between FEV(1)% and them were -0.131, -0.107 and -0.065. Among those non-smoking workers, the coefficients between benzene solubles and FVC, FEV(1) and FEV(1)% were -0.154, -0.052 and -0.176, and between benzopyrene and them were -0.121, -0.037 and -0.159. CONCLUSION: The lung ventilation function of coke oven workers has certain negative correlation with both occupational exposure and smoking.