Indole alkaloids isolated from the Nicotiana tabacum-derived Aspergillus fumigatus 0338 as potential inhibitors for tobacco powdery mildew and their mode of actions.

To explore active natural products against tobacco powdery mildew caused by Golovinomyces cichoracearum, an extract from the fermentation of endophytic Aspergillus fumigatus 0338 was investigated. The mechanisms of action for active compounds were also studied in detail. As a result, 14 indole alkaloid derivatives were isolated, with seven being newly discovered (1-7) and the remaining seven previously described (8-14). Notably, compounds 1-3 are rare linearly fused 6/6/5 tricyclic prenylated indole alkaloids, with asperversiamide J being the only known natural product of this kind. The isopentenyl substitutions at the 5-position in compounds 4 and 5 are also rare, with only compounds 1-(5-prenyl-1H-indol-3-yl)-propan-2-one (8) and 1-(6-methoxy-5-prenyl-1H-indol3-yl)-propan-2-one currently available. In addition, compounds 6 and 7 are new framework indole alkaloid derivatives bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. The purified compounds were evaluated for their activity against G. cichoracearum, and the results revealed that compounds 7 and 9 demonstrated obvious anti-G. cichoracearum activities with an inhibition rate of 82.6% and 85.2%, respectively, at a concentration of 250 µg/mL, these rates were better than that of the positive control agent, carbendazim (78.6%). The protective and curative effects of compounds 7 and 9 were also better than that of positive control, at the same concentration. Moreover, the mechanistic study showed that treatment with compound 9 significantly increased the structural tightness of tobacco leaves and directly affect the conidiospores of G. cichoracearum, thereby enhancing resistance. Compounds 7 and 9 could also induce systemic acquired resistance (SAR), directly regulating the expression of defense enzymes, defense genes, and plant semaphorins, which may further contribute to increased plant resistance. Based on the activity experiments and molecular dockings, the indole core structure may be the foundation of these compounds' anti-G. cichoracearum activity. Among them, the indole derivative parent structures of compounds 6, 7, and 9 exhibit strong effects. Moreover, the methoxy substitution in compound 7 can enhance their activity. By isolating and structurally identifying the above indole alkaloids, new candidates for anti-powdery mildew chemical screening were discovered, which could enhance the utilization of N. tabacum-derived fungi in pesticide development.

Extraction and characterization of anti-virus anthraquinones from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220.

In this study, seven novel anthraquinones (1-7) and four described anthraquinones (8-11) were purified from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220. It is worth noting that only analogs of 4 and 5 have been reported as natural products to date, while the nuclei of compounds 1-3, 6 and 7 were isolated for the first time in nature. Among them, compounds 1-3 bear an unusual anthra[2,3-b]furan-9,10-dione nucleus, 4 and 5 possess a rare 3-methyl-1H-pyrrol-2-yl substituent, and 6 and 7 are new framework anthraquinones bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. Interestingly, the in vivo assays indicated that 1, 4 and 5 had inactivation effects against tobacco mosaic virus (TMV) with inhibition rates of 41.6%, 55.4% and 38.6%, respectively, at a concentration of 50 µg/mL, which were better than that of the positive control agent, ningnanmycin (33.8%). Compounds 1, 4 and 5 also had protective effects with inhibition rates of 48.7%, 60.2% and 43.5% at the same concentration, while 4 had a better curative effect than ningnanmycin at a concentration of 100 µg/mL. In addition, mechanistic studies also revealed that a potent direct effect on TMV, the induction of SAR in tobacco plants, and the effective regulation of defense enzymes, defense genes, and defense hormones may be the reasons for the significant effects of 4 against TMV. At the same time, downregulation of the expression of total NtHsp70 protein by inhibiting the related Hsp70 genes may also be involved in tobacco resistance to TMV. To evaluate whether compounds have broader antiviral activities, the antirotavirus activities of new isolates were also evaluated and found to be highly effective with a therapeutic index (TI) value ranging from 11.6 to 17.7. This study suggests that the above anthraquinone compounds, particularly 4, have broad spectrum antiviral activities. The successful isolation and structure identification of the above anthraquinones provide new materials for the screening of anti-TMV agents and contribute to the improved utilization of N. tabacum-derived fungi.

Development of a sandwich ELISA for the detection of Chinese sacbrood virus infection.

Chinese sacbrood disease (CSBD) is a highly pathogenic infectious disease in bees that is caused by Chinese sacbrood virus (CSBV). Although several molecular detection methods have been developed for CSBV, there are no commercially available enzyme-linked immunosorbent assay (ELISA) kits. We therefore developed a sandwich ELISA to detect CSBV antigens. To this end, monoclonal antibodies were produced using VP2 as an immunogen and subsequently characterized. Hybridomas were screened for the secretion of immunoglobulin G (IgG). Using an unlabeled monoclonal antibody (mAb) for coating and a horseradish peroxidase (HRP)-labeled mAb for detection, a CSBV sandwich ELISA method was established. This method showed specificity for CSBV and did not show cross-reactivity with other bee viruses. The detection limit of the sandwich ELISA was 3.675 × 104 copies/µL. Sixty bee larvae were tested using our sandwich ELISA method, and the presence of CSBV was verified by reverse transcription polymerase chain reaction (RT-PCR). The total coincidence rate was 90%. Thus, a sandwich ELISA method with high specificity and accuracy and a detection limit of 3.675 × 104 copies/µL has been successfully developed and can be used for the clinical detection of CSBV. This method will support rapid diagnosis, real-time monitoring, and early warning of CSBD.

Establishment of a reverse transcription-recombinase polymerase amplification-lateral flow dipstick method for the dual detection of Israeli acute paralysis virus and chronic bee paralysis virus.

Introduction: As an important social insect, honey bees play crucial roles in agricultural production, sustainable development of agricultural production, and the balance of the natural environment. However, in recent years, Israeli acute paralysis virus (IAPV) and chronic bee paralysis virus (CBPV), the main pathogens of bee paralysis, have continuously harmed bee colonies and caused certain losses to the beekeeping industry. Some beekeeping farms are located in wild or remote mountainous areas, and samples from these farms cannot be sent to the laboratory for testing in a timely manner, thereby limiting the accurate and rapid diagnosis of the disease. Methods and results: In this study, we used a reverse transcription-recombinase polymerase amplification-lateral flow dipstick (RT-RPA-LFD) method for the dual detection of IAPV and CBPV. RPA primers and LFD detection probes were designed separately for their conserved genes. Primers and probes were screened, and the forward and reverse primer ratios, reaction times, and temperatures were optimized. According to the results of the optimization tests, the optimal reaction temperature for RT-RPA is 37°C, and when combined with LFD, detection with the naked eye requires <20 min. The developed RPA-LFD method specifically targets IAPV and CBPV and has no cross-reactivity with other common bee viruses. In addition, the minimum detection limit of the RT-RPA-LFD method is 101 copies/µL. Conclusion: Based this study, this method is suitable for the detection of clinical samples and can be used for field detection of IAPV and CBPV.

Interaction between the VP2 protein of deformed wing virus and host snapin protein and its effect on viral replication.

Introduction: Deformed wing virus (DWV) is one of the causative agents of colony collapse disorder. The structural protein of DWV plays a vital role in the process of viral invasion and host infection; however, there is limited research on DWV. Methods and Results: In this study, we screened the host protein snapin, which can interact with the VP2 protein of DWV, using the yeast two-hybrid system. Through computer simulation and GST pull-down and CO-IP assays, an interaction between snapin and VP2 was confirmed. Furthermore, immunofluorescence and co-localization experiments revealed that VP2 and snapin primarily co-localized in the cytoplasm. Consequently, RNAi was used to interfere with the expression of snapin in worker bees to examine the replication of DWV after the interference. After silencing of snapin, the replication of DWV in worker bees was significantly downregulated. Hence, we speculated that snapin was associated with DWV infection and involved in at least one stage of the viral life cycle. Finally, we used an online server to predict the interaction domains between VP2 and snapin, and the results indicate that the interaction domain of VP2 was approximately located at 56-90, 136-145, 184-190, and 239-242 aa and the snapin interaction domain was approximately located at 31-54 and 115-136 aa. Conclusion: This research confirmed that DWV VP2 protein could interacts with the snapin of host protein, which provides a theoretical basis for further investigation of its pathogenesis and development of targeted therapeutic drugs.

Establishment and Application of CRISPR-Cas12a-Based Recombinase Polymerase Amplification and a Lateral Flow Dipstick and Fluorescence for the Detection and Distinction of Deformed Wing Virus Types A and B.

Deformed wing virus (DWV) is one of the important pathogens of the honey bee (Apis mellifera), which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 12a fluorescence system (RPA-CRISPR-Cas12a-LFD). The limit of detection of this system was ~6.5 × 100 and 6.2 × 101 copies/µL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription-quantitative polymerase chain reaction, and the RPA-CRISPR-Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks.

Stepwise Crystalline Structural Transformation in 0D Hybrid Antimony Halides with Triplet Turn-on and Color-Adjustable Luminescence Switching.

Intelligent stimuli-responsive fluorescence materials are extremely pivotal for fabricating luminescent turn-on switching in solid-state photonic integration technology, but it remains a challenging objective for typical 3-dimensional (3D) perovskite nanocrystals. Herein, by fine-tuning the accumulation modes of metal halide components to dynamically control the carrier characteristics, a novel triple-mode photoluminescence (PL) switching was realized in 0D metal halide through stepwise single-crystal to single-crystal (SC-SC) transformation. Specifically, a family of 0D hybrid antimony halides was designed to exhibit three distinct types of PL performance including nonluminescent [Ph3EtP]2Sb2Cl8 (1), yellow-emissive [Ph3EtP]2SbCl5·EtOH (2), and red-emissive [Ph3EtP]2SbCl5 (3). Upon stimulus of ethanol, 1 was successfully converted to 2 through SC-SC transformation with enhanced PL quantum yield from ~0% to 91.50% acting as "turn-on" luminescent switching. Meanwhile, reversible SC-SC and luminescence transformation between 2 and 3 can be also achieved in the ethanol impregnation-heating process as luminescence vapochromism switching. As a consequence, a new triple-model turn-on and color-adjustable luminescent switching of off-onI-onII was realized in 0D hybrid halides. Simultaneously, wide advanced applications were also achieved in anti-counterfeiting, information security, and optical logic gates. This novel photon engineering strategy is expected to deepen the understanding of dynamic PL switching mechanism and guide development of new smart luminescence materials in cutting-edge optical switchable device.

Three new chromones from the whole plants of Chamaecrista rotundifolia and their anti-rotavirus activities.

Chamaecrista rotundifolia (C. rotundifolia) is a perennial herb of leguminosae, which increasingly being grown as a forage in China. In our search for original bioactive metabolites from Cassia plants, the phytochemical reinvestigation of the C. rotundifolia was carried out, which led to the isolation of three new (1-3) and six known (4-9) chromones. Their structures were confirmed by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1-9 were evaluated for their anti-rotavirus activities, and the results revealed that compounds 1-9 exhibited potential anti-rotavirus activities with therapeutic index (TI) valves in the range of 12.0 ∼ 20.2, respectively.

Three new isoquinoline alkaloids from the fermentation of Aspergillus sp. 0338 and their anti-MRSA activities.

There is growing evidence that bioactive substances produced by microbial endophytes have applicability in medicine, agriculture and industry. To enrich the bioactive substances, in our search for new bioactive metabolites from fungi Aspergillus, the phytochemical reinvestigation on the Aspergillus sp. 0338 was carried out, and this led to the isolation of three new (1-3) and five known alkaloids (4-8). Their structures were elucidated by spectroscopic analysis, including extensive 1D and 2D NMR techniques, as well as comparison with literature values. Additionally, compounds 1-3 were evaluated for their anti-MRSA activities. The results revealed that compounds 1-3 exhibited good inhibitions with IZD of 15.2 ± 1.8, 14.6 ± 2.0, and 13.4 ± 2.2 mm, respectively.

Real-time reverse transcription recombinase polymerase amplification for rapid detection of murine hepatitis virus.

Murine hepatitis virus (MHV) is a highly infectious murine coronavirus that has a high potential for causing harm to host animals. This study aimed to develop a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for rapid detection of MHV in laboratory mice. Methods: Specific primers and probes for RT-RPA assay were designed targeting the conserved region in the M gene of the MHV reference strain (accession no. FJ6647223) according to the TwistDx manual instructions. The specificity, sensitivity, and reproducibility of the RT-RPA method were evaluated and compared with those of the standard RT-qPCR method. The clinical applicability of this assay was evaluated using 68 field samples. Results: Amplification using the newly developed RT-RPA assay was completed within 20 min at 37°C, while that using the RT-qPCR method required nearly 60 min. The RT-RPA method exhibited an obvious time-saving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 × 101 copies/µL. The specificity was indicated by a lack of cross-reaction with MHV, pneumonia virus of mice, Sendai virus, hantavirus, minute virus of mice, and reovirus type III. The MHV detection rate of RT-RPA assays was 13.63% (9/66) and RT-qPCR assays was 15.15% (10/66). Cohen's "kappa" (κ) analysis results exhibited a very good agreement between two methods with the value of κ ≥ 0.750(since κ = 0.939) and p < 0.0005 (since p = 0.000). Conclusion: The RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.

Identification of a novel host protein interacting with the structural protein VP2 of deformed wing virus by yeast two-hybrid screening.

Deformed wing virus (DWV) interacting with Varroa destructor is a possible cause of honeybee colony mortality. VP2 is the structural protein of DWV but its function remains unknown. To clarify the function of VP2 and screen for novel binding proteins that interact with VP2, we carried out a membrane protein yeast two-hybrid screening using VP2 as bait. Subsequently, the interaction between VP2 and the host interacting protein [heat shock protein 10 (Hsp10)] was further verified using glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in cells. Furthermore, fluorescence confocal microscopy revealed that VP2 and Hsp10 were mainly co-localized in the cytoplasm. Using real-time polymerase chain reaction, we found that Hsp10 expression in DWV-infected worker honey bees were downregulated compared with that in healthy honey bees. Additionally, we showed that overexpression of VP2 protein could reduce the expression of Hsp10. These results suggest that Hsp10 plays a vital role in host immunity and antiviral effects.

Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening.

Chinese sacbrood virus (CSBV) is the major cause and lead to the collapse of Apis cerana colonies. VP1, the structural protein of CSBV, shows the highest variation in the amino acid sequences among proteins from different CSBV strains as well as exhibits excellent immunogenicity. However, its function with host protein still remains unclear. To clarify its function with host protein, we screened out host cellular proteins that interact with VP1 using the membrane protein yeast two-hybrid system. In addition, we verified interactions between heat shock protein 70 cognate 5 (Hsp70-c5) and VP1 using glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. VP1 and Hsp70-c5 were colocalized in the cytoplasm and nucleus. Using western blot and real-time polymerase chain reaction (PCR), Hsp70-c5 expression in CSBV-infected larvae was upregulated compared with that in healthy larvae. We observed that when we silenced Hsp70-c5, VP1 expression was significantly downregulated. These results demonstrate that Hsp70-c5 is involved in at least one stage(s) of the viral life cycle.

A study on the enzymatic properties and reuse of cellulase immobilized with carbon nanotubes and sodium alginate.

Cellulase has many potential applications in ethanol production, extraction of medicinal ingredients, food, brewing, oil exploration, environmental protection. However, the widespread use of cellulase is limited by its relatively high production costs and low biological activity. Therefore, we studied the enzymatic properties and reusability of cellulase immobilized on multiwalled carbon nanotubes and sodium alginate for the first time. The results showed that the optimum temperature and pH of immobilized cellulase was 40 °C and 3.0, respectively. After 1 month of storage at 4 °C, the enzyme activity of immobilized cellulase dropped to 71.2% of the baseline. Immobilized cellulase was proved to be reusable and maintained ~ 70% of its activity after 7 cycles of repeated use. Versus free cellulase, the immobilized cellulase showed good thermal stability, pH resistance, storage stability and reusability, which could be beneficial in large-scale industrial manufacturing processes.