Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the predominant pathogens associated with BRD. Our previous study involved the development of attenuated M. bovis HB150 and BoHV-1 gG-/tk- vaccine strains, which were thoroughly assessed for their safety profiles and protective efficacy in cattle. In this study, we applied a combination of vaccines in varying ratios and used a rabbit model to determine the safety and protective efficacy. We used PCR/RT-PCR to detect the postimmunization and challenge shedding of M. bovis and BoHV-1. Additionally, we measured antibody titers and the expression of IFN-β and TNF-α to evaluate the humoral and cellular immune responses, respectively. Furthermore, we performed a histopathological analysis to assess lung damage. Our study provides evidence of the safety and effectiveness of the bivalent M. bovis–BoHV-1 vaccine in rabbits, particularly when applying a combination of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain. The bivalent vaccine significantly enhanced both the long-term antibody immune response and cellular protection against the M. bovis and BoHV-1 challenge. These findings provide a valuable model for the potential application in cattle.Bovine respiratory disease (BRD) is a global prevalent multifactorial infection primarily caused by viral and bacterial coinfections. In China,  Pasteurella multocida, Mannheimia haemolytica, and Mycoplasma bovis (M. bovis) [Bovine respiratory disease (BRD), which caused approximately 75% of illness and 50–70% of mortality in feedlots, stands as a widespread and economically challenging problem for the cattle industry ,2. BRD h. bovis) .M. bovis is currently the most prevalent causative agent of BRD globally. It can induce not only pneumonia, but also a variety of other conditions, including mastitis, arthritis, skin abscesses, meningitis, otitis, and reproductive tract disorders in cattle of all ages and across different sectors [M. bovis pneumonia was first reported in 2008 among newly imported beef cattle transported over long distances throughout the country, and has since become remarkably widespread [M. bovis, our laboratory developed a single attenuated vaccine using the clinical isolate M. bovis HB0801 strain in 2014. This vaccine exhibited a remarkable protection rate of 87.7% in cattle [ sectors . In Chindespread . To combn cattle .BoHV-1 is also an important causative agent of BRD in China. It was initially identified in an imported cow in 1981 and rapidly spread throughout the country. The overall prevalence of BoHV-1 among cattle in China is 40% . In InneM. bovis HB0801 and BoHV-1 HB06, respectively. Rabbits challenged by M. bovis HB0801 showed increased rectal temperature and respiratory symptoms, and lung consolidation was observed after dissection. The rectal temperature of rabbits challenged by BoHV-1 HB06 was immediately elevated and lasted for several days, followed by an increase in nasal secretions; histopathology observation of the lungs showed obvious lesions after dissection.Based on our previous work, we established rabbit infection models for M. bovis–BoHV-1 bivalent vaccine designed for calves.Single vaccines prove insufficient to address the challenges posed by mixed infections of multiple pathogens; therefore, we assumed a combination approach, using two vaccines with different doses based on the above studies. We evaluated its safety and efficacy in a rabbit model to establish its effect for the subsequent development of an attenuated M. bovis HB0801 (GenBank accession number: CP002058.1) strain, and the M. bovis HB150 strain were maintained in the National Key Laboratory of Agricultural Microbiology. Madin–Darby bovine kidney cells (MDBK) were obtained from the China Institute of Veterinary Drug Control.Wild-type BoHV-1 HB06 (GenBank accession number: AJ004801.1), the BoHV-1 gG-/tk- strain, the M. bovis and BoHV-1 were cultured as previously reported [M. bovis HB0801 and HB150 strains were cultured in complete PPLO medium at 37 °C in a 5% CO2 incubator for 40–48 h. The BoHV-1 HB06 and BoHV-1 gG-/tk- strains were cultured using MDBK cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  at 37 °C in a 5% CO2 incubator.reported ,16. BrieM. bovis HB150 and BoHV-1 gG-/tk- were 1.0 × 108 CFU and 1.0 × 106 TCID50, respectively. In this research, we mixed them in ratios of 1:1, 1:2, and 2:1, corresponding to the following combinations: 1.0 × 108 CFU M. bovis HB150 with 1.0 × 106 TCID50 BoHV-1 gG-/tk-, 1.0 × 108 CFU M. bovis HB150 with 2.0 × 106 TCID50 BoHV-1 gG-/tk-, and 2.0 × 108 CFU M. bovis HB150 with 1.0 × 106 TCID50 BoHV-1 gG-/tk-. The study objective was to determine the most effective combination of these two vaccine strains.The initial dosages of the M. bovis, Pasteurella, and Manniella hemolysticus, and all resulted negative. Subsequently, those rabbits were divided into 11 groups, each consisting of 3 rabbits. Groups 1 and 2, 3 and 4, and 5 and 6 were immunized with 1.0 × 108 CFU M. bovis HB150 mixed with 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain (ratio of 1:1), 1.0 × 108 CFU M. bovis HB150 mixed with 2.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain (ratio of 1:2), and 2.0 × 108 CFU M. bovis HB150 mixed with 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain (ratio of 2:1), respectively; Group 7 was immunized with 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain; Group 8 was immunized with 1.0 × 108 CFU of the M. bovis HB150 strain; Groups 9 and 10 were inoculated with DMEM and the complete PPLO medium, respectively. All experimental groups were then challenged with 1.0 × 109 CFU of the M. bovis HB0801 strain or 4.0 × 107 TCID50 of the BoHV-1 HB06 strain 28 days after immunization. Group 11 was the control group. All rabbits received intranasal administration of the M. bovis HB150, BoHV-1 gG-/tk-, and BoHV-1 HB06 strains using a 2 mL sterile syringe. The M. bovis HB0801 strain was introduced via tracheal administration. Each rabbit was kept in a separate housing to avoid the risk of cross-infection. The detailed information is summarized in Thirty-three Japanese white rabbits, with an average weight of 1.5 kg, were obtained from the Experimental Animal Center at Huazhong Agricultural University. The rabbits were tested for BoHV-1, Clinical observation and rectal temperature were continuously recorded for 14 days after immunization and 14 days after the challenge. Animal weights were measured on day 0 before immunization, on day 28 before the challenge, and before euthanasia on day 56, which was 28 days after the challenge.M. bovis HB0801-challenged groups were placed and fully vortexed in tubes containing 1 mL sterile PBS, filtered through a 0.45 µm syringe filter; all samples were then stored at −20 °C for PCR/RT-PCR and M. bovis counting. Both serum and whole-blood samples were collected on days 0, 7, 14, 21, 28, 35, 42, 49, and 56 after immunization for antibody and cytokine detection. Lungs were collected and fixed in 4% paraformaldehyde for 48 h, then embedded in paraffin. Hematoxylin–eosin (HE) staining was performed on the sections, followed by histopathological examination.Nasal swabs were collected daily for 14 days postimmunization and postchallenge. Samples collected from M. bovis using the uvrC gene and BoHV-1 using gG and TK genes, as previously described [M. bovis and BoHV-1 gG and tk genes are as follows:Genomes of the treated nasal swabs were extracted, followed by PCR/RT-PCR to detect the escribed ,16. The M. bovis uvrC gene PCR reaction conditions: 95 °C for 3 min; 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min. For the BoHV-1 gG PCR reaction conditions: 95 °C for 3 min; 95 °C for 15 s, 60.5 °C for 15 s, 72 °C for 40 s, 35 cycles; 72 °C for 5 min. Similarly, for the BoHV-1 tk gene PCR, the conditions were: 95 °C for 3 min; 95 °C for 15 s, 60.5 °C for 15 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min.M. bovis HB0801 shedding following the challenge, the nasal swabs that had undergone treatment were subjected to 10-fold serial dilutions ranging from 108. Subsequently, 100μL from each dilution was introduced into a PPLO liquid medium and incubated at 37℃ within a 5% CO2 incubator for a period of 3–5 days, or until there was no further change in the color of the medium. The highest dilution that resulted in a color change was designated as 1 CCU/mL. The M. bovis HB0801 shedding counting was then calculated according to the dilution.To assess the quantity of To quantify the amount of BoHV-1 HB06 shedding after the challenge, the extracted DNA from the nasal swabs was used for the RT-PCR detection established in our lab using the gB gene. The program for the BoHV-1 gB gene was as follows: 95 °C for 30 s; 95 °C for 10 s, 60 °C for 20 s, 40 cycles; 95 °C for 15 s, 60 °C for 20 s, 95 °C for 15 s.50 in a 5% CO2 incubator at 37 °C for 1 h. The serum–virus mixture was introduced into a 96-well cell culture plate with MDBK cells. The plate was then incubated for three days at 37 °C in a 5% CO2 incubator. Neutralizing antibody titers were calculated using the Reed–Muench method, representing the highest serum dilution inhibiting the BoHV-1 virus infection.Serum samples were collected weekly throughout the study. The serum samples were subjected to 56 °C for 30 min to inactivate any potential agents. Following inactivation, the serum was serially diluted in a 96-well cell culture plate and incubated with the BoHV-1 HB06 virus at a concentration of 100TCIDM. bovis antibodies were determined by a competitive ELISA established in our lab. Briefly, the 4× diluted tested serum, positive and negative serum controls, and HRP-labeled monoclonal antibodies were added to the M. bovis p579-protein-coated plate and incubated at 37 °C for 60 min. After complete washing, 100 µL of the substrate chromogenic solution was added and incubated at room temperature in the dark for 10 min, and stop solution was added to stop the reaction. Values under the OD450nm were taken. The blocking rate  was calculated according to the following calculation formula:450nm; N = mean OD450nm of the negative control serum. Conditions for the establishment of the test: 0.65 < OD450nm, negative control < 2.0, and PI positive control > 60%. PI sample ≥ 41% means positive; PI sample < 41% means negative.PI value = (1 − S/N) × 100%, where S = sample OD450nm.The serum samples were used to detect IFN-β and TNF-α using commercial ELISA kits  according to the manufacturers’ instructions. In brief, diluted serum samples were added to the plate wells and incubated at 37 °C for 30 min. After adequate washing, the secondary antibody was added; after being incubated at 37 °C for 30 min, the plate was washed sufficiently and substrate-color-development solution was added. The reaction was stopped and read under ODt-test and a one-way ANOVA were used to compare the data of different groups; mean ± SEM was used. The comparison results of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****) were considered to have significant or highly significant statistical differences.The M. bovis HB0801 and BoHV-1 HB06, the rectal temperature reached higher than 39.5 °C from days 1–3 and 2–4, respectively, then returned to normal . On day 3–5 postchallenge, animals challenged without immunization showed a considerably higher amount of BoHV-1 HB06 shedding than the blank control group (p < 0.05).BoHV-1 HB06 shedding after the challenge was accessed using RT-PCR B. ImportM. bovis HB0801 or BoHV-1 HB06.Taken together, the 1:1 group maintained the lowest shedding amount compared to all other mixed-immunization groups after the challenges of M. bovis serum antibodies after the immunization and challenge, competitive ELISA was used to detect the changes in serum. The antibody levels of the 1:1 group were significantly higher than those of the blank control group 21 days after immunization (p < 0.001). After the challenge, the highest titer was observed in the 1:1 group throughout the entire observation period. On day 7 postchallenge, the 1:1 immunized group displayed a significantly higher antibody titer compared to all other groups, including the immunized and nonimmunized groups (p < 0.05), and peaked at day 14 after the challenge (p < 0.05). From day 21 to 28 postchallenge, all immunized groups showed significantly higher antibody levels compared to the blank control group (p < 0.01) (To investigate the level of  < 0.01) A.p < 0.05). The neutralization titer of the 1:2 group was significantly higher than that of the blank control 14 days postchallenge (p < 0.05) . The 1:1 group induced a significantly higher neutralization titer compared to the challenge-without-immunization group at 7 and 14 days postchallenge (35 and 42 days postimmunization) ( < 0.05) B.M. bovis and BoHV-1 antibody titers among all immunization groups.In summary, we believe that the 1:1 mixed-immunization group can induce the highest and most prolonged p < 0.05 and p < 0.01, respectively). By day 21 postimmunization, a sharp decline was observed in all immunization groups, except for the BoHV-1 gG-/tk- single-vaccination group, which had significantly higher expression than the blank control (p < 0.001). At 28 days postimmunization, except for the 1:1 and BoHV-1 gG-/tk- single-immunization groups, the expression of IFN-β could not be detected in the other groups . The 2:1 and 1:2 groups only induced higher TNF-α levels on days 14 and 28 postimmunization, respectively (p < 0.05). The BoHV-1 gG-/tk- single-vaccination group induced higher levels of TNF-α on days 21 and 28 postimmunization (p < 0.05)  B.The data above show that the 1:1 mixed group provoked a superior cellular immune response among all the immunized groups.M. bovis HB0801-challenged groups, the alveolar structural morphology of the 1:1 and 1:2 groups were still intact, but the alveolar wall of the 1:1 group was thicker than that of the 2:1 group, and showed a small amount of plasmacytic exudation in the alveolar with inflammatory cells in the alveolar cavity (M. bovis HB150 single-component group remained intact, with no obvious pathological alterations in the alveolar wall and alveolar cavity (According to the results, in the r cavity A,B. The r cavity C. The alr cavity D. In ther cavity E. All tir cavity F.For the animals challenged with BoHV-1 HB06, the alveolar structures in the 1:1 and 1:2 groups remained mostly intact, with only a slight thickening of the alveolar wall and a small amount of plasmacytic detected in the alveolar cavity A,B. The M. bovis HB0801 and BoHV-1 HB06. However, when considering all the mixed-immunization groups, the 1:1 group outperformed all others.The results illustrate that the 2:1 and 1:2 groups provided the most significant protection among the mixed-immunization groups following the challenges with M. bovis–BoHV-1 bivalent vaccine using a rabbit model, and the results confirm its safety for overall health. Immunological analysis and challenge experiments showed that the M. bovis–BoHV-1 bivalent vaccine can elicit the antibodies against both M. bovis and BoHV-1, successfully protecting rabbits against live M. bovis and BoHV-1 challenges. The ratio of M. bovis HB150: BoHV-1 gG-/tk- 1:1 was highly recommended among all groups.In this study, we thoroughly evaluated the attenuated During the immunization and challenge experiment, rabbits were subjected to intranasal immunization with three different vaccine ratios, which induced protective antibodies and cytokines. All bivalent-vaccine groups generally demonstrated immune responses compared to the two single-vaccine groups. After the challenge, all experimental groups experienced a temporary elevation in rectal temperature for 1 day, except for the nonimmune-challenged group, which a sustained a high temperature for three consecutive days. All bivalent vaccines effectively shielded the rabbits from fever, aligning with our previous single-vaccine research studies ,14.M. bovis, even early on day 2 postchallenge, the ratio of the 1:1 mixed vaccine could effectively suppress M. bovis HB0801 shedding, demonstrating superior protection compared to the single-vaccine-immunization group. Following 2 to 4 days of BoHV-1 incubation postchallenge, the virus replicated in the mucosal and tonsil tissues, leading to clinical signs in cattle and the subsequent shedding of the virus between days 3 and 10 following the initial infection [The shedding of pathogens during the challenge period was a vital indicator of the disease . For M. nfection . Our stuM. bovis and BoHV-1, and the expression of cytokines such as IFN-β and TNF-α to assess the humoral and cell-mediated immunity responses related to protection. The 1:1 vaccine group initially exhibited the most significant increase in IFN-β and TNF-α postimmunization. Furthermore, after the challenge, animals vaccinated with the 1:1 ratio also displayed the highest antibody titers. Previous research reported that the gene-deletion vaccine can induce high antibody titers after the BoHV-1 challenge, consistent with our findings [We measured the antibody titers against findings .8 CFU M. bovis HB150 combined with 1.0 × 106 TCID50 BoHV-1 gG-/tk- led to the best immunoprotective response among all vaccine groups.The stimulation of IFN-β transcription represents an early response to viral infection, with IFN-β expression exhibiting potent antiviral activity . In thisTNF-α is a proinflammatory cytokine that facilitates endothelial activation and the recruitment of leukocytes to infection sites . It is oHistopathological examination was also used to assess the protective efficacy. Generally, rabbits immunized with the 1:1, 1:2 mixed vaccines, and two single vaccines had nearly normal alveolar structures, while the nonimmune-challenged ones exhibited severe pulmonary pathological changes.M. bovis–BoHV-1 bivalent vaccine was safe and effective to rabbits, with the most effective combination being 1.0 × 108 CFU M. bovis HB150 mixed with 1.0 × 106 TCID50 BoHV-1 gG-/tk-. In addition to enhancing long-term immune responses, the bivalent vaccine also offered cellular protection against the M. bovis and BoHV-1 challenge. This model holds significant promise for potential application in cattle. However, further evaluation of bivalent vaccine efficacy in cattle remains an essential avenue for exploration.In summary, the M. bovis–BoHV-1 bivalent vaccine was developed for the first time. This study concluded that this vaccine elicited a robust production of serum antibodies and significantly elevated the production of both IFN-β and TNF-α, with the 1:1 antigen ratio having the best effect. These results suggest that the vaccine provides effective, adequate protection for the effective protection of rabbits, and lays the foundation for a series of subsequent experiments in cattle.Based on preliminary research, an attenuated


Piper nigrum L. fruit essential oil (EO) against Stomoxys calcitrans (stable fly), a blood-feeding fly distributed worldwide, was investigated. This study aimed to evaluate the insecticidal activity of EO based on contact and fumigant toxicity tests. Chemical analysis of the EO using gas chromatography–mass spectrometry revealed that sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%) were the major components. The results demonstrated that fly mortality increased with increasing EO concentration and time during the first 24 h of exposure. The median lethal dose was 78.37 µg/fly for contact toxicity, while the 90% lethal dose was 556.28 µg/fly. The median lethal concentration during fumigant toxicity testing was 13.72 mg/L air, and the 90% lethal concentration was 45.63 mg/L air. Our findings suggested that essential oil extracted from P. nigrum fruit could be a potential natural insecticidal agent for control of stable fly. To examine the insecticidal properties of P. nigrum fruit EO, further field trials and investigation into the efficacy of nano-formulations are required.The efficacy of  Stomoxys calcitrans (Diptera: Muscidae)], an insect belonging to the order Diptera, is distributed worldwide. Stable flies carry bovine life-threatening viral pathogens including lumpy skin disease virus, bovine herpes virus, and bovine leukosis virus , clove (Eugenia caryophyllata Thunberg) buds and leaves, and lovage  roots, had the potential to repel female flies  EO, which primarily contains limonene, had a reported LD50 of 105.88 µg/fly and an LD90 of 499.25 µg/fly for contact toxicity, and an LC50 of 13.06 mg/L air and an LC90 of 43.13 mg/L air for fumigant toxicity within 24 h [50 of rosalva (from the rose), geranyl acetone , and citronellol  were 13.10 µg/cm2, 25.20 µg/cm2, and 35.69 µg/cm2, respectively, while the LC90 values were 18.54 µg/cm2, 34.97 µg/cm2, and 50.10 µg/cm2, respectively, after 24 h exposure [The insecticidal effects of potential medicinal plant essential oils have been tested against stable fly. Catnip EO  had an LChin 24 h . AdditioPiper nigrum L. is widely employed as a food ingredient to increase taste and is found mainly in tropical zones throughout the world [he world . A previhe world . The EO he world . Anotherhe world .Aedes aegypti mosquito larvae [P. nigrum EO were studied in the adult and larval stages of the pulse beetle Callosobruchus chinensis (Coleoptera: Bruchidae) [Tribolium castaneum (Coleoptera: Tenebrionidae) [Black pepper oil contained primarily monoterpene hydrocarbons (47–64%) and sesquiterpene hydrocarbons (30–47%) . The EO o larvae . Fumiganuchidae)  and the ionidae) . HoweverP. nigrum fruit EO insecticidal activities against S. calcitrans, using in vitro contact toxicity and fumigant toxicity methods. Thereafter, the EO was analyzed for its chemical composition using gas chromatography–mass spectrometry (GC-MS).The aim of the present study was to evaluate the effects of unripe or green P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C, and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (P. nigrum EO is shown in w/v), respectively.The yield of ty index . A chromP. nigrum EO against S. calcitrans was studied at various EO concentrations. The test was validated using acetone, with no insecticidal activity, as a negative control. When EO treatments at 43.25 µg/µL, 86.50 µg/µL, 216.25 µg/µL, and 432.50 µg/µL were compared with cypermethrin, EO was found to have little to no insecticidal efficacy  = 32.61, p < 0.001; treatment, F = 244.77, p < 0.001; treatment × time, F = 4.64, p < 0.001). The LD50 and LD90 values were 15.68% and 111.23% (w/v) or 78.37 µg/fly and 556.28 µg/fly, respectively  = 75.47, p < 0.001; treatment, F = 158.11, p < 0.001; treatment × time, F = 24.20, p < 0.001). The LC50 and LC90 values were 13.72 mg/L air and 45.63 mg/L air, respectively  [Sitophilus zeamais [50 of 0.287 μL/cm3 and 0.152 μL/cm3 after 24 and 48 h exposure periods, and a contact toxicity assay reported an LC50 of 0.208 μL/cm2 and 0.126 μL/cm2 after 24 and 48 h exposure periods, respectively [P. nigrum EO demonstrated insecticidal activity via the AChE function against insect pest species T. castaneum and Sitophilus oryzae [Previous reports have described the repellent and insecticidal effects of ionidae) ,29. EOs  zeamais . A fumigectively . In addis oryzae .P. nigrum fruit EO was shown to have insecticidal effects, via contact and fumigant toxicity studies. The four most important chemical compounds identified in this study were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%), which altogether represented approximately 77.32% of all P. nigrum EO. Sabinene, limonene, and α-pinene belong to the monoterpene group, which is the predominant class of terpene found in P. nigrum oil [P. nigrum fruit EO in this study was similar to that in other reports [P. nigrum fruit cultivated in Brazil found the main volatile compounds in the monoterpene group to be α-pinene (6.91–10.67%), β-pinene (22.61–35.05%), and limonene (21.0–31.77%) [In this study, unripe grum oil . The che reports . However reports . Another–31.77%) . Thus, vlimonene .0–31.77%Xylopia aethiopica EO containing sabinene (26.1 ± 3.1%), β-pinene (17.4 ± 1.9%), and α-pinene (9.6 ± 1.6%) had contact toxicity against the house fly (Musca domestica) and exhibited an LD50 of 61.5 µg/adult and LC90 of 178.5 µg/adult in males, and an LD50 of 30.7 µg/adult and LC90 164.2 µg/adult in females [M. domestica, there was a higher LD50 of 109.7 µg/adult and LC90 of 213.8 µg/adult. This was similar to the results when α-pinene was applied to the female M. domestica, with a higher LD50 of 69.7 µg/adult and LC90 of 254.7 µg/adult. In contrast to its effects in females, the LD50 and LC90 values of sabinene decreased to 10.4 µg/adult and 117.5 µg/adult in male M. domestica, and the LD50 and LC90 of α-pinene decreased to 8.6 µg/adult and 100.2 µg/adult, respectively. This suggests that major and minor compounds, acting synergistically or antagonistically, are responsible for the insecticidal activity [Schinus areira seeds containing the monoterpenes sabinene (39.15%), α-pinene (14.96%), and β-pinene (8.75%) also showed insecticidal activity against the seed beetle, Rhipibruchus picturatus. Using a molecular docking assay, the main component from S. areira leaves was found to bind the AChE enzyme’s active site [Aloysia citriodora EO containing sabinene (22.9%), limonene (7.4%), and caryophyllene (2.4%) was evaluated against the southern green stinkbug Nezara viridula (L.), using fumigant activity in an enclosed chamber. The toxicity of A. citriodora increased with increasing concentration and exposure time. Fumigant activity had an LC50 of 13.5 μg/mL air 24 h after treatment and contact toxicity had an LC50 of 8.1 μg/cm2 [Haplopappus foliosus EO consisted of limonene (28.0%), caryophyllene (3.97%), β-pinene (1.43%), sabinene (0.93%), and α-pinene (0.89%), with an LC50 value of 4.43 mg/dm3 air against M. domestica [Medicinal plant EOs containing at least two of the major compounds sabinene, limonene, β-caryophyllene, or α-pinene have been reported to have insecticidal effects.  females . When saactivity . EO extrive site . Aloysia1 μg/cm2 . Haplopaomestica .50 of 78.37 µg/fly and an LD90 of 566.28 µg/fly 24 h after exposure, which differs slightly from the toxicity of citrus EO for the stable fly (LD50 of 105.88 µg/fly and LD90 of 499.25 µg/fly). However, the fumigant toxicity values of an LC50 of 13.72 mg/L air and an LD90 of 45.63 mg/L air were consistent with a previous report (LC50 of 13.06 mg/L air and LC90 of 43.13 mg/L air) [P. nigrum EO. Prior studies have reported the insecticidal effects of compounds found in essential oil. Sabinene is reportedly insecticidal against Aphis pomi [Tribolium castaneum [Aphis gossypii Glover (Homoptera: Aphididae) by inhibiting AChE activity, polyphenol oxidase, carboxylesterase, and glutathione S-transferases in A. gossypii [This study noted a contact toxicity LDg/L air) . Furtherg/L air) , while shis pomi , has a rastaneum , and exhastaneum . Limonenastaneum , horn flastaneum , and theastaneum . β-caryoP. nigrum EO has been investigated on a normal cell line, human fetal lung fibroblast (MRC-5), and five different tumor cell lines including human cervical carcinoma (HeLa), human myelogenous leukemia (K562), lung adenocarcinoma (A549), colon cancer (LS-174), and melanoma (FemX). The results indicate that P. nigrum EO exhibited lower cytotoxic activity, with an IC50 of 25.57–56.74 µg/mL after 48 h of treatment compared with cisplatin  as a positive control [P. nigrum EO at concentrations of 5, 10, or 50 mg/kg was evaluated for 21 days in adult male albino Swiss mice. None of the P. nigrum EO doses induced signs of toxicity, such as irritability, reaction to contact, gripping the tail, twisting, writhing, grip strength, ataxia, tremor, convulsion, lacrimation, piloerection, changes in respiratory frequency, changes in body weight, or abnormalities in biochemical parameters [90 of 556.28 (325.89–1475.26) µg/fly and the LC90 of 45.63 mg/L air obtained in the current study, we expected that the P. nigrum EO used in this study should not affect horses and livestock, the primary hosts of the stable fly.The cytotoxicity of  control . The toxrameters . Based oP. nigrum EO and the compounds sabinene, limonene, β-caryophyllene, and α-pinene should be the subjects of future studies of the EO insecticidal mechanism of action.Although this study did not investigate the molecular mechanism of EO effect, the insecticidal activities of EO have been reported. Plant-derived EOs, primarily monoterpenes, exhibit their neurotoxic effects by the inhibition of AChE in the octopamine pathway in insects, alongside the essential detoxification enzymes glutathione S-transferase and catalase. They are also capable of inhibiting gamma-aminobutyric acid receptors ,48,49. HP. nigrum fruit EO, primarily containing sabinene, limonene, β-caryophyllene, and α-pinene, as an environmentally friendly insecticidal agent against stable fly , using contact and fumigant toxicity tests. The synergistic effects of the components of this EO on the insect’s neuromuscular system should be investigated further. As there are limited reports available discssing the insecticidal activities of P. nigrum fruit EO on horse and livestock vectors such as horse fly, horn fly, and midges, the study of P. nigrum fruit EO should be expanded and its nano-formulation for open field trials should be implemented.This study demonstrated the toxicity of unripe This study protocol was approved by the Faculty of Veterinary Science, Mahidol University Animal Care and Use Committee (Ref. MUVS-2020-12-63).S. calcitrans used in this study were collected using Nzi Traps [S. calcitrans were tested and were selected from groups of undamaged flies under a stereomicroscope . Blood-fed flies were separated out of this study. The samples were preserved between 27–29 °C and 70–80% relative humidity upon arrival at the laboratory until testing.Populations of zi Traps , from a Piper nigrum L. fruit (v/w) based on the fresh plant material weight.L. fruit  was obtaThe EO’s physical properties were analyzed as follows: color was visually verified by three different individuals, pH was measured with pH-indicator strips , density was measured with a density meter , and the refractive index was calculated with a refractometer .−1. The injection port temperature was 250 °C, and the column temperature schedule was as follows: 50 °C for 2 min followed by a rise to 250 °C at a rate of 10 °C per minute, then maintained at 250 °C for 5 min. The MS included an ion source temperature of 230 °C, an ionization energy of 70 eV, and a mass scan range of 350–550 amu. Components were identified by comparing their mass spectra with data from the Wiley 7th edition mass spectrometry library. Concentrations of the principal components were calculated by comparing the peak area of the sample with the peak area of the standard.The EO’s chemical composition was determined using a GC-MS model 7890A-MS5975C  equipped with a DB-5HT capillary column . The sample was injected in split mode with a 1:10 split ratio. Helium was used as the carrier gas, with a flow rate of 1 mL·minP. nigrum L. to S. calcitrans was achieved by topical application as described by Leesombun et al., with some modifications [P. nigrum EO were prepared in a 1.5 mL microcentrifuge tube using acetone as a solvent. Five EO concentrations were used in this trial: 43.25 µg/µL, 86.50 µg/µL, 216.25 µg/µL, 432.50 µg/µL, and 865.00 µg/µL, representing 5%, 10%, 25%, 50%, and 100% (v/v) of S. calcitrans (mixed sexes). Flies were anesthetized at −20 °C for 30–45 s, and 0.5 µL of EO at each concentration was applied directly to the fly’s chest using a micropipette. Acetone and cypermethrin (1% w/v) were applied at the same volume for negative and positive controls, respectively. Each treatment was performed in three iterations on 10 flies each time. After application, the treated flies were placed in a clear plastic cup (10 flies/cup), and the cup was covered with a fine mesh fabric and fastened with rubber bands. A honey solution (10%) on cotton was layered over the mesh fabric, for the flies to consume. The flies were brought to recovery at 27–29 °C and 70–80% relative humidity. Mortality was recorded at 1, 2, 4, 6, 12, and 24 h after treatment. Flies were determined to be dead when they did not move after being mechanically stimulated with a brush.Contact toxicity of the EO from ications . PrelimiP. nigrum active against S. calcitrans was examined according to the procedures reported by Leesombun et al., with some modifications [w/v) were used as negative and positive controls, respectively. The petri dishes were placed on the bottom of the plastic container. Honey solution (10%) on cotton wool was placed on the bottom of the plastic container. Mixed sexes of S. calcitrans were used for testing. Flies were anesthetized at −20 °C for 30–45 s, placed in the plastic containers, and each container was closed. Each treatment was performed with ten flies in 3 iterations. Flies were allowed to recover and were maintained at 27–29 °C with 70–80% relative humidity. Mortality was recorded at 1, 2, 4, 6, 12, and 24 h after treatment. Flies were determined to be dead when they did not move.Fumigant toxicity of the EO from ications . This tep value < 0.05 was considered significant. Probit analysis to calculate toxicity values, mean lethal dose (LD50) and 90% lethal dose (LD90) at 24 h after treatment, and mean lethal concentration (LC50) and 90% lethal concentration (LC90) at 24 h after processing was performed using LdP series software . The software can be downloaded free of charge from http://www.ehabsoft.com/ldpline/ and was accessed on 6 November 2022.Insect mortality was calculated and the mortality data were corrected using Abbott′s formula , when th


Peridroma saucia, a worldwide pest that causes serious damage to various crops. A gene expression value  analysis revealed that seven OBPs  were highly abundant in the antennae. Next, we focused on the expression and functional characterization of PsauGOBP2. Real-time quantitative-PCR analysis demonstrated that PsauGOBP2 was predominantly expressed in the antennae of both sexes. Fluorescence binding assays showed that the recombinant PsauGOBP2 strongly binds to the female sex pheromone components Z11-16: Ac (Ki = 4.2 μM) and Z9-14: Ac (Ki = 4.9 μM) and binds moderately (6 µM ≤ Ki ≤ 13 µM) to the host plant volatiles phenylethyl acetate, β-myrcene, and dodecanol. Further 3D structural modeling and molecular docking revealed that several crucial amino acid residues are involved in ligand binding. The results not only increase our understanding of the olfactory system of P. saucia but also provide insights into the function of PsauGOBP2 that has implications for developing sustainable approaches for P. saucia management.Odorant-binding proteins (OBPs) are expressed at extremely high concentrations in the chemo-sensilla lymph of insects and have long been thought to be crucial for delivering the semiochemicals to the odorant receptors. They are represented by multiple classes: general odorant-binding proteins (GOBP1 and GOBP2) and pheromone-binding proteins. In the current study, we identified a total of 35 OBPs in the antennal transcriptome of  Insects depend on olfaction system to locate oviposition sites, food sources, and mate partners, and to avoid natural enemies . The antAntheraea polyphemus in 1981  and pheromone-binding proteins (PBPs) are numerically dominant among classic OBPs . PBPs ara exigua  and Chilressalis  have higeromones . In situ1-16: OH . In Plut-16: Ald . On the d phoxim . TherefoPeridroma saucia Hübner (Lepidoptera: Noctuidae) is a polyphagous feeder on various vegetables and field crops. Generally, P. saucia larvae sporadically damage crops during the growing season and pose great problems in the mid-summer  could efficiently attract male P. saucia moths in the field , male and female antennae, mouthparts, and legs were collected separately from 50 to 80 individuals of 2-3-day-old adult P. saucia. All of the tissue samples were kept in a −80°C freezer until used.ar water . For trade novo method (Trinity v 2.11) as we previously described (https://www.ncbi.nlm.nih.gov/orffinder). To evaluate the expression abundance, TPM values (Transcripts Per Kilobase of exon model per Million mapped reads) of candidate PsauOBP transcripts were calculated with Salmon (v 1.4.0).Total RNA from male and female antennae was extracted using Trizol reagent . The purity and quantity of the RNA were evaluated with an ND-2000 spectrophotometer . The RNA integrity was further checked with a 2100 bioanalyzer . Genomic DNA was eliminated from the total RNA with DNase I . mRNA was then isolated from ≥1 µg (concentration ≥50 ng/μL) of the total RNA with Dynabeads mRNA purification kit . RNA-seq libraries were constructed following Illumina’s library construction protocol and then sequenced on the Nova seq6000 platform  at Origingene, Shanghai, China. Sequence assembly was performed with a escribed . PutativBombyx mori, Spodoptera litura, Helicoverpa armigera, and Agrotis ipsilon was constructed with MEGA 11. The evolutionary distance was calculated with the JTT matrix-based method ; Protein parameters including the molecular weight and the theoretical isoelectric point were predicted with ProtParam (https://web.expasy.org/cgi-bin/protparam/protparam). Gene-specific primers designed against the PsauGOBP2 cDNA were listed in PCR amplification of PsauGOBP2 was carried out with Premix Taq (Takara) under the following procedure: 94°C for 3 min; 34 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 1 min. The crude PCR products were then ligated into a pGEM-T easy vector  at a molar ratio of 5: 1 (insert: plasmid). The ligation products were used to transform 2O. The RT-qPCR program was set as: 1 cycle of 94°C for 30 s; 40 cycles of 94°C for 5 s and 60°C for 10 s; followed by 1 cycle of 95°C for 5 s, 60°C for 1 min, and 50°C for 30 s. The primers designed with Primer Premier 6.0 were listed in Psauβ-actin (accession number QQ472022), using the 2−ΔΔCT method  with a mixture  of 10 μL of TB Green Fast qPCR Mix (Takara), 0.8 μL (10 μM) of each primer, 5 ng of sample cDNA, and appropriate volume of sterilized ultrapure HT method . Three bE. coli cells. Protein expression was induced by the addition of IPTG  when the OD600 value of cell culture reached about 0.8. Cells cultured for further 2–4 h at 37°C were harvested by centrifugation. PsauGOBP2 was present as inclusion bodies. 10 mL of 8 M urea and 1 mM DTT in Tris buffer  were then used to solubilise the inclusion body pellet (from 1 L of culture). The dissolved protein was dialyzed against 1 L of Tris buffer . The recombinant protein was purified on the anion exchange resins QFF, following standard protocols previously adopted for other moth OBPs  to PsauGOBP2 was obtained by processing the data with GraphPad Prism 6.0.The fluorescence binding assays were conducted on a Hitachi F-2710 with a 1 cm light path quartz cuvette. To measure the affinity of the fluorescent probe 1-NPN (N-phenyl-1-naphthylamine) to PsauGOBP2, a 2 mM solution of the protein in 50 mM Tris-HCl, pH 7.4, was titrated with aliquots of 1 mM 1-NPN in methanol to a final concentration of 20 µM. The probe was excited at 337 nm, and emission spectra were recorded between 380 and 460 nm. The dissociation constant of 1-NPN  of competitors to PsauGOBP2 were calculated using the equation: Ki = [IC50]/(1 + [1-NPN]/K1-NPN), where [IC50] is the concentration of the competitor halving (50%) the initial fluorescence value (100%), [1-NPN] is the free concentration of 1-NPN, and K1-NPN is the dissociation constant of the protein-1-NPN complex.Binding affinities of odorants to PsauGOBP2 were measured by competitive binding assays. A panel of 28 compounds (competitors) including the A 3D structure of PsauGOBP2 was modeled with Alphafold2 . The 3D P. saucia antennae was obtained using a Nova seq6000 platform. A total of 44.85 million and 35.53 million clean reads were produced in male and female samples, respectively. All clean reads from male and female data were then merged for de novo assembly, which generated a total of 151,541 unigenes with an N50 length of 1158 bp and a mean length of 683.39 bp (Transcriptomic data of 83.39 bp . MoreoveP. saucia antennae (P. saucia (this study), B. mori, H. armigera, S. litura, and A. ipsilon revealed several distinct clades, where three putative PsauPBPs (PsauPBP1/2/3) were clustered with PBPs from other species; PsauGOBP1 and PsauGOBP2 were grouped with other GOBP1s and GOBP2s, respectively , PsauPBP2 (9151.4/281.5), PsauPBP3 (525/51.9), PsauGOBP1 (1348.2/65.8), and PsauOBP18 (1529.2/2.3) showed higher levels in males than in females. While PsauOBP6 and PsauGOBP2 showed higher TPM values in female  than in male antennae . Other PsauOBPs showed relatively low TPM values and their expressions were variable in male and female antennae , followed by AipsGOBP2 of A. ipsilon (88.89%).Based on the TPM values, we focused on a highly abundant transcript in the antennae of no acids . The moln GOBP2s . PsauGOBPsauGOBP2 in P. saucia, we measured its transcript levels in different chemosensory tissues including antennae, mouthparts, and legs of both sexes. RT-qPCR results showed that the expression of PsauGOBP2 was significantly higher in the antennae than in other tissues. Moreover, PsauGOBP2 expression was slightly higher in female antennae than in male antennae; however, the difference was not significant  , which i acetate , Table 1 acetate .P. saucia. Then we investigated the expression profile, ligand affinity, and binding mechanism of PsauGOBP2.Understanding of how insects sense external chemical stimulants is important for developing effective pest management strategies. OBPs represent the first step of odorant recognition in insect chemical communication . In the P. saucia. This number is close to that identified in the antennae of other noctuid moths such as H. armigera, Mythimna separata, S. litura and S. exigua, which have 34, 32, 38, and 45 OBPs, respectively  of female P. saucia (i < 5 µM) with Z11-16: Ac and Z9-14: Ac and moderate binding affinities (6 µM ≤ Ki ≤ 13 µM) with the host plant volatiles phenylethyl acetate, β-myrcene, and dodecanol. Meanwhile, our previous research validated that PsauGOBP1 can actively bind the host plant volatiles (Z)-3-hexenyl acetate (Ki = 4.0 µM), citral (Ki = 5.6 µM), farnesol (Ki = 6.4 µM), nonanal (Ki = 6.8 µM) (Z)-3-hexen-1-ol (Ki = 8.5 µM), and benzaldehyde (Ki = 9.4 µM)  and behavioral responses of the female moths to the sex pheromone components are needed, which are useful to fully understand the functional roles of GOBP2s in moths  . Therefoeromones . Similarlepigone . Female 2-14: Ac . Of whicfinities , suggestolatiles . Furtherin moths .Z11-16: Ac, Z9-14: Ac, phenylethyl acetate, β-myrcene, and dodecanol was −6.9 kcal.mol-1, −6.7 kcal.mol-1, −6.5 kcal.mol-1, −6.1 kcal.mol-1, and −6.0 kcal.mol-1, respectively. This is in accordance with the results of the fluorescence competitive binding assay which showed that the Ki value for each ligand was 4.2 μM, 4.9 μM, 6.3 μM, 8.0 μM, and 13.0 μM, respectively. Moreover, we found several key polar and nonpolar amino acid residues involved in the binding of PsauGOBP2 to the ligands, as reported for other insect OBPs , we could spot some polar amino acid residues that are possibly involved in the binding of PsauGOBP2 to dodecanol.Previous studies have demonstrated that insect OBPs bind specific ligands with polar and nonpolar residues in a hydrophobic cavity . In moleect OBPs . FurtherP. saucia. Among the OBPs, PsauGOBP2 is abundantly expressed in the antennae of both sexes. In vitro fluorescence binding assays demonstrated that PsauGOBP2 binds to sex pheromone components as well as some host plant volatiles. Finally, 3D structural modeling and molecular docking showed several amino acid residues in PsauGOBP2 that are involved in ligand binding. The results increase our understanding of the olfactory system of P. saucia and provide insights into the function and binding mechanism of PsauGOBP2 which would be used as a target for developing olfaction-based management of P. saucia.In summary, our studies provide the expression pattern of OBPs in the antennae of


The deregulation in the Wnt/β-catenin signaling pathway is associated with many human cancers, particularly colorectal cancer (CRC) and, therefore, represents a promising target for drug development. We have screened over 300 semisynthetic and natural compounds using a Wnt reporter assay and identified a family of novel chalcone derivatives (CXs) that inhibited Wnt signaling and CRC cell proliferation. Among them, we selected CX258 for further in vitro and in vivo study to investigate the molecular mechanisms. We found that CX258 significantly inhibited β-catenin expression and nuclear translocation, inducing cell cycle arrest at the G2/M phase in CRC cells. Additionally, CX258 reduced the expression of DNA Topoisomerase II alpha (TOP2A) in CRC cells. Moreover, knocking down TOP2A by siRNAs inhibited the Wnt/β-catenin signaling pathway, a finding suggesting that CX258 inhibited Wnt/β-catenin signaling and CRC cell proliferation at least partially by modulating TOP2A. Further studies showed that CDK1 that interacts with TOP2A was significantly reduced after TOP2A knockdown. We demonstrated that CX258 significantly inhibited DLD-1 CRC cell xenografts in SCID mice. In summary, we identified CX258 as a promising candidate for colorectal cancer treatment by targeting the TOP2A/Wnt/β-catenin signaling pathway. Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality across the world ,2,3. MorChalcones are a group of natural products with antitumor activity. They have been reported to inhibit multiple stages of tumor occurrence and development ,11 by rev/v) fetal bovine serum  and 1% penicillin-streptomycin . HCT116 and HT29 colon cancer cells and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin . All cells were cultured at 37 °C with 5% CO2 in a water-jacketed incubator. Cell viability and number were analyzed using a Vi-CELL XR cell viability analyzer .HEK293T, LS174T, HCT116, DLD-1, and HT29 cell lines were purchased from the ATCC . LS174T and DLD-1 colon cancer cells were cultured in RPMI 1640  containing 10% (HEK293T cells were transfected with a plasmid containing Super 8× TOPFlash subcloned into pGL4.83 (hRlucP/Puro) . A stable HEK293T cell line containing the TOPFlash reporter was established by puromycin selection. Potential chalcone inhibitors were screened with the stable cell line by measuring firefly luciferase activities  after the treatment with DMSO or the testing of compounds in DMSO solution, and LiCl was used to activate Wnt signaling at a final concentration of 2.5 mM. In order to verify if these compounds directly acted on the Wnt/β-catenin/TCF complex, HEK293T cells were transiently transfected with either a TOPFlash or a FOPFlash reporter (Super 8× TOPFlash) and Renilla, followed by co-transfection of Wnt3A, wild-type β-catenin, β-catenin (S33A) mutants, LEF-VP16, respectively . After 24 h of culture, the cells were treated with CX258 (5 μM) or DMSO as a control. Luciferase activity was determined using a dual-luciferase reporter assay system  using a Sirius-L luminometer . Renilla luciferase activity was used as an endogenous control. All experiments were performed at least twice. 4 cells per well in 12-well plates and cultured at 37 °C for 24 h, followed by the addition of 1 μL of each compound in DMSO to each well to give a final concentration of 10 μM. DMSO was used as a control. Each experiment was performed in triplicate. After 5 days, the medium was removed, and the cells were digested with 200 µL trypsin (0.25%) for 5 min, resuspended in 800 µL phosphate-buffered saline, and counted using a cell viability analyzer . IC50 data were analyzed by GraphPad Prism 8.0.1, and the parameter was selected as log(inhibitor) vs. response.Cells were seeded at a density of 4 × 10v/v) glycerol, 50 mM NaF, 1 mM Na3VO4, 1% (v/v) Triton X-100, and protease inhibitors) for 20 min on ice. Supernatant was collected after centrifugation at 13,000 rpm for 30 min. Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and electro-blotted onto equilibrated PVDF membranes . After blocking in Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were immunoblotted with primary antibody of target proteins overnight at 4 °C. The antibodies and the dilutions for Western blots used in these studies are as follows: β-catenin , Axin2 , c-Myc , TOP2A , CDK1 , P53 , P21 , CDK4 , and GAPDH  were used in this study. After three washes in TBS containing 0.1% Tween 20 (TBST), membranes were incubated with HRP-conjugated antibodies. The following products were used in these experiments: goat anti-rabbit IgG (H + L) secondary antibody , HRP-linked anti-mouse IgG . Membranes were subsequently incubated with ECL-enhanced chemiluminescence reagent  for 1–2 min and exposed to the Bio-Rad ChemiDoc™ XRS System .The cells were lysed in an appropriate volume of lysis buffer  for 5 min and collected by centrifugation. According to the kit  instructions, the permeabilization solution (10 μL) and DNA staining solution (1 mL) were added, vortexed for 5–10 s, mixed evenly, and stained for 30 min in the dark. Cells were then sorted by fluorescence-activated cell sorter scan , according to the manufacturer’s instructions . The percentages of cells in each cell cycle phase were analyzed by ModFit LT™ 3.1 software . Each experiment was performed in triplicate. Cells were plated at a density of 5 × 104 cells/well on slides in 24-well plates and cultured at 37 °C for 24 h, followed by the addition of CX258 in DMSO to each well to give a final concentration of 5 μM. These cells were cultured for another 24 h. The slides used to analyze these lysed cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min, washed again with PBS, and permeabilized with 0.5% Triton X-100. Each well was blocked for 30 min with 10% bovine serum albumin (BSA). The primary antibody  was diluted in PBS containing 1% bovine serum albumin and incubated overnight at 4 °C. The secondary antibody  was diluted with 1% bovine serum albumin and incubated for 2 h at 25 °C. DAPI staining  was performed for 15 min. Images were captured using an ImageXpress Micro Confocal microscope .Cells were seeded at a density of 4 × 10https://string-db.org/cgi/input.pl (accessed on 2 April 2022)) [RNA-Seq was performed to evaluate the differential gene expression profiles of LS174T cells treated with 5 μM CX258 or DMSO for 24 h, and the significantly dysregulated mRNA levels were verified by qRT-PCR. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to predict the potential mechanisms of action of CX258 in colon cancer cells. The functional protein association network was constructed by STRING (l 2022)) . 6 cells/well in 6-well plates and treated with DMSO or inhibitors in DMSO. After 24 h, the cells were collected, and total RNA was extracted using the Eastep® Super Total RNA extraction kit . cDNA was synthesized from 1 μg of total RNA using a Reverse Transcription Kit , according to the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was performed using ChamQ SYBR qPCR Master Mix  on a real-time PCR system . Thermal cycling was conducted at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and then at 60 °C for 1 min. The melting program was as follows: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Relative quantification of gene expression in each sample was analyzed using the 2−ΔΔCt method. Each experiment was performed in triplicate. We designed specific primers for TOP2A , CDK1 , and GAPDH .Cells were seeded at 1 × 10siRNA-TOP2A was designed and synthesized  in order to knock down TOP2A expression. Transfection was performed using Lipofectamine 2000 , according to the manufacturer’s instructions. The sequences of the siRNAs: si-TOP2A-T765 .si-TOP2A-T2698 .si-TOP2A-T3476 .6 cells were subcutaneously injected into the back of NOD/SCID mice (2 tumors per mouse). When the subcutaneous tumor volume reached 5 × 5 mm3, the mice were divided into a control group and a treatment group (with 75 mg/kg CX258). Every day, CX258 was intraperitoneally (i.p.) administered to the treatment group at a dose of 75 mg/kg body weight. Tumor volumes were measured and recorded every 3 days. Mouse weights were measured twice a week, and the experiment was stopped on day 15. At the end of the experiment, the mice were euthanized. Tumors were isolated, weighed and fixed in 10% neutral-buffered formalin for IHC analysis.NOD/SCID mice (6–8 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. , housed under specific pathogen-free (SPF) conditions , and allowed free access to food and water during the study period. DLD-1 cells in the exponential growth phase were trypsinized and resuspended in 50% Matrigel . Next, 2 × 102O2 peroxide solution in the dark to block endogenous peroxidase. Goat antiserum was used for blocking at 25 °C for 30 min. The sections were incubated with primary antibodies  to detect the expression levels of target proteins in the tissues. The tissue slices were incubated overnight at 4 °C and washed thoroughly with PBS. The secondary antibody (Zhongshanjinqiao SP9000) was incubated at 25 °C for 1 h. A DAB chromogenic solution  was used to stain the cells. The sections were placed in hematoxylin solution to restore the nucleus. The samples were then dehydrated, sealed, observed and photographed under a microscope.Tissue sections were dewaxed, rehydrated, and heated in a microwave in sodium citrate buffer  for antigen extraction. The tissue slices were incubated with 3% Ht-test and one-way ANOVA, and p < 0.05 was considered statistically significant. Statistical significance is represented by * if p < 0.05, ** if p < 0.01, and *** if p < 0.001.Data are presented as mean ± SD. Statistical significance across the groups was calculated using Student’s two-tailed We screened 300+ synthetic, semisynthetic or naturally occurring chalcones using a Wnt reporter assay. We identified a family of chalcone derivatives that inhibited Wnt signaling reporter and Wnt signaling downstream genes, including c-Myc and Axin2 A–C. Howe50 = 0.42 ± 0.01 μM), HCT116 (IC50 = 0.34 ± 0.09 μM), HT29 (IC50 = 0.65 ± 0.08 μM) and DLD-1 (IC50 = 0.32 ± 0.04 μM)  A. CX258 0.04 μM) . The eff0.04 μM) B. In add0.04 μM) C. P53 isCRC cell lines LS174T, DLD-1, HCT116, and HT29 were treated with DMSO or CX258 (5 μM), and immunofluorescence cytochemistry was performed to examine β-catenin expression and location. CX258 reduced β-catenin levels in the nucleus of DLD-1 and HT-29 cells but not in either LS174T or HCT116 cells A. We alsTo investigate the mechanisms of CX258 on Wnt/β-catenin signaling, we performed several TOPFlash reporter assays by activating Wnt signaling with Wnt3A, wild-type β-catenin, β-catenin (S33A) mutants, and the LEF-VP16 fusion protein, respectively. We found that CX258 inhibited Wnt3A, wild-type β-catenin and β-catenin (S33A) induced TOPFlash reporter activities, but CX258 was less effective for LEF-VP16 fusion protein-induced TOPFlash reporter activity C. This fLS174T cells were treated with CX258, and the gene expression profile was analyzed by RNA-Seq . A totalTo test the effects of TOP2A on Wnt/β-catenin signaling, we knocked down TOP2A in DLD-1 and LS174T cells by siRNAs. Three siRNAs specifically targeting TOP2A  were transfected into DLD-1 and LS174T cells. The silencing effects were confirmed by qRT-PCR and western blot analyses A,B. Next3. Tumor volumes and vital signs were monitored and recorded daily. As shown in To evaluate the tumor-suppressive potential of CX258 in vivo, an NOD/SCID mouse model of colon cancer transplanted with DLD-1 cells was established. CX258 was administrated intraperitoneally (i.p.) at a dosage of 75 mg/kg/day for two weeks, at which point the tumors reached a size of 5 × 5 mmCRC is one of the leading causes of cancer-related deaths in the world. Almost all types of CRC are characterized by an unusually active Wnt/β-catenin pathway, which is considered a critical cancer initiation and driver event and an important target for CRC treatment ,36. ChalIn addition, CX258 blocked the cell cycle at the G2/M phase and inhibited CRC cell proliferation A–C. The In summary, we identified CX258 as a novel inhibitor of the TOP2A/Wnt/β-catenin signaling . TOP2A iIn accord with the University of Kentucky policy, the information in this publication was disclosed to the Office of Technology Commercialization, and a provisional patent  was jointly filed by the University of Kentucky Research Foundation and Hebei Normal University.


Eremochloa ophiuroides (Munro) Hack.) is commonly used as a low-maintenance warm-season turfgrass owing to its excellent adaptation to various soil types. A better understanding of the genetic diversity pattern of centipedegrass is essential for the efficient development and utilization of accessions. This study used fifty-five pairs of primers to detect the genetic variation and genetic structure of twenty-three wild centipedegrass accessions by Sequence-related amplified polymorphism (SRAP) markers. A total of 919 reliable bands were amplified, among which 606 (65.80%) were polymorphic and 160 (2.91%) were the monomorphic loci. The average polymorphic information content (PIC) value was 0.228. The unweighted pair group method with arithmetic mean (UPGMA) clustering analysis grouped the twenty-three accessions into two clusters. Meanwhile, the structure analysis showed that the tested accessions possessed two main genetic memberships (K = 2). The Mantel test significantly correlated the genetic and geographic distance matrices . Furthermore, geographical groups showed moderate genetic differentiation, and the highest intragroup genetic diversity was found in the Sichuan group (He = 0.201). Overall, the present research findings could promote the protection and collection of centipedegrass and provide comprehensive information to develop novel breeding strategies.Centipedegrass ( Eremochloa ophiuroides (Munro) Hack.] is a perennial warm-season diploid grass species (2n = 2x = 18) that belongs to the genus Eremochloa in the family Poaceae. Centipedegrass originated in southwest China, and the wild population is reportedly mainly distributed in the southern Yangtze River region of China , Chongqing municipality (n = 6), abroad (n = 1), and other parts of China (n = 7)  , Fig. S1 (n = 7) . We diviThe above seven morphological traits are measured as follows: For EBLL and EBLW, five mature leaves were randomly selected from upright branches and the length and width (at the widest point) of the leaves were determined using a vernier caliper. For SLL and SLW, five mature leaves were randomly selected from the stolons and the length and width(at the widest point) of the leaves were determined using vernier calipers. For GLH, randomly measure the natural height of the grass layer and repeat the measurement 5 times. For SIL and SID, random five healthy stolons were selected to determine the internode length and diameter in the middle of stolons.2O and stored at −20 °C for PCR amplification.Genomic DNA was extracted using a Plant Genomic DNA Extraction Kit. The concentration of DNA was detected by ultramicro spectrophotometer. Completely tested DNA samples were diluted to 10 ng/µL with sterile ddH−1), MIX 7.5 µL , upstream and downstream primer 0.3 µL (10 µmol L−1) each, ddH2O 3.6 µL, and Taq enzyme 0.3 µL. The SRAP-PCR reaction was performed as follows: predenaturation at 94 °C for 5 min, 5 cycles of denaturation at 94 °C for 1 min, annealing at 35 °C for 1 min, stretching at 72 °C for 1 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, 72 °C for 1 min, final extension at 72 °C for 10 min and storage at 4 °C. The PCR products were separated by 6% modified polyacrylamide gel and detected by silver staining. Gel clear photographs were used for the following analysis.A total of 215 pairs of SRAP primers were randomly combined to screen polymorphic primers for twenty-three wild centipedegrass accessions. SRAP amplification system: 15 µL SRAP reaction system: DNA template 3 µL  matrix was generated for statistical software analysis. The number of polymorphic bands (NPB), percentage of polymorphic bands (PPB), marker index (MI), and resolution (RP) were calculated to evaluate the ability of SRAP primers to identify marker differences. PIC was used to evaluate the value of markers for detecting population polymorphism. PIC was calculated by the following formula: e allele . The Gene allele  was usede allele . Otherwie allele , with po K value .Fifty-five pairs of qualified primers were screened from 215 pairs of primers, and the polymorphism of 23 wild centipedegrass accessions germplasm resources was evaluated. The results showed that the number of reliable bands amplified by each primer pair was seven (M14E07)–23(M01E07), and a total of 919 reliable bands were amplified. The polymorphic bands per primer pair ranged from 16.67% (M07E07) to 90% (M01E20 and M17E10), with an average of 65.8%. The polymorphism and recognition ability of primers were evaluated by PIC, MI and RP. The average PIC value was 0.228, and the PIC value of primer M12E19 was the highest (0.312). The average MI and RP values were 1.85 and 5.40, respectively, indicating the high utility of the primers , Fig. S2Based on the  matrix, UPGMA analysis showed that all accessions could be divided into two clusters . ClusterI = 0.312)  . The ana= 0.312) . The gen= 0.312) .r =  − 0.0003, p = 0.5093) , BIO15  , and BIO17 (precipitation in the arid region)  were highly correlated with genetic distance .Mental analysis showed no correlation between the genetic and morphological distance matrices ( 0.5093) . When thdistance . At the Compared with other molecular markers, SRAP has the advantages of simplicity, high efficiency, high yield, and good repeatability . The preIt is widely acknowledged that the primer efficiency index represents the overall utility of a specific primer during the identification of many accessions; higher values are associated with higher efficiency and amount of information on primers. In this study, the average MI (1.85) and RP (5.40) values of primers were higher than the SRAP and EST-SSR markers in prairie grass  . BesidesHemarthria compressa (vs. 0.165), which may be attributed to the collection of resources from only five provinces These provinces were from the southeast region, which led to low genetic diversity. The twenty-three wild centipedegrass accessions in the present study were collected from a wide geographical distribution, accounting for their higher genetic diversity. Accordingly, the high genetic diversity of centipedegrass germplasm may be related to its geographical distribution and biological characteristics. Indeed, centipedegrass is a perennial cross-pollination plant with self-incompatibility  than the average value (0.104) of ompressa , since ttibility . Furthertibility .Elymus nutans , indicating that geographical isolation led to genetic differentiation, similar to findings reported by Capsicum annuum. The reason may be that, first of all, the number of molecular markers obtained in this experiment is small, the number of effective loci is small, and most of the traits are usually regulated by many alleles, so there is a great chance that these loci will not be associated with traits  M15 + E03 (left), M15 + E08 (right); (B) M9 + E14 (left), M9 + E20 (right) (accessions 1 to 23 from left to right).Click here for additional data file.10.7717/peerj.15900/supp-3Figure S3Click here for additional data file.10.7717/peerj.15900/supp-4Figure S4Click here for additional data file.10.7717/peerj.15900/supp-5Figure S5Click here for additional data file.10.7717/peerj.15900/supp-6Supplemental Information 6Raw data for figure 2Click here for additional data file.10.7717/peerj.15900/supp-7Supplemental Information 7Raw data for Figure 1 and Table 1-4.Click here for additional data file.10.7717/peerj.15900/supp-8Supplemental Information 8p < 0.01), *: Significant difference (p < 0.05).Statistical results for Figure 2. Note: **: Very significant difference (Click here for additional data file.10.7717/peerj.15900/supp-9Table S1Click here for additional data file.10.7717/peerj.15900/supp-10Table S2Click here for additional data file.10.7717/peerj.15900/supp-11Table S3Click here for additional data file.10.7717/peerj.15900/supp-12Table S4Click here for additional data file.10.7717/peerj.15900/supp-13Table S5Click here for additional data file.10.7717/peerj.15900/supp-14Table S6Click here for additional data file.10.7717/peerj.15900/supp-15Table S7SC: from Sichuan province, CQ: from Chongqing municipality, OT: Other accessions except Sichuan and Chongqing.Click here for additional data file.10.7717/peerj.15900/supp-16Table S8Click here for additional data file.


Our findings offer an alternative approach for the discovery of SARS-CoV-2 antivirals, as well as application for broad-spectrum treatment of viral pathogenic infections with similar entry pathways.Since 2019, SARS-CoV-2 has evolved rapidly and gained resistance to multiple therapeutics targeting the virus. Development of host-directed antivirals offers broad-spectrum intervention against different variants of concern. Host proteases, TMPRSS2 and CTSL/CTSB cleave the SARS-CoV-2 spike to play a crucial role in the two alternative pathways of viral entry and are characterized as promising pharmacological targets. Here, we identify compounds that show potent inhibition of these proteases and determine their complex structures with their respective targets. Furthermore, we show that applying inhibitors simultaneously that block both entry pathways has a synergistic antiviral effect. Notably, we devise a bispecific compound,  TMPRSS2 and CTSL/CTSB, host proteases that facilitate SARS-CoV-2 entry, are promising drug targets. Here the authors simultaneously inhibit these host proteases and see synergistic antiviral effects, offering a broad-spectrum intervention against SARS-CoV-2 variants. However, the continuously emerging SARS-CoV-2 variants, especially Omicron subvariants, have the ability to evade the immune system when challenged by antibody-mediated neutralization or vaccine protection15. In addition, five antiviral agents have received authorization for COVID-19 treatment: remdesivir3, molnupiravir4, azvudine5, nirmatrelvir6, and ensitrelvir7. The first three target the RNA-dependent RNA polymerase (RdRp), and the last two target the main protease (Mpro). However, in vitro and in vivo studies have indicated that mutations in the SARS-CoV-2  RdRp confer resistance to remdesivir17, and Mpro mutations cause resistance to nirmatrelvir18, suggesting the inevitable risk of drug resistance is already upon us. Unlike viral proteins, which are susceptible to mutation during SARS-CoV-2 evolution, host proteins are much more conservative and could potentially be used as therapeutic targets for the development of broad-spectrum antiviral compounds. Therefore, a viable solution for persisting drug resistance is to develop chemical compounds targeting host proteins that are indispensable to the viral life cycle.The SARS-CoV-2 pandemic has imposed a severe public health burden worldwide since 2019. For the prevention and treatment of COVID-19, vaccines23, viral entry is the primary step for infection and is already recognized as an excellent target for drug development. The entry of SARS-CoV-2 depends on two distinctive pathways, cell surface entry and endosomal entry24, where the transmembrane serine protease, TMPRSS2, and endosomal cysteine proteases cathepsin L/B (CTSL/CTSB) cleave the viral spike protein26 , including Delta, TMPRSS2-dependent cell surface entry is the primary pathway31, whereas the Omicron variant uses endosomal entry as the primary pathway, which increases its infectivity on more cell types in the respiratory epithelium and thus enhances its intrinsic transmissibility29. Moreover, SARS-CoV-2 infection is sensitive to TMPRSS2 targeting inhibitors in human Calu‐3 lung adenocarcinoma cells but not human A549 epithelial lung cells27, demonstrating the polytropic preference for viral entry pathways. Therefore, to completely prevent SARS-CoV-2 infection, the simultaneous blocking of the two independent pathways for viral entry is essential33.Despite the identification of numerous host factors associated with the SARS-CoV-2 life cyclen26 Fig. . Previou212-148, which shows dual-inhibition of both TMPRSS2 and CTSL/CTSB. We also determine the structure of 212-148 bound to TMPRSS2 or CTSB. Cell-based assays show that this compound could prevent infection of both the Delta and Omicron variants. Our results validate that dual-inhibitors for SARS-CoV-2 entry targeting both TMPRSS2 and CTSL/CTSB are promising candidates for antiviral drug discovery.In this study, we establish a fluorescence resonance energy transfer (FRET) assay to measure the enzymatic activity of TMPRSS2 and CTSL/CTSB, and to carry out high-throughput screening of compounds among a library of 10,000 approved drugs, clinical-trial drug candidates, and natural products. The crystal structures of TMPRSS2 and CTSL/CTSB in complex with the potent inhibitors were determined to reveal their specific mode of interaction. Notably, antiviral assays show that the combination of two compounds, nafamostat, and K777, which simultaneously blocked TMPRSS2- and CTSL/CTSB-mediated viral entry pathways exhibited synergistic antiviral activity against SARS-CoV-2, indicating that dual-inhibition is an effective strategy for clinical development to treat COVID-19. Based on the structural and antiviral information, we designed the bispecific compound 35. The zymogen was purified and activated by enteropeptidase cleavage for activity assay. The enzymatic activity of TMPRSS2 was determined by a continuous kinetic assay based on FRET, using Boc-Gln-Ala-Arg-AMC as the fluorogenic substrate36. The catalytic efficiency (kcat/Km) for TMPRSS2 was measured to be 971,000 s−1·M−1, demonstrating the enzyme is fully active   Fig. . Nafamos38. As a result, two inhibitors, K777 and E64d were identified with an IC50 value of 5.43 and 88.85 nM for CTSL, and 174.30 and 244.26 nM for CTSB  of 1.44 Å over 378 Cα atoms, indicating the two protomers are similar. The ectodomain of TMPRSS2 can be traced in the electron density map except for several small loop regions . And the ectodomain of TMPRSS2 is comprised of three subdomains: an N-terminal low-density lipoprotein receptor type-A (LDLRA) domain (residues 118–148), a scavenger receptor cysteine-rich (SRCR) domain (residues 149–242), and a C-terminal trypsin-like serine peptidase (SP) domain (residues 256–492) which harbours the Ser-His-Asp catalytic triad41 Fig. .Fig. 2Cr35, a major difference is observed. In the original structure, the LDLRA domain is invisible but it is clearly seen in our electron density maps. It consists mainly of random coil but there are several stabilizing loops. A canonical calcium ion stabilizes the structure by coordinating six conserved residues. Two disulfide bonds are formed around the calcium-binding region to further stabilize the overall structure of the domain  = 7,829, SI is calculated as the ratio of CC50 value against EC50 value, a common measurement for comparing cytotoxicity and antiviral potency of compounds) and 239.8 nM   and for nafamostat, camostat and UK-371804, the EC50 values are 26.3 nM , 109.1 nM  and 424.6 nM (SI = 405), respectively Fig. . In cont00) Fig. , which ivelyFig. . The inhvelyFig. . In conc50 value of 0.9 nM , more than 10-fold better than individual administration of either nafamostat or K777  and 235.2 nM , respectively. Furthermore, the TCID50 assay further indicated that viral replication could be completely blocked by 212-148, a feat that none of the single-targeted inhibitors discussed above could achieve . The values are low because only the same remnant moiety of guanidinobenzoyl group occupies the active site of TMPRSS2 after the reaction, although the complete structures of nafamostat and camostat are different Fig. . These T60. Therefore, dual-inhibition of TMPRSS2 and CTSL/CTSB offers a broad-spectrum anti-coronavirus strategy that is effective not only for the current SARS-CoV-2 pandemic but also for other potential coronavirus pandemics.Previous studies have suggested that TMPRSS2 and CTSL/CTSB inhibitors effectively prevent the infection of other coronaviruses, including SARS-CoV, MERS-CoV, and human coronavirus (HCoV)-229E62, which challenges the current antiviral treatment targeting the viral protease specific to one virus. Host protease TMPRSS2-based treatment could be a feasible strategy to cure Flurona. Our crystal structure of the complete TMPRSS2 ectodomain presented here provided a solid basis for further drug design and development.Other than fighting against coronavirus, the identified TMPRSS2 inhibitors can also be used to treat other diseases related to TMPRSS2. Entry of influenza virus is dependent on the TMPRSS2-mediated cleavage of the viral surface glycoprotein precursor HA0, making TMPRSS2 a key antiviral target. Therefore, drug development targeting TMPRSS2 also provides us with alternative solutions to treat influenza. In addition, coinfection with influenza virus (Flurona) would enhance SARS-CoV-2 infectivitypro and PLpro. As evidenced in the drug development for HIV or HCV, using an inhibitor cocktail is a highly effective strategy to treat infectious diseases64. The lead compounds we have identified here can serve as promising leads for use in combination with other antivirals against SARS-CoV-2.Although we have focused on dual inhibition of TMPRSS2 and CTSL/CTSB involved in host cell entry, our strategy is also applicable to the combination of inhibitors targeting different host targets involved in host entry and host antiviral response. Our findings also apply to combining different inhibitors targeting host and targeting virus, such as RdRp, MSpodoptera frugiperda (sf9) cells by the GENEWIZ. The PCR fragment containing ectodomain of TMPRSS2 (residues 109–492) was amplified and inserted into the pFastBac1 vector using restriction sites BamH I and Xho I, possessing an N-terminal GP64 (baculovirus envelope glycoprotein) signal peptide and C-terminal 10 × His tag. The sequence responsible for TMPRSS2 autoactivation (250SSRQSR255) was substituted with the enteropeptidase cleavage site (DDDDK). The final plasmid was transformed into E. Coli (DH10 Bac strain) competent cells to generate the recombinant bacmid DNA.The coding gene for human TMPRSS2 was synthesized and codon-optimized for expression in the E. coli. The DNA segments of CTSL and CTSB were amplified and subcloned into the pET28a vector using restriction sites Nco I/BamH I and Nde I/BamH I, respectively.The genes coding human CTSL (residues 18–333) and CTSB (residues 18–333) were synthesized from GenScript and the sequence was codon-optimized for expression in 6 cells/ml using Cellfectin II Reagent (Invitrogen) according to the manufacturer’s instructions. After 72 h post-infection, P0 viral stock was collected and continually amplified for the production of higher-titer P1 to P3 viral stock. High Five cells were selected for TMPRSS2 overexpression. Four liters of High Five cells cultured in the Sf-900™ II SFM (Thermo Fisher Scientific) were infected with the P3 virus at a cell density of 2.5 × 106 cells/ml. TMPRSS2 was expressed and secreted outside cells for 4 days after baculovirus infection at 27 °C under constant shaking.The resulting bacmid was transfected into sf9 cells at a cell density of 2.0 × 10g, 15 min) to remove the cell pellet. The supernatant was concentrated to 300 mL and diluted with TBS , followed by Ni-NTA affinity chromatography purification. The protein bound to the Ni-NTA column was washed using TBS supplemented with 30 mM imidazole before elution using TBS supplemented with 250 mM imidazole. The eluted sample was concentrated to 2 mg/ml and exchanged to Reaction buffer , which is suitable for enteropeptidase (NEB) digestion to activate the TMPRSS2 zymogen. After incubation with enteropeptidase (15 U/mg TMPRSS2) and PNGase F  overnight at 20 °C, the activated sample was concentrated to load onto the Superdex 75 Increase 10/300 GL column  in gel filtration buffer . The pooled fractions containing the target protein were concentrated and stored at −80 °C for further use.Cell culture supernatant was harvested by centrifugation  cells and then bacteria were cultured in the Luria broth (LB) at 37 °C until the OD600 approached 0.6-0.8. Overexpression of proteins was induced by the addition of 0.4 mM IPTG . Then the harvested cells were resuspended in lysis buffer  and disrupted by the high-pressure homogenizer at 4 °C. After centrifugation, the inclusion bodies of CTSL/CTSB were collected and sequentially washed with buffer A  and buffer B . Subsequently, the final inclusion bodies were denatured in buffer C ) at a concentration of 2.5 mg/mL and shaken vigorously for 1 h.Both CTSL/CTSB were overpressed as inclusion bodies in the The refolding of CTSL/CTSB was performed using a sample pump with a flow rate of 0.1 mL/min to drip solubilized inclusion bodies into 3 L refolding buffer , 10 mM GSH, 1 mM GSSG) at 4 °C, stirred simultaneously overnight. Then the solution containing folded enzymes was filtered, concentrated and dialyzed against dialysis buffer  at 4 °C.4)2SO4 before purification through hydrophobic interaction chromatography (Phenyl Beads 6FF column (High Sub), Cytiva, USA) using linear gradient elution. Fractions containing CTSL were pooled and incubated in the activation buffer A  for 1 h at 37 °C to auto-activate the zymogen. For CTSB, the protein solution was loaded on HiTrap Q column equilibrated with Buffer A  and eluted with a linear gradient to Buffer B . Fractions containing CTSB were collected and adjusted to pH 3.5 by adding 1 M formic acid and pepsin at the molar ratio of 1: 100 relative to CTSB for activation. Finally, the activated CTSL/CTSB was concentrated and loaded on a Superdex 75 Increase 10/300 GL column  in gel filtration buffer . The pooled fractions containing the target protein were concentrated and stored at −80 °C for further use.After refolding, the CTSL/CTSB adopted a different strategy for sequent purification and activation. The folded CTSL solution was brought to a final concentration of 1.2 M  incubated with camostat, nafamostat, UK-371804 or The purified and activated CTSL at a concentration of 8 mg/mL was incubated with E64d or K777 at a molar ration of 5:1 for 2 h at room temperature. Crystals were obtained by sitting drop vapor diffusion at 16 °C, with the reservoir solution consisting of 100 mM citric acid, pH 3.5 and 3 M sodium chloride for CTSL-E64d complex; the reservoir solution consisting of 9% (v/v) 2-propanol, 90 mM sodium cacodylate/ hydrochloric acid pH 6.5, 180 mM zinc acetate, and 0.5%w/v n-dodecyl-N,N-dimethylamine-N-oxide  for the CTSL-K777 complex.212-148 at a molar ration of 10:1 overnight at 4 °C. Crystals were obtained by sitting drop vapor diffusion at 16 °C, with the reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate (pH 4.6) and 30% w/v polyethylene glycol 4,000 for CTSB-E64d complex; the reservoir solution consisting of 17.1 % v/v polyethylene glycol 600, 50 mM MES (pH 5.6) and 8.6 % w/v polyethylene glycol 4,000 for CTSB-K777 complex; the reservoir solution consisting of 0.2 M magnesium chloride hexahydrate, 0.1 M sodium acetate (pH 5.2) and 28 % w/v PEG 3350 for CTSB-212-148 complex. All the crystals above were cryo-protected by reservoir solutions supplemented with 20% glycerol and flash frozen in liquid nitrogen for further data collection.The purified and activated CTSB at a concentration of 5.5 mg/mL was incubated with E64d, K777 or 65. For structures of TMPRSS2 in complex with inhibitors, the phases were determined by the molecular replacement using a coordinate file of TMPRSS2 in complex with nafamostat (PDB ID: 7MEQ) as a search model in Phenix66. Similarly, the structures of CTSL/CSTB in complex with inhibitors were determined by molecular replacement using models (PDB ID: 6EZX) and (PDB ID: 5MAJ) as templates, respectively. Subsequently, all the models were subjected to iterative cycles of refinement with Phenix66. The inhibitors were built manually according to the omit map with Coot67. Data collection and structure refinement statistics are summarized in Tables The X-ray diffraction data were collected at beamlines BL18U1, BL19U1, BL02U1 and BL10U2 at the Shanghai Synchrotron Radiation Facility (SSRF), China and beamline I04 of Diamond Light Source, UK. Data were indexed, integrated and scaled with XDSKm and kcat.The enzymatic activity of TMPRSS2 was measured by a continuous kinetic assay based on FRET, using the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (GL Biochem). The excitation and emission wavelengths of the fluorogenic substrate were 340 nm and 460 nm, respectively. Fluorescence intensity was monitored with an EnVision multimode plate reader (Perkin Elmer). The reaction assay was performed at a final volume of 50 µL in the buffer containing 20 mM Tris pH 7.4, 150 mM NaCl, 0.1 mg/ml BSA. The concentration of TMPRSS2 was set to 0.5 nM. The reaction was initiated by adding TMPRSS2 into a solution containing different concentrations of substrate (10–160 µM) and their fluorescence values were recorded immediately. Initial velocities were calculated by fitting the linear portion of the curves to a straight line. The data were analyzed by non-linear regression analysis in GraphPad Prism version 9.4 (Dotmatics) to generate the kinetic parameters Potential inhibitors against human TMPRSS2 were screened by an enzymatic inhibition assay in vitro. High-throughput drug screening was performed on about 10000 compounds from five drug libraries, the Approved Drug Library (Target Mol), Clinic Compound Library (Target Mol), FDA-approved Drug Library (Selleck), Natural Product Library (Selleck), and Anti-virus Drug Library . The screening assays were performed in 384-well black microplates (PerkinElmer) at a total volume of 50 µL. Bravo Automated Liquid Handling Platform (Agilent) was used to rapidly add the compounds into the enzymatic reaction mixture. The final reaction system adopted the enzymatic assay buffer supplemented with 0.1 mg/mL BSA, 0.01% v/v Triton X-100, including 0.5 nM TMPRSS2, 10 μM substrate, and 10 μM compounds. DMSO treatment was used as a negative control. The initial velocities were changed by the addition of compounds, and compared with the control to evaluate their inhibitory effect. The compounds with inhibition over 90% were defined as hits and picked up for further testing. The CTSL/CTSB adopted a similar high-throughput drug screening approach with their specific substrate  at a final concentration of 10 μM. The reaction systems also used their enzymatic conditions supplemented with 0.1 mg/mL BSA, 0.01% v/v Triton X-100. All experimental statistic was analyzed using GraphPad Prism 9.4.50 assay buffer  and the IC50 values of TMPRSS2 inhibitors were measured using 0.5 nM TMPRSS2 and 10 μM substrate in the presence of different concentrations of compounds. Fluorescence intensity was monitored with an EnVision multimode plate reader (Perkin Elmer) and the initial velocities were obtained by fitting the linear portion of the curves to a straight line. IC50 was derived by fitting a nonlinear regression curve in GraphPad Prism version 9.4 (Dotmatics). For CTSL/CTSB, the general process is similar except the buffer was changed to 0.4 M acetic acid/sodium acetate pH 5.5, 4 mM EDTA, 5 mM DTT, 0.1 mg/mL BSA, 0.01% v/v Triton X-100.TMPRSS2 was diluted in IC68. The measurements were performed using 10 μL of a suspension of TMPRSS2  in a capillary placed in the sample holder. The temperature was increased from 20 to 90 °C at a ramp rate of 1 °C/min, with one fluorescence measurement taken at every 0.025 °C increments. The ratio of the recorded emission intensities (Em350nm/Em330nm) was plotted as a function of temperature, and the fluorescence intensity ratio and its first derivative was calculated using the manufacturer’s software . This ratio represents the change in TRP fluorescence intensity and the shift in emission maximum to higher wavelengths (“red-shift”) or lower wavelengths (“blue-shift”). For each condition, we conducted three independent measurements.Using the Prometheus NT.48 instrument from NanoTemper Technologies, we conducted the real-time simultaneous monitoring of intrinsic tryptophan fluorescence (ITF) at 330 nm and 350 nm, and the excitation wavelength is 280 nm70. In brief, the gene encoding spike with the substation of proline at residues 986 and 987 was synthesized and inserted into the pcDNA3.1 vector for expression. After expression for 3 days, the supernatant was collected, and soluble spike protein was purified by Ni-NTA affinity chromatography. Spike protein was further purified via gel filtration chromatography with a Superose 6 10/300 column  in a PBS buffer (pH 7.4). Spike protein lacking the S1/S2 site was obtained through the replacement of the 682RRAR685 with GSAS. Both spike proteins with or without the S1/S2 site were concentrated at 1.5 mg/mL and incubated with 1.5 μM TMPRSS2, respectively, in the assay buffer . For cleavage inhibition assay, TMPRSS2 was preincubated with 10 times molar 212-148 for 1 h. When digested at 0, 1,5,15, 30, and 60 min, the SDS-PAGE samples under each condition were immediately prepared and further visualized in the gels by Coomassie blue. Additionally, nafamostat served as a positive control, and SARS-CoV-2 Mpro served as a negative control in these assays.Spike protein of the SARS-CoV-2 Delta variant was cloned, expressed and purified as reported previously2, and have been tested negative for mycoplasma infection.Huh-7  cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM); Human Calu-3  cells were cultured in Minimum Essential Medium (MEM). All media were supplemented with 10% fetal bovine serum (FBS) and containing 100 IU/mL penicillin and 100 μg/mL streptomycin. All cells were cultured at 37 °C in a fully humidified atmosphere containing 5% CO50. SARS-CoV-2 Delta, Omicron BA.1.1 and BA.2 stocks used in the experiments had undergone three, four and two passages on Vero E6 cells and were stored at −80 °C, respectively. All of the infection experiments were performed at BSL-3 in Guangzhou Customs Inspection and Quarantine Technology Center (IQTC).SARS-CoV-2 Delta variant , Omicron BA.1.1 variant  and Omicron BA.2-3 variant  were propagated in Vero E6 cells . Virus titers were determined with 10-fold serial dilutions in confluent Vero E6 cells in 96-well microtitre plates. Three days after inoculation, a cytopathic effect (CPE) was scored, and the Reed-Muench formula was used to calculate the TCID4 cells in 100 μl culture medium were seeded into opaque-walled 96-well plates for 24 h and 100 μl of Cell Titer-Glo reagent was added to each well. After 5-minutes shaking and 10-minutes incubation, luminescence was measured by GloMax 20/20 (TurnerBio Systems). The half-cytotoxic concentration (CC50) was assessed in the absence of viruses after being cultured for one or two days.Cell viability was evaluated using a Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer’s instructions. In brief, 2 × 104 Huh-7 cells were seeded in a 96-well plate and pre-treated with different doses of the compounds for one hour. The compounds with different dilution concentrations were mixed with SARS-CoV-2 (650 TCID50/well), and 200 μL mixtures were inoculated onto monolayer Huh-7 cells. Chemiluminescence signals were detected twenty-four hours after the incubation of cells and virus at 37 °C with 5% CO2. The Britelite plus reporter gene assay system  and PerkinElmer Ensight luminometer were used for signal collection. The inhibition of compounds and the values of EC50 are calculated from the luciferase level of pseudotyped SARS-CoV-2. Two independent experiments were performed with triplicate infections and one representative is shown.SARS-CoV-2-S pseudotyped virus Omicron BA.2 (DR-XG-C011), Omicron BA.2.12.1 (DR-XG-C015) and Omicron BA.4/BA.5 (DR-XG-C013) were purchased from Guangzhou DARUI Biotechnology Co., Ltd. The VSV-based pseudotyped SARS-CoV-2 variants were produced by transfecting 293 T cells  with spike protein expression plasmids and simultaneously infected with G*ΔG-VSV . 2 × 105 Calu-3 cells were seeded in a 24-well plate and pre-treated with different doses of the compounds for one hour. The compounds with different dilution concentrations were mixed with SARS-CoV-2 (MOI = 1), and 500 μL mixtures were inoculated onto Calu-3 cells. Two hours after inoculation, the wells were extensively washed with PBS, and then inoculated with different dilution concentrations compounds for twenty-four hours. The supernatants were collected and virus titers were determined with 10-fold serial dilutions in confluent Vero E6 cells in 96-well microtitre plates. Three days after inoculation, CPE was scored using Celigo Image Cytometer, and the Reed-Muench formula was used to calculate the TCID50. The inhibition of compounds and the values of EC50 were calculated from SARS-CoV-2’s titers. Three independent experiments were performed with triplicate infections and one representative is shown.5 × 10212-148 was synthesized through ten steps reaction as shown in the scheme  instrument. The deuterated solvents employed were purchased from Energy Chemical. Chemical shifts were given in ppm with respect to referenced TMS peaks. Spectra were analyzed with MestReNova. High-resolution mass spectra (HRMS-ESI) were obtained on an ABsciex 4600.3 solution and dried by sodium sulfate. Solvent was removed under vacuum. The residue was purified by Flash Column Chromatography (using EA in PE from 20%–50%) to obtain the desired product 212-11171. Yield = 67.8%To a solution of boc-L-homophenylalanine  in THF (100 ml), EDCI , HOBt  and DIPEA  were added. This mixture was cooled to 0 °C and stirred for 10 min. Then N,O-Dimethylhydroxylamine hydrochloride  was added. The reaction mixture was diluted by water after stirring overnight and extracted by EA. The organic layer was washed with saturated NaHCO212-111  in dry THF, LiAlH4  was added slowly within 10 min under 0 °C. The reaction mixture was stirred for 30 min under this temperature. After quenched by H2O, diluted HCl (1 M) was added to adjust pH to 6. The mixture was extracted by EA for 3 times. The organic layer was collected and washed by saturated NaHCO3 solution, and dried by sodium sulfate. After removing the solvent by vacuum, the residue was purified by Flash Column Chromatography (using EA in PE from 20%–50%) to obtain the desired product 212-11471. Yield = 89%.To a cooled solution of 212-114 was then added to the reaction mixture and allowed to stir for a further hour. The mixture was concentrated under vacuum. The residue was diluted by H2O and extracted by EA. The organic layer was collected and washed by saturated NaHCO3 solution, brine respectively and further dried by Na2SO4. The residue was purified by Flash Column Chromatography (using EA in PE from 20%–50%) to obtain the desired product 212-11771. Yield = 53.8%To a suspension of NaH  in THF (60 ml), the solution of diethyl p-[(phenylsulfonyl)methyl]phosphonate  in THF (10 ml) was added dropwise under 0 °C. This reaction mixture was stirred for 30 min under this temperature. 212-117  in DCM (5 ml), TFA (5 ml) was added. This reaction mixture was stirred for 3 h and the solvents were removed under vacuum. The crude product was purified by HPLC. Yield = 93.6%. ESI-HRMS calcd for C17H29NO2S [(M + H)+]: 302.1215 found: 302.3073. 1H NMR  δ 7.99 – 7.93 , 7.77 – 7.70 , 7.65 , 7.25 , 7.21 – 7.14 , 7.13 , 7.01 , 6.94 , 4.03 , 2.68 – 2.53 , 2.16 , 2.11 – 1.99 . 13C NMR  δ 139.61, 139.56, 139.30, 135.73, 133.97, 129.48, 128.38, 127.99, 127.70, 126.19, 50.85, 33.81, 30.83 , triphosgene  was added in one portion under 0 °C. This reaction mixture was stirred for 15 min under this reaction. The organic layer was collected and washed with brine, dried (Na2SO4) and concentrated under vacuum. The residue was used immediately in the next step without any further purification.To a solution of L-Phenylalanine methyl ester  in the mixture of DCM (2 ml) and saturated NaHCO212-111. Yield = 42%. ESI-HRMS calcd for C17H26N3O4 [(M + H)+]: 336.1923 found: 336.3982.The crude product in THF (3 ml) was added to a solution of 1-piperazineethanol  and DIPEA  in THF (3 ml) under 0 °C. The resulting solution was stirred under room temperature overnight. THF was removed under vacuum and the residue ws purified by Flash Column Chromatography (using MeOH in DCM from 0%–10%) to obtain the desired product 212-108  in THF (10 ml), LiOH ∙ H2O  in H2O (3 ml) was added slowly. The reaction mixture was monitored by TLC. After the reaction, HCl in dioxane (4 M) was added to adjust pH to 2 under 0 °C. Solvent was removed under vacuum and the residue was washed by ethyl ester. Organic solution was collected and concentrated under vacuum. The crude product was purified by HPLC. Yield = 79.0% ESI-HRMS calcd for C16H24N3O4 [(M + H)+]: 322.1767 found: 322.3612. 1H NMR  δ 7.30 – 7.22 , 7.17 , 4.40 , 3.88 , 3.70 – 3.56 , 3.20 , 3.18 – 3.07 , 3.01 . 13C NMR  δ 177.59, 157.50, 138.57, 129.18, 127.97, 126.12, 66.78, 58.75, 57.25, 55.83, 51.78, 41.15, 37.81  in DMF (8 ml), EDCI , HOBt , and DIPEA  were added. This reaction mixture was stirred overnight and purified by HPLC directly to obtain the desired product 212-127. Yield = 32.8%. ESI-HRMS calcd for C33H41N4O5S [(M + H)+]: 605.2798 found: 605.4355. 1H NMR  δ 8.22 , 7.88 – 7.82 , 7.73 – 7.65 , 7.61 , 7.25 – 7.19 , 7.15 , 6.79 , 6.09 , 4.55 – 4.46 , 4.43 , 4.21 – 4.09 , 3.90 – 3.84 , 3.61 – 3.52 , 3.30 – 3.18 , 3.10 – 3.00 , 3.00 – 2.89 , 2.72 – 2.62 , 2.62 – 2.52 , 1.96 – 1.85 , 1.85 – 1.74 . 13C NMR  δ 173.22, 157.20, 146.25, 140.97, 140.39, 137.06, 133.41, 130.04, 129.19, 128.98, 128.27, 128.22, 128.10, 127.33, 126.73, 125.74, 58.30, 56.95, 54.78, 51.49, 51.41, 49.35, 49.26, 40.71, 37.63, 35.10, 31.56  in DMF (1 ml) was added afterwards. The reaction mixture was stirred overnight and purified by HPLC to obtain the desired product 212-129. Yield = 28.8% ESI-HRMS calcd for C51H63N7O10S [(M + H)+]: 966.4435 found: 966.4485.To a solution of 4-[[Bis[[carbonyl]amino]methylene]amino]benzoic acid  in DMF (3 ml), EDCI , DMAP  and DIPEA  were added and stirred for 10 min under room temperature. 212-129  in DMF (2 ml), HCl  was added. The reaction was stirred for 2 h and purified by HPLC to obtain the desired product 212-148. ESI-HRMS calcd for C41H48N7O6S [(M + H)+]: 766.3387 found: 766.2722. 1H NMR  δ 8.22 , 8.16 – 8.10 , 7.87 – 7.81 , 7.73 – 7.66 , 7.61 , 7.40 – 7.35 , 7.24 – 7.19 , 7.14 , 6.77 , 6.07 , 4.71 – 4.65 , 4.52 – 4.45 , 4.43 , 3.62 – 3.54 , 3.38 – 3.33 , 3.02 , 2.94 , 2.65 , 2.56 , 1.95 – 1.85 , 1.84 – 1.73 . 13C NMR  δ 173.22, 157.20, 146.25, 140.97, 140.39, 137.06, 133.41, 130.04, 129.19, 128.98, 128.27, 128.22, 128.10, 127.33, 126.73, 125.74, 58.30, 56.95, 54.78, 51.49, 51.41, 49.35, 49.26, 40.71, 37.63, 35.10, 31.56 (Supplementary Fig. To a solution of Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource data