Staphylococcus aureus and ≈99% of Escherichia coli. Furthermore, ex vivo results show that IBV‐TENG implanted under porcine tissue successfully inactivates bacteria. This antibacterial technology is expected to be a countermeasure strategy against SSIs, increasing life expectancy and healthcare quality by preventing microorganisms of deep tissue.To prevent surgical site infection (SSI), which significantly increases the rate morbidity and mortality, eliminating microorganisms is prominent. Antimicrobial resistance is identified as a global health challenge. This work proposes a new strategy to eliminate microorganisms of deep tissue through electrical stimulation with an ultrasound (US)‐driven implantable, biodegradable, and vibrant triboelectric nanogenerator (IBV‐TENG). After a programmed lifetime, the IBV‐TENG can be eliminated by provoking the on‐demand device dissolution by controlling US intensity with no surgical removal of the device from the body. A voltage of ≈4 V and current of ≈22 µA generated from IBV‐TENG under ultrasound in vitro, confirming inactivating ≈100% of  A novel strategy of inhibiting microorganisms of deep soft tissue by electrical stimulation is presented through implantable, biodegradable, and vibrant triboelectric nanogenerator for being a potential treatment and prevention method of surgical site infection, especially against antimicrobial resistance, resulting in decreasing the morbidity and mortality rate of patients. The mortality rate can increase up to 11 times if postoperative SSI happens; furthermore, around half a million SSI cases are reported each year in the United States and costing $3–10 billion each year. Inhibiting the proliferation of microorganisms is one of the essential ways to prevent SSI. Since the development of penicillin in 1928, various antibiotics have been used to control microorganisms’ proliferation. With the emergence of antimicrobial resistance (AMR), the effectiveness of the existing antibiotics has been compromised, making this one of the most significant health problems of the 21st century. Furthermore, the emergence of COVID‐19 evoked rising concerns about the limitations of conventional pharmaceutics since highly resistive microbes and variants continue to emerge while developing a suitable vaccine or a cure for them in a short time is difficult. In other words, in the future, microorganisms that cannot be eliminated by drugs, could lead to serious pandemic situations and the concerns have evoked high demands for an alternative solution. Therefore, there is a need to explore a new non‐drug antibacterial technology rather than simply relying on medicines. Various studies have been conducted to control microorganisms through methods such as photodynamic therapy (PDT) and photothermal therapy (PTT). However, these antimicrobial therapy methods have their shortcomings such as limited selectivity of treating sites, low efficiency, and causing tissue damage due to high temperature and lighting effects. Sonodynamic therapy (SDT) is triggered by ultrasound (US), which has many characteristics including deep tissue penetration, non‐invasion and non‐ionization. However, the therapeutic efficiency of SDT is limited by the hypoxic microenvironment of the deep tissues of tumors or infection sites. In addition, sonothermal therapy (STT) is a developed the ultrasonic interfacial engineering of metal/semiconductor (sonosensitizers) interface antibacterial treatment. Nevertheless, the biocompatibility and biodegradability of sonosensitizers need to be developed because of toxicity and biosafety. Electrical stimulation (ES) is also one of the non‐drug methods used to inhibit and control the growth of microorganisms. Various studies have shown that an antibiotic effect can be realized using either direct current (DC) or alternative current (AC). Nevertheless, the reported strategies primarily apply electrical energy at the suture area close to the superficial wound, limiting their capability to control the microorganisms of deep tissues. Therefore, a novel approach is required to induce low‐voltage electrical energy to prevent SSI inside the body without any tissue damage.Microorganisms cause surgical site infection (SSI) during the postoperative wound healing process, leading to physical pain and economic burden to patients.2, 3−2 power), which confirmed that up to 100% of Staphylococcus aureus and 99% of Escherichia coli were eliminated under in vitro environment. Furthermore, ex vivo evaluation indicated inhibiting the bacteria effectively under porcine tissue. Our proposed technology is composed of biodegradable materials so that the device can be degraded, absorbed, and discharged from the body after the antibacterial activity of IBV‐TENG is completed. Furthermore, the degradation rate of the stable encapsulation layer can be accelerated under high ultrasonic intensity. This on‐demand transient property of IBV‐TENG does not require a removal surgery of the device from the body. The advantages of using IBV‐TENG based on transcutaneous US technique are it efficiently removes the bacteria in the surgical site near deep tissue, hence reducing the physical and financial burden of surgical patients.Herein, we present a technology for inhibiting microorganisms under soft tissue using an implantable, biodegradable, and vibrant triboelectric nanogenerator (IBV‐TENG) driven by US to transmit and generate electric stimulation in vitro. The IBV‐TENG produced voltage of ≈4 V and current of ≈22 µA underwater at 3 mm distance from US probe  (PVA) was used as a counter friction layer due to its triboelectrically negative property compared to PHBV  analysis was used to confirm the obtained polymers  to cover for preventing oxidation of Mg foil during the coating process by PVA solution . While PHBV film was stable at lower US power (≤2.0 W cm−2), degradation started after applying high US intensity of 3 W cm−2, and the film was fully degraded at 5 W cm−2 within 120 min ‐2, 5‐diphenyltetrazolium bromide (MTT) assay tests. The MTT assay results indicate that PHBV and PVA films show high cell viability even after 1, 2, and 3 days  used in the fabrication of the IBV‐TENG was experimentally observed after immersed in PBS .30 Thes Figure .[38]n Figure . This iss Figure .Figure 5 colony‐forming units per milliliter [CFU mL−1], 500 µL) was added to ES part for various treatments. E. coli (Gram‐negative) and S. aureus (Gram‐positive) were used to investigate the antibacterial activity of IBV‐TENG and to observe the bacterial survival rate after 24 h of culture. Figure E. coli and ≈100% of S. aureus  than on E. coli (Gram‐negative). Due to fundamental structural differences in the cell envelope of these two groups of bacteria, Gram‐positive and Gram‐negative, the outer membrane plays an essential role in protecting Gram‐negative microorganisms from the environment and providing an additional stabilizing layer around the cell in comparison to Gram‐positive. Specifically, after producing the electrical charge by IBV‐TENG, the different electrical potential between the electrode surface and bacteria membrane occurs due to the condition of electrostatic attraction, making disruption in electron transfer of bacterial activities. Since the bacteria membrane has negative charge by inducing external electrical charge, the polarity of the membrane surface is changed. This polarization difference destroys the bacteria structure, subsequently, results in leakage and ultimately killing the bacteria  applied to the bacteria sample for 1 h without IBV‐TENG, and there was no significant difference compared to the original rate  for 60 min, suggesting that the antibacterial activity of IBV‐TENG is pH‐independent  was spread on the porcine tissue with 0.5 × 2.5 cm2 area. Then, IBV‐TENG was placed on the contaminated part of the tissue and covered with a skin layer of pork (≈5 mm thickness). The probe of US was fixed just above the skin where we implanted the IBV‐TENG for inducing the US and making ES in the contaminated tissue  and subsequently dried at room temperature for 24 h and peeled off. The thickness of the obtained PHBV films was 40 µm. For the fabrication of poly (PVA) film: PVA powder  was added in deionized (DI) water with 10% w/v concentration, then the solution was stirred and heated at 120 °C for 2 h on a hot plate to obtain a completely mixed solution of PVA. The PVA solution was spin‐coated on a glass plate at 1000 rpm for 10 s, then dried at room temperature for 24 h. After drying, PVA films were obtained with 20 µm thickness.For the fabrication of poly  (PHBV) film: PHBV (98:2) powder  was dissolved in chloroform solvent with 5% w/v concentration. The solution was stirred and heated at 90 °C for 3 h on a hot plate. A homogenous solution of PHBV was cast on a glass plate  was spin‐coated on the Mg electrode at a speed of 4000 rpm for 10 s and then dried for 6 h at room temperature. Then, the PVA solution  was spin‐coated on the dried PHBV layer at a speed of 1000 rpm for 10 s and put into the vacuum chamber for 24 h for vaporizing the solvents completely. After drying, a microwire was connected to an uncovered side of the Mg electrode to confirm the output performance of IBV‐TENG. Both sides of the coated Mg electrode were encapsulated with PHBV film (with 40 µm thickness), with the upper side of being used as the friction layer of the IBV‐TENG device. Then, the extra edges of PHBV films that covered the Mg electrode were cut and closed by a needle of hot solder.Mg film with 50 µm thickness was cut by a laser cutter machine. The obtained Mg electrodes had a dimension of 1 × 2 cm2 for each. For the Mg foil and PHBV, after completely immersing them in the 20 mL of PBS solution, they were taken out from the container every week, then washed with DI water and dried for 3 h in the oven at 40 °C to observe the biodegradation. Due to the fast‐dissolving PVA film, only the photos of the initial and final dissolution PVA film in the PBS were taken.The biodegradation time of Mg foil, PHBV, and PVA in the phosphate‐buffered saline  at 37 °C was assessed. The thicknesses of Mg foil, PHBV film, and PVA film were 50, 40, and 20 µm, respectively, with an area of 1 × 2 cm5 cell mL−1) were seeded in a 96‐well plate (100 µL per well) for being incubated at 37 °C for 24, 48, and 72 h. Subsequently, the culture medium was removed in the 96‐well plates (medium suction) and added 50‐µL MTT solution (5 mg/mL) and 50‐µL culture medium to each well. After incubation for 3 h at 37 °C, the MTT solution and culture medium was removed again. The samples were washed using diluted PBS solution and added 150‐µL solubilization solution (dimethyl sulfoxide) to dissolve formazan crystals. Finally, the solution of 100 µL was transferred to other 96‐well plates and measured absorbance to evaluate viability (OD = 570 nm).In vitro biocompatibility assay tests were carried out with CRL‐1502 (skin fibroblast cell) by a MTT method. Those cells  was set at 20 kHz frequency and 1 and 2 W cm−2 power density.Experimental setup of equipment for recording and displaying the output of IBV‐TENG consisted of the oscilloscope (Tektronix DPO3052) with a voltage probe  and a current probe by using low noise current amplifier . For ultrasound generation, an ultrasonic transductor and generator (Mirae MV100) was employed. Ultrasound and power density were set up and measured by a digital power meter (Yokogawa WT‐1600). The ultrasound probe frame was in cylindrical shape to reduce the noise. The ultrasound probe and device were placed in DI water during the measurements. DI water was used as an impedance matching layer to minimize the ultrasound energy loss during the experiment. The output of IBV‐TENG was demonstrated at the different time scales (2 to 60 min) at 3 mm ultrasound probe distance, 20 kHz frequency with 1 W cmE. coli (ATCC 25922) and Gram‐positive S. aureus (ATCC 25923) were obtained from Merck as the experimental samples of model organisms. In brief, the pure bacteria in LB were cultivated overnight in a rotating shaker at 37 °C. Bacteria solutions source of S. aureus and for E. coli had a concentration of 0.2–1.2 × 109 CFU mL−1 that diluted to a volume of 1:9 for three times to concentration of 0.2–1.2 × 105 CFU mL−1. 500 µL of each solution of E. coli and S. aureus were seeded on the PHBV‐covered Mg electrode. After the treatment, the experimental and control bacteria were diluted twice to a volume of 1:9 and 50 µL spread on the agar plates by using the L‐shape spreader for the CFU analysis. The samples were then placed inside the agar culture plate and cultured in the incubator at 37 °C for 24 h. One day before the experiment, 9.2 gr of nutrient agar powder (Merck) was solved in 400 mL DI water, then the bottle of agar solution with the semi‐closed cap was put in an autoclave that uses steam and pressure at 121 °C and 15 PSI for 30 min, for killing the microorganisms on the surface and in agar solution, then placed in the incubator at 75 °C. Solution agar poured in the plastic Petri dish and after drying provided for bacteria culture. The bacterium numbers were measured by Image J software, the bacteria number of original groups was set as A, and the bacteria number of sample groups after treatment was set as B. Finally, the bacterial survival rate (R) is calculated by Equation  and Gram‐positive (S. aureus) and applied different voltages to kill the bacteria. Ultrasound power was changed at a constant distance to obtain the different voltages.To observe the antibacterial effect of electrical stimulation from IBV‐TENG without the influence of directly inducing of ultrasonic heat on bacteria solution, the power generation and the electrical stimulation were divided into two parts. The device was stuck in the two plastic petri dishes by double side tape, once the petri dish contained encapsulated IBV‐TENG, and the next petri dish contained the PHBV‐covered Mg film with PHBV , which is bacteria solution location. Encapsulated IBV‐TENG and PHBV‐covered Mg electrode were connected by a microwire. Encapsulated IBV‐TENG was in the water during the experiments at 3 mm distance from the ultrasound probe with 20 kHz frequency to make electrical stimulation for killing the bacteria in the next petri dish which has PHBV‐covered Mg electrode and 500 µL of bacteria solution. The images of bacteria culture were then recorded and analyzed and calculated the number of viable bacteria grown on different samples after 24 h of culture. The antibacterial ability of IBV‐TENG was studied on Gram‐negative (2 dimension. 30 µL of bacteria solution (≈105 CFU mL−1) was dropped on the particular place of fresh tissue with 0.5 × 2.5 cm2 dimension for 5 h, then IBV‐TENG was placed on the contaminated part of the tissue and covered by skin layer of pork (≈5 mm thickness). The probe of ultrasound was fixed on the skin where IBV‐TENG is implanted, for inducing ultrasound vibration and making electrical stimulation on the contaminated tissue. After treatment, an L‐shape spreader was rubbed on the contaminated part, culturing the bacteria on the agar plate and the samples were placed inside an incubator at 37 °C for 24 h.The antibacterial ability of IBV‐TENG was evaluated from ex vivo. In the beginning, the fresh soft tissue of pork with fat layer and skin provided was cut into parts at same condition and size, thickness of ≈4 cm and 8 × 8 cmhttps://www.advancedsciencenews.com/road‐better‐data‐presentation‐dos‐donts/. Data of all experiments were evaluated as mean values ± standard deviation (SD) of at least three tests from a representative experiment (n = 3 independent samples per group). The error bars mean the standard deviations. Statistical significance of the variance was evaluated by the GraphPad prism using the two‐way analysis of variance (ANOVA) program using the Holm–Sidak method. Values of *p < 0.05, **p < 0.01, and ***p < 0.001 are considered statistically significant. The “ns” means no significant difference.Statistical analysis was referenced in detail according to checklist: The authors declare no conflict of interest.Supporting informationClick here for additional data file.


Holothuria leucospilota, are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 (Hl-CASP9) cDNA in the sea cucumber, Holothuria leucospilota. Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H2O2), nitric oxide (•NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo. Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates.Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd’s toxic effects on immune systems in the sea cucumber,  Holothuroidea) constitute a major scavenging component integral to many marine ecosystems such as coral reefs, kelp grasslands and mangroves  can be adversely affected by anthropogenic pollutants from industrial discharges. Among the most deleterious pollutants are heavy metals, namely, cadmium (Cd), mercury, lead, and arsenic . The preAs a key protein family involved with apoptosis, cysteine-dependent aspartate-directed proteases (caspases) are a family of cysteine proteases ubiquitously found in vertebrate and invertebrate species including echinoderms . TypicalParacentrotus lividus), sea cucumber (H. leucospilota and Apostichopus japonicus) and starfish (Asterina pectinifera)  or polyinosinic-polycytidylic acid [poly(I:C)] in H. leucospilota coelomocytes in vitro. Furthermore, both caspase-9 and Apaf-1 are present in the genome of Strongylocentrotus purpuratus (sea urchin), lending support to a conserved mechanism governing intrinsic apoptosis within deuterostomes  –22 (See inifera) Table 1.aspase-3 . In prevpoptosis , while Hrostomes . HoweverH. leucospilota, an ecologically relevant and commercially valuable aquaculture species in southern China (H. leucospilota caspase-9 (Hl-CASP9), we analyzed mRNA distribution profiles across developmental stages and various adult tissues of H. leucospilota, and compared transcriptional expression profiles following pharmacological challenges of immunostimulants and oxidative stressors including Cd and chemical donors of ROS/RNS (reactive oxygen/nitrogen species). Furthermore, the functional relevance of Hl-CASP9 to mitochondrial stress and apoptosis in response to Cd was also examined. Our current work thus provides fresh insights into the functional significance of sea cucumber caspase-9 in relation to environmental perturbations such as heavy metal contaminants and microbial infections, which should help stimulate interest in regulatory mechanisms underlying cell death and stress adaptation in marine invertebrates including echinoderms.In order to clarify the physiological roles of caspase-9 in echinoderms, we first cloned and characterized caspase-9 cDNA from tropical sea cucumber, rn China . To scruH. leucospilota) weighing 200 ± 10 g were obtained from Daya Bay  and temporarily reared in a seawater aquarium with filtrated and aerated seawater with salinity of 35‰ and temperature of 32°C for a week before experiments. After being placed on ice for 30 min, sea cucumbers were dissected and the tissues were collected and frozen rapidly in liquid nitrogen for storage at −80°C, until RNA extraction. Meanwhile, coelomic fluids were filtered by 100 µm sterile nylon mesh and centrifuged immediately at 1,000 × g for 10 min at 4°C to harvest coelomocytes. Then, the harvested coelomocytes were then kept in 1 mL TRIzol reagent  at −80°C. All animal experiments have been approved by the Ethics Committees of the South China Sea Institute of Oceanology, Chinese Academy of Sciences, and were conducted in accordance with guidelines thereof.Healthy adult individuals of sea cucumbers . First-strand cDNA was synthesized by using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara). Based on a transcriptomic library of H. leucospilota coelomocytes previously constructed by our lab, a partial sequence for the Hl-CASP9 homologue was verified with gene-specific primers Hl-CASP9-F and Hl-CASP9-R (α competent cells (Takara) were used for subcloning of the amplicons. Three positive clones for each amplicon were subsequently sequenced.Total RNA from coelomocytes of -CASP9-R Table S1https://www.ncbi.nlm.nih.gov/orffinder/). Structural domains of Hl-CASP9 were predicted by using the SMART (http://smart.embl-heidelberg.de/), ScanProsite (http://prosite.expasy.org/) and BLAST (http://blast.ncbi.nlm.nih.gov) programs. Molecular weight (M.W.) and theoretical isoelectric point were calculated according to deduced amino acids by ProtParam (http://web.expasy.org/protparam/). Alignment for amino acids sequences among various species was performed with the Clustal Omega program  and presented by using the Jalview program . Phylogenetic trees were constructed based on amino acid differences (p-distance) according to the neighbor-joining method (pairwise deletion) with 1,000 bootstrap replicates by using MEGA X. Three-dimensional (3-D) models were deduced with the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) and visualized by the VDM program (http://www.ks.uiuc.edu/Research/vmd/).Open reading frame (ORF) and amino acids sequences were predicted by using ORF Finder  and reverse transcription by using the PrimeScript™ RT reagent Kit (Takara) for quantitative PCR (qPCR). Specific primers QHl-CASP9-F and QHl-CASP9-R (Hl-CASP9 cDNA sequences. qPCR reactions were performed by using SYBR Premix Ex Taq™ II (Takara) in a final volume of 20 μL, with the conditions of 40 cycles of 95°C for 5 s and 60°C for 30 s. In this experiment, Hl-β-actin was used as an internal control to verify qPCR results with reference to previous modifications , an important pathogen of many aquatic animals, or alternatively, with oxidative stress inducers including H2O2 , NOC-18  and CdCl2 , respectively. After these treatments for 0, 3, 6, 12 and 24 h, coelomocytes were harvested and lysed in TRIzol reagent, followed by procedures for RNA extraction and, first-strand cDNA synthesis. Quantitative changes in mRNA expressions of Hl-CASP9 after immune and oxidative challenges were analyzed quantitatively by qPCR assays, as described above.Coelomocytes were chosen as a cell model for investigating transcriptional responses of escribed . After cHl-CASP9 was amplified by PCR by using the gene-specific primers PHl-CASP9-F and PHl-CASP9-R (BamH I and Xho I. PCR products and the pcDNA3.1(+) vector (Invitrogen) were digested with BamH I (Takara) and Xho I (Takara), and ligated by means of T4 DNA ligase  at 16°C for 1 h and then used to transform DH5α competent cells. Accuracy of the constructed plasmid was confirmed by double-enzyme (BamH I and Xho I) digestion and sequencing. A positive clone was picked and cultured in lysogen broth (LB) containing ampicillin (100 μg/mL) at 37°C with shaking at 220 rpm overnight. The amplified pcDNA3.1(+)/Hl-CASP9 recombinant plasmid was purified by using the Plasmid MiniPrep DNA Kit .Coding region of -CASP9-R Table S12 for one day. Afterwards, HEK293T cells were transfected with 3 μg pcDNA3.1(+)/Hl-CASP9 recombinant plasmid by using 3 μL lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. By using the same methods, HEK293T cells were transfected with an empty pcDNA3.1(+) vector as a control.Before transfection, HEK293T cells were seeded in a 6-well plate and cultured in Dulbecco’s Modified Eagle Medium  containing 10% fetal calf serum (FCS), penicillin (100 μg/mL) and streptomycin (100 μg/mL) at 37°C with 5% CO6 cells/mL. Subsequently, 100 μL cell suspension mixed with 5 μL Annexin V-FITC and 5 μL propyl iodide (PI) was incubated in dark for 10 min at room temperature. Then, 400 μL of 1 × binding buffer was added prior to detection. Finally, cell apoptosis proportions were measured by flow cytometry  within 1 h. Annexin V-FITC was used to stain phospholipid serine on the cell membrane at the early stage of apoptosis, wherein FITC signals indicate the rate of early apoptosis. PI penetrates membranes of damaged cells, such as late apoptotic cells or necrotic cells, and binds nuclear DNA to give red staining. Cells doubly stained by PI and Annexin V-FITC are considered late apoptotic cells or necrotic cells.HEK293T cells were incubated in the presence or absence of CCCP , a protonophore which uncouples oxidative phosphorylation in mitochondria, at 37°C for 30 min. Then, apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit . Cells were collected and washed twice with pre-cooled PBS (pH 7.4) and resuspended in 1×binding buffer, with a final cell suspension density of 1×106 cells/mL, survival rate > 95%) were seeded into 6-well plates pre-coated with poly D-lysine and grown overnight in Leiboviz’s L-15 culture medium (with 0.39 M NaCl). After treatment with different concentrations of CdCl2  for 3 h, the medium was removed. Then, 1 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA)  diluted in serum-free medium was added. After 30-min incubation, coelomocytes were washed twice in PBS (with 0.53 M NaCl), followed by observation under a fluorescent microscope . Fluorescence intensity quantification was done with ImageJ.Primary coelomocytes  according to the manufacturer’s instructions. Similarly, a GFP cDNA fragment was amplified as the negative control. Specific primers used for the synthesis of dsRNA, as shown in in vivo dsRNA interference, nine healthy individuals of H. leucospilota (each weighing about 100 g) were randomly assigned into three groups . The experimental group was injected with 100 μL dsCASP9, while the control group was injected with 100 μL RFSS and the negative control was injected with an equivalent amount of dsGFP. Following RNAi for 48 h, all sea cucumbers were dissected on the ice and their coelomocytes were isolated. Thereafter, a sample of coelomocytes was taken to assess interference efficiency of RNAi experiment by qPCR, with specific primers and reaction conditions identical to those listed in section 2.4.Double-stranded RNA (dsRNA) targeting the 6 cells/mL) from different groups  were plated into 3.5-cm glass-bottomed confocal dishes pre-coated with poly D-lysine, followed by incubation at 28°C for 18 h and subsequent treatment with CdCl2 (20 μM) for 3 h. Prior to imaging experiments, coelomocytes were loaded with the mitochondrial superoxide probe MitoSOX  for 30 min and then washed with HBSS (with 0.53 M NaCl) immediately before imaging. Images were obtained with a confocal fluorescence microscope . Fluorescence intensity quantification was done with ImageJ.Primary coelomocytes (1×10ψm) was measured by the commercial membrane potential probe JC-1 . Briefly, primary coelomocytes (1×106 cells/mL) from different groups  were seeded into 6-well plates pre-coated with poly D-lysine and grown in Leiboviz’s L-15 culture medium (with 0.39 M NaCl) overnight. Then, coelomocytes were treated with CdCl2  or CCCP , respectively. After removing culture medium, coelomocytes were incubated with JC-1 for 20 min in dark and then washed twice with PBS (with 0.53 M NaCl) before acquisition. Δψm in coelomocytes from different groups was detected by flow cytometry (Beckman Coulter).Mitochondrial membrane potential (Δ2O2 (2 μM), NOC-18 (1 μM) and CdCl2 (20 μM). After 24 h, the H. leucospilota coelomocytes were resuspended by proper pipetting and the coelomocytes proportions of undergoing apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit (Solarbio) and determined by flow cytometry (Beckman Coulter) as described above.After RNAi for 48 h, primary coelomocytes from different groups  were harvested and cultured overnight in Leiboviz’s L-15 cell culture medium (with 0.39 M NaCl), and then exposed to oxidative stress inducers including Hn = 3), followed by Fisher’s least significant difference (LSD) test with SPSS 22.0 . Statistical significance was determined at p > 0.05 , *p < 0.05, **p < 0.01 or ***p < 0.001.All data were analyzed by using one-way ANOVA and presented as mean ± SEM  of 180 bp, a 3’-UTR of 398 bp and an ORF of 1,263 bp encoding a protein of 420 a.a.  at the C-terminus were predicted in the amino acid sequence of Hl-CASP9 (215HSNFDCVALAILTHG229) and the cysteine active site motif (262KPKIIILQACRG273) were also found in the P20 subunit  and heat-inactivated V. harveyi are as shown in Hl-CASP9 mRNA from 0 to 24 h post-challenge. In contrast, Hl-CASP9 mRNA expression in primary coelomocytes were significantly upregulated under oxidative stressors including H2O2, NOC-18 and CdCl2  relative to the untreated baseline at 24 h post-challenge. When treated with NOC-18, a well-established chemical donor of nitric oxide (•NO) (Hl-CASP9 was significantly elevated at 6 h and reached a peak of 2.84-fold (p < 0.001) increase relative to the basal levels at 24 h post-challenge. In the case of Cd, Hl-CASP9 expression was strongly stimulated: a 1.94-fold (p < 0.01) increase at 3 h following CdCl2 treatment, and a peak of 3.74-fold (p < 0.001) increase at 24 h. Taken together, these results suggest that Hl-CASP9 mRNA expression seems differentially regulated in response to oxidative stressors and immunostimulants.Dynamic changes in de (•NO) , the exp2 exposure in sea cucumber coelomocytes, intracellular ROS levels in coelomocytes were detected by means of the non-fluorescent probe DCFH-DA, which is switched on in a fluorescent state as DCF on reaction with ROS  relative to the untreated baseline /Hl-CASP9 recombinant plasmid (rHl-CASP9) was transfected into HEK293T cells. CCCP was then used to test whether Hl-CASP9 could participate in apoptosis via the classical mitochondria-dependent pathway. As noted in Hl-CASP9), respectively (Hl-CASP9 significantly increased by 1.69 folds (p < 0.001) (p < 0.001) higher than that of the untransfected treatment group  rapidly induced a loss in mitochondrial membrane, as gauged by the membrane-potential sensitive dye JC-1 (2.5 μM)  , NOC-18 (1 μM) and CdCl2 (20 μM) could efficiently induce apoptosis in sea cucumber coelomocytes. Early apoptosis rates of coelomocytes under the challenges of H2O2, NOC-18 and CdCl2 in the control group (with no dsRNA) were 32.71%, 33.97% and 47.26%, respectively, whereas corresponding rates in the untreated group was 24.27% (p < 0.001) (p < 0.05) (p < 0.001) , a large catalytic subunit  and a small catalytic subunit  2 as an active caspase, which eventually commits irreversibly to apoptosis  . Both P2 10 kDa) . Upon inpoptosis .Hl-CASP9 contains a conserved caspase-9 architecture encompassing the CARD domain and CASc domain. Hl-CASP9 possesses two active sites rich in histidine and cysteine residues, which is consistent with previous reports that the active site is a catalytic dyad composed of a cysteine sulfohydryl group and a histidine imidazole ring  are amply expressed in the developing nervous system during embryonic development in zebrafish . The mitocytosis , 8. AddiHLcaspase-6 and HLcaspase-8 were positively stimulated by LPS or poly(I:C) in H. leucospilota coelomocytes , LPS and heat-inactivated V. harveyi as immune stimulants. Surprisingly, Hl-CASP9 transcript expression was virtually unaltered after these treatments, suggesting that Hl-CASP9 may not be directly responsible for host responses to infiltrating pathogens. In vertebrates, caspase-9 has been recognized as a core initiator caspase in response to oxidative stress  respond to a variety of pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) to initiate or sustain a coordinated immune response during infection or inflammation. In our previous studies, transcriptional responses of omocytes , 22. To e stress . Consequ pathway . In thism of rat , 50. In C6 cells . In brievia the mitochondria-dependent pathway. In HEK293T cells overexpressing Hl-CASP9, the results corroborate the idea that Hl-CASP9 is a mediator in mitochondria-dependent apoptosis, as in the case of intrinsic apoptosis in mammals , peroxynitrite (ONOO-) and hypochlorite (OCl-). Unrestrained ROS generation in turn may directly or indirectly alter signal transduction, modulating the outcomes of apoptosis via the mitochondria-dependent pathway 56) and Z pathway . In the H. leucospilota for functional characterization. Our findings give fresh insights into how this ancient caspase-9 mediates coelomocytic apoptosis in response to Cd toxicity via a mitochondria-dependent pathway. Evidently, Cd as an important heavy metal in marine pollution can profoundly impact the health and survival of benthic species. There is an urgent need to establish a mechanistic understanding of the organismal and cellular adaptive processes such as autophagy and apoptosis in toxic metal pollution  [GML2019ZD0402], the National Natural Science Foundation of China , Li Ka Shing Foundation [510858044 to N.W.] and the Key Deployment Project of Centre for Ocean Mega-Research of Science, Chinese Academy of Sciences [COMS2020Q03].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


H-pyrrolopyrimidine competitive inhibitors of PAK4 were investigated at the molecular level, mainly using molecular dynamics simulations and binding free energy calculations. The results show that the inhibitors had strong interactions with the hinge region, the β-sheets, and the residues with charged side chains around the 4-substituent. The terminal amino group of the inhibitor 5n was different from the other three, which could cause the enhancement of hydrogen bonds or electrostatic interactions formed with the surrounding residues. Thus, inhibitor 5n had the strongest inhibition capacity. The different halogen atoms on the 2-substituents of the inhibitors 5h, 5g, and 5e caused differences in the positions of the 2-benzene rings and affected the interactions of the hinge region. It also affected to some extent the orientations of the 4-imino groups and consequently their affinities for the surrounding charged residues. The combined results lead to the weakest inhibitory capacity of inhibitor 5e.The overexpression of p21-activated kinase 4 (PAK4) is associated with a variety of cancers. In this paper, the binding modes and inhibitory mechanisms of four 7 The p21-activated kinases (PAKs) are serine/threonine protein kinases that are widely found in eukaryotes . PAK4 isPAK4 has the common structure of protein kinases, containing an N-terminal regulatory domain and a C-terminal kinase domain . The C-tStudies in recent years showed that PAK4 inhibitors can inhibit its expression, thus preventing tumor growth, inducing tumor regression, and avoiding tumor cell metastasis, which has a good effect on the treatment of cancers . PAK4 inH-pyrrolopyrimidine derivatives, which showed good inhibitory capacity for PAK4 with no cytotoxicity pyrimidine derivatives as competitive inhibitors of p21-activated kinase 4 were studied in this paper. The results show that the main binding regions of enzymes and inhibitors were the hinge region, β1–β3 and β7 sheets, and residues with charged side chains around the 4-substituent of inhibitors. Of these, the hinge region interacted most strongly with the inhibitors of all the complexes and was the main reason for their inhibitory capacities, but not for the differences in inhibitory capacities of the inhibitors. Unlike the other three, the terminal amino group of inhibitor 5n promoted enhanced interactions with the inhibitor of residues such as Ile327, Lys350, Asp405, Asp444, and Asp458, which formed hydrogen bonds or electrostatic interactions with the inhibitor, increasing the stability of the inhibitor in the enzyme and providing the strongest inhibitory capacity in the experiment. The different 2-substituents of the inhibitors 5h, 5g, and 5e caused differences in the binding free energy of Ile327 and in the 2-benzene ring binding position, which further affected the hinge region interactions. The combined effect of these two resulted in the weakest interaction of inhibitor 5e with the protein and the weakest inhibitory capacity measured in the experiments. There was also a difference in the 2-benzene ring position between inhibitor 5h and 5g, which resulted in stronger interaction between inhibitor 5h and Met395 and Gly401 in the hinge region. More importantly, inhibitor 5h led to enhanced electrostatic interactions with charged residues such as Lys350, Glu366, Asp405, Arg589, and Arg591 due to the flipping of the 4-imino group. Therefore, the enzyme-inhibitor interaction in complex 5h was stronger than that in complex 5g, but the larger solvation energy difference between the two offsets some of this, resulting in little difference in total binding free energy between the two complexes.The binding modes and inhibitory mechanisms of four 7


The SARS-CoV-2 coronavirus, which causes COVID-19, uses a viral surface spike protein for host cell entry and the human cell-surface transmembrane serine protease, TMPRSS2, to process the spike protein. Camostat mesylate, an orally available and clinically used serine protease inhibitor, inhibits TMPRSS2, supporting clinical trials to investigate its use in COVID-19. A one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) model for camostat and the active metabolite FOY-251 was developed, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. The model predicts that 95% inhibition of TMPRSS2 is required for 50% inhibition of viral entry efficiency. For camostat 200 mg dosed four times daily, 90% inhibition of TMPRSS2 is predicted to occur but with only about 40% viral entry inhibition. For 3-fold higher camostat dosing, marginal improvement of viral entry rate inhibition, up to 54%, is predicted. Because respiratory tract viral load may be associated with negative outcome, even modestly reducing viral entry and respiratory tract viral load may reduce disease progression. This modeling also supports medicinal chemistry approaches to enhancing PK/PD and potency of the camostat molecule.IMPORTANCE Strategies to repurpose already-approved drugs for the treatment of COVID-19 has been attractive since the beginning of the pandemic. Camostat mesylate, a serine protease inhibitor approved in Japan for the treatment of acute exacerbations of chronic pancreatitis, inhibits TMPRSS1, a host cell surface serine protease essential for SARS-CoV-2 viral entry. In vitro experiments provided data suggesting that camostat might be effective in the treatment of COVID-19. Multiple clinical trials were planned to test the hypothesis that camostat would be beneficial for treating COVID-19 . The present work used a one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) mathematical model for camostat and the active metabolite FOY-251, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. This work is valuable to guide further development of camostat mesylate and possible medicinal chemistry derivatives for the treatment of COVID-19. The coronavirus disease 2019 (COVID-19) pandemic has reinforced the need for early oral treatment to prevent disease progression . Current4–Remdesivir, an inhibitor of the viral RNA-dependent, RNA polymerase, is the most widely investigated antiviral drug for the treatment of COVID-19 . In one Molnupiravir is a newer oral antiviral drug that has recently been tested in COVID-19 . Early tDespite this recent progress with molnupiravir, additional therapeutic options are needed, particularly for early treatment of SARS-CoV-2 infection, i.e., newly diagnosed individuals in the outpatient setting, as well as for post-exposure prophylaxis.Previous experimental data , 15 has in vitro, a mechanism that has been shown to be both necessary and sufficient for viral entry into respiratory epithelial cells  for the treatment of pancreatitis and esophagitis , 17. Botal cells , 15, 18.al cells . Therefoal cells , might bal cells . CamostaCamostat mesylate is currently being investigated, at the clinical dose of 200 mg every 8 h, as a treatment of COVID-19 in several clinical trials in Denmark, Israel, and the United States . The first clinical trial results of camostat in hospitalized COVID-19 patients have been recently published and, while significant adverse events were not observed, conclusions regarding efficacy appear to be negative .This study took a modeling approach to provide quantitative estimates, using published pharmacokinetic (PK)/pharmacodynamic (PD) data and experimentally inferred mechanism of action of camostat mesylate, of this compound’s anti-SARS-CoV-2 effects in humans.in vivo, we focused on FOY-251 pharmacokinetics for antiviral modeling.The pharmacological properties of camostat have been studied in detail . Due to in vivo, in the model camostat dose is considered as equimolar in amount to FOY-251.bio) was estimated to be low, approximately 5%. The volume of distribution of FOY-251 was 22.4 liters, approximating plasma volume/extracellular water space with limited tissue distribution. The terminal elimination half-life of FOY-251 was estimated to be about 0.6 h. FOY-251 volume of distribution and elimination half-life agree with published estimates  in airways . Small molecule drugs freely penetrate through the pores in the pulmonary capillary between plasma and the extracellular fluid. Alveolar epithelial cells are connected by tight junctions that potentially restricts passive diffusion between cells. To reach ELF a drug must pass through the alveolar epithelial cells themselves. The factors influencing how antibiotic drugs exposure in ELF are discussed in .The pharmacokinetic profiles of an antibiotic drug (cefdinir) in plasma and in “blister fluid” (which approximates the ELF fluid) were shown in . CompariiK parameter), and then covalent binding of the drug molecule with the serine protease (kcat parameter). As it was measured for TMPRSS2 homolog enteropeptidase incubated with camostat, the covalent complex is relatively stable, but reversible, with release of GBA molecule from the enzyme active site , with TMPRSS2 activity producing half-maximal viral entry rate Kal entry . The samThe camostat/FOY-251 PK model predictions were used as input for mechanistic PD model predicting TMPRSS2 inhibition and respective SARS-2-S-driven viral entry rate inhibition. Rapid exchange between blood plasma and extracellular fluid compartments would be expected for small zwitterionic molecules like FOY-251. Thus, FOY-251 concentration in extracellular fluid of lung tissue is assumed to be equal to plasma concentration.in vivo semi-mechanistic PK/PD model. The TMPRSS2 molecules expressing on the cell surface of a respiratory epithelial cell are depicted in active and inactive states. The in vivo PK/PD model includes TMPRSS2 protein synthesis and degradation rates. Potentially, TMPRSS2 fast turnover rate might be a limiting factor for the drug effect, because all newly synthetized enzyme molecules are in the active state. No TMPRSS2 half-life estimate was found in the literature, and the assumed half-life 12 h was used to calculate the model parameter kdeg (=log (2)/T1/2 = 0.0575 1/h).The PK/PD model simulations were done for varying camostat oral doses . The timThe effect of the TMPRSS2 half-life on PK/PD model outcomes was explored. A notable increase of viral entry rate inhibition is predicted with enzyme half-life is increased from 4 h to 12 h. Similar viral entry rate inhibitions were calculated for enzyme half-life values of 12 h and 24 h .For the case where camostat is dosed 200 mg four times daily (QID), the model predicts 90% inhibition of TMPRSS2. Importantly, however, this translated to only about 40% inhibition of the viral entry rate were predicted. In the case where a camostat dose was increased 3-fold (600 mg QID), only a moderate improvement of inhibition of viral entry rate to 54% is predicted.The effect of a drug slower absorption rate due to a hypothetical new formulation was also tested . This sctr, elf =1.0 1/h) are shown in Fig. S4A and B, and demonstrate similar TMPRSS2 and viral entry rate inhibition in both central and ELF compartments.To explore the drug exposure in ELF in more detail the PK/PD model was updated with epithelial lining fluid compartment . To describe FOY-251 PK in ELF airways, two unknown parameters were added—the drug transition rate between plasma and ELF compartments and the drug degradation rate in ELF compartment, which is hypothetically smaller than the drug elimination rate in central compartment. The PK/PD model simulations were done for four sets of parameter values describing the PK in ELF  . A faster transition rate between compartments leads to larger exposure of the drug in ELF. The impact of the drug degradation rate in ELF might be also meaningful. The structure-based prediction supports a FOY-251 fast transition rate. The PK/PD simulations with fast transition rate of the drug , into which we incorporated the known and potential pharmacological mechanisms of the drug action. Based on the results of preclinical studies were reviewed in Breining et al. , a numbein vitro data about anti-SARS-CoV-2 effect of camostat has been published separately  algorithm and performed using Monolix software . Visualization of model diagnostics and simulations were performed in R software version 3.5.1, using the mlxR and ggplot2 library packages.in vivo, in the model camostat dose is considered as equimolar amount of FOY-251, i.e., camostat to FOY-251 transition was considered implicitly.A one-compartment PK model with linear elimination describes FOY-251 (the active metabolite of camostat) concentration profiles in human blood plasma. The model includes two routes of administration of camostat: intravenous and first order absorption from the gut with bioavailability coefficient F. Due to rapid transformation of camostat into FOY-251 The ordinary differential equations (ODEs) for FOY-251 amount (in nmol) in gut (Ad) and central compartment (Ac): (1)dAd/dd.Concentration of FOY-251 in blood plasma: Cc (nM) = Ac/VThe definition of the PK model parameters is given in https://go.drugbank.com/metabolites/DBMET03117).One approach to estimate a drug exposure in ELF relative to exposure in plasma is to use drug molecular weight and octanol/water partition coefficient as key metrics . We usedUsing formula (2) and these two estimates of log(PC) for FOY-251 the values of K = 0.75 and 0.85 were obtained, respectively.In vitro experimental data were digitized from Hoffmann et al.’s Figure 4  activity inhibition as function on time of incubation with FOY-251 concentration C (nM):The following ODEs were used to describe in vitro in Hoffmann et al.’s Figure 7  with SARS-2-S bearing pseudo-type viral particles entry rate, as measured Figure 7 .(4)Virain vivo predictions of the drug effect the model was updated to include TMPRSS2 turnover.To make Without drug TMPRSS2 turnover equation is:synt = kdeg · 1 (units/h).using the assumption that at steady-state SP = 1 (unit) we obtain kin vivo:The ODEs for TMPRSS2 inhibition by FOY-251 The TMPRSS2 catalytic domain is on the target cell surface exposed to extracellular fluid. Thus, considering fast exchange between plasma and extracellular fluid, the drug concentration in plasma (Cc) was used in The definition of the PD model parameters is given in


Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate.Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho-This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’. Tryptopte (PRA) –5. Its ate (PRA) . AnPRT frculosis –12. Tuberculosis –14.The enzyme AnPRT belongs to a wider group of phosphoribosyltransferases (PRTs) that all catalyse the transfer of a phosphoribosyl moiety from PRPP to a nitrogen-based nucleophile, including purines, pyrimidines and their biosynthetic precursors . PRTs haα/β fold decorated by a highly flexible and multifunctional loop (β2-β3 loop) and a variable hood domain that play essential roles in catalysis and regulation of the corresponding PRT [α/β barrel domain containing the active site in its centre and an N-terminal α/β plait domain that serves as a cap for the barrel of the neighbouring enzyme subunit [α/β sandwich domains that contain the active site in the large cleft between them, but display two very diverse forms recruiting their own allosteric machinery [Type I PRT is the largest group and comprises mainly enzymes involved in the nucleotide salvage pathways ,20–25. Tding PRT . Type II subunit –31. Typeachinery ,33. The . 2α/β domain, with the active site located in a cleft between the two domains (α1–α4) augmented by helices α8 and α9. The AnPRT α/β domain is characterized by a large central mixed beta sheet  and eight surrounding helices  as well as three helical motifs, one just after β3 and two in the long β7-α11 loop [α15), as observed for Saccharolobus solfataricus AnPRT (SsoAnPRT) [The structures of AnPRT from several organisms, across three domains of life, have been elucidated by X-ray crystallography over the past 20 years ,34–37. T domains . The N-tα11 loop . Occasioα1, α3 and α8 of the N-terminal domain [2. Because of this, the AnPRT dimer adopts an elongated structure (110 Å long versus 40 Å across), with the two active sites void of direct inter-dimer interactions [SsoAnPRT have revealed that dimerization is important for the stability of AnPRT but does not play a direct role in catalytic activity [All AnPRT structures determined to date are homodimeric, with their dimer interface being formed by interactions among helices l domain . The intractions ,36. Mutaactivity .α/β hammerhead domain. The recently identified TrpD2 proteins are structural homologues to AnPRT but lack the strictly conserved PRPP binding motif and instead possess DNA-binding functionality. These homologues show a low, less than 20%, sequence identity to AnPRT [α/β domain alone can be found in higher plants [There are few known homologues to the characteristic AnPRT fold. The enzymes of the nucleotide phosphorylase type II are highly similar to the AnPRT monomer with the C-terminal addition of an to AnPRT . Furtherr plants .In addition to the common monofunctional AnPRTs, a fused polypeptide (TrpGD) combining the two enzymes glutamine amido transferase and AnPRT exists. This architecture can be found predominantly in Enterobacteriaceae where it forms component II of the anthranilate synthase complex ,43.. 32+ ions, but preference for a Zn2+ ion for maximum activity has been reported for Thermococcus kodakarensis AnPRT (TkoAnPRT) [AnPRT catalyses the straightforward displacement of pyrophosphate from the anomeric carbon of PRPP by anthranilate to form PRA. The reaction is thought to occur with high stereoselectivity, although epimerization in solution means both anomers are observed for PRA. The reaction requires the presence of a divalent metal ion in the active site, which plays a role in activating the leaving pyrophosphate group in the AnPRT reaction. Most AnPRT enzymes characterized to date use MgkoAnPRT) .Escherichia coli AnPRT [Salmonella tryphimurium AnPRT [MtuAnPRT [SsoAnPRT [Saccharomyces cerevisiae AnPRT (SceAnPRT) [MtuAnPRT in the presence of substrates have shown that PRPP binding is required to fully form the catalytic binding site for anthranilate, suggesting an ordered sequential mechanism where PRPP binds first followed by anthranilate binding in the catalytic position [SsoAnPRT based on structural evidence that anthranilate could bind in the catalytic position without the presence of PRPP [MtuAnPRT (Ki 45 ± 6 µM) [TkoAnPRT (inhibition observed for anthranilate concentration above 4 µM) [Kinetics studies on li AnPRT , Salmoneum AnPRT , MtuAnPRMtuAnPRT , SsoAnPRSsoAnPRT ,48 and SceAnPRT)  have revposition . By cont of PRPP . Further ± 6 µM)  and TkoAve 4 µM) .MtuAnPRT in the presence of anthranilate analogues imply a likely dissociative mechanism, as discussed in detail below [Two reaction mechanisms, namely dissociative or associative, have been proposed for AnPRT . In a diil below .Figure . 4MtuAnPRT (PDB 1ZVW) [SsoAnPRT (PDB 1ZXY) [SceAnPRT (PDB 7DSJ) [MtuAnPRT and SceAnPRT, in which it binds deep in the C-terminal domain with enclosing loops β1-α5 and β2-α6 (a). The pyrophosphate group of PRPP binds the deepest and forms an extensive network of polar interactions with the N-terminal end of helix α5 , helix α6 (G147 in MtuAnPRT) and sheet β2 (K135 in MtuAnPRT). In addition, the pyrophosphate group is coordinated to one Mg2+ ion (MG1), which is essential for catalysis. MG1 is also coordinated to a conserved Ser on helix α5 (S119 in MtuAnPRT) and a conserved Glu on loop β5-β6 (E252 in MtuAnPRT). A second Mg2+ ion (MG2) can be observed in most PRPP-bound structures of MtuAnPRT, in which it shares a coordinated water molecule and the Glu residue with MG1 and is also coordinated to a conserved Asp on β5-β6 (D251 in MtuAnPRT). The 5′-phosphate group interacts with the β1-α5 loop (G107 and G110 in MtuAnPRT), which contains a conserved ‘GTGGD’ motif and the β2-α6 loop (S142 and S143 in MtuAnPRT). The ribose moiety is held in position by polar interactions with the backbones of residues on the β2-α6 loop (e.g. N138-R139 in MtuAnPRT and G141-K142 in SceAnPRT).Clear electron densities of PRPP have been observed in crystal structures of DB 1ZVW) , SsoAnPRDB 1ZXY)  and SceADB 7DSJ) . PRPP adnd β2-α6 a. The pySsoAnPRT, in which one PRPP is coordinated to two Mg2+ ions via both its pyrophosphate and 5′-phosphate groups [2+ : PRPP complex is the singly coordinated PRPP, as observed in MtuAnPRT and SceAnPRT [PRPP was observed to bind in a different conformation in e groups . HoweverSceAnPRT .β1-α5 and β2-α6 undergo significant conformational rearrangements upon PRPP binding (b). In the substrate-free crystal structures of AnPRTs, these two loops are either observed in various conformations or disordered (e.g. Pectobacterium carotovorum AnPRT (PcaAnPRT) [SceAnPRT [Xanthomonas campestris AnPRT (PDB 4HKM) and Acinetobacter baylyi AnPRT (PDB 4GTN)). While in the presence of PRPP, significant conformational rearrangements and ordering of these two loops can be observed in MtuAnPRT [b), PcaAnPRT [SceAnPRT [The flexible loops  binding b. In thecaAnPRT) , SceAnPRSceAnPRT , XanthomMtuAnPRT  , and to account for the observation of substrate inhibition by anthranilate. Site 1 is the catalytically competent binding site for anthranilate and is in close proximity to PRPP. Anthranilate at site 1 is surrounded by residues from the closed loops β1-α5 and β2-α6, helices α8 and α9, and sheet β2 (b). The carboxylate group of anthranilate is held by the side chain of a conserved Arg on helix α8 (R193 in MtuAnPRT) and forms polar interactions with loop β2-α6 (N138 in MtuAnPRT). Amino acid substitutions at N139 and R193 of MtuAnPRT caused moderate to a significant impairment to catalysis and a significant increase in the apparent Km for anthranilate, confirming their importance in anthranilate binding and catalytic function [β1-α5 (G107 in MtuAnPRT).Multiple binding sites of anthranilate on AnPRT enzymes have been predicted by molecular modelling for MtuAnPRT  and laters bound ,46 and ite bound . Three bsheet β2 b. The cafunction . The amiSsoAnPRT [MtuAnPRT [β2-α6 and the anthranilate in site 1 (c).Site 2 is a transient site between sites 1 and 3, surrounded largely by hydrophobic residues. Occupancy of site 2 has been observed in SsoAnPRT  .Occupancy of site 3, the outermost site, has only been observed crystallographically in MtuAnPRT ,46. By s of H183 d.. 6β2-α6 observed in the substrate-free and PRPP-bound crystal structures of AnPRTs, another ‘folded’ conformation of this loop has been observed in crystal structures of wild-type and R193A variant of MtuAnPRT when anthranilate or its analogue binds only at site 3 after a short period of soaking [β2-α6 parked in the anthranilate channel, blocking access to site 2 and the catalytic binding site (site 1) of anthranilate (a).In addition to the open and closed conformations of loop  soaking ,46. Thisranilate a.FigureMtuAnPRT that is consistent with the known kinetic features (b) [β2-α6 in a folded conformation . Loop β2-α6 then rearranges to the open conformation to allow PRPP entrance to the active site , which is followed by the closure of loop β2-α6 to stabilize PRPP binding and fully form the catalytic binding site for anthranilate . Anthranilate is then shuttled to site 1 and the reaction occurs . It has been proposed that the product PRA is released before pyrophosphate, which is deeply buried in the C-terminal domain. Loop β2-α6 moves away from the closed conformation to adopt the folded conformation to release the pyrophosphate from the active site . The active site conformation is then regenerated for the next catalytic cycle.The large amount of structural information has allowed a mechanism to be proposed for features b [6,9,18ures (b) . The pro step 1, b. Loop β step 2, b, which  step 3, b. Anthra step 4, b. It has step 5, b. The acMtuAnPRT by anthranilate [MtuAnPRT when loop β2-α6 is in the closed conformation and the anthranilate channel is not obstructed (b). In this case, the loop β2-α6 cannot move back into the folded conformation as the anthranilate channel is occupied, and the pyrophosphate is probably trapped in the active site, which disrupts the catalytic cycle. It should be noted that this mechanism is only proposed for the MtuAnPRT enzyme. However, substrate inhibition by anthranilate has also been reported for TkoAnPRT [The proposed mechanism also explains the reported substrate inhibition of ranilate . In the structed b. In thiTkoAnPRT , suggest. 7M. tuberculosis to overcome the innate host immune defence [The essential nature of tryptophan biosynthesis for some pathogens has created considerable interest in exploring inhibition of AnPRT. In particular, it has been shown that the tryptophan biosynthetic pathway genes are among those that enable  defence . In addiMtuAnPRT [50 40 ± 2 µM [MtuAnPRT complexed with ACS172 reveals two distinct modes which overlap with the multiple anthranilate binding sites observed for the enzyme [Mycobacterium marinum at concentrations between 25 and 600 µg ml−1 [Mycobacterium marinum causes disease in fishes and responds to anti-TB drugs in a similar way to M. tuberculosis.Several anthranilate-like compounds have been screened as inhibitors for MtuAnPRT ,46,53,540 ± 2 µM ). The cre enzyme . Howevere enzyme . This co µg ml−1 . MycobacI) caused complete inhibition of the enzyme and was shown to bind into the catalytically relevant anthranilate binding site (IC50 = 6.8 ± 0.3 µM) [II), was reported to be a 40-fold more potent inhibitor for MtuAnPRT than the original ACS172 amino)-3-methylbenzoate ( 0.3 µM) . In addiIII, IV). While some of these inhibitors showed a modest increase in potency relative to ASC172 , crystallographic and kinetic data suggested the flexible phosphonate-containing tails were not accessing the PRPP binding sites. As this site is well defined, it may be that more-rigid phosphoribosyl-mimicking components need to be incorporated [Attempts were also made to extend binding of the bianthranilate-like inhibitor ACS172 into the PRPP binding site, and a series of phosphonate-containing compounds was synthesized (rporated .Plasmodium falciparum hypoxanthine-guanine-xanthine PRT is consistent with a dissociative reaction with an asymmetric TS with low bond order to both the departing diphosphate group and incoming N from hypoxanthine, with substantial positive charge build up on the anomeric carbon [The dissociative mechanism predicted for AnPRT parallels the catalytic mechanisms of other PRPP-using PRT enzymes, and this feature has been exploited in the design of powerful inhibitors for these enzymes . For exac carbon . This mec carbon .. 8MtuAnPRT, has illuminated many catalytic details and supports a dissociative mechanism for this enzyme. In particular, the observation of multiple binding sites for the substrate anthranilate in at least some AnPRT homologues may lead to specific, tailored inhibitors for this enzyme, when combined with detailed mechanistic information. The essential nature of tryptophan biosynthesis in some pathogens has meant there continues to be interest in the development of inhibitors for this enzyme.The transfer of the phosphoribosyl group from PRPP catalysed by the phosphoribosyltransferases is an important reaction in metabolism. Elegant and detailed mechanistic studies have defined the reaction coordinate and provided information to guide specific and potent inhibitor design. While detailed KIE measurements and mechanistic studies are yet to be performed for AnPRT, structural information from many ligand-bound structures, particularly for


Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen–IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs  Significance StatementImmunotherapy is an allergen-specific disease-modifying treatment that involves administration of increasing allergen doses to reduce IgE production, but can induce side-effects due to IgE cross-linking by allergen. Direct knowledge of IgE epitopes that bind to specific allergens will contribute to designing engineered allergens with impaired IgE reactivity resulting on diminished side-effects. The direct identification of critical, allergen-binding IgE epitopes, such as the one reported here, thanks to IgE hybridoma technology, has not been possible before due to the low frequency of IgE-producing B cells in blood. A direct mapping of IgE antibodies by X-ray crystallography will provide high resolution information of allergen epitopes bound by IgEs from the human repertoire and facilitate structure-based design of allergen vaccines.CDCircular dichroismCDRComplementarity determining region2ODDeuterated waterFabFragment antibody bindingFcɛRIHigh-affinity IgE receptorIMACImmobilized metal affinity chromatographyIgEImmunoglobulin EmAbMonoclonal antibodyPBSPhosphate Buffered SalineDSS4,4-dimethyl-4-silapentane-1-sulfonic acidrDer p 2Recombinant Der p 2nDer p 2Natural Der p 2NMRNuclear Magnetic ResonancePDBProtein Data BankrmsdRoot-mean-square deviationThree letter code (e.g. Asp)Single amino acidSDStandard deviationSHMSomatic hypermutationsSingle letter code and number (e.g. K100D)Mutationin vivo) in complex with an allergen  immune response to molecules  that are otherwise innocuous in a nonallergic population . The intallergen , 4.−7 to 7 × 10−6)  , 10. An  × 10−6) . By a si × 10−6) , 20. The  in complex with the Fab of IgE mAb 2F10 was determined at 2.1 Å resolution , was 4.74E-10 M as measured by full kinetics and 2.97E-10 M as measured by scouting analysis. These KD show a high affinity of the allergen–antibody interaction consistent with what is expected of a somatically hypermutated (and class-switched) antibody.The binding affinity of the IgE mAb 2F10 for Der p 2 was measured by surface plasmon resonance. This affinity, reported as the equilibrium dissociation constant (K2. Der p 2 is present in solution as both monomers (mostly) and dimers at high allergen concentration (2 (PDB code: 1KTJ). The dimers superpose with a rmsd value of 0.7 Å over 258 superposed residues , Val63, Ile97, Pro99, and Lys100 from the epitope. These interactions, which involve the largest area, correspond to a region of the epitope that is in contact with the heavy chain of the 2F10 antibody.On the paratope side, residues participating in formation of H-bonds originate mainly from Complementarity Determining Regions (CDRs) 2 and 3 of the heavy chain, and CDR3 of the light chain, whereas the remaining CDRs do not form any major contacts with the allergen. The largest areas of hydrophobic contacts with the allergen are mediated by Ile52, Phe55, and Leu99 from the heavy chain of the antibody.Next, we sought to analyze how the structure of the IgE–Der p 2 interaction compared with non-IgE antibodies in complex with allergens, to gain further insight into the biological basis of IgE. Currently, only one crystal structure of Der p 2 in complex with a non-IgE mAb has been determined, which is that containing the Fab fragment of murine IgG 7A1 (Protein Data Bank—PDB—code: 6OY4) . Superpo2) observed for the 17 antibody–allergen complexes  in complex with their specific allergen that have their structures determined . Accordixes Fig. . Similarxes Fig. , which ixes Fig. , hydrogexes Fig. , hydrogexes Fig. , and thexes Fig. . The patxes Fig. .D. pteronyssinus, D. farinae, and Euroglyphus maynei), as well as homologous allergens from storage mites of the Glycyphagoidea superfamily (Glycyphagus domesticus and Lepidoglyphus destructor) that share lower amino acid sequence identities with Der p 2 , and Leu61 establishes hydrophobic interactions. Due to its dual interaction with both chains, Asp59 and Leu61 were selected for site-directed mutagenesis, with the goal to significantly reduce IgE mAb 2F10 binding. Additional residues that establish main chain hydrogen bonds with the antibody are: Glu62, Asp64, Lys100, and Glu102 with the heavy chain, and Gly60 with the light chain . Howevermutant #1 (rDer p 2 D59A L61A), or (2) to Der p 2.0103 containing mutations in the adjacent IgG mAb 7A1 epitope (7A1 mut) resulting in mutant #2 (D59A L61A + 7A1 mut). The latter contains two substitutions (K96E-I97E) in the 7A1 epitope that is adjacent to the epitope for IgE mAb 2F10, which were geared to perturb a hydrogen bond network and change to the hydrophilic and negatively charged residue Glu. These substitutions were previously shown to prevent binding of IgG mAb 7A1 and binding of an IgE antibody construct obtained from a phage display library  to the wildtype allergen Der p 2.0103, resulting in  library .mutant #3). Another mutant was aimed at eliminating hydrophobic interactions by mutating the positively charged Lys100 to a negatively charged Asp that has a shorter side chain (mutant #4). Lys100 main chain residues establish main chain hydrogen bonds with the antibody, and therefore, the K100D mutation might not affect them. Finally, a 2F10 epitope triple mutant (mutant #5) was made by combining the substitutions in mutants #3 and 4. The K100D substitution was added to the D59K-L61K mutant to ensure that these three substitutions (that turned out to be effective at impairing IgE mAb 2F10 binding) were present for disruption of antibody binding. A summary of the substitutions in the mutants is shown in A third mutant was designed to change Asp59 and Leu61 to lysine instead of alanine. These changes were targeted to impair the allergen–antibody interaction by replacing small side chains with larger ones, as well as changing a negatively charged aspartate and a neutral leucine to positively charged lysine , whereas 2G1 and 1B8 inhibited IgE binding by 10.1% (range 1.2 to 19.5) and 12.7% (range 3.7 to 21.8), respectively  to inhibit binding of polyclonal IgE to Der p 2 in plasma obtained from allergic subjects who were highly sensitized to this allergen. The 2F10 antibody showed the largest inhibition among the three hybrid constructs tested, in 9 out of 10 specimens  was selected to compare with the wildtype allergen. For all 10 patients tested, there was reduced inhibition of IgE antibody binding to Der p 2 by the mutant versus the wildtype, highlighting the contribution of the 2F10 clone to the full polyclonal IgE antibody response . For some plasma, the curve for the mutant did not reach a 100% inhibition as the one for the wildtype allergen , which indicates the immunodominance of the IgE recognizing the disrupted epitope versus the total IgE polyclonal response. In contrast, other plasma such as PL20 showed similar inhibition curves for wildtype and mutant, indicating the importance of other IgE mAb for that particular IgE polyclonal antibody response. In addition, a high and significant inverse correlation  was found between the % inhibition of the polyclonal IgE binding to Der p 2 by the 2F10 epitope triple mutant and the IgE titers measured by ImmunoCAP  allergen protein to induce mast cell activation and degranulation. This model tests whether FcεRI is cross-linked by allergen and, thus whether the nonoverlapping IgE mAb pair passively transferred to mice is capable of binding the allergen. Mice were sensitized by i.v. route using purified IgE mAb pairs specific to Der p 2, 2 days prior to challenge by i.p. route with 50 µg of purified recombinant Der p 2 wildtype or mutant. Three IgE mAbs 2F10, 2G1, and 4C8 were selected because they bind to nonoverlapping epitopes . Anaphylin vivo , comparable  to the area of epitopes in other allergen–antibody complexes . In fact, Der p 2 is present in solution as both monomers (mostly) and dimers at high allergen concentration. However, whether this dimerization occurs ith Fabs , 19. Theith Fabs .in vivousing a mouse model of passive systemic anaphylaxis. First, site-directed mutagenesis analyses of the 2F10 epitope revealed three amino acids  that are important for antibody recognition. The position of these three residues is in agreement with the location of the 2F10 binding area previously predicted by NMR, which comprised residues 54 to 65, 97 to 99, 103 to 107, and 121 to 123 as likely responsible for antibody binding  the loss of their IgE binding capacity, and (2) their functional ability to cross-link IgE  binding . Howevermutant#1) produced a weaker reduction of IgE mAb 2F10 binding than to lysines , which are longer and positively charged residues and can better interfere with antibody recognition. Interestingly, a single mutation (K100D) in mutant#4 showed the same strong effect as four or two mutations in mutants#2 and #3, respectively, highlighting the importance of the hydrophobic interactions established by Lys100. In addition, mutation of the adjacent epitope for the IgG mAb 7A1 reduced the binding of 2F10 IgE mAb to Der p 2 mutant#1, with an effect comparable to the one observed for mutants#3, #4, and #5  showed a strong reduction of IgE mAb 2F10 antibody binding. This triple mutant had also a reduced capacity to inhibit polyclonal IgE antibody binding to Der p 2 compared to the wildtype allergen for the 10 subjects tested. This last result indicates that the 2F10 epitope is important, if not dominant, for the allergic response to Der p 2. In addition, a high and significant inverse correlation was found between the maximum inhibition of the polyclonal IgE binding to Der p 2 by the triple mutant and the IgE titers for the 10 subjects tested. A larger inhibition observed for lower IgE levels to Der p 2 could indicate that the response is less polyclonal at low IgE levels, and therefore, the 2F10 IgE represents a larger proportion of the polyclonal IgE response. In other words, the IgE mAb associated with the 2F10 epitope seemed to be more relevant for subjects with lower Der p 2-specific IgE levels.in vitrostandard basophil or mast cell activation tests because it is correctly folded, and contains at least two other intact IgE epitopes  in mouse anaphylaxis  differs from the intraperitoneal injection for challenging mice. The allergen doses used in the mouse model (50 μg purified recombinant Der p 2) are difficult to compare with the human allergen exposure levels, which are measured as allergen concentration in dust (µg/g) . Mite alphylaxis , 38. TheD. farinae and E. maynei), which has been recognized as the most important source of mite allergens since 1967 , with reduced capacity to bind and cross-link IgE, have been successful for immunotherapy , 40. Hownce 1967 . ModificD. farinae, 77.4% for D. pteronyssinus, 12.9% for E. maynei, and 3.2% for Blomia tropicalis  as described in The DNA encoding for antibody constructs  was synthesized by GeneArt  with codon optimization for expression in CHO cells. IgE–IgG constructs are hybrid monoclonal antibodies that comprise the IgE mAb Fabs (containing the paratope) and the Fc from an IgG as described in Recombinant Der p 2.0103 and 2F10 Fab were mixed at 1:1 molar ratio to prepare a complex that was purified by size exclusion chromatography. The purified complex was used for crystallization experiments by vapor diffusion. Data collection was performed at the Advanced Photon Source, Argonne National Laboratory  at 100 K. Full crystallization details and computational approaches are available in D. pteronyssinus spent mite cultures. Amino acid substitutions in the epitope were performed by site-directed mutagenesis. Recombinant allergens were expressed and purified, and their correct folding was assessed by NMR, CD spectra, and immunoassays. Methodological details are in Natural Der p 2 was purified from To assess the relevance of the IgE mAb 2F10, inhibition assays of human polyclonal IgE antibody binding to Der p 2 were performed using chimeric IgE–IgG constructs of 2F10 as inhibitor. Other chimeric IgE–IgG antibodies  were also used for comparison. As a control, inhibition of IgE mAb 2F10 to Der p 2 by IgE–IgG 2F10 was also performed.To assess the effects of site-directed mutagenesis on antibody binding to the mutants two kinds of assays were performed: (1) direct binding of monoclonal antibodies to Der p 2 (wildtype and mutants) coating the plates, and (2) inhibition assays in which allergens (wildtype or mutants) were preincubated with IgE mAb or polyclonal antibodies before measuring antibody binding to the wildtype allergen coating the plate. Full details of the experiments are available in tm1KntTg(FCER1A)1Bhk/J] were purchased from The Jackson Laboratory (stock 010506), bred, and genotyped. These mice carry two gene mutations: the human Fc fragment of IgE receptor α polypeptide (FCER1A) under control of the human FCER1A promoter and a mutation targeting Fcεr1atm1Knt, blocking expression of murine FCER1A. Human IgE can induce anaphylaxis in mice hemizygous for the transgene and homozygous for targeted deletion of mouse FcεRI. Transgenic mice were sensitized by i.v. injection of 100 μg total IgE and challenged by i.p. injection of 50 μg purified recombinant allergen. Changes in mouse core body temperature were monitored over 90 min using implanted temperature probes. Statistical analysis is described in Mouse studies were carried out in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Human FcεRI–transgenic mice [B6.Cg-Fcεr1apgac054_Supplemental_FileClick here for additional data file.


Fusarium equiseti was developed based on chitosan nanoparticles (CNPs) combined with Trichoderma longibrachiatum and Penicillium polonicum. Two strains of F. equiseti which were isolated from wilting tomato plant as well as three antagonistic fungi including Trichoderma longibrachiatum and two strains of Penicillium polonicum were isolated from the surrounding soil. All the isolated pathogenic and antagonistic fungi were identified using genomic DNA sequences. The antifungal activity of the three antagonistic fungi were studied against the two strains of F. equiseti. Also, CNPs which were prepared according to the ionic gelation method using sodium tripolyphosphate anions in acetic acid solution were used to enhance the antifungal activity of the three antagonistic fungi. The results exhibit that, combination of T. longibrachiatum with CNPs and P. polonicum with CNPs achieve high antifungal activity against F. equiseti by an inhibition rate equal to 71.05% and 66.7%, respectively.A safe and ecofriendly biocontrol of pathogenic  Fusarium species are considered one of the most important known soil borne plant pathogens [Fusarium species are widely distributed in many sources such as air, soil, plants, marine ecosystems, and fresh water [Fusarium species have the ability to be alive either as chlamydospores in the remains of the infected plants for about 30 years or in the alternative host roots, and cause high levels of damage for many crops such as tomato, pea, potato, bean, wheat, corn and rice with yield losses up to 30–70% [Fusarium species, F. equiseti causes wilt diseases on various plant hosts such as grafted watermelon, grape, cucumber, tomato, cowpea, bean, potato [F. equiseti was reported to cause root rot of sugar beets [F. equiseti, can produce mycotoxins such as Zearalenone which they are usually detected in combination with other fusariotoxines, such as trichothecenes and fumonisins. All are dangerous toxins to life being [Phytopathogenic fungi seriously affect large numbers of plants, seeds and damage many important crops all over the world. As a result, many crops were spoiled, which led to a decrease in agriculture, both qualitatively and quantitatively , 2. Amonathogens –5. Fusarsh water . Also, F, potato –15. In aar beets . Also, sfe being .Fusarium species especially F. equiseti is an essential requirement to maintain a safe environment for both human and animals. Although plant pathogenic fungi can be eliminated with chemical fungicides, excessive use of them has many drawbacks including being harmful to soil, causing deviation from the normal flora and fauna system, resistance to pathogens and pollution of the environment [F. equiseti with moderate inhibition rates. For instance, Azotobacter nigricans was reported as antifungal against some Fusarium species including F. sporotrichioides, F. graminearum, F. poae and F. equiseti with a growth inhibition up to 50% [Bacillus subtilis was used for controlling F. solani, F. equiseti and F. oxysporum with inhibition percentage ranged from 51, 66 and 47% after 5 days, respectively [Streptomyces bellus was applied against F. equiseti and two strains of F. fujikuroi and achieved inhibition percentage 55, 43 and 36%, respectively [In this context, effective control of ironment . Howeverp to 50% . Bacilluectively . Also, Sectively .2 [During the past few years, metal nanoparticles (MNPs) such as silver , 22, cop2  have bee2 . Althoug2 –32. As a2 .Trichoderma asperellum was effective in suppressing mycelial growth pathogenic fungi including Fusarium oxysporum and F. graminearum [Fusarium oxysporum ciceri, Pyricularia grisea, and Alternaria solani with the rate of inhibition 87%, 92%, and 72%, respectively [It is stated that biogenic CNPs from four different fungal sources in combination with minearum , 39. Aliectively .F. equiseti and the good properties of CNPs as well as the few published studies deal with the use of CNPs as antifungal against F. equiseti, we aim in this study to isolate F. equiseti from wilting tomato plant and inhibit their growth using some antagonistic fungi and CNPs. Furthermore, we aimed to study the antifungal activities of these antagonistic fungi combined with CNPs against F. equiseti.Due to the hazard issue of − 1 to 10− 5), was transferred to PDA plates, incubated for 5 days at 25 ± 27 °C. The growing hyphal tips were picked up and preserved on PDA slopes further studies. [The pathogenic and antagonistic fungi were isolated from the infected vascular tissues of tomato (roots and stem) and the surrounding soil collected from Damietta and Dakahlia governorates, Egypt. Pathogenic isolated from surface sterilized (5% hypochlorite solution) root and stem pieces (0.5–1 cm) on potato dextrose agar (PDA) containing traces of Chloramphenicol for 5 to 7 days at 25 ± 27 °C. For isolation of antagonistic fungi, the surrounding soils were air dried and saved to remove large particles. Then 1 ml from each dilution  were cut by corkborer from the peripheral end and inserted in the center of the test Petri dishes. All Petri dishes were incubated in laboratory condition at 25 °C for 7 days, and daily measurements of radial colony growth were taken until the fastest growing colony approached the plate's edge. All the treatments had three replications, and the experiment was carried out twice. By using Vincent's formula  of 7 days old culture of each strain of F. equiseti which was cut by sterilized cork borer and then was placed in PDA media in the edge of petri dish. In the opposite edge of petri dish another same sized agar disk of the antagonistic fungi was placed, then incubated in laboratory condition at 25 ± 27 °C for 5 days. The antagonistic activity was recorded after incubation by calculating the inhibition rate percentage according to Eq. , a mycelial disc (5 mm diameter) was cut by corkborer and inoculated into another two plates containing F. equiseti st.1 and F. equiseti st.2 on one side of the PDA plate, respectively. After observing full growth in the control plates, the radial growth of the pathogens was recorded. The percentage of inhibition of mycelial growth was calculated according to Eq. An in vitro study was carried out to determine the efficiency of CNPs combined with the three antagonistic fungi against the two strains of equiseti . In thisGenomic DNA from pure pathogenic and antagonistic fungal cultures were extracted using ABT DNA mini extraction kit , according to the manufacturer’s instructions. PCR was performed in Veriti™ 96-Well Thermal Cycler (Applied Biosystems). The products of the amplified PCR were submitted to Solgent Co Ltd (South Korea) for gel purification and sequencing. Fourier transform infrared analyses (FTIR) was recorded by using KBr plates on a JASCO FT/IR-4100 Fourier transform infrared spectrometer. Transmission Electron Microscopy (TEM) image for CNPs was acquired by JEOL JEM–2100 microscopy at an accelerating voltage of 200 kV. Zeta Potential analysis was carried out using Malvern Zetasizer Nano-ZS90, Malvern, UK.The results obtain from DNA sequences for both pathogenic and antagonistic fungi were trimmed and assembled in Geneious software (Biomatters). Consequently, the trimmed sequences were identified by search in basic local alignment search tool (BLAST) in GenBank and recorded with accession numbers as shown in Table−1 which are attributed to amide (-CONH2), anhydro glucosidic ring and another peak at 3450 cm−1 which is related to primary amine group (NH2). Moving into CNPs, the characteristic peaks of amide and primary amine groups are shifted to lower wavenumbers and appear at 1602 and 3425 cm−1, respectively. The decrease in stretching frequency could be due to the TPP interaction with ammonium group of chitosan and more hydrogen bonding in chitosan–TPP complex [FTIR was used to confirm the formation of CNPs through the interaction between chitosan and TTP. Figure  complex .Fig. 1FTBoth transmission electron microscopy (TEM) image and particle size distribution of CNPs are presented in Fig. Figure F. equiseti st.1 and F. equiseti st.2. The antifungal activity of CNPs appears from the affinity of its cationic amino groups to cellular components [F. equiseti. The results show that increasing the concentration of CNPs leads to increase the inhibition percentage for both F. equiseti st.1 and F. equiseti st.2. The maximum inhibition rates for F. equiseti st.1 and F. equiseti st.2 is found to equal 40.39% and 66% at CNPs concentration 0.172 mg/ml, respectively. While the minimum inhibition rates are 8.81% and 19% at CNPs concentration 0.043 mg/ml for F. equiseti st.1 and F. equiseti st.2.CNPs are used as a biodegradable polymer for the inhibition of mycelium radial growth of both mponents . All theF. equiseti which were isolated from wilting tomato plants. The measuring of antagonistic activity used the dual plate method [T. longibrachiatum, P. polonicum st.1 and P. polonicum st.2 as antagonistic fungi for inhibition of F. equiseti st.1 and F. equiseti st.2. It is clear from Tables T. longibrachiatum exhibits the higher antifungal activity against F. equiseti st.1 and F. equiseti st.2 by inhibition rates of 60% and 62.74%, respectively while P. polonicum st.1 appears less antifungal activity by inhibition rates of 40% and 51.41% for F. equiseti st.1 and F. equiseti st.2, respectively. In fact, Trichoderma spp. have long history as a biocontrol agent against several pathogenic fusaria such as Fusarium oxysporum [Tr. longibrachiatum acts as a biocontrol agent of Fusarium wilt of cucumber [The antagonistic activities are measured by inhibition the growth of the two strains e method . Table 3xysporum  and Fusaxysporum . It is acucumber .Table 3AT. longibrachiatum, P. polonicum st.1 and P. polonicum st.2 against the two strain F. equiseti, the antagonistic fungi were firstly cultivated in PDA media containing CNPs with a concentration of 0.172 mg/ml that achieves the maximum inhibition for both F. equiseti st.1 and F. equiseti st.2 to produce a combination of T. longibrachiatum with CNPs, P. polonicum st.1 with CNPs as well as P. polonicum st.2 with CNPs. After 7 days of incubation, the effect of these combinations against the two strain F. equiseti were studied using the dual plate method. The results in Table T. longibrachiatum increases its antifungal activity from 60% to 65.88% and from 62.74% to 71.05% against F. equiseti st.1 and F. equiseti st.2, respectively. Also, CNPs increases the antifungal activity of P. polonicum st.1 and P. polonicum st.2 from 40% to 42.35% and from 51.41% to 55.52% against F. equiseti st.1, respectively. In case of F. equiseti st.2, CNPs lead to increase the inhibition rate from 51.41% to 55.52% and from 58.82% to 66.7% using P. polonicum st.1 and P. polonicum st.2, respectively. Alike, it is stated that biogenic CNPs from four different fungal sources in combination with Trichoderma asperellum was effective in suppressing mycelial growth Fusarium oxysporum and other soil borne pathogenic fungi [F. equiseti.In order to enhance the antifungal activity of ic fungi . These rF. equiseti based on the use of microorganisms including bacteria, fungi and actinomycetes. Table T. longibrachiatum combined with CNPs, which achieve the maximum inhibition rate, and the other reported antifungal against F. equiseti. According to Table T. longibrachiatum with CNPs and Talaromyces strain DYM25 [F. equiseti.There are a few studies dealing with biocontrol of in DYM25  exhibit F. equiseti can be controlled using CNPs, T. longibrachiatum, P. polonicum st.1 and P. polonicum st.2 with high inhibition rate. The use of CNPs is ecofriendly, biodegradable and anti- F. equiseti with higher inhibition rate compared with antagonistic fungi. Moreover, the combination of CNPs with T. longibrachiatum or P. polonicum strains enhance their antifungal activity against F. equiseti.In conclusion, the growth of tomato wilt pathogen,


However, no statistically significant differences were observed for becoming exposed to ARV between villages (p < 0.05). The backyard chickens tested in this study had no previous history of vaccination, outbreaks or typical clinical signs of IB and AR diseases. Therefore, the presence of antibodies to IBV and ARV was considered clear evidence that the birds have been naturally exposed to those two infectious agents, and the infection was of subclinical type. It is concluded that IBV and ARV are widespread in backyard chickens in the studied area. These obtained data are essential for design and implementation of chicken health development programmes.Infectious bronchitis virus (IBV) and avian reovirus (ARV) cause significant losses in the poultry industry throughout the world. A cross-sectional study was conducted in four villages in Manjacaze district, Southern Mozambique, to determine the seroprevalence of IBV and ARV. A total of 467 serum samples from adult unvaccinated backyard chickens were screened using commercial and competitive enzyme-linked immunoabsorbent assay kits. Our results showed anti-IBV and anti-ARV antibodies in all surveyed households and villages. The overall seroprevalence was 89.5%  and 95.7% (95% CI: 88.0–99.2) for IBV and ARV, respectively. The risk of becoming exposed to IBV was lower in Chidenguele village compared with the other three villages (The epidemiology of IBV and ARV of backyard chicken in Mozambique is unknown. This study determined the seroprevalence of IBV and ARV in backyard chicken health. The obtained data are essential for design and implementation of chicken health development programmes. Coronaviridae and subfamily Coronavirinae within the genera of Gammacoronaviridae. Infectious bronchitis virus is an enveloped virus with single-stranded positive-sense linear ribonucleic acid (RNA) with the genome of about 27 kB (Jackwood Infectious bronchitis (IB) is a highly contagious disease with severe economic consequences in the chicken industry worldwide (Cavanagh 2007). The etiologic agent, infectious bronchitis virus (IBV), belongs to the family Jackwood . Birds iJackwood . In layiJackwood . MorbidiJackwood . At necrJackwood . In backJackwood , South AJackwood , BotswanJackwood , NigeriaJackwood , EthiopiJackwood , Ivory CJackwood , Mexico Jackwood , SwitzerJackwood , BangladJackwood  and IranJackwood . There aGallus gallus) and a member of the genus Orthoreovirus in the Reoviridae family. The ARV genome contains 10 segments of double-stranded RNA that encode at least eight structural proteins  and four nonstructural proteins   is a pathogenic agent in chickens  and anti-ARV antibodies  according to the manufacturer’s instructions. Optical reading was performed at 450 nm, and the cut-off values used were according to the manufacturer’s instructions.Serum samples were analysed using commercial enzyme-linked immunosorbent assay (ELISA) kits for the presence of anti-IBV antibodies  and exported to Stata® version 12.1 (Stata IC 12.1 for Windows) software  for analysis. Prevalence data were analysed using the chi-square test (χ2-test). Logistic regression models were used to compute odds ratios (OR) to identify the risk for being infected as dichotomous dependent variable and independent variable (location). In all chi-square tests, p < 0.05 was considered statistically significant.Data were entered into a Microsoft ExcelEthical review and approval were granted by the Scientific Board of the Faculty of Veterinary Medicine, Eduardo Mondlane University .p < 0.05). Narrowed seroprevalence variations between villages were seen for ARV. Seroprevalences of ARV did not exhibit significant differences between villages (p = 0.05).Antibodies to IBV and ARV were detected in chickens from all villages included in the study. Serological results are presented in This study is the first cross-sectional survey conducted to determine the seroprevalence of IBV and ARV among backyard chickens at the village level in Southern Mozambique. In unvaccinated flocks, positive serological results are clear evidence that the birds have been exposed to the infectious agent under consideration, although without identifying the infectious strains  , which agrees with reports from western Turkey (86.53%) (Erol & Sengül In backyard flocks from the present study sites, biosecurity measures are not employed, flocks are often composed of a mixture of ages and species and new chickens are continually added. The authors speculate that these husbandry practices and the backyard chickens–wild birds contact may explain the high seroprevalence reported in this study for both IBV and ARV (Wille & Holmes In summary, it is concluded that IBV and ARV are circulating among indigenous chickens in the investigated area. Therefore, the infected chickens may represent a threat in the transmission of those viruses to wild birds, broiler or layer chickens in that region. However, additional research is warranted to identify the circulating strains and the epidemiology of these diseases.