Cpd-A1 alleviates acute kidney injury by inhibiting ferroptosis.

Acute kidney injury (AKI) is defined as sudden loss of renal function characterized by increased serum creatinine levels and reduced urinary output with a duration of 7 days. Ferroptosis, an iron-dependent regulated necrotic pathway, has been implicated in the progression of AKI, while ferrostatin-1 (Fer-1), a selective inhibitor of ferroptosis, inhibited renal damage, oxidative stress and tubular cell death in AKI mouse models. However, the clinical translation of Fer-1 is limited due to its lack of efficacy and metabolic instability. In this study we designed and synthesized four Fer-1 analogs (Cpd-A1, Cpd-B1, Cpd-B2, Cpd-B3) with superior plasma stability, and evaluated their therapeutic potential in the treatment of AKI. Compared with Fer-1, all the four analogs displayed a higher distribution in mouse renal tissue in a pharmacokinetic assay and a more effective ferroptosis inhibition in erastin-treated mouse tubular epithelial cells (mTECs) with Cpd-A1 (N-methyl-substituted-tetrazole-Fer-1 analog) being the most efficacious one. In hypoxia/reoxygenation (H/R)- or LPS-treated mTECs, treatment with Cpd-A1 (0.25 µM) effectively attenuated cell damage, reduced inflammatory responses, and inhibited ferroptosis. In ischemia/reperfusion (I/R)- or cecal ligation and puncture (CLP)-induced AKI mouse models, pre-injection of Cpd-A1 (1.25, 2.5, 5 mg·kg-1·d-1, i.p.) dose-dependently improved kidney function, mitigated renal tubular injury, and abrogated inflammation. We conclude that Cpd-A1 may serve as a promising therapeutic agent for the treatment of AKI.

Madecassoside alleviates acute kidney injury by regulating JNK-mediated oxidative stress and programmed cell death.

BACKGROUND: Acute kidney injury (AKI) has high morbidity and mortality, which is manifested by inflammation and apoptosis. Effective treatment methods for AKI are currently lacking. OBJECTIVE: This study demonstrated the protecting effects of Madecassoside (MA) in the cisplatin- and hypoxia-reoxygenation-induced renal tubular epithelial cells in vitro and AKI mice in vivo. METHODS: In vivo AKI mouse models were established by inducing them with cisplatin and renal ischemia-reperfusion. In vitro injury models of mouse renal tubular epithelial cells were established by inducing them with cisplatin and hypoxia and reoxygenation, respectively. The mechanism of MA effects was further explored using molecular docking and RNA-sequencing. RESULTS: MA could significantly reduce kidney injury in the cisplatin-and renal ischemia-reperfusion (IRI)-induced AKI. Further validation in the two cellular models also showed that MA had protect effects. MA can alleviate AKI in vitro and in vivo by inhibiting inflammation, cell apoptosis, and oxidative stress. MA exhibited high permeability across the Caco-2 cell, can enter cells directly. Through RNA-seq and molecular docking analysis, this study further demonstrated that MA inhibits its activity by directly binding to JNK kinase, thereby inhibiting c-JUN mediated cell apoptosis and improving AKI. In addition, MA has better renal protective effects compared to curcumin and JNK inhibitor SP600125. CONCLUSION: The results demonstrate that MA might be a potential drug for the treatment of AKI and act through the JNK/c-JUN signaling pathway.

O2-Generating Fluorescent Carbon Dot-Decorated MnO2 Nanosheets for "Off/On" MR/Fluorescence Imaging and Enhanced Photodynamic Therapy.

Imaging-guided photodynamic therapy (PDT) has emerged as a promising protocol for cancer theragnostic. However, facile preparation of such a theranostic system for simultaneously achieving tumor location, real-time monitoring, and high-performance reactive oxygen species generation is highly desirable but remains challenging. Herein, we developed a reasonable tumor-targeting strategy based on carbon dots (CDs)-decorated MnO2 nanosheets (HA-MnO2-CDs) with an active magnetic resonance (MR)/fluorescence imaging and enhanced PDT effect. Under light irradiation, the addition of HA-MnO2-CDs increased the production of 1O2 by 2.5 times compared with CDs, providing favorable conditions for the PDT treatment effect on breast cancer. Moreover, HA-MnO2-CDs exhibited excellent performance in producing O2 in the presence of endogenous H2O2, which alleviated hypoxia in tumors and improved the therapeutic effect of PDT. In the presence of glutathione (GSH), the degraded MnO2 nanosheets released CDs and Mn2+ from HA-MnO2-CDs, restoring their fluorescence imaging function and increasing T1 relaxivity (r1) by 23 times. In vivo fluorescence and MR imaging suggested the excellent tumor-targeting property of HA-MnO2-CDs. By combining the complementary properties of nanoprobes and tumor microenvironments, the in vivo PDT therapeutic effect was significantly improved under the action of HA-MnO2-CDs. Overall, our reasonably designed HA-MnO2-CDs may inspire the future development of the next generation of high-performance tumor-responsive diagnostic and therapeutic agents to further enhance the targeted therapy effect of tumors.

Protein acetylation and related potential therapeutic strategies in kidney disease.

Kidney disease can be caused by various internal and external factors that have led to a continual increase in global deaths. Current treatment methods can alleviate but do not markedly prevent disease development. Further research on kidney disease has revealed the crucial function of epigenetics, especially acetylation, in the pathology and physiology of the kidney. Histone acetyltransferases (HATs), histone deacetylases (HDACs), and acetyllysine readers jointly regulate acetylation, thus affecting kidney physiological homoeostasis. Recent studies have shown that acetylation improves mechanisms and pathways involved in various types of nephropathy. The discovery and application of novel inhibitors and activators have further confirmed the important role of acetylation. In this review, we provide insights into the physiological process of acetylation and summarise its specific mechanisms and potential therapeutic effects on renal pathology.

Coating influence on inner shell water exchange: An underinvestigated major contributor to SPIONs relaxation properties.

Superparamagnetic iron oxide nanoparticles (SPIONs) are heavily studied as potential MRI contrast enhancing agents. Every year, novel coatings are reported which yield large increases in relaxivity compared to similar particles. However, the reason for the increased performance is not always well understood mechanistically. In this review, we attempt to relate these advances back to fundamental models of relaxivity, developed for chelated metal ions, primarily gadolinium. We focus most closely on the three-shell model which considers the relaxation of surface-bound, entrained, and bulk water molecules as three distinct contributions to total relaxation. Because SPIONs are larger, more complex, and entrain significantly more water than gadolinium-based contrast agents, we consider how to adapt the application of classical models to SPIONs in a predictive manner. By carefully considering models and previous results, a qualitative model of entrained water interactions emerges, based primarily on the contributions of core size, coating thickness, density, and hydrophilicity.

Novel insights into STAT3 in renal diseases.

Signal transducer and activator of transcription 3 (STAT3) is a cell-signal transcription factor that has attracted considerable attention in recent years. The stimulation of cytokines and growth factors can result in the transcription of a wide range of genes that are crucial for several cellular biological processes involved in pro- and anti-inflammatory responses. STAT3 has attracted considerable interest as a result of a recent upsurge in study because of their role in directing the innate immune response and sustaining inflammatory pathways, which is a key feature in the pathogenesis of many diseases, including renal disorders. Several pathological conditions which may involve STAT3 include diabetic nephropathy, acute kidney injury, lupus nephritis, polycystic kidney disease, and renal cell carcinoma. STAT3 is expressed in various renal tissues under these pathological conditions. To better understand the role of STAT3 in the kidney and provide a theoretical foundation for STAT3-targeted therapy for renal disorders, this review covers the current work on the activities of STAT3 and its mechanisms in the pathophysiological processes of various types of renal diseases.

Hyperbranched polyamidoamine-chitosan polyelectrolyte gels crosslinking by polyacrylic acid and alginate for removal of anionic dyes.

Difficult-to-treat wastewater discharged from printing and dyeing industries has posed an environmental risk. Three-dimensional porous structures exhibit excellent dye adsorption properties. In this study, chitosan (CTS) was introduced in situ on polyamidoamine (PAMAM). Then, the composite gels containing sodium alginate (SA)/CTS in situ grown on PAMAM/polyacrylic acid (SCPP-Gn (n = 0,1,2,3)) were prepared with a controllable structure using a semi-dissolution acidification sol-gel transition method (SD-A-SGT). Owing to the stronger mechanical strength and larger specific surface area, the SCPP-G2 composite gel has the higher stability and the greater adsorption capacity for dyes, with maximum adsorption capacities of 325.21 mg/g for rose bengal (RB) and 222.40 mg/g for sunset yellow (SY). The mechanism study showed that the adsorption toward RB and SY was dominated by chemical adsorption. After five adsorption-desorption cycles, it was found that 70 % of the initial adsorption capacity could be retained. Our study demonstrates the great potential of the SCPP-G2 composite gel as an adsorbent in water treatment.

Targeted Delivery of DNA Topoisomerase Inhibitor SN38 to Intracranial Tumors of Glioblastoma Using Sub-5 Ultrafine Iron Oxide Nanoparticles.

Effectively delivering therapeutics for treating brain tumors is hindered by the physical and biological barriers in the brain. Even with the compromised blood-brain barrier and highly angiogenic blood-tumor barrier seen in glioblastoma (GBM), most drugs, including nanomaterial-based formulations, hardly reach intracranial tumors. This work investigates sub-5 nm ultrafine iron oxide nanoparticles (uIONP) with 3.5 nm core diameter as a carrier for delivering DNA topoisomerase inhibitor 7-ethyl-10-hydroxyl camptothecin (SN38) to treat GBM. Given a higher surface-to-volume ratio, uIONP shows one- or three-folds higher SN38 loading efficiency (48.3 ± 6.1%, mg/mg Fe) than those with core sizes of 10 or 20 nm. SN38 encapsulated in the coating polymer exhibits pH sensitive release with <10% over 48 h at pH 7.4, but 86% at pH 5, thus being protected from converting to inactive glucuronide by UDP-glucuronosyltransferase 1A1. Conjugating αv ß3 -integrin-targeted cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) as ligands, RGD-uIONP/SN38 demonstrates targeted cytotoxicity to αv ß3 -integrin-overexpressed U87MG GBM cells with a half-maximal inhibitory concentration (IC50 ) of 30.9 ± 2.2 nm. The efficacy study using an orthotopic mouse model of GBM reveals tumor-specific delivery of 11.5% injected RGD-uIONP/SN38 (10 mg Fe kg-1 ), significantly prolonging the survival in mice by 41%, comparing to those treated with SN38 alone (p < 0.001).

Biochemical characterization and redesign of the coenzyme specificity of a novel monofunctional NAD+-dependent homoserine dehydrogenase from the human pathogen Neisseria gonorrhoeae.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD+ dependent, with an approximate 479-fold (kcat/Km) preference for NAD+ over NADP+, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD+-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP+, caused a shift in coenzyme preference from NAD+ to NADP+ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD+-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.

Ultracompact Iron Oxide Nanoparticles with a Monolayer Coating of Succinylated Heparin: A New Class of Renal-Clearable and Nontoxic T1 Agents for High-Field MRI.

Herein, we present a new magnetic iron oxide nanoparticle (MION) with a succinylated heparin monolayer coating, which exhibits the highest T1 relaxivity at 7 T and the lowest r2/r1 reported for any MION at these high-field conditions. While the recent proliferation of 7 T MRI instruments in hospitals worldwide has enabled widespread access to higher quality, more finely detailed, diagnostic imaging, clinically available contrast agents have not kept pace due to the general phenomenon of reduced efficacy of T1 relaxation as magnetic field strength is increased. Development of new MION agents is one strategy to address this need, and to this end, we demonstrate the in vitro magnetic properties of the MIONs reported here to extend to in vivo applications, providing greatly increased contrast in tumor imaging in a murine xenograft subject at 7 T. While MION-based contrast agents can have side effects in clinical application, these are generally thought to be less than those of gadolinium-based agents and here are further reduced by the small size allowing direct glomerular filtration from the blood followed by renal-excretion. Finally, we show the succinylated heparin monolayer coating to provide class leading magnetic properties over a homologous series of particles with core size ranging from 2 to 18 nm and show the properties to be strongly related to the surface area. We suggest the increased porosity and hydrophilicity of the coating to increase water accessibility to the surface resulting in the increased magnetic properties.

Immunological evaluation of virulence-deficient Listeria monocytogenes strains in C57BL/6 mice.

Attenuated Listeria monocytogenes (L. monocytogenes), which has unique advantages in presenting foreign antigens, was widely used in tumor immunotherapy research. As a live vaccine vector, attenuated L. monocytogenes was required to not only have certain invasiveness but also ensure safety, while the lack of different virulence factors may cause L. monocytogenes to show different safety and invasiveness. To evaluate the potential of virulence-deficient L. monocytogenes strains as a vaccine vector, four mutant strains EGD-eΔactA, EGD-eΔactA/inlB, EGD-eΔhly, and EGD-eΔprfA were used to infect C57BL/6 mice for determining related immune indexes. Compared with EGD-e, mutant strains showed significantly decreased invasion in C57BL/6 mice and caused relatively minor damage to spleen and liver. However, EGD-eΔactA and EGD-eΔactA/inlB were superior to EGD-eΔhly and EGD-eΔprfA in the comprehensive evaluation of inflammatory factor transcription level, immune cell differentiation and antibody level, which proved that they have a stronger adjuvant effect as a vaccine vector.

Going even smaller: Engineering sub-5 nm nanoparticles for improved delivery, biocompatibility, and functionality.

The rapid development and advances in nanomaterials and nanotechnology in the past two decades have made profound impact in our approaches to individualized disease diagnosis and treatment. Nanomaterials, mostly in the range of 10-200 nm, developed for biomedical applications provide a wide range of platforms for building and engineering functionalized structures, devices, or systems to fulfill the specific diagnostic and therapeutic needs. Driven by achieving the ultimate goal of clinical translation, sub-5 nm nano-constructs, in particular inorganic nanoparticles such as gold, silver, silica, and iron oxide nanoparticles, have been developed in recent years to improve the biocompatibility, delivery and pharmacokinetics of imaging probes and drug delivery systems, as well as in vivo theranostic applications. The emerging studies have provided new findings that demonstrated the unique size-dependent physical properties, physiological behaviors and biological functions of the nanomaterials in the range of the sub-5 nm scale, including renal clearance, novel imaging contrast, and tissue distribution. This advanced review attempts to introduce the new strategies of rational design for engineering nanoparticles with the core sizes under 5 nm in consideration of the clinical and translational requirements. We will provide readers the update on recent discoveries of chemical, physical, and biological properties of some biocompatible sub-5 nm nanomaterials as well as their demonstrated imaging and theranostic applications, followed by sharing our perspectives on the future development of this class of nanomaterials. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Implantable Materials and Surgical Technologies > Nanomaterials and Implants.

Attenuated Listeria monocytogenes as a Vaccine Vector for the Delivery of OMPW, the Outer Membrane Protein of Aeromonas hydrophila.

Listeria monocytogenes (LM) is a gram-positive facultative intracellular pathogen that could stimulate host to produce inflammatory response, cell-mediated immunity, and humoral immunity. In this study, an attenuated live vector vaccine for Aeromonas hydrophila (AH) named EGDeABdd-dat-ompW was successfully constructed using an attenuated vector named EGDeABdd, in which dal, dat, actA, and inlB genes were deleted from wild-type LM-EGDe. To construct EGDeABdd-dat-ompW, a recombinant plasmid pERL3-dat-ompW obtained by inserting the dat gene from EGDe and outer membrane protein gene ompW from AH into pERL3 plasmid was transformed into EGDeABdd cell. The safety and immunogenicity of EGDeABdd-dat-ompW as an attenuated vector vaccine for delivery of OMPW were assessed through analyzing invasion to Caco-2 cells and mice, cytokine production of macrophagocyte and mouse splenocytes, and T-cell proliferation of mouse splenocytes. Serum titers against AH and the immunoprotective effect of the vaccine to mice were also measured after intravenous injection with vaccine for four times. The results showed that the live vector vaccine EGDeABdd-dat-ompW for AH exhibited high attenuation in invading Caco-2 cells and mice than did EGDe. Real-time PCR (RT-PCR) showed that cytokines (e.g., TNF-α, IL-6, and IL-1ß from macrophages; and IL-6 and IFN-γ from mouse splenocytes) had significantly increased after immunization by EGDeABdd-dat-ompW. Meanwhile, the vaccine could induce the production of CD3+CD4+ and CD3+CD8+ T-cell proliferation of mice and generate effective immunoprotection against lethal challenge of 20 × LD50 AH. All these results indicated that the attenuated EGDeABdd-dat could be used as a live vector for the delivery of the exogenous gene, not only possessing safety but also providing high immunogenicity. The successful application in the AH vaccine further showed that it could be used in other fields such as vaccines in cancer or infectious diseases.

Succinylated heparin monolayer coating vastly increases superparamagnetic iron oxide nanoparticle T2 proton relaxivity.

Superparamagnetic iron oxide nanoparticles (SPIONs) have a history of clinical use as contrast agents in T2 weighted MRI, though relatively low T2 relaxivity has caused them to fall out of favor as new faster MRI techniques have gained prominence. We demonstrate that SPIONs coated with a monolayer of succinylated heparin (Su-HP-SPIONs) exhibit over four-fold increased T2 relaxivity (460 mM-1 s-1) as compared to the clinically approved SPION-based contrast agent Feridex (98.3 mM-1 s-1) due to greatly increased water interaction from increased hydrophilicity and thinner coating as supported by our proposed parametric model. In vivo, the performance increase of the Su-HP-SPIONs in T2 MRI imaging of xenograft tumors is ten-fold that of our in-house synthesized Feridex analogue, due to better tumor localization from the smaller size imparted by the thinner coating. In addition to these significantly improved magnetic properties, the succinylated heparin coating also exhibits favorable synthetic reproducibility, solution stability, and biocompatibility. These findings demonstrate the untapped potential of SPIONs as possible high performance clinical T2 contrast agents.

Reactive oxygen species inhibits Listeria monocytogenes invasion into HepG2 epithelial cells.

Listeria monocytogenes (Lm) can colonize human gastrointestinal tract and subsequently cross the intestinal barrier. Reactive oxygen species (ROS) are produced by NADPH oxidase. However, the role of ROS in bacterial invasion remains to be less understood. Herein, we investigated the impact of ROS on Lm invasion to HepG2 using NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the ROS scavenger, N-acetyl cysteine (NAC). Our results showed that inhibiting ROS increased the invasive capability of Lm. Moreover, after Lm infection, inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1ß) in HepG2 were significantly upregulated. However, after inhibiting ROS, the expression levels of TNF-α and IL-1ß were downregulated, indicating a failure of host cells to activate the immune mechanism. Taken together, ROS in Lm might be as a signal for host cells to sense Lm invasion and then stimulate cells to activate the immune mechanism.

Exploration of the bacterial invasion capacity of Listeria monocytogenes in ZF4 cells.

Despite the results from zebrafish challenged model have demonstrated that Listeria monocytogenes (Lm) has strong adjuvant effects when this attenuated pathogenic bacteria is viewed as aquaculture vaccine vector, the underlying mechanism is not clear and extensive investigations are required. To further explore the potential of Lm in the field of aquaculture vaccine, zebrafish embryonic fibroblast cell line (ZF4) was used to evaluate the invasion ability of Lm. The data from cellular level showed that Lm had the lower invasion tendentiousness in ZF4 cells while bacterial invasion capacity was compared between zebrafish embryos cell line and human intestinal epithelial cell line. In ZF4 cells, there is no significant difference in bacterial invasion capacity between wild strain EGD-e and double-deleted strain ΔactA/inlB, which suggested that this attenuated effect was not showed in zebrafish cells. In addition, translation analysis indicated that the expressions of CD4 and CD8a in ZF4 cells increased after 2-h infection of the two Lm strains. These results further demonstrated that Lm presented multiple advantages including lower pathogenicity and antigen presentation when attenuated stain was viewed as aquaculture vaccine vector.

Downregulating Heparanase-Induced Vascular Normalization: A New Approach To Increase the Bioavailability of Chemotherapeutics in Solid Tumors.

Downregulating heparanase has been shown to reduce tumor angiogenesis and prevent chemoresistance, and it is becoming an appealing approach to treat solid tumors. However, little attention has been given to its underlying antitumor mechanisms, especially the relationship between heparanase and vascular development in solid tumors, which is not yet fully understood. In this study, we found that downregulating heparanase through orthotopic injection of heparanase small interfering RNA not only could reduce vascular density but, more importantly, lead to vascular normalization in solid tumors. Consequently, this may lead to a more efficient delivery of chemotherapeutic agents. These findings provide the basis for developing new approaches to treat solid tumors with a combination of heparanase inhibitors and chemotherapeutics.

Evaluation of Caco-2 cells response to Listeria monocytogenes virulence factors by RT-PCR.

Listeria monocytogenes expresses various virulence factors enabling the invasion and multiplying in host cells, and together induces cytokines transcription. In order to explore the relationship between virulence factors of L. monocytogenes wild-type EGD-e and cellular response in human colonic epithelial cell line(Caco-2), we constructed mutant strains with in-frame deletions of critical virulence genes of inlA, inlB, hly, actA and virulence regulatory factor prfA from EGD-e, respectively. Compared with EGD-e, mutant strains showed significantly decreased invasion and apoptosis in Caco-2 cells. However, mutant strains were capable to evoke cytokines transcription of interleukin-8 (IL-8), mononuclear chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and CXCL-2 production in Caco-2 cells. Interestingly, EGD-e Δhly-infected Caco-2 cells showed a significant decrease of IL-6, IL-8 and MCP-1 transcription compared with EGD-e at 1 h post-infection. Simultaneously, EGD-e ΔinlB-infected cells showed a decrease in IL-6 transcription, while EGD-e ΔactA-infected cells reflected a decrease in MCP-1 transcription. Virulence genes play a role in inflammatory transcription, but the interaction between pathogenic bacteria and the host cells predominates in inflammatory transcription. Overall, the data showed cellular response of Caco-2 cells infected with EGD-e mutant strains.

Fluorescein Isothiocyanate Labeling Antigen-Based Immunoassay Strip for Rapid Detection of Acidovorax citrulli.

A simple and fast immunoassay strip to detect Acidovorax citrulli (Ac) using fluorescein isothiocyanate as a marker was developed. Fluorescein isothiocyanate (FITC) was added to sample culture medium for bacteria incubation, and the bacteria could emit a yellow-green fluorescence under ultraviolet light and become a fluorescent probe. This immunofluorescence strip (IFS) was based on the binding between fluorescent bacteria and the unlabeled monoclonal antibody (McAb) immobilized on the test area in nitrocellulose membrane. The detection limit of the strip was 106 CFU/ml with a result that could be observed within 10 min. The IFS could detect eight strains of Ac and display no cross-reactions with 30 other pathogenic strains. The detection results would not be affected by impurities in plant or unknown microorganisms in natural field samples and were consistent with PCR results, indicating that the IFS has high accuracy. This is the first report of using only one unlabeled McAb to develop a direct-type immunofluorescence strip for the rapid detection of Ac. The IFS reduced detection time and simplified operation procedures compared with the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. In addition, this simple and inexpensive method will play a significant role in monitoring plant pathogens on field detection.