Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation.

Targeted protein degradation technology holds great potential in biomedicine, particularly in treating tumors and other protein-related diseases. Research on intracellular protein degradation using molecular glues and PROTAC technology is leading, while research on the degradation of membrane proteins and extracellular proteins through the lysosomal pathway is still in the preclinical stage. The scarcity of useful targets is an immense limitation to technological advancement, making it essential to explore novel, potentially effective approaches for targeted lysosomal degradation. Here, we employed the glucose transporter Glut1 as an innovative lysosome-targeting receptor and devised the Glut1-Facilitated Lysosomal Degradation (GFLD) strategy. We synthesized potential Glut1 ligands via reversible addition-fragmentation chain transfer (RAFT) polymerization and acquired antibody-glycooligomer conjugates through bioorthogonal reactions as lysosome-targeting protein degradation molecules, utilized in the management of PD-L1 high-expressing triple-negative breast cancer. The glucose transporter Glut1 as a lysosome-targeting receptor exhibits potential for the advancement of a broader array of medications in the future.

Amphiphilic Block Copolymers Containing Benzenesulfonyl Azide Groups as Visible Light-Responsive Drug Carriers for Image-Guided Delivery.

Nanoparticles (NPs) containing light-responsive polymers and imaging agents show great promise for controlled drug delivery. However, most light-responsive NPs rely on short-wavelength excitation, resulting in poor tissue penetration and potential cytotoxicity. Moreover, excessively sensitive NPs may prematurely release drugs during storage and circulation, diminishing their efficacy and causing off-target toxicity. Herein, we report visible-light-responsive NPs composed of an amphiphilic block copolymer containing responsive 4-acrylamide benzenesulfonyl azide (ABSA) and hydrophilic N,N'-dimethylacrylamide (DMA) units. The polymer pDMA-ABSA was loaded with the chemotherapy drug dasatinib and zinc tetraphenylporphyrin (ZnTPP). ZnTPP acted as an imaging reagent and a photosensitizer to reduce ABSA upon visible light irradiation, converting hydrophobic units to hydrophilic units and disrupting NPs to trigger drug release. These NPs enabled real-time fluorescence imaging in cells and exhibited synergistic chemophotodynamic therapy against multiple cancer cell lines. Our light-responsive NP platform holds great promise for controlled drug delivery and cancer theranostics, circumventing the limitations of traditional photosensitive nanosystems.

Identification of key potential infection processes and risk factors in the computed tomography examination process by FMEA method under COVID-19.

PURPOSE: To identify the key infection processes and risk factors in Computed Tomography (CT) examination process within the standard prevention and control measures for the COVID-19 epidemic, aiming to mitigate cross-infection occurrences in the hospital. METHOD: The case hospital has assembled a team of 30 experts specialized in CT examination. Based on the CT examination process, the potential failure modes were assessed from the perspective of severity (S), occurrence probability (O), and detectability (D); they were then combined with corresponding risk prevention measures. Finally, key infection processes and risk factors were identified according to the risk priority number (RPN) and expert analysis. RESULTS: Through the application of RPN and further analysis, four key potential infection processes were identified, including "CT request form (A1)," "during the scan of CT patient (B2)," "CT room and objects disposal (C2)," and "medical waste (garbage) disposal (C3)". In addition, eight key risk factors were also identified, including "cleaning personnel does not wear masks normatively (C32)," "nurse does not select the vein well, resulting in extravasation of the peripheral vein for enhanced CT (B25)," "patient cannot find the CT room (A13)," "patient has obtained a CT request form but does not know the procedure (A12)," "patient is too unwell to continue with the CT scan (B24)," "auxiliary staff (or technician) does not have a good grasp of the sterilization and disinfection standards (C21)," "auxiliary staff (or technician) does not sterilize the CT machine thoroughly (C22)," and "cleaning personnel lacks of knowledge of COVID-19 prevention and control (C33)". CONCLUSION: Hospitals can publicize the precautions regarding CT examination through various channels, reducing the incidence of CT examination failure. Hospitals' cleaning services are usually outsourced, and the educational background of the staff employed in these services is generally not high. Therefore, during training and communication, it is more necessary to provide a series of scope and training programs that are aligned with their understanding level. The model developed in this study effectively identifies the key infection prevention process and critical risk factors, enhancing the safety of medical staff and patients. This has significant research implications for the potential epidemic of major infectious diseases.

Dual Recognition of H3K4me3 and DNA by the ISWI Component ARID5 Regulates the Floral Transition in Arabidopsis.

Chromatin remodeling and histone modifications are important for development and floral transition in plants. However, it is largely unknown whether and how these two epigenetic regulators coordinately regulate the important biological processes. Here, we identified three types of Imitation Switch (ISWI) chromatin-remodeling complexes in Arabidopsis (Arabidopsis thaliana). We found that AT-RICH INTERACTING DOMAIN5 (ARID5), a subunit of a plant-specific ISWI complex, can regulate development and floral transition. The ARID-PHD dual domain cassette of ARID5 recognizes both the H3K4me3 histone mark and AT-rich DNA. We determined the ternary complex structure of the ARID5 ARID-PHD cassette with an H3K4me3 peptide and an AT-containing DNA. The H3K4me3 peptide is combinatorially recognized by the PHD and ARID domains, while the DNA is specifically recognized by the ARID domain. Both PHD and ARID domains are necessary for the association of ARID5 with chromatin. The results suggest that the dual recognition of AT-rich DNA and H3K4me3 by the ARID5 ARID-PHD cassette may facilitate the association of the ISWI complex with specific chromatin regions to regulate development and floral transition.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Recognition of H3K9me1 by maize RNA-directed DNA methylation factor SHH2.

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation pathway, which has extensive cross-talk with histone modifications. Here, we report that the maize RdDM regulator SAWADEE HOMEODOMAIN HOMOLOG 2 (SHH2) is an H3K9me1 reader. Our structural studies reveal that H3K9me1 recognition is achieved by recognition of the methyl group via a classic aromatic cage and hydrogen-bonding and salt-bridge interactions with the free protons of the mono-methyllysine. The di- and tri-methylation states disrupt the polar interactions, decreasing the binding affinity. Our study reveals a mono-methyllysine recognition mechanism which potentially links RdDM to H3K9me1 in maize.

Role of type IV secretion system genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae strain RS-2.

Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4-62.0% in biofilm formation and 37.7-47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2.

Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification.

Reverse transcription recombinase polymerase amplification (RT-RPA) for detection of Cucumber green mottle mosaic virus (CGMMV) was developed in this study. It was found to be specific, with a limit of detection of 0.5 pg of total CGMMV RNA. CGMMV on inoculated leaves was tested using RT-RPA assay, suggesting that this method is suitable for CGMMV detection from leaves.

Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis.

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Degradation dynamics of glyphosate in different types of citrus orchard soils in China.

Glyphosate formulations that are used as a broad-spectrum systemic herbicide have been widely applied in agriculture, causing increasing concerns about residues in soils. In this study, the degradation dynamics of glyphosate in different types of citrus orchard soils in China were evaluated under field conditions. Glyphosate soluble powder and aqueous solution were applied at 3000 and 5040 g active ingredient/hm2, respectively, in citrus orchard soils, and periodically drawn soil samples were analyzed by high performance liquid chromatography. The results showed that the amount of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in soils was reduced with the increase of time after application of glyphosate formulations. Indeed, the amount of glyphosate in red soil from Hunan and Zhejiang Province, and clay soil from Guangxi Province varied from 0.13 to 0.91 µg/g at 42 days after application of aqueous solution. Furthermore, the amount of glyphosate in medium loam from Zhejiang and Guangdong Province, and brown loam from Guizhou Province varied from less than 0.10 to 0.14 µg/g, while the amount of AMPA varied from less than 0.10 to 0.99 µg/g at 42 days after application of soluble powder. Overall, these findings demonstrated that the degradation dynamics of glyphosate aqueous solution and soluble powder as well as AMPA depend on the physicochemical properties of the applied soils, in particular soil pH, which should be carefully considered in the application of glyphosate herbicide.

[Genetic characterization of Vibrio parahaemolyticus O3: K6 serovariant isolated in Shenzhen].

OBJECTIVE: To characterize the O3: K6 serovariant of Vibrio parahaemolyticus on virulence gene and molecular typing, and analyze the genetic relationship between O3: K6 and O3: K6 serovariants. METHODS: PFGE was performed on 115 strains of V.parahaemolyticus which were collected from the anal swab of cases of foodbrone diseases in Shenzhen during 2006-2012. According to isolation times and locations, 7 strains of O3: K6 were selected as control strains. Tdh gene, trh gene, orf8 gene were detected, GS-PCR, multi-locus sequence typing (MLST) were used to chracterize 7 strains of O3: K6 and O3: K6 serovariants. RESULTS: PFGE indicated that 58.3% (67/115) of V. parahaemolyticus strains shared a high similarity of band pattern (similarity > 80%) , which comprised of O3: K6 (44/67), O1: KUT(4/67), and O3: K6 serovariants(19/67). Among the O3: K6 serovariants, O1: K25 accounted for 7% (5/67), O4: K68 accounted for 10% (7 /67), O11: K36 accounted for 10% (7 /67). They all carried both tdh and trh gene, and 53% (10/19) was GS-PCR positive and carried orf8 gene, 26% (5/19) was both GS-PCR and orf8 gene negative, 21% (4/19) was GS-PCR negative, orf8 gene positive, 89% (17/19) was assigned to ST-3, 11% (2/19) was assigned to ST-305. Seven strains of O3: K6 was GS-PCR positive, carried orf8 gene, assigned to ST-3. ST-305 and ST-3 had differences in 2 housekeeping genes, which was dtdS gene and pntA gene. In the 305th base of dtdS gene, ST-305(147 allele profile) was T, while ST-3(4 allele profile) was C. In the 33th base of pntA gene, ST-305(93 allele profile) was T, while ST-3(29 allele profile ) was C. CONCLUSION: O4: K68,O1: K25 and O11: K36 were highly similar in virulencec gene carriage, MLST type of O3: K6, and aslo shared a close genetic relationship with O3: K6, thus were considered as O3: K6 serovirants.

[Epidemiological investigation of fatty liver disease in Northwest China].

OBJECTIVE: To describe the incidence and related risk factors of fatty liver disease in the general population of Northwest China. METHODS: The study was a cross-sectional survey with multiplestage stratified cluster and random sampling. All participants were 18 years or older and resided in northwest provinces of China.Demographic and behavioral data was gathered by questionnaire.Clinical data such as height, body weight, waist circumference, biochemical function (standard tests) and liver status (ultrasonographic examination) were also collected. RESULTS: Among the 2 300 total study participants, 1 523 were habitual drinkers (total drinking rate: 66.2%). This population of drinkers was composed almost exclusively of males. There were 201 cases of alcoholic liver disease (total prevalence:8.7%), represented by mild alcoholic injury (prevalence: 4.2%), alcoholic fatty liver (3.8%), alcoholic hepatitis (0.5%), and alcoholic cirrhosis (0.3%).When examined according to provinces of residence, the prevalence of alcoholic liver disease followed this pattern: Shaanxi (14.0%), Gansu (8.6%), and Xinjiang autonomous region (5.0%). Of the total 337 cases of nonalcoholic fatty liver disease (total prevalence: 14.7%), the prevalence by province was: Shaanxi (18.5%), Gansu (10.3%), and Xinjiang autonomous region (16.6%). Individuals with alcoholic liver disease had significantly higher mean age (years), body weight, body mass index (BMI), waist circumference, average daily alcohol intake, and level of aspartate aminotransferase (AST), and alanine aminotraasferase (ALT) (vs. Those with no liver disease, P less than 0.05). Individuals with nonalcoholic fatty liver disease had significantly higher mean age (years), height, body weight, BMI, waist circumference, level of AST and ALT, and presence of obesity, abdominal obesity, hyperlipidemia, diabetes mellitus and hypertension (vs. Those with no liver disease, P less than 0.05). Multiple logistic regression demonstrated that alcoholic liver disease was closely related to age, sex, and average daily alcohol intake. The prevalence of nonalcoholic fatty liver disease was positively correlated to age, female sex, hyperlipidemia, diabetes mellitus, and BMI. CONCLUSION: The drinking-rate and prevalence of fatty liver disease is high in Northwest China, but the most prevalent type is nonalcoholic fatty liver disease.Fatty liver disease is closely related to age, sex, average daily alcohol intake, hyperlipidemia, diabetes mellitus, and BMI.

Different concentrations of hyaluronic acid eye drops for dry eye syndrome: a systematic review and Meta-analysis.

AIM: To compare high or low concentration of hyaluronic acid eye drops (HY) for dry eye syndromes (DES). METHODS: Randomized controlled trials (RCTs) comparing various concentrations of HY were searched in PubMed, Embase, Web of Science, Cochrane, SinoMed, CNKI, Wanfang Database, CQVIP, and Chinese journals databases between inception and July 2023. Pooled standardized mean differences (SMD) or weighted mean difference (WMD) with 95% confidence intervals (CI) from RCTs evaluating Schirmer's I test (SIT), corneal fluorescein staining score (CFS), tear breakup time (TBUT), DES score (DESS), and Ocular Surface Disease Index (OSDI) were calculated. Sensitivity analysis, Egger's test and Meta-regression analysis were performed for all indicators. RESULTS: We conducted a Meta-analysis of 10 RCTs that met the inclusion criteria, involving 1796 cases. High-concentrations group significantly improved the outcome of CFS according to random effects modelling (SMD, -3.37; 95%CI, -5.25 to -1.48; P=0.0005). The rest of the results were not statistically significant, including indicators such as SIT, TBUT, DESS and OSDI. CONCLUSION: For dry eyes with positive corneal staining, a high concentration of HY is recommended, whereas in other cases, a high concentration of HY does not offer a more pronounced advantage over a low concentration of HY in the treatment of dry eyes.

Development of a precision tumor bone metastasis model by a magnetic micro-living-motor system.

An ideal bone metastasis animal model is critical and fundamental for mechanistic research and following development of new drug and treatment. Caudal artery (CA) injection allows bone metastasis in the hindlimb, while in-depth targeted and quantitative studies of bone metastasis require a new model to overcome its limitations. Here, we developed a targeted, quantitative, and highly consistent method for the modeling of bone metastasis with cell-based magnetic micro-living-motor (MLM) system created by effectively combining Fe3O4-PDA-Au with biosafety. The MLM system can achieve efficient migration, target site colonization and control tumorigenesis in bone precisely with the application of a magnetic field. In vivo, day 3 post cell injection, tumor bone metastasis signals were observed locally in the injected femur among 82.76% mice of the MLM group as compared to the 56.82% in the CA group, and the signal intensity was 45.1 and 95.9 times stronger than that in the left and right lower limbs of the CA group, respectively. Post-injection day 28, metastasis in vital organs was reduced by approximately 90% in the MLM group compared to the CA group. Our innovative use of the MLM system in the field of tumor modeling opens a new avenue for exploring the mechanisms of tumor bone metastasis, recurrence and drug resistance.

Copper oxide nanoparticles: an effective suppression tool against bacterial leaf blight of rice and its impacts on plants.

BACKGROUND: To address the challenges of food security for the ever-increasing population, the emergence of nanotechnology provides an alternate technology of choice for the production of safer pesticides which serves as a substitute for conventional fertilizer. The antidrug resistance of Xanthomonas oryzae pv. oryzae (Xoo) and build-up of chemicals in the environment has made it necessary to find alternative safe techniques for effective disease management. Hence, in this study, copper oxide nanoparticles (CuONPs) were produced by green synthesis using a Hibiscus rosa-sinensis L. flower extract. RESULTS: The characterization of CuONPs using ultraviolet-visible spectrophotometry, scanning electron microscopy with an energy-dispersive spectrum profile, Fourier transform infrared spectroscopy, and X-ray diffraction ascertained the presence of CuONPs, which were nanorods of 28.1 nm. CuONPs significantly obstructed the growth and biofilm development of Xoo by 79.65% and 79.17% respectively. The antibacterial mechanism of CuONPs was found to result from wounding the cell membrane, giving rise to an exodus of intracellular content and generation of oxidative reactive oxygen species that invariably inhibited Xoo respiration and growth. A toxicity study under greenhouse conditions revealed that CuONPs significantly increased growth variables and the biomass of rice, and reduced bacterial leaf blight. Application of CuONPs on Arabidopsis improved the chlorophyll fluorescence parameters; the ΦPSII was significantly increased by 152.05% in comparison to the control. CONCLUSION: Altogether, these results suggest that CuONPs in low concentration (200.0 µg mL-1 ) are not toxic to plants and can serve as nano-fertilizers and nano-pesticides. © 2023 Society of Chemical Industry.

Induction chemotherapy regimes in first-line treatment for locoregionally advanced nasopharyngeal carcinoma: A network meta-analysis and cost-effectiveness analysis.

OBJECTIVE: The aim of this study is to evaluate the efficacy and cost-effectiveness of various induction chemotherapy (IC) regimens as first-line treatment for Locoregionally advanced nasopharyngeal carcinoma (LA-NPC), aiming to provide clinicians and patients with informed insights to aid in treatment decision-making. PATIENTS AND METHODS: We conducted a network meta-analysis (NMA) and cost-effectiveness analysis (CEA) based on data from 10 clinical trials investigating IC regimens for the treatment of LA-NPC. A Bayesian NMA was performed, with the primary outcomes being hazard ratios (HRs) for disease-free survival (DFS) and overall survival (OS). To model the disease progression of LA-NPC, we developed a dynamic partitioned survival model consisting of three disease states: progression-free survival (PFS), progression disease (PD), and death. The model was run on a 3-week cycle for a research period of 10 years, with quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs) serving as outcome measures. RESULTS: According to the surface under the cumulative ranking curve (SUCRA) estimates derived from the NMA, TPC and TP, as IC regimens, appear to exhibit superior efficacy compared to other treatment modalities. In terms of CEA, concurrent chemoradiotherapy (CCRT), TPF + CCRT, and GP + CCRT were found to be dominated (more costs and less QALYs). Comparatively, TPC + CCRT emerged as a cost-effective option with an ICER of $1260.57/QALY when compared to PF + CCRT. However, TP + CCRT demonstrated even greater cost-effectiveness than TPC + CCRT, with an associated increase in costs of $3300.83 and an increment of 0.1578 QALYs per patient compared to TPC + CCRT, resulting in an ICER of $20917.62/QALY. CONCLUSION: Based on considerations of efficacy and cost-effectiveness, the TP + CCRT treatment regimen may emerge as the most favorable first-line therapeutic approach for patients with LA-NPC.

Possible role of mast cells and neuropeptides in the recovery process of dextran sulfate sodium-induced colitis in rats.

OBJECTIVE: To clarify the role of mast cells and neuropeptides substance P (SP), somatostatin (SS), and vasoactive intestinal peptide (VIP) in dextran sulfate sodium (DSS)-induced colitis in rats. METHODS: Experimental colitis was induced in Sprague-Dawley rats (180-200 g, n=20) by oral ingestion of 4% (w/v) DSS in drinking water for 7 days. Control rats (n=5) drank water and were sacrificed on day 0. Mast cell number, histamine levels in whole blood and tissue, tissue levels of SP, SS and, VIP in the distal colon of the rats were measured on day 8, day 13, and day 18 of experimentation. RESULTS: Oral administration of 4% DSS solution for 7 days resulted in surface epithelial loss and crypt loss in the distal colon. Mast cell count increased on day 8 (1.75±1.09/mm vs. 0.38±0.24/mm, P<0.05) and day 13 (1.55±1.01/mm vs. 0.38±0.24/mm, P<0.05) after DSS treatment. Whole blood histamine levels were increased on day 8 (266.93±35.62 ng/mL vs. 76.87±32.28 ng/mL, P<0.01) and gradually decreased by day 13 and day 18 after DSS treatment. Histamine levels in the distal colon were decreased on day 8 (1.77±0.65 ng/mg vs. 3.06±0.87 ng/mg, P<0.05) and recovered to control levels by day 13 after DSS treatment. SP level in the distal colon gradually increased and were raised significantly by day 13 (8777.14±3056.14 pg/mL vs. 4739.66±3299.81 pg/mL, P<0.05) after DSS treatment. SS and VIP levels in the distal colon were not changed. CONCLUSIONS: Mast cell degranulation followed by histamine release may play an important role in the pathogenesis of colitis induced by DSS. SP may be a significant substance in the progression of inflammation and the recovery process of DSS-induced colitis.

Development of a reverse-transcription droplet digital PCR method for quantitative detection of Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is a re-emerging threat to the production of greenhouse cucumber and other Cucurbitaceae crops worldwide. This seed-borne virus can easily spread from a contaminated seed to seedlings and adjacent plants by mechanical contact between the foliage of diseased and healthy plants, causing extensive yield losses. An accurate method for detecting and quantifying this virus is urgently needed to ensure the safety of the global seed trade. Here, we report the development of a reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR)-based method for specific and high-sensitive detection of CGMMV. By testing three primer-probe sets and optimizing reaction conditions, we showed that the newly developed RT-ddPCR method is highly specific and sensitive, with a detection limit of 1 fg/µL (0.39 copy/µL). The sensitivity of the RT-ddPCR method was compared with that of real-time fluorescence quantitative RT-PCR (RT-qPCR) using a series of plasmid dilutions and total RNAs extracted from infected cucumber seeds, and the detection limit of RT-ddPCR was 10 times higher than RT-qPCR with plasmid dilutions and 100 times higher than RT-qPCR for detecting CGMMV from infected cucumber seeds. The RT-ddPCR method was further assessed for detecting CGMMV from a total of 323 samples of Cucurbitaceae seeds, seedlings, and fruits as compared with the RT-qPCR method. We found that the infection rate of CGMMV on symptomatic fruits was as high as 100%, whereas infection rates were lower for seeds and lowest for seedlings. Notably, the results of two methods in detecting CGMMV from different cucurbit tissues showed the high consistency with Kappa value from 0.84 to 1.0, demonstrating that the newly developed RT-ddPCR method is highly reliable and practically useful for large-scale CGMMV detection and quantification.

Biocontrol of Soft Rot Dickeya and Pectobacterium Pathogens by Broad-Spectrum Antagonistic Bacteria within Paenibacillus polymyxa Complex.

Polymyxin-producing bacteria within the Paenibacillus polymyxa complex have broad-spectrum activities against fungi and bacteria. Their antibacterial activities against soft rot Dickeya and Pectobacterium phytopathogens containing multiple polymyxin-resistant genes were not clear. Here, we selected nine strains within the P. polymyxa complex having broad-spectrum antagonistic activities against phytopathogenic fungi and a polymyxin-resistant D. dadantii strain causing stem and root rot disease of sweet potato and did antagonistic assays on nutrient agar and sweet potato tuber slices. These strains within the P. polymyxa complex showed clear antagonistic activities against D. dadantii in vitro and in vivo. The most effective antagonistic strain P. polymyxa ShX301 showed broad-spectrum antagonistic activities against all the test Dickeya and Pectobacterium strains, completely eliminated D. dadantii from sweet potato seed tubers, and promoted the growth of sweet potato seedlings. Cell-free culture filtrate of P. polymyxa ShX301 inhibited D. dadantii growth, swimming motility, and biofilm formation and disrupted D. dadantii plasma membranes, releasing nucleic acids and proteins. Multiple lipopeptides produced by P. polymyxa ShX301 may play a major role in the bactericidal and bacteriostatic actions. This study clarifies that the antimicrobial spectrum of polymyxin-producing bacteria within the P. polymyxa complex includes the polymyxin-resistant Dickeya and Pectobacterium phytopathogens and strengthens the fact that bacteria within the P. polymyxa complex have high probability of being effective biocontrol agents and plant growth promoters.