Afterglow Nanoprobe-Enabled Quantitative Lateral Flow Immunoassay by a Palm-Size Device for Household Healthcare.

Lateral flow immunoassay (LFIA), a classical point-of-care testing (POCT) technique, plays an important role in disease screening and healthcare monitoring. However, traditional LFIA is either designed for qualitative analysis or requires expensive equipment for quantification, limiting its use in household diagnosis. In this study, we proposed a new generation of LFIA for household health monitoring by using ultralong organic phosphorescence (UOP) nanomaterials as afterglow nanoprobes with a self-developed palm-size sensing device. The UOP nanoprobes exhibit a phosphorescence signal with a second-level lifetime, which completely avoids the interference from excitation light and biological background fluorescence. Therefore, an ultraminiaturized and low-cost UOP nanosensor was successfully designed by eliminating the complex optical path and filtering systems. We chose an inflammatory factor, C-reactive protein (CRP), for household POCT validation. The whole analysis was completed within 9 min. A limit of detection (LOD) of 0.54 ng/mL of CRP antigen was achieved with high stability and good specificity, which is comparable to laboratory instruments and fully satisfying the clinical diagnosis requirement.

Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

Chromosome recombination and modification by LoxP-mediated evolution in Vibrio natriegens using CRISPR-associated transposases.

Chromosome rearrangement by LoxP-mediated evolution has emerged as a powerful approach to studying how chromosome architecture impacts phenotypes. However, it relies on the in vitro synthesis of artificial chromosomes. The recently reported CRISPR-associated transposases (CASTs) held great promise for the efficient insertion of abundant LoxP sites directly onto the genome of wild-type strains. In this study, with the fastest-growing bacterium Vibrio natrigens (V. natriegens) as an object, a multiplex genome integration tool derived from CASTs was employed to achieve the insertion of cargo genes at eight specific genomic loci within 2 days. Next, we introduced 30 LoxP sites onto chromosome 2 (Chr2) of V. natriegens. Rigorously induced Cre recombinase was used to demonstrate Chromosome Rearrangement and Modification by LoxP-mediated Evolution (CRaMbLE). Growth characterization and genome sequencing showed that the ~358 kb fragment on Chr2 was accountable for the rapid growth of V. natriegens. The enabling tools we developed can help identify genomic regions that influence the rapid growth of V. natriegens without a prior understanding of genome mechanisms. This groundbreaking demonstration may also be extended to other organisms such as Escherichia coli, Pseudomonas putida, Bacillus subtilis, and so on.

Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering.

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified.  • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Comparative analysis of microbial composition and functional characteristics in dental plaque and saliva of oral cancer patients.

BACKGROUND: The oral cavity is home to various ecological niches, each with its own unique microbial composition. Understanding the microbial communities and gene composition in different ecological niches within the oral cavity of oral cancer (OC) patients is crucial for determining how these microbial populations contribute to disease progression. METHODS: In this study, saliva and dental plaque samples were collected from patients with OC. Metagenomic sequencing was employed to analyze the microbial community classification and functional composition of the different sample groups. RESULTS: The results of the study revealed significant differences in both the function and classification of microbial communities between saliva and dental plaque samples. The diversity of microbial species in saliva was found to be higher compared to  that in plaque samples. Notably, Actinobacteria were enriched in the dental plaque of OC patients. Furthermore, the study identified several inter-group differential marker species, including Prevotella intermedia, Haemophilus parahaemolyticus, Actinomyces radius, Corynebacterium matruchitii, and Veillonella atypica. Additionally, 1,353 differential genes were annotated into 23 functional pathways. Interestingly, a significant correlation was observed between differentially labeled species and Herpes simplex virus 1 (HSV-1) infection, which may be related to the occurrence and development of cancer. CONCLUSIONS: Significant differences in the microbial and genetic composition of saliva and dental plaque samples were observed in OC patients. Furthermore, pathogenic bacteria associated with oral diseases were predominantly enriched in saliva. The identification of inter-group differential biomarkers and pathways provide insights into the relationship between oral microbiota and the occurrence and development of OC.

The co-regulation of the gut microbiome and host genes might play essential roles in metformin gastrointestinal intolerance.

Metformin is commonly used, but approximately 20% of patients experience gastrointestinal intolerance, leading to medication discontinuation for unclear reasons and a lack of effective management strategies. In this study, the 18 fecal and blood samples were analyzed using 16S rRNA and mRNA transcriptome, respectively. These samples included 3 fecal and 4 blood from metformin-tolerant T2D patients before and after metformin treatment (T and Ta), 3 fecal and 5 blood from metformin-intolerant T2D patients before and after treatment (TS and TSa), and 6 fecal samples from healthy controls. The results showed that certain anti-inflammatory gut bacteria and gene, such as Barnesiella (p = 0.046), Parabacteroides goldsteinii (p = 0.016), and the gene JUND (p = 0.0002), exhibited higher levels in metformin-intolerant patients, and which decreased after metformin treatment (p < 0.05). This potentially invalidates patients' anti-inflammatory effect and intestinal mucus barrier protection, which may lead to alterations in intestinal permeability, decreased gut barrier function, and gastrointestinal symptoms, including diarrhea, bloating, and nausea. After metformin treatment, primary bile acids (PBAs) production species: Weissella confusa, Weissella paramesenteroides, Lactobacillus brevis, and Lactobacillus plantarum increased (p < 0.05). The species converting PBAs to secondary bile acids (SBAs): Parabacteroides distasonis decreased (p < 0.05). This might result in accumulation of PBAs, which also may lead to anti-inflammatory gene JUND and SQSTM1 downregulated. In conclusion, this study suggests that metformin intolerance may be attributed to a decrease in anti-inflammatory-related flora and genes, and also alterations in PBAs accumulation-related flora. These findings open up possibilities for future research targeting gut flora and host genes to prevent metformin intolerance.

Customized multiple sequence alignment as an effective strategy to improve performance of Taq DNA polymerase.

Engineering Taq DNA polymerase (TaqPol) for improved activity, stability and sensitivity was critical for its wide applications. Multiple sequence alignment (MSA) has been widely used in engineering enzymes for improved properties. Here, we first designed TaqPol mutations based on MSA of 2756 sequences from both thermophilic and non-thermophilic organisms. Two double mutations were generated including a variant H676F/R677G showing a decrease in both activity and stability, and a variant Y686R/E687K showing an improved activity, but a decreased stability. Mutations targeted on coevolutionary residues of Arg677 and Tyr686 were then applied to rescue stability or activity loss of the double mutants, which achieved a partial success. Sequence analysis revealed that the two mutations are abundant in non-thermophilic sequences but not in thermophilic homologues. Then, a small-scale MSA containing sequences from only thermophilic organisms was applied to predict 13 single variants and two of them, E507Q and E734N showed a simultaneous increase in both stability and activity, even in sensitivity. A customized MSA was hence more effective in engineering a thermophilic enzyme and could be used in engineering other enzymes. Molecular dynamics simulations revealed the impact of mutations on the protein dynamics and interactions between TaqPol and substrates. KEY POINTS: • The pool of sequence for alignment is critical to engineering Taq DNA polymerase. • The variants with low properties can be rescued by mutations in coevolving network. • Improving binding with DNA can improve DNA polymerase stability and activity.

Genetically Engineering Escherichia coli to Produce Xylitol from Corncob Hydrolysate without Lime Detoxification.

Before fermentation with hemicellulosic hydrolysate as a substrate, it is generally necessary to detoxify the toxic substances that are harmful to microorganism growth. Cyclic AMP receptor protein (CRP) is a global regulator, and mutation of its key sites may have an important impact on E. coli virulence tolerance. Using corncob hydrolysate without ion-exchange or lime detoxification as the substrate, shake flask fermentation experiments showed that CRP mutant IS5-dG (I112L, T127G, A144T) produced 18.4 g/L of xylitol within 34 h, and the OD600 was 9.7 at 24 h; these values were 41.5% and 21.3% higher than those of the starting strain, IS5-d, respectively. This mutant produced 82 g/L of xylitol from corncob hydrolysate without ion-exchange or lime detoxification during fed-batch fermentation in a 15-L bioreactor, with a productivity of 1.04 g/L/h; these values were 173% and 174% higher than the starting strain, respectively. To our knowledge, this is the highest xylitol concentration and productivity produced by microbial fermentation using completely non-detoxified hemicellulosic hydrolysate as the substrate to date. This study also showed that alkali neutralization, high temperature sterilization, and fermentation of the hydrolysate had important effects on the xylose loss rate and xylitol production.

Preparation of Coupling Catalyst HamZIF-90@Pd@CALB with Tunable Hollow Structure for Efficient Dynamic Kinetic Resolution of 1-Phenylethylamine.

Chiral amines are essential components for many pharmaceuticals and agrochemicals. However, the difficulty in obtaining enantiomerically pure amines limits their application. In this study, hollow amorphous ZIF-90 (HamZIF-90) materials were prepared by template engraving, and chemical-enzyme coupling catalysts (HamZIF-90@Pd@CALB) were constructed for the chiral resolution of 1-phenylethylamine. Different from conventional materials, HamZIF-90 had tunable hollow structures by altering its central node zinc ion concentrations, and the embedded hydrogel template gave it more pore structures, which facilitated the loading of enzyme molecules and Pd nanoparticles (NPs). The establishment of the coupling catalysts shortened the mass transfer distance of the reactant molecules between the metal nanoparticles and the enzyme catalyst in the dynamic kinetic resolution (DKR) reaction, resulting in 98% conversion of 1-phenylethylamine and 93% selectivity of Sel.R-amide. The proposal of this idea provided a good idea for future tailor-made MOFs loaded with chemical and enzyme coupled catalyst.

A Simple Screening and Optimization Bioprocess for Long-Chain Peptide Catalysts Applied to Asymmetric Aldol Reaction.

Peptides have demonstrated their efficacy as catalysts in asymmetric aldol reactions. But the constraints inherent in chemical synthesis have imposed limitations on the viability of long-chain peptide catalysts. A noticeable dearth of tools has impeded the swift and effective screening of peptide catalysts using biological methods. To address this, we introduce a straightforward bioprocess for the screening of peptide catalysts for asymmetric aldol reactions. We synthesized several peptides through this method and obtained a 15-amino acid peptide. This peptide exhibited asymmetric aldol catalytic activity, achieving 77% ee in DMSO solvent and 63% ee with over an 80.8% yield in DMSO mixed with a pH 9.0 buffer solution. The successful application of our innovative approach not only represents an advancement but also paves the way for currently unexplored research avenues.

Use of Intravenous Anakinra for Management of Pediatric Cytokine Storm Syndromes at an Academic Medical Center.

Background: Off-label intravenous (IV) route of anakinra is increasingly recognized to enable higher and faster maximal plasma concentrations than subcutaneous route for treatment of cytokine storm syndromes. Objective: To describe off-label indications of IV anakinra, corresponding dosing and safety profiles, particularly during the coronavirus disease 19 (COVID-19) pandemic. Methods: A retrospective, single-cohort study was conducted at an academic medical center to evaluate use of IV anakinra in hospitalized pediatric patients (age ≤21 years). Institutional Review Board review was considered exempt. The primary endpoint was the primary indication(s) for IV anakinra. The key secondary endpoints were dosing of IV anakinra, previous immunomodulatory therapies, and adverse events. Results: Of 14 pediatric patients, 8 (57.1%) received IV anakinra for treatment of multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19, whereas 3 and 2 were treated for hemophagocytic lymphohistiocytosis (HLH) and flares of systemic onset juvenile idiopathic arthritis (SoJIA), respectively. The initial dosing regimen of IV anakinra for MIS-C associated with COVID-19 was a median dose of 2.25 mg/kg/dose with a median dosing interval of 12 hours for a median initial treatment duration of 3.5 days. Eleven (78.6%) patients received previous immunomodulatory therapies (IV immune globulin [n = 10; 71.4%] and steroids [n = 9; 64.3%]). No adverse drug events were documented. Conclusion: IV anakinra was used off-label for treatment of MIS-C associated with COVID-19, HLH and SoJIA flares in critically ill patients with no adverse drug events documented. This study helped ascertain the off-label indications of IV anakinra and corresponding patient characteristics.

Mutability-Landscape-Guided Engineering of l-Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C-C Bond Formation.

l-threonine aldolase (LTA) catalyzes C-C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C-C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C-C bond-forming enzymes.

The prognosis biomarkers based on m6A-related lncRNAs for myeloid leukemia patients.

BACKGROUND: Chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) are two common malignant disorders in leukemia. Although potent drugs are emerging, CML and AML may still relapse after the drug treatment is stopped. N6-methyladenosine (m6A) and lncRNAs play certain roles in the occurrence and development of tumors, but m6A-modified LncRNAs in ML remain to be further investigated. METHODS: In this study, we extracted and analyzed the TCGA gene expression profile of 151 ML patients and the clinical data. On this basis, we then evaluated the immune infiltration capacity of ML and LASSO-penalized Cox analysis was applied to construct the prognostic model based on m6A related lncRNAs to verify the prognostic risk in clinical features of ML. Quantitative reverse transcription PCR was used to detect the expression level of LncRNA in in ML cell lines K562, MOLM13 and acute monocytic leukemia cell line THP-1. RESULTS: We found 70 m6A-related lncRNAs that were related to prognosis, and speculated that the content of stromal cells and immune cells would correlate with the survival of patients with ML. Next, Prognostic risk model of m6A-related lncRNAs was validated to have excellent consistency in clinical features of ML. Finally, we verified the expression levels of CRNDE, CHROMR and NARF-IT1 in ML cell lines K562, MOLM13 and acute monocytic leukemia cell line THP-1, which were significant. CONCLUSIONS: The research provides clues for the prognosis prediction of ML patients by using the m6A-related lncRNAs model we have created, and clarifies the accuracy and authenticity of it.

Secukinumab for PASS syndrome: A new choice for therapeutic challenge?

PASS syndrome is a rare inflammatory disease characterized by a chronic-relapsing course of pyoderma gangrenosum, acne vulgaris, hidradenitis suppurativa, and spondyloarthritis, which is lack of any biological or genetic marker. Moreover, the optimal therapeutic management remains unclear. We herein describe a Yi Chinese man with PASS syndrome who was treated with secukinumab and showed a remarkable response with almost complete clinical improvement at the 2-year follow-up.

Evaluation of piggyBac-mediated anti-CD19 CAR-T cells after ex vivo expansion with aAPCs or magnetic beads.

Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.

Electrochemically Promoted Asymmetric Transfer Hydrogenation of 2,2,2-Trifluoroacetophenone.

The study reported an electrochemically promoted asymmetric hydrogen transfer reaction of 2,2,2-trifluoroacetophenone with a chiral Ru complex. (R)-α-(Trifluoromethyl) benzyl alcohol with a 96% yield and 94% ee could be obtained with only a 0.5 F mol-1 charge amount at room temperature and normal pressure.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

First Report of Maize Stalk Rot Caused by Fusarium kyushuense in China.

During 2017 to 2019, a field survey for maize stalk rot was conducted in 21 counties (districts) across the Guangxi province of China. This disease caused yield losses ranging from 20% to 30%. Maize plants with stalk rot were collected during the late milk stage and pieces of diseased pith tissue were cultured as previously described (Shan et al. 2017). Fungal colonies and mycelia with morphological characteristics of Fusarium species were subcultured onto fresh potato dextrose agar (PDA) and carnation leaf agar (CLA) plates. Based on morphological characteristics and molecular detection by amplification of Fusarium genus-specific primers (Duan et al. 2016), 39 Fusarium isolates were identified. Among them, five isolates from Du'an, Pingguo, Debao, and Daxin had abundant, pale orange to yellow aerial mycelium with deep red pigments when grown on PDA (Fig. 1A; 1B). The average growth rate was 8.0 to 12.0 mm per day at 25°C in the dark. The fungi produced two types of spores on CLA. Microconidia were ovoid to clavate, generally 0- to 3-septate, and 4.6 to 9.4 µm in length (n = 30) (Fig. 1D); Macroconidia were slightly curved with an acute apical cell, mostly 3- to 4- septate, and 19.4 to 38.2 µm in length (n = 30) (Fig. 1C). No chlamydospores were observed. These five isolates were initially identified as Fusarium kyushuense based on morphological features. PCR was performed to amplify three phylogenetic genes (TEF1-α, RPB1, and RPB2) (O'Donnell et al. 1998) and species specific primers kyuR1F/kyuR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3/5-TCCAATGGACTGGGCAGCCAAAACACC-3), kyuR2F/kyuR2R (5-CAGATATACATTTGCCTCGACAC-3/5-TACTTGAGCACGGAGCTTG-3) were used to confirm species identity. The obtained sequences were deposited in GenBank under the accession numbers MT997084, MT997080, MT997081 (TEF1-α); MT550012, MT997085, MT997086 (RPB1); MT550009, MT997089, and MT997090 (RPB2), respectively. Using BLAST, sequences of TEF1-α, RPB1, and RPB2 of the isolates were 99.33% (MH582297.1) to 100% (MG282364.1) similar to those of F. kyushuense strains (Supplementary Table 1). Based on phylogenetic analysis with maximum likelihood methods using tools of the website of CIPRES (http://www.phylo.org), isolates GX27, GX167, and GX204 clustered with F. kyushuense with 100% bootstrap support (Fig. 2). The pathogenicity of the three isolates was tested using young seedlings and adult plants as previously described with modification (Ye et al. 2013; Zhang et al. 2016). The primary roots of three-leaf-old seedlings were inoculated by immersing the roots into a 1 × 106 macroconidia solution, incubating for 6 h at 25°C, and transferring to normal growth conditions (26°C, 16 h light/22°C, 8 h dark). The second or third internode above the soil surface of flowering stage plants grown in a greenhouse was bored with a Bosch electric drill to make a hole (ca. 8 mm in diameter) and inoculated with 0.5 mL of mycelia plug then sealed with petrolatum. The inoculum was created by homogenizing five plates of flourish hyphal mats (approximately 125 mL) with kitchen blender and adjusting to a final volume of 200 mL with sterilized ddH2O. No symptoms were observed in the seedlings or adult plants that were mock-inoculated with PDA plugs. Three days post-inoculation (dpi), roots of the infected seedling turned dark-brown and shrunk and the leaves wilted (Fig. 1E). Typical stalk rot symptoms observed in the inoculated plants were premature wilting of entire plant and hollow and weak stalks, leading to lodging; the longitudinal section of the internodes exhibited obvious dark brown necrosis and reddish discoloration at 14 dpi and 30 dpi, respectively (Fig. 1F). Fusarium kyushuense was re-isolated from the inoculated stalk lesions but not from the control. This is the first record of stalk rot caused by F. kyushuense on maize plants in China. However, F. kyushuense is known to cause maize ear rot in China (Wang et al. 2014) and can produce type A and type B trichothecene mycotoxins in kernels (Aoki and O'Donnell 1998). The occurrence of maize stalk rot and ear rot caused by F. kyushuense should be monitored in China due to the potential risk for crop loss and mycotoxin contamination.

First Report of Fusarium thapsinum Causing Maize Stalk Rot in China.

Maize (Zea mays L.) is the most widely grown crop in China, which was planted 41.28 million hectares in 2019 (http://data.stats.gov.cnw/easyquery.htm?cn=C01&zb=A0D0F&sj=2019). Several fungal diseases of maize are reported in which stalk rot has become one of the most destructive diseases in China. The average yield losses affected by the disease are estimated at 10% to 20% (Yu et al. 2016). From 2017 to 2019, a survey was conducted to determine the population diversity of Fusarium species associated with maize diseases in 18 cities across Henan province. Fusarium stalk rot of maize with disease incidence more than 25% was observed in two continuous maize fields at Xuchang city. The diseased stem tissues from junctions in health and disease were chopped into small pieces (3 × 8 mm), superficially disinfected (70% ethyl alcohol for 1 min), placed onto potato dextrose agar (PDA) amended with L-(+)-Lactic-acid (1 g/L), poured in petri plates and incubated at 25°C for 4 days. Mycelia showing morphological characteristic of Fusarium spp. were sub-cultured from single conidium. The pure fungal isolates produced fluffy colonies, white aerial mycelium with yellow pigment in agar. The radial mycelial growth was measured and calculated at an average growth rate 10.9 mm/day at 25°C (Fig. 1A; 1B). Macroconidia produced on carnation leaf agar (CLA) were relatively slender, slightly curved and thick-walled, mostly 3 to 5 marked septa, with a curved and tapering apical cell and poorly developed foot cell, 46.9 ± 5.6 µm × 4.9 ± 0.2 µm (Fig. 1C). Microconidia formed abundantly and were generally oval on CLA, 8.2 ± 0.5 µm × 3.4± 0.1 µm (Fig. 1D). No chlamydospores were observed. Morphological characteristics of the isolates matched the description of Fusarium thapsinum (Leslie and Summerell 2006). To further get the phylogenetic evidence, TEF1-α (translation elongation factor), RPB1 (the largest subunit of RNA polymerase II) and RPB2 (the second largest subunit of RNA polymerase II) were amplified with primer pairs EF1/EF2 (O'Donnell et al. 1998), thapR1F (5'-TTTTCCTCACAAAGGAGCAAATCATG-3')/thapR1R (5'-GTTCACCCAAGATATGGTCGAAAGCC-3'), and thapR2F (5'-ACTCTTTCACATTTGCGCCGAAC-3')/thapR2R (5'-CGGAGCTTTCGTCCAGTGTGAC-3'), and sequenced, respectively. The BLAST search of the sequences of EF1-α, RPB1 and RPB2 shared 99.87% to 100% identity with those of F. thapsinum strains deposited in the GenBank (Supplementary Table 1). Sequences from two isolates (XCCG-3-B-1 and XCCG-3-A-1) were deposited in GenBank (Accession No. MT550014, MT997082 for EF-1α; MT550011, MT997087 for RPB1 and MT550008, MT997091 for RPB2). The phylogenetic relationships based on analysis of the partial sequences showed the representive isolates clustered together with F. thapsinum at 96% bootstrap values (Fig. 2). Combined with the results of morphological characteristics and phylogenetic analysis, the strain designated as Fusarium thapsinum. To complete Koch's postulates, the pathogenicity of the isolates was tested using the silking-stage plants in a greenhouse based on previously described method with modification (Zhang et al. 2016). An 8 mm in diameter wound hole was created at the second or third internode of the plant above the soil surface and injected with 0.5 ml of mycelia plug. The inoculated stalk exhibited internal dark brown necrotic regions and the brown area elongated obviously around the insertion at 14 dpi (days post inoculation). At 30 dpi, the stalks turned soft, hollow and even lodging of the plants for those severe ones, which are similar to those observed on naturally infected maize plants in the field (Fig. 1F). When the roots of the three-leaf-stage seedlings were inoculated with 1×106 macroconidia solution (Ye et al. 2013), the root rot and leaf wilting symptoms were observed (Fig. 1E). While the control plants that were inoculated with only sterile water showed no disease symptoms. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed by the morphological characters. Fusarium thapsinum had been described as causal agent of maize stalk rot in Pakistan (Tahir et al. 2018). To our knowledge, this is the first report of F. thapsinum associated with maize stalk rot in China. The discovery will strengthen the theoretical foundation of maize stalk rot disease management.

Development of celecoxib-derived antifungals for crop protection.

Selective COX-2 inhibitor celecoxib was found directly inhibiting the growth of tested phytopathogenic fungi with the inhibitory rate ranging from 30 to 40% at 100 µg/ml. Lead optimization of celecoxib led to the identification of compound 12 among its derivatives as the most active antifungal candidate. The antifungal effect of compound 12 was supposed to be independent of COX-2 inhibition. Transcriptome profiling analysis of Fusarium graminearium (PH-1) treated with compound 12 brought about 406 up-regulated and 572 down-regulated differentially express genes (DEGs) respectively.