Occurrence of Neopestalotiopsis clavispora Causing Leaf Blight on Acer oblongum in China.

Acer oblongum is native to Southwest China and is also distributed in Nepal and Northern India. It is an excellent garden ornamental tree species, suitable for solitary planting in courtyards and parks. From June to August 2022, severe leaf blight occurred on A. oblongum in Baihe Wetland Park (32°5'42" N, 112°28'13" E) in Nanyang City, China. The foliar disease rate reached 59% (n=100). Early signs were yellow spots on the leaves, mainly on the middle and edge parts. Then, the lesions gradually expanded, became amorphous, and turned yellowish brown, eventually led to necrosis on leaves and branches. Twenty diseased leaves were collected and the junction areas between infected and healthy tissues were cut into 5 x 5 mm2 pieces. The collected plant materials were sterilized in 75% ethanol and 1% NaClO for 30 s and 1 minute, respectively, followed by rinsing in sterile water, and placing on a potato dextrose agar (PDA) plate supplemented with 50 µg ml-1 streptomycin at 25 °C for 3 days. Colony edges were cut and transferred to new PDA plates for purification culture. A total of 18 purified fungal strains were obtained, which showed similar phenotypes in morphological characteristics. All colonies had spread radially with wavy surfaces, and dense cream to white aerial hyphae. After 14 days in culture, black fruiting bodies appeared. Conidia were fusiform to slightly clavate, with five cells and two or three setae, 4.2 to 7.9 µm × 17.5 to 25.4 µm in diameter (n = 100). The apical and basal cells and setae were colorless, three median cells were brown, and the middle cell was dark brown. Morphological characteristics of all 18 strains were consistent with the genus description of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). Further molecular identification showed that the ITS region sequences of all strains have extremely high homology with Neopestalotiopsis spp. The ß-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified for molecular identification (Shu et al. 2020). The sequences of three representative strains (FE-05, 09, 16) from different regions were deposited in GenBank with accession Nos. OQ867279, OQ867288, OQ867289 (ITS), OQ870207, OQ870208, OQ870209 (TUB), and OQ870204, OQ870205, OQ870206 (TEF1). BLASTn analysis of these sequences showed 99 to 100% identity to Neopestalotiopsis clavispora strains (OK655673, MZ648263 for ITS, ON000362, MZ286974 fr TUB, MH423941, MK512481 for TEF1). These morphological features and molecular identification indicated that the pathogen has the same characteristics as N. clavispora. Pathogenicity was tested on ten healthy 3-month-old seedlings using the three representative strains through in vivo experiments. For each strain, the conidial suspension (106 conidia ml-1) in absorbent cotton balls (50 µl of inoculum) were inoculated onto the healthy leaves of two seedlings, while a total of two other plants were served with sterile water as a blank control. The plants were potted in a climate incubator at 28°C and a relative humidity of approximately 90%. Symptoms consistent with natural lesions were observed on the inoculated leaves after 5 days while the control plants remained healthy. The strains of N. clavispora were reisolated from the symptomatic inoculated leaves, fulfilling Koch's postulates. N. clavispora is known to cause disease in a variety of plants in China, such as Dendrobium officinale (Cao et al., 2022), Fragaria ananassa (Shi et al., 2022), and Garcinia mangostana (Qiu et al., 2019). To the best of our knowledge, this is the first report of N. clavispora causing leaf blight on A. oblongum in China. The yellowing and falling off of leaves would seriously affects the garden landscape. It is necessary to further clarify the host range of the pathogen to select appropriate landscape matching plants in future planning.

The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Assessing the impact of counterion types on the sustained release characteristics of high-payload drug-ion complex: A case study on tetracycline hydrochloride.

Complexation of ionized hydrophilic drugs with counterions (e.g. polyelectrolytes, ionic amphiphiles, multivalent salt ions) represents a well-established formulation approach to produce sustained release of highly soluble drugs while maintaining a high drug payload. This renders the drug-ion complex an attractive alternative to the conventional polymer matrix systems. The effects of the counterion's type on the sustained release characteristics of drug-ion complexes, however, have not been investigated before under the same dissolution environment. Using antibiotic tetracycline hydrochloride (TC•HCl) as the model hydrophilic drug, we investigated the effects of three types of counterions, sodium dextran sulfate (DXT), sodium dodecyl sulfate (SDS), and K2HPO4, on (1) the sustained release characteristics, (2) long-term storage stability, (3) preparation efficiency (i.e. yield, payload), and (4) antibiotic activity of the resultant (TC•HCl)-ion complexes. The results showed that the three complexes exhibited comparable TC•HCl payloads at approximately 80% (w/w) and yield between 40 and 60% (w/w). They also exhibited good storage stability after 18 months and uncompromised antibiotic activity compared to the native drug. In the intestinal fluid, all three complexes could produce sustained drug release profiles, albeit at different rates ((TC•HCl)-DXT > (TC•HCl)-SDS > (TC•HCl)-HPO4), whereas in the gastric fluid, only the (TC•HCl)-DXT complex could produce a sustained release profile suitable for oral delivery. The different sustained release profiles among the complexes were attributed to their different solid forms (amorphous versus crystalline), hydrophobicity, solubility, and drug release mechanisms. The present work highlighted the importance of selecting the most suitable counterion to achieve the desired sustained drug release profile.

[Molecular cloning and characterization of a novel metagenome-derived bacterial perhydrolase].

The aim of this study is to obtain bacterial perhydrolases with chlorination activity, expands the resources of perhydrolases, and lays a foundation for it's industrial applications. We constructed a metagenomic library using environmental DNA isolated from sludge samples of a paper mill of Tanghe county, and identified a per822 gene encoding a bacteria perhydrolase via activity-based functional screening. Then, we overexpressed Per822 heterologously in Escherichia coli, and characterized the recombinant enzyme after purification. Finally, we further investigated the ability of Per822 to produce peracetic acid (PAA). Sequence analysis revealed that per822 encoded a protein of 273 amino acids. The recombinant Per822 had the activity of peroxidase, esterase and halogenase respectively, and thus was regarded as a typical representative of multifunctional enzymes. The purified Per822 exhibited maximal chlorination activity (hyperhydrolysis) at 55 °C and pH 4.5 with monochlorodimedone as substrate, and the enzyme was stable in the pH range of 3.5-8.0 and below 70 °C. Also, the chlorination activity of this enzyme could be activated by Fe²âº. In addition, the enzyme displayed high ability to generate PAA using ethyl acetate as cosubstrate. The highly soluble expression, catalytic versatility and good PAA production capacity of Per822 make it a potential candidate in organic synthesis, wastewater treatment, disinfection and biomass pretreatment, etc.

Carboxymethyl cellulose is a superior polyanion to dextran sulfate in stabilizing and enhancing the solubility of amorphous drug-polyelectrolyte nanoparticle complex.

Amorphous drug-polyanion nanoplex represents an effective solubility enhancement strategy of weakly-basic poorly-soluble drugs. While dextran sulfate (DXT) was chosen in most studies as the polyanion for nanoplex formation, drug-DXT nanoplex demonstrated poor long-term physical stability for drugs with high crystallization propensity, such as ciprofloxacin (CIP). Herein we hypothesized that amorphous form stability of CIP nanoplex could be improved by substituting DXT with carboxymethyl cellulose (CMC) known for its crystallization inhibiting activity. The optimal preparation condition of the CIP-CMC nanoplex was determined by investigating the effects of CMC/CIP charge ratio and pH on the resultant nanoplex's physical characteristics and preparation efficiency. At the optimal condition, the CIP-CMC nanoplex possessed size, zeta potential, and CIP payload of approximately 200 nm, -49 mV, and 76%, respectively. Its preparation was highly efficient with CIP utilization rate and overall yield of roughly 89% and 46%, respectively. Compared to the CIP-DXT nanoplex, the CIP-CMC nanoplex was larger and with higher CIP payload attributed to CMC's higher chain stiffness. The CIP-CMC nanoplex exhibited superior physical stability after twelve-month storage and improved solubility enhancement capability (30% higher), despite its slower dissolution. These results clearly established CMC as the superior polyanion to DXT for nanoplex formation of weakly-basic drugs.

Enhancing the physical stability and supersaturation generation of amorphous drug-polyelectrolyte nanoparticle complex via incorporation of crystallization inhibitor at the nanoparticle formation step: A case of HPMC versus PVP.

Amorphous drug-polyelectrolyte nanoparticle complex (or nanoplex in short) has emerged as a highly attractive solubility enhancement strategy of poorly-soluble drugs attributed to its simple and highly efficient preparation. The existing nanoplex formulation, however, exhibits poor amorphous form stability during long-term storage for drugs with high crystallization propensity. Using ciprofloxacin (CIP) and sodium dextran sulfate (DXT) as the model drug-polyelectrolyte nanoplex, we investigated the feasibility of incorporating crystallization inhibiting agents, i.e. hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP), at the nanoplex formation step to improve the physical stability of the CIP nanoplex. The effects of the HPMC or PVP additions on the nanoplex's physical characteristics (i.e. size, zeta potential, CIP payload), CIP utilization rate, dissolution rate, and supersaturation generation were also examined. The results showed that the additions of HPMC or PVP increased the CIP nanoplex size (from 300 to 500 nm) and CIP utilization rate (from 65% to 90% w/w) with minimal impacts on the CIP payload (70-80% w/w). Their additions had opposite impacts on the nanoplex's colloidal stability due to surfactant nature of PVP. Significantly, unlike the CIP-DXT and CIP-DXT-PVP nanoplexes, the CIP-DXT-HPMC nanoplex remained amorphous after three-month accelerated storage, while also exhibited superior solubility enhancement (15-30% higher).

Expression and characterization of an esterase belonging to a new family via isolation from a metagenomic library of paper mill sludge.

A new bacterial lipolytic enzyme Est903 was obtained from paper mill sludge via metagenomic approach. Est903 displayed moderate similarities to two lipolytic enzymes from Rhodopirellula islandica and contained a distinctive pentapeptide motif (GFSAG) that differed from those of all known fourteen families of bacterial lipolytic enzymes. Est903 was regarded as from a new bacterial lipolytic enzyme family through multiple sequence alignment and phylogenetic analysis. The recombinant Est903 showed the highest activity for ρ-nitrophenol butyrate. The pH optimum and temperature optimum of the recombinant enzyme was 9.0 and 51 °C, respectively. Also, this enzyme displayed moderate thermostability, high activity under alkaline conditions, and good tolerance against several organic solvents. In addition, the compatibility test and washing performance analysis revealed that Est903 had good compatibility with commercial laundry detergent and high cleaning ability of oil stains. These good properties make Est903 a potential candidate in organic synthesis or detergent industry.

Proof-of-concept preparation and characterization of dual-drug amorphous nanoparticle complex as fixed-dose combination of poorly soluble drugs.

OBJECTIVES: To carry out a proof-of-concept study on the development of dual-drug amorphous nanoparticle complex (nanoplex in short) as a potential formulation platform for fixed-dose combination (FDC) of poorly-soluble drugs. SIGNIFICANCE: FDC has been proven effective in improving patient compliance for treatment that requires complex multidrug regimen. Currently, there is growing interest to develop FDC of poorly-soluble drugs due to the increased number of drugs exhibiting poor solubility thus low bioavailability. METHODS: The dual-drug nanoplex was prepared by electrostatically-driven co-complexation of drug molecules with oppositely charged dextran sulfate, using ciprofloxacin (CIP) and itraconazole (ITZ) as the model poorly-soluble drugs. RESULTS: We first verified that the co-complexation products were dual-drug CIP-ITZ nanoplex, and not binary mixtures of the single-drug CIP and ITZ nanoplexes, by demonstrating their distinct thermal behaviors and dissolution characteristics. Depending on the preparation condition, the dual-drug nanoplex exhibited size and zeta potential of 160-410 nm and -35-50 mV, respectively. The individual drug payloads were readily manipulated by varying the CIP/ITZ mass ratio in the feed, resulting in CIP and ITZ payloads in the range of 60-30% and 15-45%, respectively. The CIP-ITZ nanoplex, however, exhibited diminished CIP supersaturation generation, thus lower CIP solubility enhancement, compared to the single-drug CIP nanoplex. The CIP-ITZ nanoplex, nonetheless, remained capable of generating high ITZ supersaturation level. CONCLUSION: Dual-drug nanoplex was successfully prepared with a high degree of control over its physical characteristics. Nevertheless, whether dual-drug nanoplex always exhibits diminished solubility enhancement compared to its single-drug counterparts needs to be investigated using different poorly-soluble drugs.

Radioprotective activity of curcumin-encapsulated liposomes against genotoxicity caused by Gamma Cobalt-60 irradiation in human blood cells.

PURPOSE: While the radioprotective activity of curcumin against genotoxicity has been well established, its poor oral bioavailability has limited its successful clinical applications. Nanoscale formulations, including liposomes, have been demonstrated to improve curcumin bioavailability. The objective of the present work was (1) to prepare and characterize curcumin-encapsulated liposomes (i.e. size, colloidal stability, encapsulation efficiency, and payload), and (2) subsequently to evaluate their radioprotective activity against genotoxicity in human blood cells caused by Gamma Cobalt-60 irradiation. MATERIALS AND METHODS: The curcumin-encapsulated liposomes were prepared by lipid-film hydration method using commercial phosphatidylcholine (i.e. Phospholipon® 90G). The blood cells were obtained from healthy male donors (n = 3) under an approved ethics protocol. The cell uptake and the radioprotective activity of the curcumin-encapsulated liposomes were characterized by fluorescence microscopy and micronucleus assay, respectively. RESULTS: Nanoscale curcumin-encapsulated liposomes exhibiting good physical characteristics and successful uptake by the human blood cells were successfully prepared. The radioprotective activity of the curcumin-encapsulated liposomes was found to be dependent on the curcumin concentration, where an optimal concentration existed (i.e. 30 µg/mL) independent of the irradiation dose, above which the radioprotective activity had become stagnant (i.e. no more reduction in the micronuclei frequency). CONCLUSIONS: The present results established for the first time the radioprotective activity of curcumin-encapsulated liposomes in human blood cells, which coupled by its well-established bioavailability, boded well for its potential application as a nanoscale delivery system of other radioprotective phytochemicals.

Direct comparison between millifluidic and bulk-mixing platform in the synthesis of amorphous drug-polysaccharide nanoparticle complex.

Amorphous drug-polysaccharide nanoparticle complex (or drug nanoplex) had emerged as an ideal supersaturating delivery system of poorly-soluble drugs attributed to its many attractive characteristics. Herein we presented for the first time direct comparison between two nanoplex synthesis platforms, i.e. millifluidics and bulk mixing, representing continuous and batch production modes, respectively. They were compared by the resultant nanoplex's (1) physical characteristics (size, zeta potential, and payload), (2) preparation efficiency, (3) storage stability, (4) dissolution rate/supersaturation generation, and (5) production consistency. The effects of key variables in drug-polysaccharide complexation (pH, charge ratio) were investigated in both platforms. Perphenazine and dextran sulfate were used as the drug and polysaccharide models, respectively. The results showed that both platforms shared similar dependences on pH and charge ratio with similar optimal preparation conditions, where the pH was the governing variable through its influence on size and zeta potential, Nanoplexes having mostly similar characteristics (size ≈70-90nm, zeta potential ≈-50mV) were produced by both platforms, except for the payload where bulk mixing resulted in lower payload (65% versus 85%). The lower payload, however, resulted in its superior supersaturation generation. Nevertheless, millifluidics was favored attributed to its superior production consistency and scalability.

Amorphous nanoparticle complex of perphenazine and dextran sulfate as a new solubility enhancement strategy of antipsychotic perphenazine.

OBJECTIVE: The objective of this study is to develop a new solubility enhancement strategy of antipsychotic drug - perphenazine (PPZ) - in the form of its amorphous nanoparticle complex (or nanoplex) with polyelectrolyte dextran sulfate (DXT). SIGNIFICANCE: Poor bioavailability of PPZ necessitated the development of fast-dissolving PPZ formulations regardless of delivery routes. Existing fast-dissolving formulations, however, exhibited low PPZ payload. The high-payload PPZ-DXT nanoplex represents an attractive fast-dissolving formulation, as dissolution rate is known to be proportional to payload. METHODS: The nanoplex was prepared by electrostatically driven complexation between PPZ and DXT in a simple process that involved only ambient mixing of PPZ and DXT solutions. We investigated the effects of key variables in drug-polyelectrolyte complexation (i.e. pH and charge ratio RDXT/PPZ) on the physical characteristics and preparation efficiency of the nanoplex produced. Subsequently, we characterized the colloidal and amorphous state stabilities, dissolution enhancement, and supersaturation generation of the nanoplex prepared at the optimal condition. RESULTS: The physical characteristics of nanoplex were governed by RDXT/PPZ, while the preparation efficiency was governed by the preparation pH. Nanoplex having size of ≈80 nm, zeta potential of ≈(-) 60 mV, and payload of ≈70% (w/w) were prepared at nearly 90% PPZ utilization rate and ≈60% yield. The nanoplex exhibited superior dissolution than native PPZ in simulated intestinal juice, resulting in high and prolonged apparent solubility with good storage stabilities. CONCLUSIONS: The simple yet efficient preparation, excellent physical characteristics, fast dissolution, and high apparent solubility exhibited by the PPZ-DXT nanoplex established its potential as a new bioavailability enhancement strategy of PPZ.

A supersaturating delivery system of silibinin exhibiting high payload achieved by amorphous nano-complexation with chitosan.

The therapeutic potentials of silibinin - a phytochemical isolated from milk thistle plants - have not been fully realized due to its poor oral bioavailability caused by the low aqueous solubility. Existing solubility enhancement strategies of silibinin by nanonization were limited by their low payload. Herein we developed a supersaturating delivery system of silibinin exhibiting a high payload (≈76%) in the form of amorphous silibinin-chitosan nanoparticle complex (or silibinin nanoplex in short) prepared by self-assembly drug-polysaccharide complexation. The effects of (1) pH and (2) charge ratio of chitosan to silibinin on the nanoplex's physical characteristics (i.e. size, zeta potential, and payload) and preparation efficiency (i.e. silibinin utilization, overall yield) were investigated. The formation of nanoplex (≈240nm) was feasible only in a narrow pH range (5.1-5.8) and favored charge ratio below unity. At the optimal condition (pH 5.8 and charge ratio of 0.30), the nanoplex preparation exhibited 87% silibinin utilization rate and 63% yield signifying its high efficiency. The amorphous state and colloidal stabilities of the nanoplex during storage, and prolonged supersaturation generation (3h) at more than 10× of the saturation solubility were successfully demonstrated.

[Expression and characterization of Coprinus cinereus peroxidase].

OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

Gene synthesis method based on overlap extension PCR and DNAWorks program.

Gene synthesis by chemical methods provides a powerful tool for modifying genes and exploring their structure, expression, and function in the post-genomic era. However, a bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. Here, we describe a simple, rapid, and low-cost gene synthesis method based on overlap extension PCR (OE-PCR) and the DNAWorks program. This method enables DNA sequences with sizes ranging from 200 bp to 3 kb to be synthesized with few errors, and these errors can be easily corrected by site-directed mutagenesis. Thus, it is amenable to automation for the multiplexed synthesis of different genes and has a potential for high-throughput gene synthesis.

An improved method of gene synthesis based on DNA works software and overlap extension PCR.

A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully.