First Report of Neofusicoccum parvum causing leaf spot on Camellia japonica in China.

Camellia japonica is an important garden landscape plant in southern China. In April 2022, leaf spot symptoms were observed at the camellia garden of Jiaying University (24°32'83″N, 17 116°12'31″E) in Meizhou city, Guangdong Province, China. The initial symptoms were grayish brown spots on the leaves, as the disease progressed, the lesions were enlarged and affected the whole leaf and eventually led to the loss of its ornamental value. The disease incidence was above 15%. Leaf pieces (5 × 5 mm) from 3 diseased Camellia leaves were sterilized in 75% ethanol for 1 min, then in 1% NaOCl for 1 min; and rinsed three times with sterile water. Leaf pieces were inoculated on potato dextrose agar (PDA) medium and incubated at 25 °C. Three days later, fungal colonies initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. Conidia were single-celled, hyaline, ellipsoidal and without septa. Dimensions of conidia (n≥50) were 14.27 to 20.65 × 4.28 to 6.56 µm. The morphological characteristics matched the genus Neofusicoccum (Pavlic et al. 2009). For molecular identification, the rDNA internal transcribed spacer (ITS1, 5.8S and ITS2) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) of a representative isolate SC6-2 were amplified using the primer pairs ITS1/ITS4, EF1/EF2 and BT2a/-BT2b, respectively (Golzar and Burgess，2011). The sequences obtained were deposited in GenBank (accession nos. PP064173, PP479650 and PP082457 for ITS, tef1-α and tub2, respectively). Nucleotide BLAST analysis showed a 99.81% homology with N. parvum (519/520 bp, OQ509869; 519/520 bp, KF294003; 518/519 bp KF293989) for ITS, 100% homology with N. parvum (398/398 bp, MN318108; 398/398 bp, MK294085; 398/398 bp, MH936021) for tub2, and >99% homology with N. parvum (259/259 bp, 100%, MW390561; 263/265 bp, 99.25%，MN175952; 263/265 bp, 99.25%, MK781982) for tef1-α. The combined phylogenetic analyses (ITS, tef1-α, and tub2) showed that the sequence of the tested isolate and the corresponding sequence of N. parvum (CMW9081, SHSJ1-2) in GenBank grouped in the same branch of the phylogenetic tree. Based on morphological characters, DNA sequencing, and the phylogenetic tree, it can be determined that the pathogen was Neofusicoccum parvum. Inoculation on Camellia leaves was performed to confirm pathogenicity. Nine healthy camellia leaves were pin-pricked with a sterile needle and inoculated with mycelial plugs of isolate SC6-2. Nine other healthy leaves were pin-pricked and inoculated with noncolonized PDA plugs as control leaves. The inoculated leaves were maintained on water agar solid medium at 25°C. To keep a high-humidity environment the inoculation sites were covered by moistened cotton for 2 days. The experiment was repeated three times. Five days after inoculation, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 6 days. The fungal isolates recovered from inoculated leaves were morphologically identical to the N. parvum isolates originally recovered from symptomatic leaves collected in the field, fulfilling Koch's postulates. Neofusicoccum parvum is an aggressive pathogen that causes severe disease on important tree and woody species (Liddle et al. 2019). It has been reported that N. parvum can infect the leaves and branches of grapes (Otoya-Martinez et al. 2023), dieback on Camellia japonica (Pintos, et al. 2012), Brazilian pepperwood (Bertetti et al. 2022), mango (Giancarlo et al. 2023) and other plants. To our knowledge, this is the first report of N. parvum causing leaf spot on Camellia japonica in China.

Preparation and in vitro evaluation of hesperidin nanoparticles by antisolvent recrystallization in a double homogenate system.

Hesperidin nanoparticles (HNPs) were made for the first time employing an antisolvent recrystallization technique in a double homogenate system with positive and negative clockwise rotation in order to completely use the underutilized nutritional components in citrus peel. Dimethyl sulfoxide (DMSO), ethanol, and deionized water were used as the solvents and antisolvents in the hesperidin solution preparation. Hesperidin solution concentration of 60.26 mg/mL, homogenization speed of 8257 rpm, antisolvent-to-solvent volume ratio of 6.93 mL/mL, and homogenization time of 3.15 min were the ideal experimental conditions. HNPs have to be at least 72.24 nm in size. The structures of the produced hesperidin samples and the raw hesperidin powder were identical, according to the findings of the FTIR, XRD, and TG characterization tests. The HNP sample had an in vitro absorption rate that was 5.63 and 4.23 times greater than that of the raw hesperidin powder, respectively. It was discovered that DMSO was more suited than ethanol for creating HNP particles. In the realms of dietary supplements, therapeutic applications, and health promotion, the HNPs produced by the ARDH technology would be a potential formulation on increasing uses for a wider range of nutraceuticals (synergistic).

Extracting myricetin and dihydromyricetin simultaneously from Hovenia acerba seed by Ultrasound-Assisted extraction on a lab and small Pilot-Scale.

The flavonoids myricetin and dihydromyricetin are significant components of Hovenia acerba seed. In this work, myricetin and dihydromyricetin were extracted from Hovenia acerba seed using an ultrasound-assisted technique, and the extraction parameters were adjusted using the response surface design approach. HPLC was used to assess the yield of myricetin and dihydromyricetin. According to the data, myricetin and dihydromyricetin yields were 0.53 mg/g and 4.06 mg/g at a 60 % ethanol solution concentration, 180 W of ultrasonic irradiation power, a 20 mL/g ratio of liquid to solid, and a 40 °C optimal extraction temperature. The aforementioned findings are virtually in agreement with the experimental findings suggested by the model. The study mentioned above thus offers a means of separating and developing useful components of natural goods.

Genistein microparticles prepared by antisolvent recrystallization with low-speed homogenization process.

To create genistein particles, a brand-new antisolvent recrystallization technique was employed. The response surface approach was utilized to optimize the single factor test findings, which were acquired via the preliminary tests. The ideal liquid-to-liquid ratio was 9, the solution concentration was 21 mg/mL, the nozzle diameter was 700 µm, the feed rate was 39.65 mL/min, and the homogenization rate was 1500 rpm. The smallest mean particle size measured was 202.782 nm. SEM was used to study the powder's morphology, while thermal analysis and infrared imaging were used to evaluate its characteristics. The homogeneous antisolvent recrystallization method-prepared GMP has a better dissolving rate and stronger antioxidant activity when compared to genistein powder. The antisolvent recrystallization approach used in this study, which uses low-speed homogenizing instead of conventional grinding and homogenizing, can successfully reduce particle size, improve bioavailability, and use less energy. This topic may thus be made popular because it has real-world applications.

Naringenin Ultrafine Powder Was Prepared by a New Anti-Solvent Recrystallization Method.

Raw naringenin directly isolated from plants is significantly limited by its poor dissolution rate and low bioavailability for clinical and in vivo studies. This study reported a method for the preparation of naringenin ultrafine powder (NUP) using a novel anti-solvent recrystallization process; preliminary experiments were conducted using six single-factor experiments. The response surface Box-Behnken (BBD) design was used to optimize the level of factors. The optimal preparation conditions of the DMP were obtained as follows: the feed rate was 40.82 mL/min, the solution concentration was 20.63 mg/mL, and the surfactant ratio was 0.62%. The minimum average particle size was 305.58 ± 0.37 nm in the derived optimum conditions. A scanning electron microscope was used to compare and analyze the appearance and morphology of the powder before and after preparation. The characterization results of FTIR, TG and XRD showed that no chemical change occurred in the powder before and after preparation. Through the simulated gastrointestinal juice digestion experiment, it was confirmed that the absorption rate of NUP was 2.96 times and 4.05 times higher than raw naringenin, respectively. Therefore, the results showed that the reduction in the particle size through the use of low-speed recrystallization could improve the absorption rate and provided a feasible approach for the further applications.

Dual inhibition of EGFR and IL-6-STAT3 signalling by miR-146b: a potential targeted therapy for epithelial ovarian cancer.

Epidermal growth factor receptor (EGFR) signalling and the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) are aberrantly activated in ovarian cancer. However, inhibition of EGFR signalling in ovarian cancer patients resulted in a disappointing clinical benefit. In this study, we found that EGFR could activate IL-6-STAT3 pathway in ovarian cancer cells. However, we also demonstrated that EGFR knockdown could increase STAT3 phosphorylation in HO8910 and OVCAR-3 ovarian cancer cells. Interestingly, we further demonstrated that the non-coding RNA miR-146b could simultaneously block both the EGFR and IL-6-STAT3 pathways. Finally, our data demonstrated that miR-146b overexpression resulted in a greater suppression of cell migration than STAT3 pathway inhibition alone.These results suggest a complex and heterogeneous role of EGFR in ovarian cancer. Combined blockade of EGFR and IL-6-STAT3 pathways by miR-146b might be a strategy for improving the clinical benefit of targeting the EGFR pathway in ovarian cancer patients in the future.

Identifying key genes and drug screening for preeclampsia based on gene expression profiles.

Preeclampsia (PE) is characterized by gestational hypertension and proteinuria, and is a leading cause of maternal death and perinatal morbidity globally. Although the exact cause of PE remains unclear, several studies have suggested a role for abnormal expression of multiple genes. The aim of the present study was to identify key genes and related pathways, and to screen for drugs that regulate these genes for potential PE therapy. The GSE60438 dataset was acquired from the Gene Expression Omnibus database to analyze differentially expressed genes (DEGs). By constructing a protein-protein interaction network and performing reverse transcription-quantitative PCR verification, proteasome 26S subunit, non-ATPase 14, prostaglandin E synthase 3 and ubiquinol-cytochrome c reductase core protein 2 were identified as key genes in PE. In addition, PE was found to be associated with 'circadian rhythm', 'fatty acid metabolism', 'DNA damage response detection of DNA damage', 'regulation of DNA repair' and 'endothelial cell development'. Through connectivity map analysis of DEGs, furosemide and droperidol were suggested to be therapeutic drugs that may target the hub genes for PE treatment. Results analysis of GSEA were included in the discussion section of this article. In conclusion, the current study identified novel key genes associated with the onset of PE and potential drugs for PE treatment.

miR-146b promotes cell proliferation and increases chemosensitivity, but attenuates cell migration and invasion via FBXL10 in ovarian cancer.

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy. However, the molecular mechanisms remain unclear. In this study, we found that miR-146b was downregulated in EOC and its expression level was negatively correlated with the pathological staging. Follow-up functional experiments illustrated that overexpression of miR-146b significantly inhibited cell migration and invasion, and increased cell proliferation, but it also improved the response to chemotherapeutic agents. Mechanistically, we demonstrated that miR-146b exerted its function mainly through inhibiting F-box and leucine-rich repeat protein 10 (FBXL10), and upregulated the Cyclin D1, vimentin (VIM), and zona-occludens-1 (ZO-1) expression in EOC. These findings indicate that miR-146b-FBXL10 axis is an important epigenetic regulation pathway in EOC. Low miR-146b may contribute to cancer progression from primary stage to advanced stage, and may be the promising therapeutic target of EOC.

Identification and Characterization of Phospholipase D Genes Putatively Involved in Internal Browning of Pineapple during Postharvest Storage.

Phospholipase D (PLD) in plants plays vital roles in growth, development, and stress responses. However, the precise role of PLDs in pineapple remains poorly understood. In this study, 10 putative PLD genes, designated as AcPLD1-AcPLD10, were identified based on the pineapple genome database. The 10 AcPLDs could be clustered into five of the six known PLD families according to sequence characterization. Their deduced amino acid sequences displayed similarities to PLDs from other plant species. Expression analyses of PLD mRNAs from pineapple pulp were performed. The 10 PLDs exhibited differential expression patterns during storage periods of fruits treated with hexaldehyde (a specific PLD inhibitor) which could alleviate internal browning (IB) of pineapple after harvest. Functional subcellular localization signaling assays of two PLD proteins (AcPLD2 and AcPLD9) were performed by fluorescence microscopy. To further detect the potential action mechanism underlying PLD involved in the IB defense response, PLD, hydrogen peroxide (H2O2) and H2O2 associated with antioxidative enzymes such as superoxide dismutase, catalase, NADPH, and ascorbate peroxidase were quantified by enzyme-linked immunosorbent assay. This report is the first to provide a genome-wide description of the pineapple PLD gene family, and the results should expand knowledge of this family.

Mechanism of internal browning of pineapple: The role of gibberellins catabolism gene (AcGA2ox) and GAs.

Internal browning (IB), a physiological disorder (PD) that causes severe losses in harvested pineapple, can be induced by exogenous gibberellins (GAs). Over the years, studies have focused on roles of Gibberellin 2-oxidase (GA2oxs), the major GAs catabolic enzyme in plants, in the regulation of changes in morphology or biomass. However, whether GA2oxs could regulate PD has not been reported. Here, a full-length AcGA2ox cDNA was isolated from pineapple, with the putative protein sharing 23.59% to 72.92% identity with GA2oxs from five other plants. Pineapples stored at 5 °C stayed intact, while those stored at 20 °C showed severe IB. Storage at 5 °C enhanced AcGA2ox expression and decreased levels of a GAs (GA4) 'compared with storage at 20 °C. However, at 20 °C, exogenous application of abscisic acid (ABA) significantly suppressed IB. ABA simultaneously upregulated AcGA2ox and reduced GA4. Ectopic expression of AcGA2ox in Arabidopsis resulted in reduced GA4, lower seed germination, and shorter hypocotyls and roots, all of which were restored by exogenous GA4/7. Moreover, in pineapple, GA4/7 upregulated polyphenol oxidase, while storage at 5 °C and ABA downregulated it. These results strongly suggest the involvement of AcGA2ox in regulation of GAs levels and a role of AcGA2ox in regulating IB.

Transcriptome characterization and expression profiles of the related defense genes in postharvest mango fruit against Colletotrichum gloeosporioides.

Anthracnose, caused by Colletotrichum gloeosporioides, is a major disease of the postharvest mango (Mangifera indica L.) fruit. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying the mango fruit defense response. Here, we studied the host responses of the mango fruit against C. gloeosporioides using Illumina paired-end sequencing technology, and expression profiles of 35 defense-related genes were further analyzed by qRT-PCR. The results indicated that 5.9Gigabase pair clean reads were assembled into a total of 131,750 unigenes, of which 89,050 unigenes found to be homologous to genes in the NCBI GenBank database and 61,694 unigenes annonated in the Swiss-Prot database. Orthologous analyses showed that 47,770 unigenes were assigned with one or more Gene Ontology terms and 44,145 unigenes were classified into 256 Kyoto Encyclopedia of Genes and Genomes pathways. Moreover, qRT-PCR of 35 defense-related unigenes, including 17 ethylene response factors (ERFs), 6 nucleotide binding site-leucine-rich repeats (NBS-LRRs), 6 nonexpressor of pathogenesis-related genes (NPRs) and 6 pathogenesis-related protein (PRs), revealed that most of these defense-related genes were up-regulated after C. gloeosporioides infection. Taken together, our study provides a platform to discover new candidate genes in mango fruit in relation to pathogen resistance.

Activation of the AMPK-ULK1 pathway plays an important role in autophagy during prion infection.

AMPK is a serine/threonine protein kinase that acts as a positive regulator of autophagy, by phosphorylating ULK1 at specific sites. A previous study demonstrated activation of the macroautophagic system in scrapie-infected experimental rodents and in certain human prion diseases, in which the essential negative regulator mTOR is severely inhibited. In this study, AMPK and ULK1 in the brains of hamsters infected with scrapie strain 263 K and in the scrapie-infected cell line SMB-S15 were analysed. The results showed an up-regulated trend of AMPK and AMPK-Thr172, ULK1 and ULK1-Ser555. Increases in brain AMPK and ULK1 occurred at an early stage of agent 263 K infection. The level of phosphorylated ULK1-Ser757 decreased during mid-infection and was only negligibly present at the terminal stage, a pattern that suggested a close relationship of the phosphorylated protein with altered endogenous mTOR. In addition, the level of LKB1 associated with AMPK activation was selectively increased at the early and middle stages of infection. Knockdown of endogenous ULK1 in SMB-S15 cells inhibited LC3 lipidation. These results showed that, in addition to the abolishment of the mTOR regulatory pathway, activation of the AMPK-ULK1 pathway during prion infection contributes to autophagy activation in prion-infected brain tissues.

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrP(Sc) in prion infected cell line via protein interaction.

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP(Sc) level in a dose-dependent manner, but had minimal effect on the expression of PrP(C) in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP(Sc). We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP(Sc) in the brain tissues.

Expression of ACO1, ERS1 and ERF1 genes in harvested bananas in relation to heat-induced defense against Colletotrichum musae.

The aim of this study was to investigate the connection between heat-induced ethylene signal changes and enhanced disease resistance. Heat enhanced ripening and elevated MaACO1 expression in naturally ripened bananas (NRB), while it delayed ripening and reduced MaACO1expression in the ethephon-treated bananas (ETB). However, in both cases, heat reduced lesion sizes infected by Colletotrichum musae. This indicates that heat-induced disease resistance in bananas was independent of ripening rate. The expression of MaERS1 gene was inhibited by heat treatment in both NRB and ETB, implying that heat as a physical signal could be sensed by banana fruits through the inhibition of ethylene receptor gene expression. The intensity of MaERF1 transcript signals was elevated in heated bananas, suggesting that the enhanced accumulation of MaERF1 transcript following heat treatment could play an important role in activation of the defense system. In ETB, inhibition of JA biosynthesis by application of IBU down-regulated the expression of MaERF and significantly weakened disease resistance, suggesting involvement of endogenous JA in induction of the gene expression, which was reconfirmed by the fact that exposure to exogenous MeJA following the combination of heat plus IBU treatment restored part of the gene expression. On the other hand, in NRB, application of IBU elevated level of MaERF1 expression at 24h and enhanced disease resistance, suggesting that, when banana was not exposed to ethephon, the expression of MaERF1 gene was not JA dependent, which was verified by the fact that MeJA application did not enhance MaERF1 gene expression. In conclusion, heat-induced disease resistance in harvested bananas could involve down-regulation of MaERS1 expression and up-regulation of MaERF1 expression and JA pathway could be involved in heat activation of the defense system in bananas exposed to ethephon.