Iridium-Catalyzed Enantioselective Transfer Hydrogenation of 1,1-Dialkylethenes with Ethanol: Scope and Mechanism.

Despite half a century's advance in the field of transition-metal-catalyzed asymmetric alkene hydrogenation, the enantioselective hydrogenation of purely alkyl-substituted 1,1-dialkylethenes has remained an unmet challenge. Herein, we describe a chiral PCNOx-pincer iridium complex for asymmetric transfer hydrogenation of this alkene class with ethanol, furnishing all-alkyl-substituted tertiary stereocenters. High levels of enantioselectivity can be achieved in the reactions of substrates with secondary/primary and primary/primary alkyl combinations. The catalyst is further applied to the redox isomerization of disubstituted alkenols, producing a tertiary stereocenter remote to the resulting carbonyl group. Mechanistic studies reveal a dihydride species, (PCNOx)Ir(H)2, as the catalytically active intermediate, which can decay to a dimeric species (κ3-PCNOx)IrH(µ-H)2IrH(κ2-PCNOx) via a ligand-remetalation pathway. The catalyst deactivation under the hydrogenation conditions with H2 is much faster than that under the transfer hydrogenation conditions with EtOH, which explains why the (PCNOx)Ir catalyst is effective for the transfer hydrogenation but ineffective for the hydrogenation. The suppression of di-to-trisubstituted alkene isomerization by regioselective 1,2-insertion is partly responsible for the success of this system, underscoring the critical role played by the pincer ligand in enantioselective transfer hydrogenation of 1,1-dialkylethenes. Moreover, computational studies elucidate the significant influence of the London dispersion interaction between the ligand and the substrate on enantioselectivity control, as illustrated by the complete reversal of stereochemistry through cyclohexyl-to-cyclopropyl group substitution in the alkene substrates.

A Programmable Microfluidic Paper-Based Analytical Device for Simultaneous Colorimetric and Photothermal Visual Sensing of Multiple Enzyme Activities.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (µPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this µPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO2) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H2O2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Novel kinase 1 regulates CD8+T cells as a potential therapeutic mechanism for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rare, chronic and progressively worsening lung disease that poses a significant threat to patient prognosis, with a mortality rate exceeding that of some common malignancies. Effective methods for early diagnosis and treatment remain for this condition are elusive. In our study, we used the GEO database to access second-generation sequencing data and associated clinical information from IPF patients. By utilizing bioinformatics techniques, we identified crucial disease-related genes and their biological functions, and characterized their expression patterns. Furthermore, we mapped out the immune landscape of IPF, which revealed potential roles for novel kinase 1 and CD8+T cells in disease progression and outcome. These findings can aid the development of new strategies for the clinical diagnosis and treatment of IPF.

Research Progress of Hippocampal Dopamine System Changes in Perioperative Neurocognitive Disorders.

Perioperative neurocognitive disorders (PND) are a cognitive impairment that occurs after anesthesia, especially in elderly patients and significantly affects their quality of life. The hippocampus, as a critical region for cognitive function and an important location in PND research, has recently attracted increasing attention. However, in the hippocampus the impact of anesthesia and its underlying mechanisms remain unclear. This review focuses on investigation of the effects of anesthesia on the hippocampal dopamine (DA) system and explores its potential association with PND. Through comprehensive review of existing studies, it was found that anesthesia affects the hippocampus through various pathways involved in metabolism, synaptic plasticity and oxygenation. Anesthesia may also influence the DA neurotransmitter system in the brain which plays a role in emotions, rewards, learning and memory functions. Specifically, anesthesia may participate in the pathogenesis of PND by affecting the DA system within the hippocampus. Future studies should explore the molecular mechanisms of these effects through techniques such as neuroimaging to study real-time effects to improve animal models to better simulate clinical observations. For clinical application, it is recommended that physicians exercise caution when selecting and managing anesthetic drugs by adopting comprehensive cognitive assessment methods to reduce post-anesthesia cognitive risk. Overall, this review provides a better understanding of the relationship between the hippocampal DA system and perioperative neurocognitive function and provides valuable guidance for prevention and treatment strategies for PND.

Phylogeny and Fungal Community Structures of Helotiales Associated with Sclerotial Disease of Mulberry Fruits in China.

Mulberry fruit sclerotiniose is a prevalent disease caused by the fungal species Ciboria shiraiana, C. carunculoides, and Scleromitrula shiraiana of the order Helotiales, and severely affects the production of mulberry. However, these species have only been identified using morphological and rDNA-ITS sequence analyses, and their genetic variation is unclear. To address this, morphological and two-locus (ITS and RPB2) phylogenetic analyses were conducted using culture-dependent and independent methods for 49 samples from 31 orchards across four provinces in China. Illumina MiSeq sequencing was used to assess the fungal communities obtained from fruits varying in disease severity and color from an orchard in Wuhan. Conidial suspensions of C. shiraiana and C. carunculoides isolated from diseased fruits, diseased fruits affected with hypertrophy and pellet sorosis sclerotiniose, and mycelia of Sclerotinia sclerotiorum were determined to be pathogenic to the mulberry cultivar YSD10. However, fruits inoculated with S. sclerotiorum mycelia exhibited nontypical disease symptoms, and mycelia and conidia obtained from C. carunculoides and S. shiraiana strains were not pathogenic. Maximum parsimony and Bayesian analyses using the sequences of the assessed loci indicated species variability with no evidence of geographic specialization. Metagenomic analysis revealed that the diversity of fungal communities was reduced with disease progression. Furthermore, within a single fruit, the presence of two Ciboria spp. was detected. These results provide novel insights into Ciboria spp., revealing the secondary infections caused by conidia in diseased fruits, genetic variations of the pathogens, and the occurrence of coinfection. This improved understanding of fungal pathogens will aid in developing effective disease control strategies.

Characteristics and complete genome analysis of the novel virulent phage Bfsp1 infecting Cytobacillus firmus.

We isolated, identified, and characterised Bfsp1, a novel virulent phage of Cytobacillus firmus. Morphologically, Bfsp1 is similar to phi29-like phages. The linear, double-stranded DNA genome of Bfsp1 is 22,320 bp in length, has a GC content of 36.06%, and has 10-bp inverted terminal repeats. The genome contains 33 open reading frames, and functions of 15 of them were predicted. Comparative genome analysis showed that Bfsp1 is distinct from other known phages, and this was confirmed by phylogenetic analysis. Morphological, genomic, and phylogenetic data indicated that Bfsp1 is a novel member of the family Salasmaviridae.

SNS-023 sensitizes hepatocellular carcinoma to sorafenib by inducing degradation of cancer drivers SIX1 and RPS16.

Hepatocellular carcinoma (HCC) remains challenging due to the lack of efficient therapy. Promoting degradation of certain cancer drivers has become an innovative therapy. The nuclear transcription factor sine oculis homeobox 1 (SIX1) is a key driver for the progression of HCC. Here, we explored the molecular mechanisms of ubiquitination of SIX1 and whether targeting SIX1 degradation might represent a potential strategy for HCC therapy. Through detecting the ubiquitination level of SIX1 in clinical HCC tissues and analyzing TCGA and GEPIA databases, we found that ubiquitin specific peptidase 1 (USP1), a deubiquitinating enzyme, contributed to the lower ubiquitination and high protein level of SIX1 in HCC tissues. In HepG2 and Hep3B cells, activation of EGFR-AKT signaling pathway promoted the expression of USP1 and the stability of its substrates, including SIX1 and ribosomal protein S16 (RPS16). In contrast, suppression of EGFR with gefitinib or knockdown of USP1 restrained EGF-elevated levels of SIX1 and RPS16. We further revealed that SNS-023 (formerly known as BMS-387032) induced degradation of SIX1 and RPS16, whereas this process was reversed by reactivation of EGFR-AKT pathway or overexpression of USP1. Consequently, inactivation of the EGFR-AKT-USP1 axis with SNS-032 led to cell cycle arrest, apoptosis, and suppression of cell proliferation and migration in HCC. Moreover, we showed that sorafenib combined with SNS-032 or gefitinib synergistically inhibited the growth of Hep3B xenografts in vivo. Overall, we identify that both SIX1 and RPS16 are crucial substrates for the EGFR-AKT-USP1 axis-driven growth of HCC, suggesting a potential anti-HCC strategy from a novel perspective.

First Report of Neocosmospora mori Causing Root Rot and Stem Blight of Mulberry in Nanzhang, Hubei, China.

Mulberry (Morus alba L.) has been cultivated for thousands of years in many temperate regions in East Asia and is commonly used to feed silkworms. In May 2021, 5 to 8% incidence of stem blight on 4-year-old mulberry 'Nongsang 14' was observed in several orchards in Nanzhang County, Hubei Province, China. The roots and stems showed symptoms of vascular discoloration, and the tender new shoots, surrounded by white hyphae, were detached easily. Symptomatic stem tissues (5 mm × 5 mm) were excised from the border between diseased and healthy tissues, surface sterilized in a 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 1.5 min, washed three times in sterile distilled water, then placed on potato dextrose agar (PDA, 250 g potatoes, 2% dextrose, 1.6% agar), and incubated at 25°C in darkness. Two isolates (Bq2 and Bq3) were subcultured using the single-spore method. On PDA, colonies were cottony, with whitish aerial mycelium and the daily growth rate was 4.25 to 5.50 mm/day at 25°C in darkness. On carnation leaf agar, macroconidia were fusiform with slightly curved apical cells and foot-shaped basal cells, three to five septate, measuring 47.5 to 80.3 × 3.6 to 5.6 µm (average 68.7 × 4.7 µm, n = 30). On spezieller nährstoffarmer agar, microconidia were produced in false heads on monophialides, mostly 0-septate, oval, obovoid, or reniform in shape, measuring 5.1 to 10.7 × 2.7 to 5.3 µm (average 8.5 × 3.3 µm, n = 30). Chlamydospores were 4.9 to 11.0 µm in diameter (average 6.8 µm, n = 30), round shaped, thick-walled, and produced individually or in pairs or in chains. For molecular identification, the ribosomal internal transcribed spacers (ITS), translation elongation factor 1α (EF-1α), 28S large subunit nrDNA (LSU), and calmodulin (CAM) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1H/EF2T (O'Donnell et al. 1998), LR0R/LR5 ( Vilgalys and Hester 1990; Vilgalys and Sun 1994), and CL1/CL2A (Geiser et al. 2021; Wang et al. 2011), respectively. The sequences were deposited in GenBank (OQ711943-OQ711944 for ITS, OQ722438- Q722439 for EF-1α, OQ722441-OQ722442 for CAM, and OR116152-OR116153 for LSU). A maximum-likelihood phylogenetic analysis based on multilocus sequences was conducted using MEGA7, which showed that the two isolates grouped into a clade with Neocosmospora mori (previously Fusarium solani species complex) supported by a high bootstrap value (85%), and hence, they were identified as N. mori based on morphological and molecular analyses (Brooks et al. 2022; Crous et al. 2021; Lombard et al. 2015; Zeng and Zhuang 2023). To complete Koch's postulates, three healthy 2-month-old seedlings grown in sterile peat mix were removed from pots and the roots were washed in sterile water. Each plant was inoculated by dipping wounded and unwounded roots in a spore suspension (1 × 107 conidia/ml) for 20 min, and then 10 mL of the spore suspension was poured over the roots of each seedling after transplanting. Three plants were treated with sterilized water as a control. The tested plants were then kept in a plastic box containing sterile water and incubated at 25°C in a 12 h/12 h light/dark cycle. The pathogenicity assay was repeated three times for each isolate. Root and stem blight was observed 10 days after inoculation, while the control plants were asymptomatic. Furthermore, fungi with morphological characteristics of N. mori were only reisolated from the symptomatic stems and sequences of LSU matched those of isolates Bq2 and Bq3. This pathogen has been reported previously causing stem blight on mulberry trees in Japan and South Korea (Sandoval-Denis et al. 2019), but to our knowledge, this is the first report of N. mori causing root rot and stem blight of mulberry in China. This report will facilitate the development of effective control strategies for the disease.

Theoretical Study on the Origin of Abnormal Regioselectivity in Ring-Opening Reaction of Hexafluoropropylene Oxide.

That nucleophiles preferentially attack at the less sterically hindered carbon of epoxides under neutral and basic conditions has been generally accepted as a fundamental rule for predicting the regioselectivity of this type of reaction. However, this rule does not hold for perfluorinated epoxides, such as hexafluoropropylene oxide (HFPO), in which nucleophiles were found to attack at the more hindered CF3 substituted ß-C rather than the fluorine substituted α-C. In this contribution, we aim to shed light on the nature of this intriguing regioselectivity by density functional theory methods. Our calculations well reproduced the observed abnormal regioselectivities and revealed that the unusual regiochemical preference for the sterically hindered ß-C of HFPO mainly arises from the lower destabilizing distortion energy needed to reach the corresponding ring-opening transition state. The higher distortion energy required for the attack of the less sterically hindered α-C results from a significant strengthening of the C(α)-O bond by the negative hyperconjugation between the lone pair of epoxide O atom and the antibonding C-F orbital.

Application of Cyclodextrin for Cancer Immunotherapy.

Tumor immunotherapy, compared with other treatment strategies, has the notable advantage of a long-term therapeutic effect for preventing metastasis and the recurrence of tumors, thus holding great potential for the future of advanced tumor therapy. However, due to the poor water solubility of immune modulators and immune escape properties of tumor cells, the treatment efficiency of immunotherapy is usually significantly reduced. Cyclodextrin (CD) has been repeatedly highlighted to be probably one of the most investigated building units for cancer therapy due to its elegant integration of an internal hydrophobic hollow cavity and an external hydrophilic outer surface. The application of CD for immunotherapy provides new opportunities for overcoming the aforementioned obstacles. However, there are few published reviews, to our knowledge, summarizing the use of CD for cancer immunotherapy. For this purpose, this paper provides a comprehensive summary on the application of CD for immunotherapy with an emphasis on the role, function, and reported strategies of CD in mediating immunotherapy. This review summarizes the research progress made in using CD for tumor immunotherapy, which will facilitate the generation of various CD-based immunotherapeutic delivery systems with superior anticancer efficacy.

Color doppler ultrasound analysis of pathological myopia induced changes in retrobulbar blood flow and its relationship with characteristic changes in myopia.

Objective: To analyze changes in retrobulbar blood flow in patients with pathological myopia using color doppler ultrasound (CDU), and to explore the relationship of these changes with the characteristic changes resulting from myopia. Methods: One hundred and twenty patients who met the selection criteria in the ophthalmology department of He Eye Specialist Hospital from May 2020 to May 2022 were included in this study. Patients with normal vision (n=40) were considered Group-A, patients with low and moderate myopia (n=40) were considered Group-B, and patients with pathological myopia (n=40) were considered Group-C. All three groups underwent ultrasonography. The peak systolic blood flow velocity (PSV), end-diastolic blood flow velocity (EDV), and resistance index (RI) of the ophthalmic artery, central retinal artery, and posterior ciliary artery were recorded and compared, and the characteristics of these parameters and myopia severity were analyzed. Results: Pathological myopia resulted in significantly lower PSV and EDV of the ophthalmic artery, central retinal artery and posterior ciliary artery and higher RI values than patients with normal vision and low/moderate myopia (P<0.05). Pearson correlation analysis showed that retrobulbar blood flow changes were significantly correlated with age, eye axis, best corrected visual acuity, and retinal choroidal atrophy. Conclusion: CDU can objectively evaluate the retrobulbar blood flow changes in pathological myopia, and such blood flow changes are significantly correlated with the characteristic changes of myopia.

A Feature Selection Approach Guided an Early Prediction of Anthocyanin Accumulation Using Massive Untargeted Metabolomics Data in Mulberry.

Identifying the early predictive biomarkers or compounds represents a pivotal task for guiding a targeted agricultural practice. Despite the various available tools, it remains challenging to define the ideal compound combination and thereby elaborate an effective predictive model fitting that. Hence, we employed a stepwise feature selection approach followed by a maximum relevance and minimum redundancy (MRMR) on the untargeted metabolism in four mulberry genotypes at different fruit developmental stages (FDSs). Thus, we revealed that 7 out of 226 differentially abundant metabolites (DAMs) explained up to 80% variance of anthocyanin based on linear regression model and stepwise feature selection approach accompanied by an MRMR across the genotypes over the FDSs. Among them, the phosphoenolpyruvate, d-mannose and shikimate show the top 3 attribution indexes to the accumulation of anthocyanin in the fruits of these genotypes across the four FDSs. The obtained results were further validated by assessing the regulatory genes expression levels and the targeted metabolism approach. Taken together, our findings provide valuable evidences on the fact that the anthocyanin biosynthesis is somehow involved in the coordination between the carbon metabolism and secondary metabolic pathway. Our report highlights as well the importance of using the feature selection approach for the predictive biomarker identification issued from the untargeted metabolomics data.

The lysosomal membrane protein Sidt2 is a vital regulator of mitochondrial quality control in skeletal muscle.

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

Success and Safety of Needle Knife Papillotomy and Fistulotomy Based on Papillary Anatomy: A Prospective Controlled Trial.

BACKGROUND: Needle knife papillotomy (NKP) and fistulotomy (NKF) are the two most commonly used rescue techniques for patients with difficult biliary cannulation (DBC). Anatomy of the major duodenal papillae (MDP) influences the optimal precut technique for biliary access. However, comparative studies of the success and safety of NKP and NKF based on the anatomy of MDP have been scarce. METHODS: Patients with intact MDPs for therapeutic endoscopic retrograde cholangio-pancreatography (ERCP) in our center were enrolled. Early needle knife precuts were uniformly applied to patients with DBC. Difficult MDPs were classified into one of five types based on their endoscopic anatomy. Each type of MDP was allocated to NKP or NKF treatment. Patients with types 1 and 2 papillae always received NKF, 3 and 4 received NKP, and 5 could receive either. The safety and efficacy were analyzed between NKP and NKF, and among different types of MDPs. RESULTS: A total of 188 out of 1674 patients undergoing ERCP satisfied the criteria for early precutting: 75 patients were assigned to the NKP group and 113 to the NKF group. The total initial success rate of biliary cannulation (ISRBC) of the precut techniques (both NKP and NKF) for patients with DBC was 91.5%. The ISRBC of patients of the NKP group was similar to that of the NKF group (90.7% vs 92.0%, P > 0.05). The ISRBC of the patients in the swollen MDP subgroup (96.1%) was higher than that of patients in the distorted MDP subgroup (81.8%, P = 0.030). The total and specific complications of the patients of the NKP group were similar to those of the NKF group (P > 0.05). CONCLUSIONS: NKP and NKF, as selected on the basis of MDP anatomy, are equally safe and highly efficient for patients with DBC to allow biliary cannulation. Patients with swollen MDPs had a higher ISRBC than patients with distorted MDPs. Selecting a precut method based on MDP anatomy is an effective and safe strategy for patients with DBC.

Stimulating the expression of sphingosine kinase 1 (SphK1) is beneficial to reduce acrylamide-induced nerve cell damage.

Sphingosine kinase 1 (SphK1) is an important signaling molecule for cell proliferation and survival. However, the role of SphK1 in acrylamide (ACR)-induced nerve injury remains unclear. The purpose of this study was to investigate the role and potential mechanism of SphK1 in ACR-induced nerve injury. Liquid chromatography triple quadrupole tandem mass spectrometry (LC-MS/MS) and reverse transcription-quantitative PCR (RT-qPCR) were used to detect sphingosine 1-phosphate (S1P) content in serum and SphK1 content in whole blood from an occupational work group exposed to ACR compared to a non-exposed group. For in vitro experiments, SphK1 in human SH-SY5Y neuroblastoma cells was activated using SphK1-specific activator phorbol 12-myristate 13-acetate (PMA). Our research also utilized cell viability assays, flow cytometry, western blots, RT-qPCR and related protein detection to assess activity of the mitogen activated protein kinase (MAPK) signaling pathway. The results of the population study showed that the contents of SphK1 and S1P in the ACR-exposed occupational contact group were lower than in the non-exposed group. The results of in vitro experiments showed that expression of SphK1 decreased with the increase in ACR concentration. Activating SphK1 improved the survival rate of SH-SY5Y cells and decreased the apoptosis rate. Activating SphK1 in SH-SY5Y cells also regulated MAPK signaling, including enhancing the phosphorylation of extracellular signal-regulated protein kinases (ERK) and inhibiting the phosphorylation of c-Jun N-terminal kinase (JNK) and p38. These results suggest that activating SphK1 can protect against nerve cell damage caused by ACR.

First Report of Colletotrichum aenigma Causing Anthracnose on Mulberry Leaves in China.

Mulberry (Morus alba L.) has been grown worldwide as a crop for silkworm rearing for over five thousand years (Jiao et al. 2020). In July 2021, a leaf spot disease was observed on mulberry leaves in Wuhan city (114°33'E, 30°48'N), Hubei province, China, with approximately 40% of leaves (about 300 trees) affected. Early symptoms were light brown, with small lesions subsequently expanding to larger sometimes irregular dark brown or black spots surrounded by yellow-brown margins, with easily perforated necrotic lesions. Leaf tissues (5 mm×5 mm) were excised from the border between diseased and healthy tissues, surface sterilized with 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 2 min, washed thrice in sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 25°C in darkness. Four isolates (C1, C9, CHS2, and CHS6) were subcultured using the single-spore method. On PDA, colonies were cottony, pale white from above, and white to grayish-green on the reverse side. Conidia were aseptate, hyaline, subcylindrical with broadly rounded ends, 8.4 to 18.3×4.1 to 7.7 µm (mean = 13.9×5.5 µm, n = 30). Appressoria were typically elliptic or irregular with a few lobes, dark brown, 5.9 to 9.6×4.2 to 8.1 µm (mean = 7.9 ×5.7 µm, n = 30). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolates were further identified by analysis of the ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), and ß-tubulin 2 (TUB2) genes, amplified respectively with ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GSF1/GSR, and Bt2a/Bt2b (Glass and Donaldson 1995; Weir et al. 2012; White et al. 1990). The sequences were deposited in GenBank (ON492187-ON492214). Concatenated sequences of the seven genes in addition to Colletotrichum species sequences from GenBank were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The four isolates were grouped into a clade with Colletotrichum aenigma supported by a high bootstrap value (89%), and hence, they were identified as C. aenigma based on the morphological and molecular analyses. To confirm Koch's postulates, wounded leaves of six healthy 2-month-old seedlings made by a sterile needle were inoculated with each isolate by spraying 10 ml of conidial suspensions (105 conidia/ml) on each plant, and the control plants were treated with sterile distilled water. All the treated plants were kept in a plastic box containing sterile water and incubated at 28°C in a 12 h/12 h light/dark cycle. The test was performed three times. After 7 days, typical anthracnose lesions appeared on all inoculated leaves, whereas control plants remained asymptotic. Furthermore, C. aenigma was only reisolated from the symptomatic leaves. Previous studies reported five Colletotrichum species (C. morifolium, C. fioriniae, C. brevisporum, C. karstii, and C. kahawae subsp. ciggaro) to cause this disease on mulberry in China (Tian, 1981; Xue et al. 2019). To our knowledge, this is the first report of C. aenigma causing anthracnose on mulberry in China. The finding will facilitate epidemiological studies and the development of effective control strategies for the disease.

The Role of PnTCP2 in the Lobed Leaf Formation of Phoebe neurantha var. lobophylla.

A lobed leaf is a common trait in plants, but it is very rare in Lauraceae plants, including species of Phoebe. In the study of germplasm resources of Phoebe neurantha, we found lobed leaf variant seedlings, and the variation could be inherited stably. Studying the lobed leaf mechanism of P. neurantha var. lobophylla can offer insight into the leaf development mechanism of woody plants. RNA-seq and small RNA-seq analysis results showed that a total of 8091 differentially expressed genes (DEGs) and 16 differentially expressed miRNAs were identified in P. neurantha var. lobophylla. Considering previous research results, a leaf margin morphological development related miRNA, pne-miRNA319a, was primary identified as a candidate miRNA. Target gene prediction showed that a total of 2070 genes were predicted to be the target genes of differentially expressed miRNAs. GO enrichment analysis of differentially expressed target genes suggested that PnTCP2 is related to lobed leaf formation. The TRV-VIGS gene silencing of PnTCP2 led to lobed leaves in P. neurantha seedlings. The downregulation of PnTCP2 led to lobed leaves. The yeast two-hybrid test and bimolecular fluorescence complementation test confirmed that the PnTCP2 protein interacted with the PnLBD41 protein. Based on the expression analysis of gene-silenced leaves and RNA-seq and small RNA-seq analysis results, pne- miRNA319a and PnLBD41 might also play important roles in this process. In conclusion, PnTCP2 plays an important and vital role in the formation of the lobed leaves of P. neurantha var. lobophylla.

Screening and Verification of Photosynthesis and Chloroplast-Related Genes in Mulberry by Comparative RNA-Seq and Virus-Induced Gene Silencing.

Photosynthesis is one of the most important factors in mulberry growth and production. To study the photosynthetic regulatory network of mulberry we sequenced the transcriptomes of two high-yielding (E1 and E2) and one low-yielding (H32) mulberry genotypes at two-time points (10:00 and 12:00). Re-annotation of the mulberry genome based on the transcriptome sequencing data identified 22,664 high-quality protein-coding genes with a BUSCO-assessed completeness of 93.4%. A total of 6587 differentially expressed genes (DEGs) were obtained in the transcriptome analysis. Functional annotation and enrichment revealed 142 out of 6587 genes involved in the photosynthetic pathway and chloroplast development. Moreover, 3 out of 142 genes were further examined using the VIGS technique; the leaves of MaCLA1- and MaTHIC-silenced plants were markedly yellowed or even white, and the leaves of MaPKP2-silenced plants showed a wrinkled appearance. The expression levels of the ensiled plants were reduced, and the levels of chlorophyll b and total chlorophyll were lower than those of the control plants. Co-expression analysis showed that MaCLA1 was co-expressed with CHUP1 and YSL3; MaTHIC was co-expressed with MaHSP70, MaFLN1, and MaEMB2794; MaPKP2 was mainly co-expressed with GH9B7, GH3.1, and EDA9. Protein interaction network prediction revealed that MaCLA1 was associated with RPE, TRA2, GPS1, and DXR proteins; MaTHIC was associated with TH1, PUR5, BIO2, and THI1; MaPKP2 was associated with ENOC, LOS2, and PGI1. This study offers a useful resource for further investigation of the molecular mechanisms involved in mulberry photosynthesis and preliminary insight into the regulatory network of photosynthesis.

The Ethylene Response Factor ERF5 Regulates Anthocyanin Biosynthesis in 'Zijin' Mulberry Fruits by Interacting with MYBA and F3H Genes.

Ethylene promotes ripening in fruits as well as the biosynthesis of anthocyanins in plants. However, the question of which ethylene response factors (ERFs) interact with the genes along the anthocyanin biosynthesis pathway is yet to be answered. Herein, we conduct an integrated analysis of transcriptomes and metabolome on fruits of two mulberry genotypes ('Zijin', ZJ, and 'Dashi', DS, with high and low anthocyanin abundance, respectively) at different post-flowering stages. In total, 1035 upregulated genes were identified in ZJ and DS, including MYBA in the MBW complex and anthocyanin related genes such as F3H. A KEGG analysis suggested that flavonoid biosynthesis and plant hormone signaling transduction pathways were significantly enriched in the upregulated gene list. In particular, among 103 ERF genes, the expression of ERF5 showed the most positive correlation with the anthocyanin change pattern across both genotypes and in the post-flowering stages, with a Pearson correlation coefficient (PCC) of 0.93. Electrophoresis mobility shift assay (EMSA) and luciferase assay suggested that ERF5 binds to the promoter regions of MYBA and F3H and transcriptionally activates their gene expression. We elucidated a potential mechanism by which ethylene enhances anthocyanin accumulation in mulberry fruits and highlighted the importance of the ERF5 gene in controlling the anthocyanin content in mulberry species. This knowledge could be used for engineering purposes in future mulberry breeding programs.