The Mitogen-Activated Protein Kinase Hog1 Regulates Fungal Development, Pathogenicity, and Stress Response in Botryosphaeria dothidea.

The high-osmolarity glycerol mitogen-activated protein kinase (HOG-MAPK) pathway plays a central role in environmental stress adaptation in eukaryotes. However, the biological function of the HOG-MAPK pathway varies in different fungi. In this study, we investigated the HOG-MAPK pathway by inactivation of the core element Hog1 in Botryosphaeria dothidea, the causal agent of Botryosphaeria canker and apple ring rot. Targeted deletion of BdHOG1 resulted in the loss of conidiation ability and significant reduction of virulence. In addition, the ΔBdHog1 mutant exhibited hypersensitivity to osmotic stress but resistance to phenylpyrrole and dicarboximide fungicides. Comparative transcriptome analysis revealed that inactivation of BdHog1 influenced multiple metabolic pathways in B. dothidea. Taken together, our results suggest that BdHog1 plays a crucial role in development, virulence, and stress tolerance in B. dothidea, which provides a theoretical basis for the development of target-based fungicides.

A new point mutation (D1158N) in histidine kinase Bos1 confers high-level resistance to fludioxonil in field gray mold disease.

Gray mold, caused by the fungus Botrytis cinerea, is one of the most important plant diseases worldwide that is prone to developing resistance to fungicides. Currently, the phenylpyrrole fungicide fludioxonil exhibits excellent efficacy in the control of gray mold in China. In this study, we detected the fludioxonil resistance of gray mold disease in Shouguang City of Shandong Province, where we first found fludioxonil-resistant isolates of B. cinerea in 2014. A total of 87 single spore isolates of B. cinerea were obtained from cucumbers in greenhouse, and 3 of which could grow on PDA plates amended with 50 µg/mL fludioxonil that was defined as high-level resistance, with a resistance frequency of 3.4%. Furthermore, the 3 fludioxonil-resistant isolates also showed high-level resistance to the dicarboximide fungicides iprodione and procymidone. Sequencing comparison revealed that all the 3 fludioxonil-resistant isolates had a point mutation at codon 1158, GAC (Asp) â†’ AAC (Asn) in the histidine kinase Bos1, which was proved to be the reason for fludioxonil resistance. In addition, the fludioxonil-resistant isolates possessed an impaired biological fitness compared to the sensitive isolates based on the results of mycelial growth, conidiation, virulence, and osmotic stress tolerance determination. Taken together, our results indicate that the high-level resistance to fludioxonil caused by the Bos1 point mutation (D1158N) has emerged in the field gray mold disease, and the resistance risk is relatively high, and fludioxonil should be used sparingly.

Novel 6-amino-1,3,5-triazine derivatives as potent BTK inhibitors: structure-activity relationship (SAR) analysis and preliminary mechanism investigation.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of B-cell related malignancies. Irreversible inhibition of BTK by a covalent inhibitor has been proved to be a clinically effective therapy. However, most irreversible BTK inhibitors also inhibit other kinases including JAK3 and EGFR, leading to some adverse events. Herein, we reported the structure-based design and optimization of a series of irreversible BTK inhibitors bearing the 6-amino-1,3,5-triazine scaffold. Most of the synthesized compounds demonstrated considerable BTK inhibition and improved anti-proliferative activity against Raji and Ramos cells. Among them, compound C11 exhibited potent BTK inhibition (BTK IC50 = 17.0 nM) and a desirable selectivity profile especially over EGFR. Moreover, C11 effectively blocked activation of BTK and downstream signaling, arrested the cell cycle in G0/G1 phase and induced apoptosis in Raji cells. Its irreversible binding mode was further investigated by both molecular modeling and a washout experiment. Collectively, C11 is a novel selective irreversible BTK inhibitor worthy of further in-depth research.

X-ray crystallography study and optimization of novel benzothiophene analogs as potent selective estrogen receptor covalent antagonists (SERCAs) with improved potency and safety profiles.

Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.

Structural optimization of tetrahydroisoquinoline-hydroxamate hybrids as potent dual ERα degraders and HDAC inhibitors.

Both estrogen receptor α (ERα) and histone deacetylases (HDACs) are valid therapeutic targets for anticancer drug development. Combination therapies using diverse ERα antagonists or degraders and HDAC inhibitors have been proven effective in endocrine-resistant ER + breast cancers based on the crosstalk between ERα and HDAC pathway. In this study, we reported the optimization of a series of methoxyphenyl- or pyridinyl- substituted tetrahydroisoquinoline-hydroxamates, which were optimized from 31, a dual ERα degrader/HDAC inhibitor previously reported by our group. Most of the synthesized compounds displayed potent ERα degradation efficacy and antiproliferative activity. Among them, A04 demonstrated the best anti-proliferation activity (MCF-7 IC50 = 1.96 µM) and HDAC6 inhibitory activity (HDAC6 IC50 = 25.96 nM), which is slightly more potent than the lead compound 31 (MCF-7 IC50 = 4.38 µM, HDAC6 IC50 = 63.03 nM). In addition, compound A04 exerted ERα-independent HDAC6-inhibiting effect without agonistic activity in endometrial cells. These results demonstrated that A04 is a novel and promising dual ERα degrader/HDAC inhibitor worthy of further development.

Detection of Cytb Point Mutation (G143A) that Confers High-Level Resistance to Pyraclostrobin in Glomerella cingulata Using LAMP Method.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emerging disease that results in severe defoliation and fruit spots in apples. In China, the compound of pyraclostrobin and tebuconazole was registered to control GLS in 2018 and has achieved excellent control efficiency. In this study, we showed that the high-level resistant isolates of G. cingulata to pyraclostrobin, caused by the point mutation at codon 143 (GGT→GCT, G143A) in the cytochrome b gene, has appeared in apple orchards in Shandong Province in 2020, and the resistance frequency was 4.8%. Based on the genotype of the resistant isolates, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of the pyraclostrobin resistance. The LAMP assay was demonstrated to have good specificity, sensitivity, and repeatability, and it exhibited high accuracy in detecting pyraclostrobin resistance in the field. This study reported the resistance status of GLS to pyraclostrobin in Shandong Province and developed a molecular tool for the detection of pyraclostrobin resistance, which is of practical significance for the scientific control of GLS.

Benzothiophene derivatives as selective estrogen receptor covalent antagonists: Design, synthesis and anti-ERα activities.

Estrogen receptor α emerged as a well validated therapeutic target of breast cancer for decades. However, approximately 50% of patients who initially responding to standard-of-care (SoC), such as undergo therapy of Tamoxifen, generally inevitably progress to an endocrine-resistance ER+ phenotype. Recently, selective estrogen receptor covalent antagonists (SERCAs) targeted to ERα have been demonstrated as a therapeutic alternative. In the present study, series of novel 6-OH-benzothiophene (BT) derivatives targeting ERα and deriving from Raloxifene were designed, synthesized, and biologically evaluated as covalent antagonists. Driven by the antiproliferative efficacy in ER+ breast cancer cells, our chemical optimization finally led to compound 19d that with potent antagonistic activity in ER+ tumor cells while without agonistic activity in endometrial cells. Moreover, the docking simulation was carried out to elucidate the binding mode, revealing 19d as an antagonist and covalently binding to the cysteine residue at the 530 position of ER helix H11.

Synthesis and evaluation of novel thiosemicarbazone and semicarbazone analogs with both anti-proliferative and anti-metastatic activities against triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive cancer with high mortality and recurrence rates. Hecogenin, a steroidal sapogenin, is reported as a potential anti-tumor agent against breast cancer. However, the moderate activity limits its further application in clinical. With the aim to identify novel analogues that are especially efficacious in therapy of TNBC, a series of novel hecogenin thiosemicarbazone and semicarbazone derivatives were designed, synthesized and biologically evaluated. Screening of cytotoxicity revealed that 4c could potently inhibit the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cells), lung cancer cells (A549) and colon cancer cells (HT-29) at low µM level. Importantly, further mechanism studies indicated the ability of 4c in inducing apoptosis of MDA-MB-231 cells by arresting the cell cycle. Moreover, 4c notably suppressed the migration and invasion of MDA-MB-231 cells compared to its parent hecogenin at the equal concentration.

Supramolecular Nanofibers Ameliorate Bleomycin-Induced Pulmonary Fibrosis by Restoring Autophagy.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease, with limited therapeutic options available. Impaired autophagy resulting from aberrant TRB3/p62 protein-protein interactions (PPIs) contributes to the progression of IPF. Restoration of autophagy by modulating the TRB3/p62 PPIs has rarely been reported for the treatment of IPF. Herein, peptide nanofibers are developed that specifically bind to TRB3 protein and explored their potential as a therapeutic approach for IPF. By conjugating with the self-assembling fragment (Ac-GFFY), a TRB3-binding peptide motif A2 allows for the formation of nanofibers with a stable α-helix secondary structure. The resulting peptide (Ac-GFFY-A2) nanofibers exhibit specific high-affinity binding to TRB3 protein in saline buffer and better capacity of cellular uptake to A2 peptide. Furthermore, the TRB3-targeting peptide nanofibers efficiently interfere with the aberrant TRB3/p62 PPIs in activated fibroblasts and fibrotic lung tissue of mice, thereby restoring autophagy dysfunction. The TRB3-targeting peptide nanofibers inhibit myofibroblast differentiation, collagen production, and fibroblast migration in vitro is demonstrated, as well as bleomycin-induced pulmonary fibrosis in vivo. This study provides a supramolecular method to modulate PPIs and highlights a promising strategy for treating IPF diseases by restoring autophagy.

Curcumin nicotinate increases LDL cholesterol uptake in hepatocytes through IDOL/LDL-R pathway regulation.

BACKGROUND: Curcumin nicotinate (Curtn), derived from curcumin and niacin, reduces serum LDL-C levels, partly due to its influence on PCSK9. This study investigates IDOL's role in Curtn's lipid-lowering effects. OBJECTIVE: To elucidate Curtn's regulation of the IDOL/LDLR pathway and potential molecular mechanisms in hepatocytes. METHODS: Differential metabolites in Curtn-treated HepG2 cells were identified via LC-MS. Molecular docking assessed Curtn's affinity with IDOL. Cholesterol content and LDLR expression effects were studied in high-fat diet Wistar rats. In vitro evaluations determined Curtn's influence on IDOL overexpression's LDL-C uptake and LDLR expression in hepatocytes. RESULTS: Lipids were the main differential metabolites in Curtn-treated HepG2 cells. Docking showed Curtn's higher affinity to IDOL's FERM domain compared to curcumin, suggesting potential competitive inhibition of IDOL's binding to LDLR. Curtn decreased liver cholesterol in Wistar rats and elevated LDLR expression. During in vitro experiments, Curtn significantly enhanced the effects of IDOL overexpression in HepG2 cells, leading to increased LDL-C uptake and elevated expression of LDL receptors. CONCLUSION: Curtn modulates the IDOL/LDLR pathway, enhancing LDL cholesterol uptake in hepatocytes. Combined with its PCSK9 influence, Curtn emerges as a potential hyperlipidemia therapy.

The Phosphatase Cascade Nem1/Spo7-Pah1 Regulates Fungal Development, Lipid Homeostasis, and Virulence in Botryosphaeria dothidea.

Protein phosphatase complex Nem1/Spo7 plays crucial roles in the regulation of various biological processes in eukaryotes. However, its biological functions in phytopathogenic fungi are not well understood. In this study, genome-wide transcriptional profiling analysis revealed that Nem1 was significantly upregulated during the infection process of Botryosphaeria dothidea, and we identified and characterized the phosphatase complex Nem1/Spo7 and its substrate Pah1 (a phosphatidic acid phosphatase) in B. dothidea. Nem1/Spo7 physically interacted with and dephosphorylated Pah1 to promote triacylglycerol (TAG) and subsequent lipid droplet (LD) synthesis. Moreover, the Nem1/Spo7-dependently dephosphorylated Pah1 functioned as a transcriptional repressor of the key nuclear membrane biosynthesis genes to regulate nuclear membrane morphology. In addition, phenotypic analyses showed that the phosphatase cascade Nem1/Spo7-Pah1 was involved in regulating mycelial growth, asexual development, stress responses, and virulence of B. dothidea. IMPORTANCE Botryosphaeria canker and fruit rot caused by the fungus Botryosphaeria dothidea is one of the most destructive diseases of apple worldwide. Our data indicated that the phosphatase cascade Nem1/Spo7-Pah1 plays important roles in the regulation of fungal growth, development, lipid homeostasis, environmental stress responses, and virulence in B. dothidea. The findings will contribute to the in-depth and comprehensive understanding of Nem1/Spo7-Pah1 in fungi and the development of target-based fungicides for disease management.

Influence of avalanche transistor switching mode on waveform characteristics of solid-state pulse source.

The design of the Marx circuit based on avalanche transistors (ATs) is one of the effective techniques for developing solid-state pulse sources to generate nanosecond pulses. However, the influence of the avalanche transistor as a switching device on the output pulse characteristics is still unclear. In this study, investigating the switching mechanism of the AT with a mixed-mode simulation of the semiconductor device has been accomplished. An experiment has checked the simulation model's transient switching characteristics. The switching mechanisms of ATs in the Marx circuit were divided into base triggering mode (BTM) and voltage ramp mode (VRM). This paper proposes a modified circuit for adjusting the output pulse parameters of solid-state pulse sources. The results show that to satisfy the simulation accuracy, the width parameter of the AT model in the BTM must be 100 µm, much less than the actual physical size. Because of the higher electric field when the initial impact ionization occurs, the AT operates at a higher switching speed in the VRM than in the BTM. In addition, since the carriers of initial impact ionization locate at the p-n0 interface or the n0-n+ interface, the AT switching process will oscillate in the VRM. All ATs in the modified Marx circuit switch to operate in the BTM. The leading edge of the output pulse increases from 275 to 1125 ps, and the pulse trailing oscillation has disappeared. The research results provide an important technical means for optimizing the output waveform of solid-state pulse sources.

Involvement of the Autophagy Protein Atg1 in Development and Virulence in Botryosphaeria dothidea.

Botryosphaeria canker and fruit rot caused by the fungus Botryosphaeria dothidea is one of the most destructive diseases of apple worldwide. Autophagy is an evolutionarily conserved self-degradation process that is important for maintaining homeostasis to ensure cellular functionality. To date, the role of autophagy in B. dothidea is not well elucidated. In this study, we identified and characterized the autophagy-related protein Atg1 in B. dothidea. The BdAtg1 deletion mutant ΔBdAtg1 showed autophagy blockade and phenotypic defects in mycelial growth, conidiation, ascosporulation and virulence. In addition, ΔBdAtg1 exhibited an increased number of nuclei in the mycelial compartment. Comparative transcriptome analysis revealed that inactivation of BdAtg1 significantly influenced multiple metabolic pathways. Taken together, our results indicate that BdAtg1 plays an important role in vegetative differentiation and the pathogenicity of B. dothidea. The results of this study will provide a reference for the development of new target-based fungicides.

Direct chemical reprogramming of human cord blood erythroblasts to induced megakaryocytes that produce platelets.

Reprogramming somatic cells into megakaryocytes (MKs) would provide a promising source of platelets. However, using a pharmacological approach to generate human MKs from somatic cells remains an unmet challenge. Here, we report that a combination of four small molecules (4M) successfully converted human cord blood erythroblasts (EBs) into induced MKs (iMKs). The iMKs could produce proplatelets and release functional platelets, functionally resembling natural MKs. Reprogramming trajectory analysis revealed an efficient cell fate conversion of EBs into iMKs by 4M via the intermediate state of bipotent precursors. 4M induced chromatin remodeling and drove the transition of transcription factor (TF) regulatory network from key erythroid TFs to essential TFs for megakaryopoiesis, including FLI1 and MEIS1. These results demonstrate that the chemical reprogramming of cord blood EBs into iMKs provides a simple and efficient approach to generate MKs and platelets for clinical applications.

Design and synthesis of novel benzothiophene analogs as selective estrogen receptor covalent antagonists against breast cancer.

Endocrine therapy (ET) has benefited patients with estrogen receptor alpha (ERα) positive breast cancer for decades. Selective estrogen receptor modulator (SERM) such as Tamoxifen represents the clinical standard of care (SoC). Despite the therapeutic importance of current SoC agents, 30-50% of prolonged treatment patients inevitably generated resistant tumor cells, usually eventually suffered tumor relapse and developed into metastatic breast cancer (MBC), which was the leading cause of female cancer-related mortality. Among these, most resistant tumors remained dependent on ERα signaling, which reignited the need for the next generation of ERα related agents. We hypothesized that selective estrogen receptor covalent antagonists targeting ERα would provide a therapeutic alternative. In the current work, series of novel benzothiophene hybrids bearing electrophile moieties were synthesized and biologically evaluated. The representative analogue 15c exhibited potent anti-proliferative effect in MCF-7 cell lines in vitro, and further mechanism studies confirmed the necessity of covalent bonding. More importantly, 15c could attenuate the expression of TFF-1, GREB-1 and downregulate the levels of cellular ERα protein.