Inhibitory Activity Against Rhizoctonia Solani and Chemical Composition of Extract from Moutan Cortex.

Rice sheath blight (RSB), caused by Rhizoctonia solani, is a significant disease of rice. The negative effects of chemical fungicides have created an urgent need for low-toxicity botanical fungicides. Our previous research revealed that the ethanol crude extract of Moutan Cortex (MC) exhibited superior antifungal activity against R. solani at 1000 µg/mL, resulting in a 100 % inhibition rate. The antifungal properties were mainly found in the petroleum ether extract. However, the active ingredients of the extract are still unclear. In this study, gas chromatography-mass spectrometry (GC-MS) was utilised for the analysis of its chemical components. The mycelium growth rate method was utilized to detect the antifungal activity. The findings indicated that paeonol constituted the primary active component, with a content of more than 96 %. Meanwhile, paeonol was the most significant antifungal active ingredient, the antifungal activity of paeonol (EC50=44.83 µg/mL) was much higher than that of ß-sitosterol and ethyl propionate against R. solani. Observation under an optical microscope revealed that paeonol resulted in abnormal mycelial morphology. This study provided theoretical support for identifying monomer antifungal compounds and developing biological fungicides for R. solani.

In Addition to Damaging the Plasma Membrane, Phenolic Monoterpenoid Carvacrol can Bind to Minor Groove of DNA of Phytopathogenic Fungi to Potentially Control Tea Leaf Spot Caused by Lasiodiplodia theobromae.

Carvacrol expresses a wide range of biological activities, but the studies of its mechanisms focused on bacteria, mainly involving the destruction of the plasma membrane. In this study, carvacrol exhibited strong antifungal activities against several phytopathogenic fungi and determined a novel antifungal mechanism against Lasiodiplodia theobromae. RNA sequencing indicated that many genes of L. theobromae hyphae were predominately induced by carvacrol, particularly those involved in replication and transcription. Hyperchromic, hypsochromic, and bathochromic effects in the UV-visible absorption spectrum were observed following titration of calf thymus DNA (ctDNA) and carvacrol, which indicated the formation of a DNA-carvacrol complex. Circular dichroism (CD) spectroscopy indicated the response of DNA to carvacrol was similar to 4', 6-diamidino-2-phenylindole (DAPI), but different from that of ethidium bromide (EB), implying the ionic bonds between carvacrol and ctDNA. Fluorescence spectrum (FS) analysis indicated that carvacrol quenched the fluorescence of double-stranded DNA more than single-stranded DNA, indicating that carvacrol mainly bound to double-stranded DNA. A displacement assay showed that carvacrol reduced the fluorescence intensity of the DNA-DAPI complex through competing with DAPI, but this did not occur for DNA-EB. FS assay revealed that carvacrol bound to AAA sequence on the minor groove of ds-oligonucleotides. The hydroxyl of carvacrol was verified to bind to ctDNA through a comparative test in which structural analogs of carvacrol, including thymol, 4-ethyl-1,2-dimethyl, etc. were analyzed. The current study indicated carvacrol can destruct plasma membranes and bind to the minor groove of DNA, which inhibited the fungal proliferation by disturbing the stability of dsDNA.

Mode of Action of Heat Shock Protein (HSP) Inhibitors against Viruses through Host HSP and Virus Interactions.

Misfolded proteins after stress-induced denaturation can regain their functions through correct re-folding with the aid of molecular chaperones. As a molecular chaperone, heat shock proteins (HSPs) can help client proteins fold correctly. During viral infection, HSPs are involved with replication, movement, assembly, disassembly, subcellular localization, and transport of the virus via the formation of macromolecular protein complexes, such as the viral replicase complex. Recent studies have indicated that HSP inhibitors can inhibit viral replication by interfering with the interaction of the virus with the HSP. In this review, we describe the function and classification of HSPs, the transcriptional mechanism of HSPs promoted by heat shock factors (HSFs), discuss the interaction between HSPs and viruses, and the mode of action of HSP inhibitors at two aspects of inhibiting the expression of HSPs and targeting the HSPs, and elaborate their potential use as antiviral agents.

Integrated Transcriptome and Proteome Analysis Reveals that the Antimicrobial Griseofulvin Targets Didymella segeticola Beta-Tubulin to Control Tea Leaf Spot.

Because effective control measures are lacking, tea leaf spot caused by Didymella segeticola results in huge tea (Camellia sinensis) production losses on tea plantations in Guizhou Province, southwestern China. Screening for natural antimicrobial agents with higher control effects against this pathogen and studying their modes of action may contribute to disease management. Here, Penicillium griseofulvum-derived antimicrobial griseofulvin (GSF) can inhibit the hyphal growth of D. segeticola strain GZSQ-4, with a half-maximal effective concentration of 0.37 µg/ml in vitro and a higher curative efficacy at a lower dose of 25 µg/ml for detached tea twigs. GSF induces deformed and slightly curly hyphae with enlarged ends, with protoplasts agglutinated in the hyphae, and higher numbers of hyphal protuberances. GSF alters hyphal morphology and the subcellular structure's order. The integrated transcriptome and proteome data revealed that the transport of materials in cells, cellular movement, and mitosis were modulated by GSF. Molecular docking indicated that beta-tubulin was the most potent target of GSF, with a binding free energy of -13.59 kcal/mol, and microscale thermophoresis indicated that the dissociation constant (Kd) value of GSF binding to beta-tubulin 1, compared with beta-tubulin 2, was significantly lower. Thus, GSF potentially targets beta-tubulin 1 to disturb the chromosomal separation and fungal mitosis, thereby inhibiting hyphal growth.

Anti-Fungal Activity of Moutan cortex Extracts against Rice Sheath Blight (Rhizoctonia solani) and Its Action on the Pathogen's Cell Membrane.

Rice sheath blight (RSB) caused by Rhizoctonia solani is one of the most destructive diseases of rice (Oryza sativa). Although chemical fungicides are the most important control methods, their long-term unreasonable application has brought about problems such as environmental pollution, food risks, and non-target poisoning. Therefore, considering the extraction of fungistatic substances from plants may be an alternative in the future. In this study, we found that the Moutan cortex ethanol extract has excellent antifungal activity against R. solani, with a 100% inhibition rate at 1000 µg/mL, which aroused our great exploration interest. In-depth exploration found that the antifungal active ingredients of M. cortex were mainly concentrated in the petroleum ether extract of the M. cortex ethanol extract, which still maintained a 100% inhibition rate with 250 µg/mL, and its effective medium concentration (EC50) was 145.33 µg/mL against R. solani. Through the measurement of extracellular relative conductivity and OD260, the petroleum ether extract induced leakage of intracellular electrolytes and nucleic acids, indicating that the cell membrane was ruined. Therefore, we preliminarily determined that the cell membrane may be the target of the petroleum ether extract. Moreover, we found that petroleum ether extract reduced the content of ergosterol, a component of the cell membrane, which may be one of the reasons for the cell membrane destruction. Furthermore, the increase of MDA content would lead to membrane lipid peroxidation, further aggravating membrane damage, resulting in increased membrane permeability. Also, the destruction of the cell membrane was observed by the phenomenon of the mycelium being transparent and broken. In conclusion, this is the first report of the M. cortex petroleum ether extract exhibiting excellent antifungal activity against R. solani. The effect of the M. cortex petroleum ether extract on R. solani may be on the cell membrane, inducing the disorder of intracellular substances and metabolism, which may be one of the antifungal mechanisms against R. solani.

First Report of Stagonosporopsis caricae Causing Chayote Leaf Spot in Guizhou Province, China.

At present, chayote (Sechium edule (Jacq.) Swartz) have been widely planted in Guizhou Province, southwestern China, and the cultivation area in Huishui county ranks first among all the counties or cities in Guizhou Province. Chayote leaf spot was firstly observed in Huishui County (25.99°N, 106.64°E) from April to June in 2019. The disease incidence ranged from 52% to 58%, and the severity of leaf symptoms ranged from 34 to 41% across nine chayote plantations. Such levels disease development lead to considerable enocomic losses. Leaf lesions initially occurred at the leaf margins, and the lesions expanded gradually, becoming dark brown and irregularly shaped. To identify the leaf spot-associated pathogen, the samples were cut from lesion margins, sterilized with 75% ethanol followed by 0.5% sodium hypochlorite for 30 s, rinsed with sterile water three times, and transferred onto potato dextrose agar (PDA). They were then incubated at 25°C in darkness for 5 days. The hyphal tips from the margins of growing colonies were successively transferred to fresh PDA plates for obtaining isolates. All strains grew with a similar morphology on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates, and the colonies presented smooth margins and abundant mycelia on all three media. The colonies were gray to light green on PDA and gray on MEA and OA at 5 days post-inoculation. At 11 days post-inoculation on 10% V8 medium at 25oC with a cycle of 14 h/ultraviolet light and 10 h/night, sexual morph was observed, ascomata pseudothecioid, subglobose, 121 × 142 µm, ostiolate, walls of brown textura angularis, and smooth. Asci were bitunicate, cylindrical to clavate, 7 × 90 µm, 8-spored, ascospores elliptical, straight to slightly curved, 5 × 17 µm, 1-septate, constricted at the septum, sub-hyaline, and smooth. Conidiomata were pycnidial, subglobose, 166 × 258 µm, ostiolate, wall of dark brown to black textura angularis, smooth. Conidia were short, cylindrical or slightly reniform, 6.18 ± 0.67 × 3.51 ± 0.33 µm (n = 50), 0-1 septate, hyaline, smooth. Chlamydospores were subhyaline to dark brown, verruculose or incidentally tuberculate, and solitary or in chains, and 14.16 ± 1.23 × 5.92 ± 0.49 µm (n = 50). The morphological characteristics of the strains were identical to those of Stagonosporopsis caricae (Aveskamp et al. 2010; Sivanesan 1990). The genes or DNA sequences of the partial 28S large subunit rDNA, the internal transcribed spacer, RNA polymerase II second largest subunit, and beta-tubulin were amplified (Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester, 1990; White et al. 1990; Woudenberg et al. 2009). The sequences were further deposited in GenBank (ITS: MZ619042-MZ619044, LSU: MZ620651-MZ620653, RPB2: MZ673652-MZ673654, and TUB: MZ673649-MZ673651). A phylogenetic analysis confirmed these strains to be identical to S. caricae reference strains CBS 248.90, CBS 282.76, and PD 06/03082531. Pathogenicity tests were performed on potted chayote and five-year-old chayote in the field. Mycelial plugs (6 mm diameter) were applied on wounded chayote leaves. Brown spots appeared on the wounded sites of chayote leaves after inoculation with mycelial plugs. No symptoms were observed on the leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the representative strains used for inoculation. To our knowledge, this is the first report of S. caricae causing leaf spot on chayote in China, and our findings will be useful for its management and further research.

Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold Potentially Targeting Fungal Succinate Dehydrogenase: Antifungal Activity and Mechanism of Action.

Succinate dehydrogenase (SDH) is known as an ideal target for the development of novel fungicides. Over the years, a series of novel pyrazole carboxamides containing a diarylamine scaffold have been reported as potent SDH inhibitors (SDHIs) in our laboratory. Among them, compound SCU3038 (EC50 = 0.016 mg/L) against in vitro Rhizoctonia solani was better than fluxapyroxad (EC50 = 0.033 mg/L). However, its mechanism of action is still unclear. In this paper, in pot tests, bioactivity evaluation indicated that in vivo antifungal activity of compound SCU3038 (EC50 = 0.95 mg/L) against R. solani was better than that of fluxapyroxad (EC50 = 2.29 mg/L) and thifluzamide (EC50 = 1.88 mg/L). In field trials, control efficacy of compound SCU3038 (74.10%) at 200 g ai/ha against rice sheath blight was better than that of thifluzamide (71.40%). Furthermore, target evaluation showed that compound SCU3038 could inhibit the fungal SDH from R. solani and fix in the binding site of SDH by molecular docking, thereby it could dissolve and reduce mitochondria of R. solani as observed by electron microscopy. In addition, transcriptome results showed that compound SCU3038 affected the TCA cycle pathway in mitochondria, and this was manifested in the downregulation of eight genes and upregulation of one gene. The most important phenomenon was the repressed expression of SDH2 confirmed by qRT-PCR. It was observed that compound SCU3038 was a potent SDHI, and these results afforded further research on pyrazole carboxamides.

Identification of APC Mutation as a Potential Predictor for Immunotherapy in Colorectal Cancer.

To date, anticancer immunotherapy has presented some clinical benefits to most of advanced mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer (CRC) patients. In addition to MSI status, we aimed to reveal the potential predictive value of adenomatous polyposis coli (APC) gene mutations in CRC patients. A total of 238 Chinese CRC patients was retrospectively identified and analyzed for clinical features and gene alternations in APC-mutant type (MT) and APC-wild-type (WT) groups. Clinical responses were then evaluated from the public TCGA database and MSKCC immunotherapy database. Although programmed cell death ligand 1 (PD-L1) level, MSI status, loss of heterogeneity at the human leukocyte antigen (HLA LOH), and tumor neoantigen burden (TNB) level were not statistically different between the APC-MT group and APC-WT group, tumor mutation burden (TMB) level was significantly higher in APC-MT patients (P < 0.05). Furthermore, comutation analysis for APC mutations revealed co-occurring genomic alterations of PCDHB7 and exclusive mutations of CTNNB1, BRAF, AFF3, and SNX25 (P < 0.05). Besides, overall survival from MSKCC-CRC cohort was longer in the APC-WT group than in the APC-MT group (HR 2.26 (95% CI 1.05-4.88), P < 0.05). Furthermore, most of patients in the APC-WT group were detected as high-grade immune subtypes (C2-C4) comparing with those in the APC-MT group. In addition, the percentages of NK T cells, Treg cells, and fibroblasts cells were higher in APC-WT patients than in APC-MT patients (P < 0.05). In summary, APC mutations might be associated with poor outcomes for immunotherapy in CRC patients regardless of MSI status. This study suggested APC gene mutations might be a potential predictor for immunotherapy in CRC.

Genomic correlates of programmed cell death ligand 1 (PD-L1) expression in Chinese lung adenocarcinoma patients.

BACKGROUND: Although PD-L1 expression is a crucial predictive biomarker for immunotherapy, it can be influenced by many factors. METHODS: A total of 248 Chinese patients with lung adenocarcinoma was retrospectively identified. Data for clinical features, gene alternations, signaling pathways and immune signatures was analyzed among negative expression group (TPS < 1%, n = 124), intermediate expression group (1% ≤ TPS < 50%, n = 93), and high expression group (TPS ≥ 50%, n = 38). Clinical outcomes among different expression groups were also evaluated from public database. RESULTS: Firstly, high tumor mutation burden was significantly associated with high PD-L1 expression in these Chinese patients with lung adenocarcinoma. In addition, gene alternations including TP53, PRKDC, KMT2D, TET1 and SETD2 apparently occurred in high PD-L1 expression group. Moreover, pathway analysis showed that mutations involving in DDR pathway, TP53 pathway, cell-cycle pathway and NOTCH pathway were obviously varied among three PD-L1 expression groups. Besides, most of patients in high PD-L1 expression group from TCGA database were determined as high-grade immune subtypes (C2-C4), showing significant higher proportions of IFN-gamma, CD8+ T-cells, NK cells, NK CD56 dim cells, Th1 cells, Th2 cells (P < 0.0001). Moreover, SETD2 mutation slightly correlated with overall survival from MSKCC cohort (HR 1.92 [95%CI 0.90-4.10], P = 0.085), and the percentage of IFN-gamma was significantly higher in SETD2 mutant group than in wild-type group (P < 0.01). CONCLUSIONS: This study illustrated in-depth genomic correlates of PD-L1 expression in Chinese lung adenocarcinoma patients and relevant immune signatures from public database, which might interpret more potential molecular mechanisms for immunotherapy in NSCLC.

The efficacies and biomarker investigations of antiprogrammed death-1 (anti-PD-1)-based therapies for metastatic bone and soft tissue sarcoma.

OBJECTIVE: Sarcomas are a group of rare malignancies with various subtypes. Patients with metastatic sarcoma who have failed traditional treatments can possibly achieve better prognoses from using novel therapies, including anti-programmed death-1 (PD-1)-based therapies. METHODS: We retrospectively analyzed clinical data of 24 metastatic sarcoma patients from June 15, 2016 to December 30, 2019. These patients mainly received angiogenesis inhibitors combined with anti-PD-1 therapy after they became resistant to traditional treatments. Furthermore, 8 patients underwent panel DNA and whole transcript sequencing. RESULTS: Six patients received 2 cycles of anti-PD-1 therapy and were included in the safety evaluation only group. The median follow-up time was 5.77 months. The median progression-free survival was 7.59 months, the overall response rate was 16.7% and the disease control rate was 55.6%. Based on whole exome and transcript sequencing data, there was no association between TMB, TNB, MSI, HLA-LOH, and PD-L1 expressions and sarcoma types with clinical responses. Immunotherapy efficacy and bioinformatics analyses indicated higher intratumoral heterogeneity (ITH) in progressive disease (PD) patients and lower ITH in partial response (PR) and stable disease patients. A higher percentage of immune cell infiltration, especially monocytes, was observed in PR patients. Active stromal gene expression was increased in PD patients but decreased in PR patients. Enrichment analysis revealed that an increased TGF-ß signaling pathway was reversely correlated with anti-PD-1 efficacy, while a decreased inflammatory response signaling pathway was positively correlated with anti-PD-1 efficacy. CONCLUSIONS: Our study showed PD-1 inhibitors combined with anti-angiogenesis agents were effective and well-tolerated. ITH, monocyte ratio, stroma subtypes, and the status of immune-associated signaling pathways may be related with anti-PD-1 based therapy.

Durable Response to Immunotherapy With Antiangiogenic Drug in Large-Cell Lung Carcinoma With Multiple Fulminant Postoperative Metastases: A Case Report.

Immunotherapy alone or chemo-immunotherapy has recently been recommended for treating advanced lung carcinoma in patients without driver mutations. However, the efficacy of immunotherapy and molecular mechanism in large-cell lung cancer (LCLC) remains unclear. Here, we reported a rare case of multiple fulminant postoperative body and mouth metastases in LCLC treating with combination immunotherapy. Initially, the patient was diagnosed as early stage LCLC and underwent a radical resection of the right lower lobe. Just one month later, multiple fulminant body and mouth lesions appeared in the right upper arm, right elbow, right waist, and tongue root. Meanwhile, serum neuron specific enolase (NSE) concentration dramatically increased from 12.12 to 30.14 ng/ml. Immumohistochemistry findings demonstrated moderate PD-L1 expressions with tumor proportion score (TPS), while next-generation sequencing indicated moderate tumor mutational burden (TMB) levels and gene mutations in PBRM1 L1230P and TP53 L194R of both foci. Besides, loss of heterozygosity (LOH) at human leukocyte antigen (HLA) class I (HLA-A*02:03, HLA-B*55:02 and HLA-C*12:03) were detected in the right upper arm metastasis, which may facilitate malignant postoperative metastases in this case. Notably, this patient received combination therapy with anti-PD-1 antibody sintilimab plus anlotinib, and achieved a partial response for at least 12 months. Using an integrated computational method, the mutant peptide TEIPENDIPL derived from PBRM1 L1230P was predicted to be a specific neoantigen and could still be presented by HLA-B*40:01. This case suggests that immunotherapy plus antiangiogenic drug may provide an alternative therapeutic option for advanced LCLC patients without common gene mutations.

Systematic analysis of molecular characterization and clinical relevance of m6A regulators in digestive system pan-cancers.

Digestive system tumors, which mainly include esophagus, stomach, colorectum, liver, pancreas, bile duct, and some other tumors, often have a poor prognosis. N6-methyladenosine (m6A) has critical functions in development and tumorigenesis and may help improve the molecular mechanisms of digestive system tumors. However, current understanding of the reconstitution of m6A in digestive system tumors is far from comprehensive. Herein, this study systematically analyzed multi-layered genomic characteristics and clinical relevance of m6A regulators in 1906 patients involving seven digestive system tumor types. We discovered that m6A regulators showed extensive genetic changes and highly consistent expression regulation. The m6A expression was closely related to the activity of cancer pathways. At the same time, we also identified m6A regulators significantly related to the common cancer pathways of digestive system tumors and specific cancer pathways of digestive tract and digestive glands. These cancer pathways may explain the prognostic differences of patients with digestive tract tumors. In addition, m6A regulators demonstrated strong potential in prognostic stratification and drug development, especially in multiple research cohorts on pancreatic cancer, pointing to a strong prognostic stratification capability of m6A regulators. Finally, a m6A scoring model significantly related to highly active ubiquitin-mediated proteolysis, mismatch repair, cell cycle, ebasal transcription factors was constructed and had a strong prognostic stratification ability in digestive gland tumors. The score showed a significant negative correlation with the tumor immune microenvironment. This study demonstrated that the similarities and difference of the action mechanism m6A regulators in the digestive tract and digestive gland tumor progression could guide potential drug development.

Unraveling the polypharmacology of a natural antifungal product, eugenol, against Rhizoctonia solani.

BACKGROUND: Rice sheath blight caused by Rhizoctonia solani is a devastating disease of rice in China. However, indiscriminate use of chemical fungicides applied to control the disease raise major environmental and food safety issues. Ecofriendly biocontrol alternatives are urgently needed. Eugenol, one of the main ingredients in Syzygium aromaticum, has attracted much attention owing to its antifungal properties. However, its mode of action is still not clear. Herein, the antifungal activity and mode of action of eugenol against R. solani were investigated. RESULTS: Results confirmed that the mycelia of R. solani treated with eugenol shrank and became dehydrated, the cytoplasmic wall separated, and the vacuoles and mitochondria decreased or dissolved. Moreover, we found that eugenol downregulated expression of C-4 methyl sterol oxidase, inhibited synthesis of ergosterol, increased membrane permeability and impaired the transportation of amino acids and glucose across the cell membrane. In addition, eugenol decreased the mitochondrial membrane potential and initiated an oxidative stress reaction by increasing reactive oxygen species and malondialdehyde, which together with membrane damage contribute to the antifungal activity of eugenol. Meanwhile, eugenol might inhibit R. solani by affecting oxidative phosphorylation and the tricarboxylic acid cycle (TCA cycle). CONCLUSION: In view of its multitarget properties against R. solani, eugenol provides an alternative approach to chemical control strategies against rice sheath blight. © 2021 Society of Chemical Industry.

Good Tumor Response to Chemoradioimmunotherapy in dMMR/MSI-H Advanced Colorectal Cancer: A Case Series.

Purpose: Immune checkpoint blockade has led to a significant improvement of patient survival in metastatic colorectal cancer (CRC) with DNA mismatch repair-deficiency (dMMR)/microsatellite instability-high (MSI-H). However, not all these patients are sensitive to monoimmunotherapy. We firstly presented a case series of advanced dMMR/MSI-H CRCs treating with PD-1 inhibitor-based chemoradioimmunotherapy (CRIT). Methods and Materials: We assessed the short-term efficacy and safety of CRIT in advanced dMMR/MSI-H CRCs, and also did next-generation sequencing (NGS) assays. Results: Our analysis included five advanced dMMR/MSI-H CRCs who have received toripalimab-based CRIT. Toripalimab was given 240mg every three weeks, and the radiation dose was 45-50 gray in 25 fractions. Chemotherapy regimens consisted of CAPOX in three patients, capecitabine in one patient, and mFOLFOX6 in one patient. Initially, two patients displayed complete response (CR), and three patients achieved partial response (PR) on imaging findings. Afterwards, one PR patient was confirmed pathological complete response after surgery, leading to three CR cases in total. Hematological toxicity was the most common adverse effect, and only two patients developed mild immune-related adverse effects besides. All the treatment-related adverse events were under control. Based on the NGS results, the median intratumor heterogeneity was 0.19 (range 0-0.957), which was less in CR patients than PR patients (P = 0.019). Genetic mutations at DNA damage repair genes and the JAK1 gene were also observed. Conclusions: For advanced dMMR/MSI-H CRC, anti-PD-1 based CRIT is effective and safe. Further studies are required to better clarify the potential role and mechanism of CRIT as a viable therapeutic strategy in this population.

Case Report: Low-Dose Decitabine Plus Anti-PD-1 Inhibitor Camrelizumab for Previously Treated Advanced Metastatic Non-Small Cell Lung Cancer.

Background: Although the programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have markedly changed the strategies of cancer treatment, most patients with advanced non-small cell lung cancer (NSCLC) do not respond to PD-1/PD-L1 monotherapy. Epigenetic drugs have been hypothesized to possess the potential to sensitize PD-1/PD-L1 inhibitors. Case Presentation: Three patients with advanced metastatic NSCLC failed to respond to first-line systemic therapy and had a low tumor mutation burden, low tumor neoantigen burden, low microsatellite instability, and HLA loss of heterozygosity according to their target lesion biopsies, all of which were considered unfavorable factors for PD-1/PD-L1 blockage. However, all three patients responded to low-dose decitabine, an epigenetic drug, in combination with camrelizumab (anti-PD-1 antibody), with only controllable adverse events, indicating that low-dose decitabine can sensitize PD-1/PD-L1 inhibitors. Summary: We report a novel therapy with low-dose decitabine plus camrelizumab for advanced NSCLC on the basis of successful treatment of three patients, emphasizing the potential of epigenetic drugs to regulate PD-1/PD-L1 inhibitors in advanced NSCLC.

Synthesis and Biological Evaluation of Novel Benodanil-Heterocyclic Carboxamide Hybrids as a Potential Succinate Dehydrogenase Inhibitors.

In order to discover new antifungal agents, twenty novel benodanil-heterocyclic carboxamide hybrids were designed, synthesized, and characterized by 1H NMR and HRMS. In vitro, their antifungal activities against four phytopathogenic fungi were evaluated, as well as some of the target compounds at 50 mg/L demonstrated significant antifungal activities against Rhizoctonia solani. Especially, compounds 17 (EC50 = 6.32 mg/L) and 18 (EC50 = 6.06 mg/L) exhibited good antifungal activities against R. solani and were superior to the lead fungicide benodanil (a succinate dehydrogenase inhibitor, SDHI) (EC50 = 6.38 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media with the addition of compound 17 grew abnormally as compared with the negative control with tenuous, wizened, and overlapping colonies, and compounds 17 (IC50 = 52.58 mg/L) and 18 (IC50 = 56.86 mg/L) showed better inhibition abilities against succinate dehydrogenase (SDH) than benodanil (IC50 = 62.02 mg/L). Molecular docking revealed that compound 17 fit in the gap composed of subunit B, C, and D of SDH. Furthermore, it was shown that the main interaction, one hydrogen bond interaction, was observed between compound 17 and the residue C/Trp-73. These studies suggested that compound 17 could act as a potential fungicide to be used for further optimization.

Mechanism of Action of Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold against Rhizoctonia solani.

In the last few decades, Rhizoctonia solani causing rice sheath blight has resulted in a lot of economic losses in the world. Therefore, many novel pyrazole carboxamide fungicides have been intensively researched and employed to fight against it. In this regard, in recent years, our group reported a novel pyrazole carboxamide containing a diarylamine scaffold with good antifungal activity against rice sheath blight in the pot test and field trial. Following this project, the antifungal mechanism of action of the pyrazole carboxamide has been elucidated in this work. The antifungal result showed that compound SCU2028, N-[2-[(3-chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was equivalent to the commercial fungicide thifluzamide and its EC50 value was 0.022 mg/L against R. solani. Also, the observation results by scanning electron microscopy and transmission electron microscopy showed that it could destroy the fungus' cell walls or membranes and result in the leakage of contents and increase of the number of mitochondria and abnormal morphology. Meanwhile, the result on the mitochondrial membrane potential (MMP) showed that it could decrease R. solani's MMP. Furthermore, the results by label-free quantitative proteomic analysis showed that 1153 proteins were found after R. solani was treated with compound SCU2028, including 212 proteins in the control group and 257 proteins in the treatment group. A total of 142 differential proteins were obtained, of which 92 proteins were upregulated and 50 proteins were downregulated. The differentially expressed proteins affected a series of physiological and biochemical pathways in the mitochondria, endoplasmic reticulum, ribosome, and other related GO and KEGG pathways. In particular, the inhibition of the respiratory chain caused by the TCA cycle and oxidative phosphorylation KEGG pathway indicated that complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase) might be compound SCU2028's main action targets. In addition, multiple experiments of qRT-PCR, enzyme activity detection, and molecular docking confirmed complex II and complex IV as targets. It could be seen that these findings provided a theoretical support for further research and development of the pyrazole carboxamide fungicides.

Efficacy and biomarker analysis of nivolumab plus gemcitabine and cisplatin in patients with unresectable or metastatic biliary tract cancers: results from a phase II study.

BACKGROUND: The prognosis of patients with unresectable or metastatic biliary tract cancer (BTC) is unacceptably low. This study aimed to determine the efficacy, safety and predictive biomarkers of the immune checkpoint inhibitor nivolumab in combination with chemotherapy in advanced BTCs. METHODS: In this open-label, single-arm, phase II trial, a chemotherapy and immunotherapy combination consisting of gemcitabine 1000 mg/m2, cisplatin 75 mg/m2 and nivolumab 3 mg/kg was administered every 3 weeks for up to six cycles. Maintenance treatment with gemcitabine plus nivolumab was administered to patients achieving disease control following the combination therapy. The primary outcome was the objective response rate. Secondary outcomes included safety, disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). The exploratory objective was to assess biomarkers for predicting clinical response and prognosis. RESULTS: Thirty-two patients with a median age of 60 (range 27-69) years were enrolled. As of September 31, 2019, the median follow-up was 12.8 (95% CI 10.8 to 14.8) months. Twenty-seven response-evaluable patients received a median of 4 (IQR, 3-6) cycles of combination therapy, of whom 15 (55.6%) patients achieved an objective response, including 5 (18.6%) with a complete response (CR), and the DCR was 92.6%. Of the six patients in cohort A who were resistant to gemcitabine-based or cisplatin-based chemotherapy, one achieved CR and one achieved partial response. Thirteen of 21 chemotherapy-naive patients (61.9%) in cohort B achieved an objective response. The median PFS of all patients in cohorts A+B was 6.1 months. The median OS was 8.5 months, with a 33.3% 12-month OS rate. The most frequent grade 3 or higher adverse events were thrombocytopenia (56%) and neutropenia (22%). Fitness might be a biomarker for predicting clinical response. On-therapy changes in serum soluble FasL, MCP-1 and interferon-γ were correlated with prognosis. CONCLUSIONS: Nivolumab in combination with gemcitabine and cisplatin offers promising efficacy and a manageable safety profile for patients with advanced BTCs. TRIAL REGISTRATION NUMBER: NCT03311789.