Spatial distribution and seasonal variation of trace hazardous elements contamination in the coastal environment.

Groundwater is the second largest water source for daily consumption, only next to surface water resources. Groundwater has been extensively investigated for its pollution level in urban areas. The groundwater quality assessments in industrial areas associated with every urban landscape are still lacking. In order to examine the spatial distribution characteristics, pollution levels, and sources of trace metals in the densely populated Chennai coastal region of Tamilnadu, India, physicochemical parameters and trace element concentrations have been determined in groundwater. 55 groundwater samples from Tamil Nadu's coastal region were collected and analyzed for physicochemical parameters such as pH, (EC), (TDS), and (TH) during the pre-monsoon (June 2015) and post-monsoon (January 2016) seasons. We used trace elements and analyzed them in this study (Mg, Zn, Pb, Ni, Co, Cu, Cr, and Fe). Furthermore, anthropogenic input from industries and power plants exacerbates the pollution of Ni, Mg, Fe, and Mn. Due to evaporites and anthropogenic input, samples with excessive salinity, total hardness, and water quality are considered unsuitable for irrigation or drinking. The results demonstrated that seasonal, geogenic, and anthropogenic influences all have a significant impact on the heterogeneous chemistry of groundwater.

Study and application of coupling relationship between water-salt-resistivity-soil structure in saline soils in arid and semi-arid areas of northwest China.

Widespread saline soils in Northwest China pose a serious threat to the region's ability to use infrastructure safely because they are prone to soil structure damage when subjected to external environmental fluctuations, which in turn affects the stability of the foundations for buildings. The non-destructive approach of measuring resistivity can be used to swiftly reflect the subsoil body's state and make assumptions about its safety. However, the electrical resistivity of the underground soil body can be used to quickly identify unstable areas because the resistivity is influenced by the water content, salt content, and structural characteristics of the soil body. To do this, it is necessary to understand the coupling relationship between various factors. In this study, we first constructed samples with various water, salt, and soil structure characteristics, and then used indoor tests, such as soil resistivity measurement and thermogravimetric analysis, to analyze the multiple factors affecting the resistivity characteristics of the soil. The relationship between soil resistivity and actual saline soil diseases in Northwest China was then further discussed in conjunction with the results of the indoor tests and analyses. subsequently, the resistivity and soil properties have been measured in the field at specific locations in Northwest China where railway roadbeds are diseased. The study's findings can theoretically support a deeper comprehension of the law and mechanism of soil resistivity change, as well as provide assistance for building infrastructure in Northwest China.

Humanized mouse models for inherited thrombocytopenia studies.

Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal platelet function, which can subsequently lead to impaired hemostasis. In the past decades, humanized mouse models (HMMs), that are mice engrafted with human cells or genes, have been widely used in different research areas including immunology, oncology, and virology. With advances of the development of immunodeficient mice, the engraftment, and reconstitution of functional human platelets in HMM permit studies of occurrence and development of platelet disorders including IT and treatment strategies. This article mainly reviews the development of humanized mice models, the construction methods, research status, and problems of using humanized mice for the in vivo study of human thrombopoiesis.

Humanized mouse models (HMMs) refer to immunodeficient mice that have been used for the investigation of human hematopoiesis and immunity for years. With engrafted human hematopoietic stem cells (HSCs), the differentiation process of HSCs and re-construction of platelets can be monitored in the mice. Until now, several strains of HMMs have been used in the studies of inherited thrombocytopenia (IT), a genetic disorder associated with low platelet count in the blood. In this study, we reviewed the development of these HMMs in IT studies, compared the different sources of HSCs transplanted into HMMs and summarize the strategies of HSC transplantation in HMMs. The Kit^−/− immunodeficient mice showed effectively long-term and stable implantation of human HSC without irradiation and higher implantation levels, and they also support multilinear differentiation of human HSC, such as platelets and red blood cells. The source and count of HSCs and the transplantation strategy may also impact the result. This study provides a basis information for HMMs used in IT and will help other investigators in this field choosing the right research plan.

Bioactivity and Metabolome Mining of Deep-Sea Sediment-Derived Microorganisms Reveal New Hybrid PKS-NRPS Macrolactone from Aspergillus versicolor PS108-62.

Despite low temperatures, poor nutrient levels and high pressure, microorganisms thrive in deep-sea environments of polar regions. The adaptability to such extreme environments renders deep-sea microorganisms an encouraging source of novel, bioactive secondary metabolites. In this study, we isolated 77 microorganisms collected by a remotely operated vehicle from the seafloor in the Fram Strait, Arctic Ocean (depth of 2454 m). Thirty-two bacteria and six fungal strains that represented the phylogenetic diversity of the isolates were cultured using an One-Strain-Many-Compounds (OSMAC) approach. The crude EtOAc extracts were tested for antimicrobial and anticancer activities. While antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium was common for many isolates, only two bacteria displayed anticancer activity, and two fungi inhibited the pathogenic yeast Candida albicans. Due to bioactivity against C. albicans and rich chemical diversity based on molecular network-based untargeted metabolomics, Aspergillus versicolor PS108-62 was selected for an in-depth chemical investigation. A chemical work-up of the SPE-fractions of its dichloromethane subextract led to the isolation of a new PKS-NRPS hybrid macrolactone, versicolide A (1), a new quinazoline (-)-isoversicomide A (3), as well as three known compounds, burnettramic acid A (2), cyclopenol (4) and cyclopenin (5). Their structures were elucidated by a combination of HRMS, NMR, [α]D, FT-IR spectroscopy and computational approaches. Due to the low amounts obtained, only compounds 2 and 4 could be tested for bioactivity, with 2 inhibiting the growth of C. albicans (IC50 7.2 µg/mL). These findings highlight, on the one hand, the vast potential of the genus Aspergillus to produce novel chemistry, particularly from underexplored ecological niches such as the Arctic deep sea, and on the other, the importance of untargeted metabolomics for selection of marine extracts for downstream chemical investigations.

Potential biomarkers for inherited thrombocytopenia 2 identified by plasma proteomics.

Inherited thrombocytopenia 2 (THC2) is difficult to diagnose due to the lack of specific clinical characteristics and diagnostic methods. To identify potential plasma protein biomarkers for THC2, we collected the plasma samples from a THC2 family (9 THC2 and 15 non-THC2 members), enriched the medium and low abundant proteins using Proteominer and analyzed the protein profiles using the liquid chromatography-mass spectrometry in data independent acquisition mode. Initially, we detected 784 proteins in the plasma samples of this family and identified 27 up-regulated and 36 down-regulated in the THC2 group compared to the non-THC2 group (|log2 ratio| >1 and p-value <0.05). To improve the predictive power, top eight dysregulated proteins (B7Z2B4, LTF, HP, ERN1, IGHV1-8, A0A0X9V9C4, VH6DJ, and D3JV41) were selected by an area under the curve-based random forest process to construct a clinical model. Multivariate analysis with random forest and support vector machine showed that the prediction model provided high discrimination ability for THC2 diagnosis (AUC: 1.000 and 0.967, respectively). The potential plasma protein biomarkers will be tested in more THC2 patients and other thrombocytopenia patients to further validate their specificity and sensitivity.

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Femtosecond laser semi-assisted Descemet stripping endothelial keratoplasty: 2-year outcomes of endothelial cell loss and graft survival.

PURPOSE: To assess 2-year endothelial cell loss and graft survival after femtosecond laser semi-assisted Descemet stripping endothelial keratoplasty (FLS-DSEK). METHODS: In this prospective and noncomparative study carried out at Eye Hospital of Shandong First Medical University, 85 eyes (84 patients) with endothelial dysfunction receiving FLS-DSEK (n=62, 75.9%) or FLS-DSEK combined with phacoemulsification cataract surgery and intraocular lens implantation (n=23, 27.1%) from 2013 through 2016 were included. The graft endothelial cell loss, endothelial graft thickness, visual acuity, and complications after surgery were evaluated. RESULTS: Thin endothelial grafts were all successfully prepared, with no occurrence of perforation. The rate of endothelial cell loss was 17.4%, 18.8%, 19.9%, and 26.7%, and the central graft thickness was 113±54 µm, 102±40 µm, 101±28 µm, and 96±23 µm at 3, 6, 12, and 24 months, respectively. The median best-corrected visual acuity was 0.4 logMAR (range, 0-2 logMAR) at 24 months, demonstrating a significant difference from that before surgery (2 logMAR; range, 0.2-3 logMAR) (T=187.5, P<.001). Partial graft dislocation was the most common postoperative complication, with an occurrence rate of 14% (n=12), and it was associated with an abnormal iris-lens diaphragm (r=.35, P<.001). The other complications included a high intraocular pressure (n=5, 6%), endothelial graft rejection (n=4, 5%), and pupillary block (n=1, 1%). Endothelial graft decompensation occurred in the two eyes, and 98% (n=83) of the grafts survived at 24 months. CONCLUSIONS: Data of the study suggest that the treatment using FLS-DSEK seems to be promising and might be considered a feasible choice in patients with endothelial dysfunction. TRIAL REGISTRATION: 1. Date of registration: 2021-02-18 2. TRIAL REGISTRATION NUMBER: ChiCTR2100044091 3. Registration site: https://www.chictr.org.cn/.

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi.

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A-C (1-3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher's ester method were used in the structure elucidation of compounds 5 and 6. The 3'R,5'R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3'S,5'R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 µM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

CRIF1 as a potential target to improve the radiosensitivity of osteosarcoma.

Resistance to ionizing radiation (IR), which is a conventional treatment for osteosarcoma that cannot be resected, undermines the efficacy of this therapy. However, the mechanism by which IR induces radioresistance in osteosarcoma is not defined. Here, we report that CR6-interacting factor-1 (CRIF1) is highly expressed in osteosarcoma and undergoes nuclear-cytoplasmic shuttling of cyclin-dependent kinase 2 (CDK2) after IR. Osteosarcoma cells lacking CRIF1 show increased sensitivity to IR, which is associated with delayed DNA damage repair, inactivated G1/S checkpoint, and mitochondrial dysfunction. CRIF1 interacts with the DNA damage checkpoint regulator CDK2, and CRIF1 and CDK2 colocalize in the nucleus after IR. Nuclear localization of CDK2 is associated with phosphorylation changes that promote DNA repair and activation of the G1/S checkpoint. CRIF1 knockdown synergized with IR in an in vivo osteosarcoma model, leading to tumor regression. Based on these findings, we identify CRIF1 as a potential therapeutic target in osteosarcoma that can increase the efficacy of radiotherapy. More broadly, our findings may provide insights into the mechanism for other types of radioresistant cancers and be exploited for therapeutic ends.

Chemistry, Chemotaxonomy and Biological Activity of the Latrunculid Sponges (Order Poecilosclerida, Family Latrunculiidae).

Marine sponges are exceptionally prolific sources of natural products for the discovery and development of new drugs. Until now, sponges have contributed around 30% of all natural metabolites isolated from the marine environment. Family Latrunculiidae Topsent, 1922 (class Demospongiae Sollas, 1885, order Poecilosclerida Topsent, 1928) is a small sponge family comprising seven genera. Latrunculid sponges are recognized as the major reservoirs of diverse types of pyrroloiminoquinone-type alkaloids, with a myriad of biological activities, in particular, cytotoxicity, fuelling their exploration for anticancer drug discovery. Almost 100 pyrroloiminoquinone alkaloids and their structurally related compounds have been reported from the family Latrunculiidae. The systematics of latrunculid sponges has had a complex history, however it is now well understood. The pyrroloiminoquinone alkaloids have provided important chemotaxonomic characters for this sponge family. Latrunculid sponges have been reported to contain other types of metabolites, such as peptides (callipeltins), norditerpenes and norsesterpenes (trunculins) and macrolides (latrunculins), however, the sponges containing latrunculins and trunculins have been transferred to other sponge families. This review highlights a comprehensive literature survey spanning from the first chemical investigation of a New Zealand Latrunculia sp. in 1986 until August 2020, focusing on the chemical diversity and biological activities of secondary metabolites reported from the family Latrunculiidae. The biosynthetic (microbial) origin and the taxonomic significance of pyrroloiminoquinone related alkaloids are also discussed.

Molecular Networking-Guided Isolation of New Etzionin-Type Diketopiperazine Hydroxamates from the Persian Gulf Sponge Cliona celata.

The Persian Gulf is a unique and biologically diverse marine environment dominated by invertebrates. In continuation of our research interest in the chemistry and biological activity of marine sponges from the Persian Gulf, we selected the excavating sponge Cliona celata for detailed metabolome analyses, in vitro bioactivity screening, and chemical isolation studies. A UPLC-MS/MS (MS2) molecular-networking-based dereplication strategy allowed annotation and structural prediction of various diketopiperazines (DKPs) and etzionin-type diketopiperazine hydroxamates (DKPHs) in the crude sponge extract. The molecular-networking-guided isolation approach applied to the crude extract afforded the DKPH etzionin (1) and its two new derivatives, clioetzionin A (2) and clioetzionin B (3). Another new modified DKP (4) was identified by MS/MS analyses but could not be isolated in sufficient quantities to confirm its structure. The chemical characterization of the purified DKPHs 1-3 was performed by a combination of 1D and 2D NMR spectroscopy, HRMS, HRMS/MS, and [α]D analyses. Compounds 1 and 2 exhibited broad antibacterial, antifungal, and anticancer activities, with IC50 values ranging from 19.6 to 159.1 µM. This is the first study investigating the chemical constituents of a C. celata specimen from the Persian Gulf. It is also the first report of full spectroscopic data of etzionin based on extensive spectroscopic analyses.

Detection and Biological Characteristics of Alternaria alternata Resistant to Difenoconazole from Paris polyphylla var. chinensis, an Indigenous Medicinal Herb.

Black spot caused by Alternaria alternata (BSAA) is one of the most common diseases of Paris polyphylla var. chinensis, causing yield losses in China. Demethylation inhibitors (DMIs) have been used to control this disease in China for decades. Some farmers have complained about the decreased efficacy of DMIs against BSAA. The objective of this study was to detect and characterize the resistance of A. alternata against difenoconazole from P. polyphylla var. chinensis during 2018. Of the 22 isolates of A. alternata obtained from Sichuan Province in the southwest of China, 20 were resistant to difenoconazole. Mycelial growth rates and sporulation of the difenoconazole-resistant (DfnR) isolates were not different from those of the difenoconazole-sensitive (DfnS) isolates. No cross resistance between difenoconazole and tebuconazole or propiconazole was observed. Mutations were identified at gene AaCYP51 of DfnR isolates based on the sequence alignment of the DfnR and DfnS isolates. All of the mutations could be divided into three resistant genotypes, I (K715R + Y781C), II (K715R + D1140G + T1628A), and III (no mutation). The docking total score of the DfnS isolates was 5.6020, higher than the resistant genotype I (4.4599) or the resistant genotype II (3.8651), suggesting that the DMI resistance of A. alternata may be caused by the decreased affinity between AaCYP51 and difenoconazole.

Detection and Fitness of Dicarboximide-Resistant Isolates of Alternaria alternata from Dendrobium officinale, a Chinese Indigenous Medicinal Herb.

Black spot, caused by Alternaria alternata, poses a severe threat to the industry of Dendrobium officinale, a Chinese indigenous medicinal herb. Dicarboximide fungicides (DCFs) have been intensively used to control this disease for decades in China, and offer excellent efficacy. The resistance of phytopathogenic pathogens against DCFs are reportedly selected in fields; however, the DCF resistance of A. alternata from D. officinale is not well understood. The isolates of A. alternata with low procymidone resistance (ProLR) were detected in the commercial orchards of D. officinale in China in 2018 and biochemically characterized in this study. The result showed that the ProLR isolates were selected in the commercial orchards with a resistance frequency of 100%, and no significant difference in mycelial growth, sporulation, and virulence was observed among the ProLR and procymidone-sensitive (ProS) isolates. A positive cross-resistance pattern was exhibited between procymidone and iprodione. Results of amino acid sequence alignment of AaOS-1 from the tested isolates showed that all of the ProLR genotypes could be categorized into two groups, including group I (mutations at AaOs-1) and group II (no mutation). Under procymidone (5.0 µg/ml) treatment conditions, the AaOs-1 expression levels increased in the ProS isolates and ranged from approximately 2.94- to 3.69-fold higher than those under procymidone-free conditions, while the AaOs-1 expressions of the ProLR isolates were significantly lower than those in the ProS isolates under the same conditions. The data indicated that the mutations at AaOs-1 are involved in the DCF resistance of A. alternata selected in the D. officinale orchards.

Tridiscorhabdin and Didiscorhabdin, the First Discorhabdin Oligomers Linked with a Direct C-N Bridge from the Sponge Latrunculia biformis Collected from the Deep Sea in Antarctica.

Guided by LC-MS/MS molecular networking-based metabolomics and cytotoxic activity, two new discorhabdin-type alkaloids, tridiscorhabdin (1) and didiscorhabdin (2), were isolated from the sponge Latrunculia biformis, collected from the Weddell Sea (Antarctica) at -291 m depth. Their structures were established by HRESIMS, NMR, [α]D, and ECD data coupled with DFT calculations. Both compounds bear a novel C-N bridge (C-1/N-13) between discorhabdin monomers, and 1 represents the first trimeric discorhabdin molecule isolated from Nature. Tridiscorhabdin (1) exhibited strong cytotoxic activity against the human colon cancer cell line HCT-116 (IC50 value 0.31 µM).

New Discorhabdin B Dimers with Anticancer Activity from the Antarctic Deep-Sea Sponge Latrunculia biformis.

Latrunculia sponges represent a rich source of discorhabdin-type pyrroloiminoquinone alkaloids, a few of which comprise a dimeric structure. The anticancer-activity-guided isolation of the n-hexane subextract of the Antarctic deep-sea sponge Latrunculia biformis yielded the known compound (-)-(1R,2R,6R,8S,6'S)-discorhabdin B dimer (1) and two new derivatives, (-)-(1S,2R,6R,8S,6'S)-discorhabdin B dimer (2) and (-)-(1R,2R,6R,8S,6'S)-16',17'-dehydrodiscorhabdin B dimer (3). The chemical structures of compounds 1-3 were elucidated by means of HR-ESIMS, NMR, [], ECD spectroscopy, and a comparison with the previously reported discorhabdin analogs. Compounds 1 and 2 showed significant in vitro anticancer activity against the human colon cancer cell line (HCT-116), with IC50 values of 0.16 and 2.01 µM, respectively. Compared to monomeric discorhabdins, dimeric discorhabdins are very rare in Nature. This study adds two new discorhabdin dimers (2 and 3) to this small pyrroloiminoquinone subfamily. This is also the first report of compound 1 as a natural product and the first assessment of its in vitro anticancer activity.

Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea.

The geographic position, highly fluctuating sea temperatures and hypersalinity make Persian Gulf an extreme environment. Although this unique environment has high biodiversity dominated by invertebrates, its potential in marine biodiscovery has largely remained untapped. Herein, we aimed at a detailed analysis of the metabolome and bioactivity profiles of the marine sponge Axinella sinoxea collected from the northeast coast of the Persian Gulf in Iran. The crude extract and its Kupchan subextracts were tested in multiple in-house bioassays, and the crude extract and its CHCl3-soluble portion showed in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium (Efm). A molecular networking (MN)-based dereplication strategy by UPLC-MS/MS revealed the presence of phospholipids and steroids, while 1H NMR spectroscopy indicated the presence of additional metabolites, such as diketopiperazines (DKPs). Integrated MN and 1H NMR analyses on both the crude and CHCl3 extracts combined with an antibacterial activity-guided isolation approach afforded eight metabolites: a new diketopiperazine, (-)-cyclo(L-trans-Hyp-L-Ile) (8); a known diketopiperazine, cyclo(L-trans-Hyp-L-Phe) (7); two known phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphocholine (1) and 1-O-octadecanoyl-sn-glycero-3-phosphocholine (2); two known steroids, 3ß-hydroxycholest-5-ene-7,24-dione (3) and (22E)-3ß-hydroxycholesta-5,22-diene-7,24-dione (4); two known monoterpenes, loliolide (5) and 5-epi-loliolide (6). The chemical structures of the isolates were elucidated by a combination of NMR spectroscopy, HRMS and [α]D analyses. All compounds were tested against MRSA and Efm, and compound 3 showed moderate antibacterial activity against MRSA (IC50 value 70 µg/mL). This is the first study that has dealt with chemical and bioactivity profiling of A. sinoxea leading to isolation and characterization of pure sponge metabolites.

Pyrenosetins A-C, New Decalinoylspirotetramic Acid Derivatives Isolated by Bioactivity-Based Molecular Networking from the Seaweed-Derived Fungus Pyrenochaetopsis sp. FVE-001.

Marine algae represent a prolific source of filamentous fungi for bioprospecting. In continuation of our search for new anticancer leads from fungi derived from the brown alga Fucus vesiculosus, an endophytic Pyrenochaetopsis sp. FVE-001 was selected for an in-depth chemical analysis. The crude fungal extract inhibited several cancer cell lines in vitro, and the highest anticancer activity was tracked to its CHCl3-soluble portion. A bioactivity-based molecular networking approach was applied to C18-SPE fractions of the CHCl3 subextract to predict the bioactivity scores of metabolites in the fractions and to aid targeted purification of anticancer metabolites. This approach led to a rapid isolation of three new decalinoylspirotetramic acid derivatives, pyrenosetins A-C (1-3) and the known decalin tetramic acid phomasetin (4). The structures of the compounds were elucidated by extensive NMR, HR-ESIMS, FT-IR spectroscopy, [α]D and Mosher's ester method. Compounds 1 and 2 showed high anticancer activity against malignant melanoma cell line A-375 (IC50 values 2.8 and 6.3 µM, respectively), in line with the bioactivity predictions. This is the first study focusing on secondary metabolites of a marine-derived Pyrenochaetopsis sp. and the second investigation performed on the member of the genus Pyrenochaetopsis.

Pyrenosetin D, a New Pentacyclic Decalinoyltetramic Acid Derivative from the Algicolous Fungus Pyrenochaetopsis sp. FVE-087.

The fungal genus Pyrenochaetopsis is commonly found in soil, terrestrial, and marine environments, however, has received little attention as a source of bioactive secondary metabolites so far. In a recent work, we reported the isolation and characterization of three new anticancer decalinoyltetramic acid derivatives, pyrenosetins A-C, from the Baltic Fucus vesiculosus-derived endophytic fungus Pyrenochaetopsis sp. FVE-001. Herein we report a new pentacyclic decalinoylspirotetramic acid derivative, pyrenosetin D (1), along with two known decalin derivatives wakodecalines A (2) and B (3) from another endophytic strain Pyrenochaetopsis FVE-087 isolated from the same seaweed and showed anticancer activity in initial screenings. The chemical structures of the purified compounds were elucidated by comprehensive analysis of HR-ESIMS, FT-IR, [a]D, 1D and 2D NMR data coupled with DFT calculations of NMR parameters and optical rotation. Compounds 1-3 were evaluated for their anticancer and toxic potentials against the human malignant melanoma cell line (A-375) and the non-cancerous keratinocyte cell line (HaCaT). Pyrenosetin D (1) showed toxicity towards both A-375 and HaCaT cells with IC50 values of 77.5 and 39.3 µM, respectively, while 2 and 3 were inactive. This is the third chemical study performed on the fungal genus Pyrenochaetopsis and the first report of a pentacyclic decalin ring system from the fungal genus Pyrenochaetopsis.

Mutations and Overexpression of CYP51 Associated with DMI-Resistance in Colletotrichum gloeosporioides from Chili.

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

Involvement of the cysteine protease BcAtg4 in development and virulence of Botrytis cinerea.

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.