First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022). From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of tea Puer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 µm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS and TEF) from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

Optimization of lipid extraction and analytical protocols for UHPLC-ESI-HRMS-based lipidomic analysis of adherent mammalian cancer cells.

Lipidomics, which reveals comprehensive characterization of molecular lipids, is a rapidly growing technology used in biomedical research. Lipid extraction is a critical step in lipidomic analysis. However, the effectiveness of different lipid extract solvent systems from cellular samples still remains unclear. In the current study, the protocol of reverse-phase liquid chromatography mass spectrometry (LC/MS)-based lipidomics was optimized for extraction and detection of lipids from human pancreatic cancer cell line PANC-1. Four different extraction methods were compared, including methanol/methyl-tert-butyl ether (MTBE)/H2O, methanol/chloroform, methanol/MTBE/chloroform, and hexane/isopropanol. Data were acquired using high-resolution mass spectrometry in positive and negative ion modes respectively. The number of total detected and identified lipids was assessed with the aid of automated lipid identification software LipidSearch. Results demonstrated that methanol/MTBE/H2O provided a better extraction efficiency for different lipid classes, which was chosen as the optimized extraction solvent system. This validated method enables highly sensitive and reproducible analysis for a variety of cellular lipids, which was further applied to an untargeted lipidomic study on human pancreatic cancer PANC-1 cell lines. Moreover, this optimized extraction solvent system can be further applied to other cancer cell lines with similar chemical and physical properties. Graphical abstract Optimized UHPLC-ESI-HRMS-based lipidomic analysis of cancer cells.

Metabolic mapping of Schisandra sphenanthera extract and its active lignans using a metabolomic approach based on ultra high performance liquid chromatography with high-resolution mass spectrometry.

Schisandra sphenanthera, the dried ripe fruit of Schisandra sphenanthera Rehd. et Wils, is widely used as a restorative, tonic and nutrition in many countries. Wuzhi tablet, an ethanol extract preparation of Schisandra sphenanthera, is a well-known herbal medicine widely used in China. Our previous studies show that Wuzhi tablet and its active lignans significantly protect liver injury. However, its metabolic profile remains unknown in vivo and in vitro. In this study, ultra high performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry based metabolomics was employed to decipher the metabolic map of Wuzhi tablet and its active lignans. Serum (2 h) and urine (24 h) samples after a 700 mg/kg single oral dose of Wuzhi tablet, and mice liver microsome samples after incubation with its active lignans were collected and analyzed. The data were further analyzed using metabolomics and metabolite identification software. In total, 33 metabolites in vivo and 34 metabolites in vitro were identified, and six among them were new metabolites. The major metabolic reactions encompassed demethylation, hydroxylation, dehydrogenation, and epoxidation. Taken together, in vitro and in vivo studies revealed the metabolic profile of Wuzhi tablet and its active lignans and demethylation and hydroxylation were their major metabolic pathways.

Binding of single/double stranded ct-DNA with graphene oxide­silver nanocomposites in vitro: A multispectroscopic approach.

The fundamental binding of single-stranded (ssDNA) and double-stranded DNA (dsDNA) with graphene oxide-Ag nanocomposites (GO-AgNCPs) has been systematically investigated by multi spectroscopic methods, i.e. ultraviolet-visible (UV-vis) absorption, fluorescence spectroscopy, and circular dichroism (CD). The experimental and theoretical results demonstrate that both ssDNA and dsDNA can be adsorbed onto the GO-AgNCPs surface. All of the evidence indicated that there were relatively strong binding of ssDNA/dsDNA with GO-AgNCPs. The article compares the differences in binding between the two types of DNA and the nanomaterials using spectroscopic and thermodynamic data. UV-vis absorption spectroscopy experiments indicate that the characteristic absorbance intensity of both ss DNA and ds DNA increases, but the rate of change in absorbance is different. The fluorescence results revealed that ss/dsDNA could interact with the GO-AgNCPs surface, in spite of the different binding affinities. The Ka value of ssDNA binding with GO-AgNCPs is greater than that of dsDNA at each constant temperature, indicating that the affinity of dsDNA toward GO-AgNCPs is comparatively weak. Molecular docking studies have corroborated the mentioned experimental results. The calculated thermodynamic parameters showed that the binding process was thermodynamically spontaneous, van der Waals force and hydrogen bonding played predominant roles in the binding process. The mechanism of ss/ds DNA binding with GO-AgNCPs was also investigated, and the results indicated that GO-AgNCPs directly binds to the minor groove of ss/ds DNA by replacing minor groove binders.

Integrated system architecture with mixed-reality user interface for virtual-physical hybrid swarm simulations.

This paper introduces a hybrid robotic swarm system architecture that combines virtual and physical components and enables human-swarm interaction through mixed reality (MR) devices. The system comprises three main modules: (1) the virtual module, which simulates robotic agents, (2) the physical module, consisting of real robotic agents, and (3) the user interface (UI) module. To facilitate communication between the modules, the UI module connects with the virtual module using Photon Network and with the physical module through the Robot Operating System (ROS) bridge. Additionally, the virtual and physical modules communicate via the ROS bridge. The virtual and physical agents form a hybrid swarm by integrating these three modules. The human-swarm interface based on MR technology enables one or multiple human users to interact with the swarm in various ways. Users can create and assign tasks, monitor real-time swarm status and activities, or control and interact with specific robotic agents. To validate the system-level integration and embedded swarm functions, two experimental demonstrations were conducted: (a) two users playing planner and observer roles, assigning five tasks for the swarm to allocate the tasks autonomously and execute them, and (b) a single user interacting with the hybrid swarm consisting of two physical agents and 170 virtual agents by creating and assigning a task list and then controlling one of the physical robots to complete a target identification mission.

1H-NMR-based metabolic profiling of rat urine to assess the toxicity-attenuating effect of the sweat-soaking method on Radix Wikstroemia indica.

Radix Wikstroemia indica (L.) C.A. Mey. (RWI) is a toxic medicinal species primarily present in the Miao area of China. The toxicity of RWI is effectively reduced whilst maintaining the therapeutic effect when processed using the 'sweat-soaking method', which is a common method of Traditional Chinese Medicine preparation. However, there is a lack of scientific and medical evidence to explain the potential mechanisms by which the toxicity of RWI is reduced after preparation using this method, and the endogenous systemic metabolic effect of RWI remains uncertain. The aim of the present study was to explore the endogetnous metabolic alterations caused by RWI and to examine the possibility of reducing the toxicity of RWI using the sweat-soaking method using proton nuclear magnetic resonance (NMR) metabolomic analysis in rats. Principal Component Analysis, Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA were used to assess individual proton NMR spectra. A total of 34 metabolic products were altered after delivering raw RWI, and 32 endogenous metabolites were induced by processed RWI. The metabolic pathways that lead to a significant impact on energy and carbohydrate, amino acid, organic acids and lipid metabolism following raw and processed RWI use were identified. The mitochondria of hepatic and renal tubules of rats were injured in the raw RWI group, whereas the processed product reduced or interfered with energy substrate, carbohydrate and amino acid metabolism, whilst reducing the levels of metabolic markers of hepatotoxicity and nephrotoxicity, without causing damage to the mitochondria. Our previous study showed that the median lethal dose (LD50) value of raw RWI was 4.05 g/kg in rats after oral administration; however, the LD50 value of the processed RWI could not be measured. The maximum tolerated dose and minimum lethal dose were 20 and 30 g/kg for the processed RWI, respectively, corresponding to 109 and 164 times the clinical daily dose (0.029 g/kg). Thus, the sweat-soaking method reduced the toxicity of RWI. Moreover, after processing, the toxic component YH-10 was converted into a YH-10 + OH compound, reducing the content of the toxic YH-10 by 48%, whilst also reducing the contents of the toxic components YH-12 and YH-15 by 44 and 65%, respectively. In conclusion, the present study showed that the sweat-soaking method reduced the toxicity of RWI, as evidenced by the reduction of the levels of metabolic markers and the activity of metabolic pathways, thus providing a basis for processing of RWI for clinical use.

Erythropoietin Preconditioning Mobilizes Endothelial Progenitor Cells to Attenuate Nephron-Sparing Surgery-Induced Ischemia-Reperfusion Injury.

The purpose of this study was to examine the role of endothelial progenitor cells (EPCs) in protection against ischemic-reperfusion injury (IRI) in a nephron-sparing surgery (NSS) rat model using erythropoietin (EPO) preconditioning. Fifty-four male Sprague-Dawley rats were randomly divided into 3 groups for right kidney nephrectomy treatment: sham group (exposure without clamp treatment), NSS group (3 days of peritoneal phosphate buffered saline [PBS] injection before renal blood vessels were clamped for 40 mins and NSS was performed), and EPO group (3 days of EPO abdomen injections prior to renal blood vessel clamping for 40 min before NSS was performed). After 12, 24, and 72 hours, inferior vena cava blood and renal tissues were harvested. The extent of renal injury was assessed, along with EPC number, cell proliferation, angiogenesis, and vascular growth factor expression. EPO preconditioning significantly improved renal function and histologic morphology, indicated by reduced blood urea nitrogen (BUN) ([33.12 ± 1.88] vs [16.03 ± 0.91], P < .05) and serum creatinine (Scr) ([190.2 ± 20.23] vs [77.23 ± 5.82], P < .05) levels and histologic injury scores ([3.20 ± 0.78] vs [1.70 ± 0.67], P < .05). Angiogenesis in peritubular capillaries markedly increased in the EPO group. EPC numbers increased in the kidneys at 24 hours following reperfusion in the EPO group, compared to the NSS group. Furthermore, EPO preconditioning also increased SDF-1α and CXCR7 expression at 24 hours following reperfusion relative to the NSS group. These findings suggest that EPO pretreatment can reduce renal injury in rats caused by IRI. Mechanistically, this may be related to EPC mobilization and recruitment to injured renal tissues by SDF-1α and CXCR7.

Exploring the Q-marker of "sweat soaking method" processed radix Wikstroemia indica: Based on the "effect-toxicity-chemicals" study.

BACKGROUND: Radix Wikstroemia indica (RWI), named "Liao Ge Wang" in Chinese, is a kind of toxic Chinese herbal medicine (CHM) commonly used in Miao nationality of South China. "Sweat soaking method" processed RWI could effectively decrease its toxicity and preserve therapeutic effect. However, the underlying mechanism of processing is still not clear, and the Q-markers database for processed RWI has not been established. PURPOSE: Our study is to investigate and establish the quality evaluation system and potential Q-markers based on "effect-toxicity-chemicals" relationship of RWI for quality/safety assessment of "sweat soaking method" processing. METHODS: The variation of RWI in efficacy and toxicity before and after processing was investigated by pharmacological and toxicological studies. Cytotoxicity test was used to screen the cytotoxicity of components in RWI. The material basis in ethanol extract of raw and processed RWI was studied by UPLC-Q-TOF/MS. And the potential Q-markers were analyzed and predicted according to "effect-toxicity-chemical" relationship. RESULTS: RWI was processed by "sweat soaking method", which could preserve efficacy and reduce toxicity. Raw RWI and processed RWI did not show significant difference on the antinociceptive and anti-inflammatory effect, however, the injury of liver and kidney by processed RWI was much weaker than that by raw RWI. The 20 compounds were identified from the ethanol extract of raw product and processed product of RWI using UPLC-Q-TOF/MS, including daphnoretin, emodin, triumbelletin, dibutyl phthalate, Methyl Paraben, YH-10 + OH and matairesinol, arctigenin, kaempferol and physcion. Furthermore, 3 diterpenoids (YH-10, YH-12 and YH-15) were proved to possess the high toxicity and decreased by 48%, 44% and 65%, respectively, which could be regarded as the potential Q-markers for quality/safety assessment of "sweat soaking method" processed RWI. CONCLUSION: A Q-marker database of processed RWI by "sweat soaking method" was established according to the results and relationship of "effect-toxicity-chemicals", which provided a scientific evidence for processing methods, mechanism and the clinical application of RWI, also provided experimental results to explore the application of Q-marker in CHM.

Is phospholipid-saturated alkyl column a convenient replacement for immobilized-artificial-membrane?

Three phosphatidylcholine (PC)-saturated C(8)/C(18) stationary phases prepared using biologically representative membrane lipids (purchasable L-alpha-PC and pure 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) have been developed to compare with IAM (immobilized-artificial-membrane) and C(8)/C(18) columns. These PC-coated stationary phases were found to be stable and reproducible for retention experiments. The retention characteristics of nucleobases on these coated phases deviate significantly from those on the IAM counterpart, but surprisingly similar to those of the underlying C(8)/C(18) columns. An inter-phase model has been proposed and explored for interpretation of the results.

The retention properties of nucleobases in alkyl C8-/C18- and IAM-chromatographic systems in relation to log Pow.

In order to evaluate the differences in the partition properties of 35 structurally congeneric nucleobases of biological interests in octanol-water biphasic, alkyl C(8)/C(18), and IAM systems, a comparative chromatographic study was performed. Comparing with the reversed-phase C(8)/C(18) retention data, most of the purines possessed weaker IAM retention except for those with specific H-bond and/or electrostatic interactions. Quantitative correlations between the experimental log P(ow) literature values and the IAM, C(8), and C(18) log k were evaluated (R(2)=0.943, 0.794, and 0.767, respectively). Although IAM retention correlated significantly better (larger R(2) value) with the log P(ow) values statistically, the latter was revealed apparently behaving more like (slope approaching unity) alkyl C(8)/C(18) retention and hence also has the same shortcoming in under-representing analytes capable of forming short-term H-bond/electrostatic interactions with polar head-groups of phospholipids. A chemically meaningful structure-retention model (q(2)=0.824 and R(2)=0.968) was derived, in which the hydrophobic interaction is identified as the underlying factor for the retention of purines in IAM system modulated non-trivially by H-bond/electrostatic interactions.

Complex retention behavior of pyrimidines on biomembrane-mimic immobilized-artificial-membrane phase.

The influence of the chemical substitutions on the interfacial interactions of pyrimidines with the phospholipid-mimic immobilized-artificial-membrane (IAM) chromatographic stationary phase was evaluated. Monocyclic pyrimidine nucleic acid bases (nucleobases) were revealed behaving differently from their bicyclic purine counterparts substantially. The computed electrostatic potential surfaces for both the IAM phase and the interacting nucleobases are intuitive in deconvoluting the retention patterns of pyrimidines molecularly. A structure-retention model has also been derived using quantitative 3D-QSAR methodology pertinent to the IAM-retention of pyrimidines for the potential use in molecular design. IAM phase is found particularly suitable in assessing the retention of pyrimidines with bulky or elongated exocyclic substituents in the biological context than the alkyl-based chromatographic counterparts.