The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

In Vitro and In Vivo Activities, Absorption, Tissue Distribution, and Excretion of OBP-4, a Potential Anti-Clostridioides difficile Agent.

Clostridioides difficile infection (CDI) is considered a major concern of the health care system globally, with an increasing need for alternative therapies. OBP-4, a new oxazolidinone-fluoroquinolone hybrid with excellent in vitro activities and good safety, shows promising features as an antibacterial agent. Here, we further evaluated the in vitro and in vivo activities of OBP-4 against C. difficile and its absorption (A), distribution (D), and excretion (E) profiles in rats. In vitro assays indicated that OBP-4 was active against all tested C. difficile strains, with MICs ranging from 0.25 to 1 mg/liter. In addition, OBP-4 showed complete inhibition of spore formation at 0.5× MIC. In the mouse model of CDI, 5-day oral treatment with OBP-4 provided complete protection from death and CDI recurrence in infected mice. However, cadazolid (CZD) and vancomycin (VAN) showed less protection of infected mice than did OBP-4 in terms of diarrhea and weight loss, especially VAN. Subsequently, ADE investigations of OBP-4 with a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method showed extremely low systemic exposure and predominantly fecal excretion, resulting in a high local concentration of OBP-4 in the intestinal tract-the site of CDI. These results demonstrated that OBP-4 possesses good activity against C. difficile and favorable ADE characteristics for oral treatment of CDI, which support further development of OBP-4 as a potential anti-CDI agent.

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

Mating-type loci of Ustilago esculenta are essential for mating and development.

Ustilago esculenta is closely related to the smut fungus Ustilago maydis and, in an endophytic-like life in the plant Zizania latifolia, only infects host stems and causes swollen stems to form edible galls called Jiaobai in China. In order to study its different modes of invasion and sites of symptom development from other smut fungi at the molecular level, we first characterized the a and b mating-type loci of U. esculenta. The a loci contained three a mating-type alleles, encoding two pheromones and one pheromone receptor per allele. The pheromone/receptor system controlled the conjugation formation, the initial step of mating, in which each pheromone was specific for recognition by only one mating partner. In addition, there are at least three b alleles identified in U. esculenta, encoding two subunits of heterodimeric homeodomain transcription factors bE and bW, responsible for hyphal growth and invasiveness. Hyphal formation, elongation and invasion after mating of two compatible partners occurred, only when a heterodimer complex was formed by the bE and bW proteins derived from different alleles. We also demonstrated that even with only one paired pheromone-pheromone receptor, the active b locus heterodimer triggered hyphal growth and infection.

Cloning and disruption of the UeArginase in Ustilago esculenta: evidence for a role of arginine in its dimorphic transition.

BACKGROUND: Ustilago esculenta, a typical dimorphic fungus could infect Zizania latifolia and induce host stem swollen to form an edible vegetable called Jiaobai in China. The strains differentiation especially in the mating ability and pathogenicity is closely related to different phenotypes of Jiaobai formed in the fields. Dimorphic switching, a tightly regulated processes, is essential for the pathogenetic development of dimorphic fungi. In responses to environment cues, dimorphic switching can be activated through two conserved cell signaling pathways-PKA and MAPK pathways. Previous study indicated that exogenous arginine could induce hyphal formation in several dimorphic fungi through hydrolysis by arginase, but inhibit the dimorphic transition of U. esculenta. We conducted this study to reveal the function of arginine on dimorphic transition of U. esculenta. RESULTS: In this study, we found that arginine, but not its anabolites, could slow down the dimorphic transition of U. esculenta proportionally to the concentration of arginine. Besides, UeArginase, predicated coding arginase in U. esculenta was cloned and characterized. UeArginase mutants could actually increase the content of endogenous arginine, and slow down the dimorphic transition on either nutritious rich or poor medium. Either adding exogenous arginine or UeArginase deletion lead to down regulated expressions of UePkaC, UePrf1, mfa1.2, mfa2.1, pra1 and pra2, along with an increased content of arginine during mating process. CONCLUSION: Results of this study indicated a direct role of arginine itself on the inhibition of dimorphic transition of U. esculenta, independent of its hydrolysis by UeArginase.

Structure-guided design of antibacterials that allosterically inhibit DNA gyrase.

A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.

Synthesis, Antibacterial Activities, Mode of Action and Acute Toxicity Studies of New Oxazolidinone-Fluoroquinolone Hybrids.

To combat bacterial resistance, a series of new oxazolidinone-fluoroquinolone hybrids have been synthesized and characterized. All synthetic hybrids were preliminarily evaluated for their in vitro antibacterial activities against 6 standard strains and 3 clinical isolates. The majority of hybrids displayed excellent activities against Gram-positive bacteria, but limited activities against Gram-negative bacteria. Hybrids OBP-4 and OBP-5 were found to be the most promising compounds. Further, in vitro antibacterial activities, mode of action and acute toxicity in mice of hybrids OBP-4 and OBP-5 were investigated. Hybrids OBP-4 and OBP-5 exhibited potent activities against Gram-positive bacteria, including drug-resistant strains. Correspondingly, studies on the mode of action of hybrids OBP-4 and OBP-5 indicated a strong inhibitory activity on protein synthesis by binding the active site of 50S subunit, but a weak inhibitory action on DNA synthesis. In addition, LD50 values of hybrids OBP-4 and OBP-5 in the acute oral toxicity were larger than 2000 mg/kg, suggesting a good safety profile.

Cloning and Characterization of Two MAPK Genes UeKpp2 and UeKpp6 in Ustilago esculenta.

Ustilago esculenta, resembling a fungal endophyte in Zizania latifolia, inhibits the host plant flowering and induces the host stems to swell and form edible galls. It is well believed that when and how the fungus infects and proliferates in the host plants during the host development is of importance in the edible gall formation. Mitogen-activated protein kinases (MAPKs) have been found to play an important role in sensing environment cues and regulating infection. Two MAPK genes UeKpp2 and UeKpp6 from U. esculenta were cloned and suggested to be involved in the Fus3/Kss1 pathway by a phylogenetic analysis with the neighbor-joining method. Quantitative RT-PCR (qRT-PCR) analyses indicated that expression of UeKpp2 and UeKpp6 were induced during mating and infection processes, and their expression patterns displayed differentially under different carbon and nitrogen sources. In addition, subcellular localization of UeKpp2 or UeKpp6 fused with the reporter green fluoresce protein was observed by confocal laser scanning microscope, and yeast two-hybrid assays were carried out. Results showed that both UeKpp2 and UeKpp6 were located in cytoplasm and interacted with UePrf1, indicating their involvement in hyphal growth and host-pathogen regulation. Only UeKpp2 but not UeKpp6 interacted with the upstream MAPK kinase UeFuz7, implying an additional MAPK pathway, in which UeKpp6 involved, existed.

Anticancer activity of safranal against colon carcinoma is due to induction of apoptosis and G2/M cell cycle arrest mediated by suppression of mTOR/PI3K/Akt pathway.

PURPOSE: Colon cancer is among the deadliest malignancies and is responsible for a significant number of deaths across the globe. The treatment options for colon cancer are limited and with lot of side effects. In this study we evaluated the potential anticancer effects of safranal against colo-205 colon cancer cells along with evaluation of its effects on apoptosis, cell cycle phase distribution, reactive oxygene species (ROS) and PI3K/AKT/m-TOR signalling pathway. METHODS: Cell viability was examined by MTT assay. Apoptosis was checked by DAPI staining, comet assay and annexin V/propidium iodide (PI) staining involving the use of fluorescence microscopy and flow cytometry. ROS, mitochondrial membrane potential (MMP) and cell cycle phase distribution were checked by flow cytometry using different fluorescent probes. Effects of safranal on the protein expression of PI3K/AKT/m-TOR were studied by western blot assay. RESULTS: The results revealed that safranal suppressed the proliferation of colo-205 cancer cells with an IC50 of 20 µM. Furthermore, the anticancer effects of safranal were found to be due to accretion of ROS and decrease in the MMP, ultimately leading to apoptosis of colo-205 cancer cells. In addition, safranal caused increase in the expression of Bax in parallel with concomitant reduction in the expression of Bcl-2. Safranal also triggered G2/M cell cycle arrest and inhibition of PI3K/AKT/mTOR signalling pathway. CONCLUSION: In conclusion, the above results indicate that safranal could prove a potential lead molecule in the treatment of colon cancer, provided further in vivo studies are carried out.

Investigation on the differentiation of two Ustilago esculenta strains - implications of a relationship with the host phenotypes appearing in the fields.

BACKGROUND: Ustilago esculenta, a pathogenic basidiomycete fungus, infects Zizania latifolia to form edible galls named Jiaobai in China. The distinct growth conditions of U. esculenta induced Z. latifolia to form three different phenotypes, named male Jiaobai, grey Jiaobai and white Jiaobai. The aim of this study is to characterize the genetic and morphological differences that distinguish the two U. esculenta strains. RESULTS: In this study, sexually compatible haploid sporidia UeT14/UeT55 from grey Jiaobai (T strains) and UeMT10/UeMT46 from white Jiaobai (MT strains) were isolated. Meanwhile, we successfully established mating and inoculation assays. Great differences were observed between the T and MT strains. First, the MT strains had a defect in development, including lower teliospore formation frequency and germination rate, a slower growth rate and a lower growth mass. Second, they differed in the assimilation of nitrogen sources in that the T strains preferred urea and the MT strains preferred arginine. In addition, the MT strains were more sensitive to external signals, including pH and oxidative stress. Third, the MT strains showed an infection defect, resulting in an endophytic life in the host. This was in accordance with multiple mutated pathogenic genes discovered in the MT strains by the non-synonymous mutation analysis of the genome re-sequencing data between the MT and T strains (GenBank accession numbers of the genome re-sequencing data: JTLW00000000 for MT strains and SRR5889164 for T strains). CONCLUSION: The MT strains appeared to have defects in growth and infection and were more sensitive to external signals compared to the T strains. They displayed an absolutely stable endophytic life in the host without an infection cycle. Accordingly, they had multiple gene mutations occurring, especially in pathogenicity. In contrast, the T strains, as phytopathogens, had a complete survival life cycle, in which the formation of teliospores is important for adaption and infection, leading to the appearance of the grey phenotype. Further studies elucidating the molecular differences between the U. esculenta strains causing differential host phenotypes will help to improve the production and formation of edible white galls.

A host plant genome (Zizania latifolia) after a century-long endophyte infection.

Despite the importance of host-microbe interactions in natural ecosystems, agriculture and medicine, the impact of long-term (especially decades or longer) microbial colonization on the dynamics of host genomes is not well understood. The vegetable crop 'Jiaobai' with enlarged edible stems was domesticated from wild Zizania latifolia (Oryzeae) approximately 2000 years ago as a result of persistent infection by a fungal endophyte, Ustilago esculenta. Asexual propagation via infected rhizomes is the only means of Jiaobai production, and the Z. latifolia-endophyte complex has been maintained continuously for two centuries. Here, genomic analysis revealed that cultivated Z. latifolia has a significantly smaller repertoire of immune receptors compared with wild Z. latifolia. There are widespread gene losses/mutations and expression changes in the plant-pathogen interaction pathway in Jiaobai. These results show that continuous long-standing endophyte association can have a major effect on the evolution of the structural and transcriptomic components of the host genome.

4-Terpineol exhibits potent in vitro and in vivo anticancer effects in Hep-G2 hepatocellular carcinoma cells by suppressing cell migration and inducing apoptosis and sub-G1 cell cycle arrest.

PURPOSE: The main purpose of this study was to demonstrate the anticancer effects of 4-terpineol against Hep-G2 hepatocellular carcinoma (HCC) cells by evaluating its effect on apoptosis induction, cell migration, DNA fragmentation and cell cycle phase distribution. METHODS: MTT assay was used to evaluate the cytotoxic effect of 4-terpineol on Hep-G2 cells, while fluorescence microscopy and flow cytometry were used to study apoptosis induction. Wound healing assay was used to study the effects of 4-terpineol on cell migration, while gel electrophoresis was performed to evaluate the effects on DNA fragmentation. Flow cytometry using propidium iodide (PI) as a probe was used to evaluate the effects on cell cycle arrest. Cells treated with dimethylsulfoxide (DMSO) only served as controls. BALB/c nude mice weighing about 35 g each were used for in vivo studies using 10 and 20 mg/kg of 4-terpineol dose. RESULTS: 4-terpineol induced dose-dependent cytotoxicity in Hep-G2 hepatocellular carcinoma cells. Gel electrophoresis indicated that DNA fragmentation was associated with increasing dose of 4-terpineol. It was also observed that a wound scratch in the vehicle-treated control cells was practically entirely closed after 48 hrs of incubation. However, treatment with 0, 25, 50 and 100 µM dose of 4-terpineol resulted in inhibition of wound healing in a dose-dependent manner. The percentage of apoptotic cells increased from 2.5% in the control cells to 10.3, 64.6 and 78.9% in cells treated with 25, 50 and 100 µM of 4-terpineol respectively. 4-terpineol-treated cells exhibited increased percentage of cells in sub-G1 phase of the cell cycle. The in vivo mouse results indicated that 10 and 20 mg/kg of 4-terpineol decreased the tumor weight and tumor volume in a dose-dependent manner. CONCLUSION: The results of this study showed that 4-terpineol exhibits anticancer effects in Hep-G2 cells by inducing apoptosis, DNA fragmentation, inhibition of cell migration and sub-G1 cell cycle arrest.

[Effects of Tanyu Tongzhi recipe on hemorheology, blood lipid and inflammatory factors in rats with mycardial ischemia-reperfusion injury and hyperlipidemia].

OBJECTIVE: To investigate the influence of Tanyu Tongzhi (TYTZ) recipe on chemorheology, blood lipid and inflammatory factors of hyperlipidemia and myocardial ischemia-reperfusion injury in rats. METHOD: Sixty SD male rats were divided into 5 groups randomly, sham-operated group, model group, high dose group of reproduced by ligation of left descending artery for 30 min followed by releasing the TYTZ and low group of TYTZ. The model of MI/RI injury of the myocardium was ligation for 2 hours in rats. Serum contents of CHO, TG, HDL-L, LDL-L and whole blood viscosity, plasma viscosity and ICAM-1, TNF-alpha, IL-10 were measured after myocardial reperfusion injury. RESULT: Compared with sham-operated group, the levels of CHO, TG, LDL-L, whole blood viscosity (1.0,3.0) plasma viscosity and the contents of ICAM-1 were significantly higher, however, HDL-L, IL-10 levels were lower in model group (P < 0.01 and P < 0.05). CHO, TG, whole blood viscosity (1.0, 3.0, 30) and expression of ICAM-1, TNF-alpha were obviously lower in low group than the model group (P < 0.01 and P < 0.05). CONCLUSION: The TYTZ recipe can relieve reperfusion injury through regulating blood lipid, improving hemorheological characteristic and inhibiting inflammatory reaction.

Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms.

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants.

The Novel Effector Ue943 Is Essential for Host Plant Colonization by Ustilago esculenta.

The smut fungus Ustilago esculenta obligately parasitizes Zizania latifolia and induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase of U. esculenta but not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted by U. esculenta and localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. The Ue943 deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing gene Ue943 or Ue943:GFP. Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943. We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.

Synthesis and Characterization of High-Purity, High-Entropy Diboride Ceramic Powders by a Liquid Phase Method.

A nano-dual-phase powder with ultra-fine grain size was synthesized by the liquid precursor method at 1200 °C. A series of single-phase high-entropy ceramic powders ((Ti, Zr, Hf, Nb)B2, (Ti, Zr, Hf, Nb, Ta)B2, (Ti, Zr, Hf, Nb, Mo)B2, (Ti, Zr, Hf, Nb, Ta, Mo)B2) with high purity (C content less than 0.9 wt% and O content less than 0.7 wt%) and ultrafine (average grain sizes of 340-570 nm) were successfully synthesized at 1800 °C. The sample of (TiZrHfNbTa)B2 exhibited a hexagonal close-packed (HCP) structure, and the metal elements were uniformly distributed at the nanoscale, microscale, and macroscale. This method did not apply to the preparation of all high-entropy ceramic powders and was unfavorable for the formation of single-phase high-entropy borides when the size difference factor exceeded 3.9%. The present work provides a guide for the development of ceramic-based composites through precursor impregnation pyrolysis.

Experimental study on anaphylaxis of qingkailing injection and its components on Beagle dogs.

OBJECTIVE: To study the anaphylaxis of Qingkailing injection (QI) and its components. METHODS: Experimental anaphylactoid and allergic reactions were used. Changes in the behaviors of Beagles and serum levels of histamine, immunoglobulin (Ig)E, IgG, IgM, eosinophil cationic protein (ECP), and interleukin (IL)-4, as well as blood pressure, after injecting QI and its components on the forelimb veins of Beagles were observed. RESULTS: According to comprehensive determination of abnormal behavior scores and changes in serum levels of histamine, IgE, IgG, IgM, ECP, and IL-4, as well as in blood pressure, radix isatidis and hyodeoxycholic acid caused anaphylactoid reactions, and honeysuckle, radix isatidis, hydrolysate, cholic acid and Gardenia jasminoides caused allergic reactions. The anaphylaxis of QI involved anaphylactoid and allergic reactions. CONCLUSION: QI and its components need to be refined further to improve the safety, efficacy, and quality of its use in clinical settings.

An investigation of the anti-diabetic effects of an extract from Cladonia humilis.

The effect of Cladonia humilis on glycaemic metabolism was researched in this studied. The blood glucose, insulin secretion and glycogen synthesis of the hyperglycemic mice induced by alloxan were analyzed respectively. The gluconeogenesis and the sugar tolerance of the normal mice were also analyzed in this paper. After the hyperglycemic mice were orally administrated with Cladonia humilis extract, the blood glucose was decreased (p<0.05), the level of insulin secretion and glycogen synthesis were elevated (p<0.05, p<0.01, respectively). In addition, Cladonia humilis extract could inhibit the gluconeogenesis (p<0.01) and improve the sugar tolerance in normal control mice. These results maybe account for the causes of Cladonia humilis extract-induced significant decreases of the blood glucose in hyperglycemic mice.

Numerical and Field Investigations of Acoustic Emission Laws of Coal Fracture under Hydro-Mechanical Coupling Loading.

Taking coal under hydro-mechanical coupling as the research object, the discrete element software PFC3D (particle flow code) was used to analyze the relationships among the force, acoustic emission (AE), and energy during coal fracture. Based on the moment tensor (MT) inversion, we revealed the AE event distribution and source type during crack initiation and propagation until the final failure of coal. Meanwhile, we examined the relationships among the stress, number and type of cracks, magnitude, KE, and b value of AE under different water and confining pressures. The results show that the numerical simulation can effectively determine the microscopic damage mechanism of coal under different conditions. Moreover, the rupture type of the numerical simulation is consistent with the field investigations, which verifies the rationality of the simulation. These research results can provide reference for safety production evaluation of water inrush mines.

Transcriptome Comparison between Two Strains of Ustilago esculenta during the Mating.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.