The phospholipase effector Tle1Vc promotes Vibrio cholerae virulence by killing competitors and impacting gene expression.

Vibrio cholerae utilizes the Type VI secretion system (T6SS) to gain an advantage in interbacterial competition by delivering anti-prokaryotic effectors in a contact-dependent manner. However, the impact of T6SS and its secreted effectors on physiological behavior remains poorly understood. In this study, we present Tle1Vc, a phospholipase effector in atypical pathogenic V. cholerae E1 that is secreted by T6SS via its interaction with VgrG1E1. Tle1Vc contains a DUF2235 domain and belongs to the Tle1 (type VI lipase effector) family. Bacterial toxicity assays, lipase activity assays and site-directed mutagenesis revealed that Tle1Vc possessed phospholipase A1 activity and phospholipase A2 activity, and that Tle1Vc-induced toxicity required a serine residue (S356) and two aspartic acid residues (D417 and D496). Cells intoxication with Tle1Vc lead to membrane depolarization and alter membrane permeability. Tli1tox-, a cognate immunity protein, directly interacts with Tle1Vc to neutralize its toxicity. Moreover, Tle1Vc can kill multiple microorganisms by T6SS and promote in vivo fitness of V. cholerae through mediating antibacterial activity. Tle1Vc induces bacterial motility by increasing the expression of flagellar-related genes independently of functional T6SS and the tit-for-tat (TFT) response, where Pseudomonas aeruginosa uses its T6SS-H1 cluster to counterattack other offensive attackers. Our study also demonstrated that the physical puncture of E1 T6SS can induce a moderate TFT response, which is essential to the Tle1Vc-mediated strong TFT response, maximizing effector functions. Overall, our study characterized the antibacterial mechanism of phospholipase effector Tle1Vc and its multiple physiological significance.

Study on the Preparation of Network Ti-N/Ti Composites by Nitridation of Ti Powders.

Composite structure design is an important way to improve reinforcement strengthening efficiency. The dispersion of the external reinforcement is often not uniform enough, however, and it is agglomerated in the matrix, which cannot uniformly and effectively bear the load. The interconnected reinforcement network prepared by the in-situ self-growth method is expected to obtain higher material properties. In this paper, the TiN shell was formed on the surface of Ti powder by the in-situ nitriding method, and then the network TiN/Ti composites were prepared by sintering. In the control group, TiN was dispersed by mechanical ball milling, and it was found that TiN powder was coated on the surface of Ti particles, and the sintered TiN/Ti composites formed a discontinuous structure with a great deal of TiN agglomeration. A uniform TiN nitride layer of 5~7 µm was formed on the surface of Ti powder by the in-situ nitriding method, and a connected TiN network was formed in the sintered Ti-N/Ti composites. The composites prepared by nitriding have higher compressive strength, hardness, and plasticity. The hardness of the Ti-N/Ti composite is 685.7 HV and the compressive strength is 1468.5 MPa. On this basis, the influence of the connected TiN structure on the material properties was analyzed, which provided theoretical guidance for the structural design of the network structure-reinforced titanium matrix composites.

Microstructure and Mechanical Properties of Core-Shell B4C-Reinforced Ti Matrix Composites.

Composite material uses ceramic reinforcement to add to the metal matrix to obtain higher material properties. Structural design is an important direction of composite research. The reinforcement distribution of the core-shell structure has the unique advantages of strong continuity and uniform stress distribution. In this paper, a method of preparing boron carbide (B4C)-coated titanium (Ti) powder particles by ball milling and preparing core-shell B4C-reinforced Ti matrix composites by Spark Plasma Sintering was proposed. It can be seen that B4C coated on the surface of the spherical Ti powder to form a shell structure, and B4C had a certain continuity. Through X-ray diffraction characterization, it was found that B4C reacted with Ti to form layered phases of titanium boride (TiB) and titanium carbide (TiC). The compressive strength of the composite reached 1529.1 MPa, while maintaining a compressive strain rate of 5%. At the same time, conductivity and thermal conductivity were also characterized. The preparation process of the core-shell structure composites proposed in this paper has high feasibility and universality, and it is expected to be applied to other ceramic reinforcements. This result provides a reference for the design, preparation and performance research of core-shell composite materials.

Discovery of potent immune-modulating molecule taccaoside A against cancers from structures-active relationships of natural steroidal saponins.

BACKGROUND: In recent years, the T-cell therapy and immune checkpoint inhibitors toward CTLA-4 and PD-1/PD-L1 axis antibody therapy have acquired encouraging success. However, most of patients were still not benefited with lots of troubles, such as low penetration of tissues/cells, strong immunogenicity and cytokine release syndrome, and long manufacturing process and expensive costs. By contrast, the immune-modulating small molecules possessed natural advantages to overcome these obstacles and might achieve greater success. PURPOSE: Exploring the potent immune-modulating natural small molecules and revealing what kinds of molecules or structures with the immunomodulatory activity against cancers. METHODS: A novel non-cytotoxic T-cell immunomodulating screening model was used to identify the cytotoxic/selective/immunomodulatory bioactivity for 148 natural steroidal saponins. The structure-activity relationships (SARs) research was used to reveal the key groups for immunomodulation/cytotoxicity/selectivity. The negative selection was used to isolate and purify the T-cell. The cell viability assay was used to measure the anti-cancer effect in vitro. The ELISA assay was used to detect the cytokines for IL-1ß, IL-6, TNF-α, IFN-γ, IL-12, perforin and granzyme B (GZMB). The western blotting assay was used to research the immunomodulatory mechanism. The siRNA knockdown was used to generate the IFN-γ resistant melanoma cells. The NOG immune-deficient mice were used to evaluate the anti-tumor efficacy in vivo. The peripheral blood samples from 10 cancer patients were used to detect the broad population anti-tumor efficacy. RESULTS: It was reported that the correlation among structures and immunomodulation/ cytotoxicity/selectivity, in which opening ring-F with 26-O-glucopyranosyl, disaccharide and trisaccharide chains at C-3, steric hindrance and polarity of C-22 were key immunomodulatory groups. Moreover, taccaoside A was identified as the most potent candidate against cancer cells, including non-small cell lung cancer, triple negative breast cancer, and the IFN-γ resistant melanoma, partly through enhancing T lymphocyte mTORC1-Blimp-1 signal to secrete GZMB. Besides, 10 patients derived T-cell also would be modulated against cancer cells in vitro. Moreover, the overall survival was great extended (>140 days vs 93 days) with nearly 100% tumor burden disappearance (0 mm3vs 1006 ± 79.5 mm3) in mice. CONCLUSION: This work demonstrated one possibility for this concerned purpose, and identified a potent immune-modulating natural molecule taccaoside A, which might contribute to cancer immunotherapy in future.

Calmodulin binds to Drosophila TRP with an unexpected mode.

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Purification of Endogenous Drosophila Transient Receptor Potential Channels.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cß/No receptor potential A (PLCß/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Bioassay-guided isolation of anti-inflammatory diterpenoids with highly oxygenated substituents from kidney tea (Clerodendranthus spicatus).

The whole plant of Clerodendranthus spicatus (Thunb.) is one of popular functional food in south of China, named as "kidney tea" and used to ameliorate renal inflammation. In order to verify this potential function and explore the accurate compounds responsible for inflammation, the ethanol extract, fractions, and subfractions of this plant were prepared to evaluate anti-inflammation effect on xylene-induced acute inflammatory mice model, and the results indicated that two subfractions from EtOAc fraction show potential activities. Subsequent bioassay-guided isolation of the bioactive subfractions led to isolation of 25 compounds. Among them, compounds 2, 4, 5, 9-11, 13, 16, 17, and 20-22 inhibited the productions of pro-inflammation factors TNF-α, IL-1ß, and IL-8 in lipopolysaccharide (LPS) -induced renal epithelia (HK-2) cells, respectively. Further anti-inflammation evaluation in vivo indicated that the major bioactive compounds 1, 2, 5-7, 17, 21, and 22 from C. spicatus were even better than aspirin. PRACTICAL APPLICATIONS: C. spicatus as a healthy tea has been available in the Chinese market and as a medicine for various disorders such as nephritis, rheumatism, inflammation, gout, and diabetes. Previous pharmacological investigation of the plant revealed the potential anti-inflammatory activities, but the material basis of anti-inflammatory activity remains to be elucidated. In our study, the anti-inflammatory fractions and compounds were obtained by the bioassay-guide isolation and the results showed that the highly oxygenated diterpenoids were major anti-inflammatory compounds, in which 1, 2, 5-7, 17, 21, and 22 were even better than aspirin. This information supported kidney tea as a functional food for treatment of renal inflammation reasonably and may add a new dimension to biological activity of this plant in the field of agriculture as a functional food were cultivated.

Atrial and Ventricular Response to Treatment of Premature Ventricular Complexes.

BACKGROUND: Premature ventricular complexes (PVC) may cause ventricular dyssynchrony and lead to left atrium and ventricle mechanical abnormalities. Although ventricular cardiomyopathy due to PVCs has been well studied, little is known about atrial adaptation to PVCs. OBJECTIVES: To assess atrial and ventricular responses to PVC therapy. METHODS: All patients with PVC burden > 5000 beats/day on Holter monitoring were enrolled. Baseline demographics, comorbidities, social habits, Holter parameters, and echocardiography profiles were recorded. Follow-up Holter electrocardiography (ECG) and echocardiography data were compared between PVC-treated and non-treated patients. RESULTS: Two hundred and eighty-six patients were enrolled, of whom 139 received PVC treatment. Among the treated patients, 125 who underwent follow up Holter ECG or echocardiography were included in the final analysis. The mean follow-up times of Holter ECG and echocardiography were 9.40 ± 6.70 and 9.40 ± 5.52 months, respectively. Ventricular arrhythmic burden was significantly reduced in the treatment group (16.46% vs. 13.41%, p = 0.041) but was significantly increased in the observation group (7.58% vs. 14.95%, p = 0.032). A significant increase in left atrial (LA) diameter (36.94 mm vs. 39.46 mm, p = 0.025) and reduction in left ventricular ejection fraction (LVEF) (57.26% vs. 53.8%, p = 0.040) were noted in the observation group. There were no significant differences in supraventricular arrhythmic burden in the observation group and LA diameter and LVEF in the treatment group. CONCLUSIONS: PVC therapy effectively reduced ventricular arrhythmic burden in the treatment group on follow-up. Our data suggest that PVC treatment may prevent LA dilation and LVEF decline.

"Kidney Tea" and Its Bioactive Secondary Metabolites for Treatment of Gout.

Clerodendranthus spicatus, popularly known as "kidney tea" in China, is consumed traditionally as a functional food for treatment of renal inflammation, dysuria, and gout. We evaluated the effects of C. spicatus on gout by assessing activities of antihyperuricemia, anti-gouty arthritis, and analgesia in vivo, and the results indicated that the ethyl acetate fraction shows potential activities. Subsequent phytochemical investigation of this fraction led to the isolation of 32 compounds, consisting of 20 diterpenoids (including the new orthosiphonones E and F), 2 triterpenoids, 6 flavonoids, 2 lignanoids, and 2 phenolic acid derivatives. Pharmacological investigation of the pure compounds in the cellular model of hyperuricemia indicated that 12 compounds could promote the excretion of uric acid at 10 µg/mL, and compounds 3, 4, 5, and 21 had better effects than that of benzbromarone, a famous uricosuric drug. Furthermore, compounds 4, 6, 7, 9, 14, 15, 23, 26, and 31 showed significant anti-gouty arthritis activity in monosodium urate (MSU)-induced joint swelling at the dose of 50 mg/kg, while compounds 4, 5, 7, 9, and 26 exhibited significant inhibition of pain induced by acetic acid. Our findings provided scientific justification to support the traditional application of "kidney tea" for treating gout and suggested its good application prospects in the future.

The characteristics and prescription patterns of Chinese herbal medicine in clinical practice for the treatment of anemia.

OBJECTIVE: Chinese herbal medicine (CHM) is frequently applied to patients to improve the symptoms and signs associated with anemia. The aim of this study is to use the claims data from the National Health Insurance Research Database (NHIRD) in Taiwan to analyze CHM prescription patterns and to identify the frequency and combinations of CHM commonly used to treat anemia. MATERIALS AND METHODS: A total of 41,028 patients were diagnosed with anemia in Taiwan within the defined study period. After randomly equal matching for age and sex, data from 7682 patients characterized as CHM users and non-users were analyzed. Network analyses of the 30 most frequently applied herbs and formulas were used to indicate CHM combinations in patients with anemia. RESULTS: Those patients with anemia who were older, office workers, and lived in central areas of Taiwan had higher tendencies toward CHM usage. Based on considerations of comorbidities, anemia patients associated with chronic kidney diseases, diabetes mellitus, and hypertensive diseases preferred Western medical management and demonstrated a lesser likelihood of combining treatment with CHM; by contrast, those with coronary artery disease demonstrated a higher tendency for CHM use. Notably, Astragalus membranaceus (AM) and Gui-Pi-Tang (GPT) were the most commonly prescribed CHM single herb and formula, respectively. The core prescription pattern consisted of AM, Salvia miltiorrhiza (SM), Angelica sinensis (AS), GPT, and Si-Wu-Tang (SWT), as indicated by the associations and frequency of CHM utilization by traditional Chinese medicine (TCM) physicians. CONCLUSION: This study demonstrates that CHM may be applied as an integral element of treatment for patients with anemia. It also provides insight regarding individual therapy and common clinical practices of TCM physicians in the treatment of anemia. Further research is required to explore potential interactions and possible mechanisms at play with CHM management of anemia.

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here, we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases.

Comparison of inducible versus constitutive expression of plectasin on yields and antimicrobial activities in Pichia pastoris.

BACKGROUND: Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation. OBJECTIVE: To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities. METHODS: Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZαA) and constitutive (pGAPZαA) vectors in order to create Pichia pastoris GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE. RESULTS: After 120 h of fermentation, the yield of constitutive plectasin (370 µg/ml) was much lower than that from inducible vector (880 µg/ml) (P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield (P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 µg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 µg/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P ï¼ž 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass. CONCLUSIONS: Although P. pastoris GS115 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.