Bioactive compounds from dichloromethane extract of Artemisia rupestris L. alleviates CCl4/ConA-induced acute liver injury by inhibiting PI3K-AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia rupestris L. (AR) is a traditional medicinal herb commonly used in the Uyghurs and Kazakhs; it was first documented in the Supplement to Compendium of Materia Medica written by Zhao Xuemin in the Qing Dynasty of China and is used clinically to treat colds, hepatitis, and allergic diseases. AIM OF THE STUDY: The material basis and mechanisms of AR in acute liver injury (ALI) remain unclear. The purpose of this study was to reveal the possible active components involved in liver protection in AR and to preliminarily explore their pharmacological mechanisms. MATERIALS AND METHODS: The chemical composition of the ethanolic extract (ARA) was identified by UPLC-Q-Exactive-MS/MS and confirmed by 32 reference standards. The pharmacodynamic results were utilized to screen the active part within the ARA that contribute to the amelioration of CCl4/ConA-induced ALI. The main active components and core targets were predicted by network pharmacology and verified by molecular docking combined with qPCR and Western blotting. RESULTS: A total of 131 chemical components were identified in the ARA. The extraction parts of ARA had different therapeutic effects on ALI, among which the dichloromethane extract (ARA-D), which might constitute the main effective fraction of ARA, had significant anti-ALI effects. The network pharmacology results showed that targets including PIK3R1, AKT1, and EGFR, as well as 7 compounds, such as artemetin, vitexicarpin and rupestonic acid may play pivotal roles in treating CCl4/ConA-induced ALI. GO and KEGG pathway enrichment analyses revealed that the PI3K-AKT signaling pathway was the main pathway involved. In each model, ARA-D dose-dependently reduced the increase in ALT levels. High-dose ARA-D markedly decreased ALT activity from 196.79 ± 24.82 to 66.37 ± 16.19 U/L in the CCl4 model group and from 178.00 ± 28.39 to 50.67 ± 7.39 U/L in the ConA model group. Further studies revealed that ARA-D significantly inhibited TNF-α, IL-1ß, and IL-6 expression and inhibited the protein expression of PI3K, p-PI3K, and p-AKT in CCl4/ConA-induced ALI. CONCLUSION: ARA-D exhibits protective effects against ALI induced by CCl4/ConA, potentially through inhibition of the PI3K-AKT signaling pathway. These findings may help to determine the material basis and mechanisms of action of ARA-D for liver protection and provide ideas for future comprehensive studies.

Construction of an alternative NADPH regeneration pathway improves ethanol production in Saccharomyces cerevisiae with xylose metabolic pathway.

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield. However, glucose and xylose share flux in the pentose phosphate pathway (PPP) and glycolysis pathway (EMP), with glucose having a competitive advantage in the shared metabolic pathways. In this work, we knocked down ZWF1 to preclude glucose from entering the PPP. This reduced the [NADPH] level and disturbed growth on both glucose or xylose, confirming that the oxidative PPP, which begins with Zwf1p and ultimately leads to CO2 production, is the primary source of NADPH in both glucose and xylose. Upon glucose depletion, gluconeogenesis is necessary to generate glucose-6-phosphate, the substrate of Zwf1p. We re-established the NADPH regeneration pathway by replacing the endogenous NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene TDH3 with heterogenous NADP + -GAPDH genes GDH, gapB, and GDP1. Among the resulting strains, the strain BZP1 (zwf1Δ, tdh3::GDP1) exhibited a similar xylose consumption rate before glucose depletion, but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001, and the ethanol yield for total consumed sugars of BZP1 was 13.5% higher than BSGX001. This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO2 release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1, therefore, to determine the competitive equilibrium between glucose-xylose co-metabolism. This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.

4F-Indole Enhances the Susceptibility of Pseudomonas aeruginosa to Aminoglycoside Antibiotics.

Infections caused by multidrug-resistant bacteria are becoming increasingly serious. The aminoglycoside antibiotics have been widely used to treat severe Gram-negative bacterial infections. Here, we reported that a class of small molecules, namely, halogenated indoles, can resensitize Pseudomonas aeruginosa PAO1 to aminoglycoside antibiotics such as gentamicin, kanamycin, tobramycin, amikacin, neomycin, ribosomalin sulfate, and cisomicin. We selected 4F-indole as a representative of halogenated indoles to investigate its mechanism and found that the two-component system (TCS) PmrA/PmrB inhibited the expression of multidrug efflux pump MexXY-OprM, allowing kanamycin to act intracellularly. Moreover, 4F-indole inhibited the biosynthesis of several virulence factors, such as pyocyanin, type III secretion system (T3SS), and type VI secretion system (T6SS) exported effectors, and reduced the swimming and twitching motility by suppressing the expression of flagella and type IV pili. This study suggests that the combination of 4F-indole and kanamycin can be more effective against P. aeruginosa PAO1 and affect its multiple physiological activities, providing a novel insight into the reactivation of aminoglycoside antibiotics. IMPORTANCE Infections caused by Pseudomonas aeruginosa have become a major public health crisis. Its resistance to existing antibiotics causes clinical infections that are hard to cure. In this study, we found that halogenated indoles in combination with aminoglycoside antibiotics could be more effective than antibiotics alone against P. aeruginosa PAO1 and preliminarily revealed the mechanism of the 4F-indole-induced regulatory effect. Moreover, the regulatory effect of 4F-indole on different physiological behaviors of P. aeruginosa PAO1 was analyzed by combined transcriptomics and metabolomics. We explain that 4F-indole has potential as a novel antibiotic adjuvant, thus slowing down the further development of bacterial resistance.

QseB/QseC: a two-component system globally regulating bacterial behaviors.

QseB/QseC is a two-component system that is involved in the regulation of multiple bacterial behaviors by regulating quorum sensing, bacterial pathogenicity, and antibiotic resistance. Thus, QseB/QseC could provide a target for new antibiotic development. Recently, QseB/QseC has been found to confer survival advantages to environmental bacteria under stress conditions. The molecular mechanistic understanding of QseB/QseC has become an active area of research and revealed some emerging themes, including a deeper understanding of QseB/QseC regulation in different pathogens and environmental bacteria, the functional difference of QseB/QseC among species, and the possibility of analyzing QseB/QseC evolution. Here, we discuss the progression of QseB/QseC studies and describe several unresolved issues and future directions. Resolving these issues is among the challenges of future QseB/QseC studies.

Pseudomonas aeruginosa Resists Phage Infection via Eavesdropping on Indole Signaling.

Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA, pilB, and pilQ, which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

Characterization and expression analysis of MATEs in Cannabis sativa L. reveals genes involving in cannabinoid synthesis.

The medicinal plant Cannabis sativa L. (C. sativa) accumulates plant cytotoxic but medicinally important cannabinoids in glandular trichomes and flowers of female plants. Although the major biosynthetic pathway of cannabinoids has been revealed, their transportation mechanism is still unknown. Multidrug and toxic compound extrusion proteins (MATEs) can transport plant metabolites, ions and phytohormones intra and inter-cellularly. MATEs could have the potential to translocate cannabinoids or their synthetic intermediates to cellular compartment, thus protecting them from unwanted modifications and cytotoxicity. In this study, we performed a genome-wide identification and expression analysis of Cannabis sativa MATEs (CsMATEs) and revealed 42 CsMATEs that were classified phylogenetically into four conserved subfamilies. Forty-two CsMATEs were unevenly distributed on 10 chromosomes, with 50% CsMATEs were physically adjacent to at least one another CsMATEs and 83% CsMATEs localized on plasma membrane. Tandem duplication is the major evolutionary driving force for CsMATEs expansion. Real-time quantitative PCR revealed CsMATE23, CsMATE28 and CsMATE34 mainly expressed in flower, whereas CsMATE17 and CsMATE27 showed strong transcription in root. Light responsive cis-acting element was most abundant in promoters of CsMATE23, CsMATE28 and CsMATE34. Finally, the contents of cannabinoids and corresponding biosynthetic intermediates as well as expressions of CsMATE28 and CsMATE34 were determined under UV-B treatment, among which strong correlation was found. Our results indicates that CsMATEs might involve in biosynthesis of cannabinoids and has the potential to be used in heterologous production of cannabinoids.

Developing a new treatment for superficial fungal infection using antifungal Collagen-HSAF dressing.

Fungal pathogens are common causes of superficial clinical infection. Their increasing drug resistance gradually makes existing antifungal drugs ineffective. Heat stable antifungal factor (HSAF) is a novel antifungal natural product with a unique structure. However, the application of HSAF has been hampered by very low yield in the current microbial producers and from extremely poor solubility in water and common solvents. In this study, we developed an effective mode of treatment applying HSAF to superficial fungal infections. The marine-derived Lysobacter enzymogenes YC36 contains the HSAF biosynthetic gene cluster, which we activated by the interspecific signaling molecule indole. An efficient extraction strategy was used to significantly improve the purity to 95.3%. Scanning electron microscopy images revealed that the Type I collagen-based HSAF (Col-HSAF) has a transparent appearance and good physical properties, and the in vitro sustained-release effect of HSAF was maintained for more than 2 weeks. The effective therapeutic concentration of Col-HSAF against superficial fungal infection was explored, and Col-HSAF showed good biocompatibility, lower clinical scores, mild histological changes, and antifungal capabilities in animals with Aspergillus fumigatus keratitis and cutaneous candidiasis. In conclusion, Col-HSAF is an antifungal reagent with significant clinical value in the treatment of superficial fungal infections.

MomL inhibits bacterial antibiotic resistance through the starvation stringent response pathway.

Antibiotic resistance in gram-negative pathogens has become one of the most serious global public health threats. The role of the N-acyl homoserine lactone (AHL)-mediated signaling pathway, which is widespread in gram-negative bacteria, in the bacterial resistance process should be studied in depth. Here, we report a degrading enzyme of AHLs, MomL, that inhibits the antibiotic resistance of Pseudomonas aeruginosa through a novel mechanism. The MomL-mediated reactivation of kanamycin is highly associated with the relA-mediated starvation stringent response. The degradation of AHLs by MomL results in the inability of LasR to activate relA, which, in turn, stops the activation of downstream rpoS. Further results show that rpoS directly regulates the type VI secretion system H2-T6SS. Under MomL treatment, inactivated RpoS fails to regulate H2-T6SS; therefore, the expression of effector phospholipase A is reduced, and the adaptability of bacteria to antibiotics is weakened. MomL in combination with kanamycin is effective against a wide range of gram-negative pathogenic bacteria. Therefore, this study reports a MomL-antibiotic treatment strategy on antibiotic-resistant bacteria and reveals its mechanism of action.

Ellagic Acid Inhibits Trichophyton rubrum Growth via Affecting Ergosterol Biosynthesis and Apoptotic Induction.

BACKGROUND: Trichophyton rubrum, among other dermatophytes, is a major causative agent for superficial dermatomycoses like onychomycosis and tinea pedis, especially among pediatric and geriatric populations. Ellagic acid (EA) and shikonin (SK) have been reported to have many bioactivities, including antifungal activity. However, the mechanism of EA and SK on Trichophyton rubrum has not yet been reported. OBJECTIVES: The purposes of this study were to evaluate the antifungal activities of EA and SK against Trichophyton rubrum and to illuminate the underlying action mechanisms. METHODS: The effect of EA (64, 128, and 256 µg/mL) and SK (8, 4, and 2 µg/mL) on Trichophyton rubrum was investigated with different doses via detecting cell viability, ultrastructure with using a scanning electron microscope (SEM), cell apoptosis and necrosis by using the flow cytometry instrument technique (FCIT), and the ergosterol biosynthesis pathway-related fungal cell membrane key gene expressions in vitro. RESULTS: SEM detection revealed that the T. rubrum cell surface was shrivelled, folded, and showed deformation and expansion, visible surface peeling, and broken hyphae, and cell contents overflowed after being treated with EA and SK; the cell apoptosis rate was significantly increased in dose-dependent manner after T. rubrum was treated with EA and SK; the qPCR results showed that mRNA expression of MEP4 and SUB1 was downregulated in EA- and SK-treated groups. CONCLUSIONS: Overall, our results revealed the underlying antifungal mechanism of EA and SK, which may be related to the destruction of the fungal cell membrane and inhibition of C14 demethylase and the catalytic rate of squalene cyclooxidase in the ergosterol biosynthesis pathway via downregulation of MEP4 and SUB1, suggesting that EA and SK have the potential to be developed further as a natural antifungal agent for clinical use.

Enhanced Activity against Multidrug-Resistant Bacteria through Coapplication of an Analogue of Tachyplesin I and an Inhibitor of the QseC/B Signaling Pathway.

Tachyplesin I (TPI) is a cationic ß-hairpin antimicrobial peptide with broad-spectrum, potent antimicrobial activity. In this study, the all d-amino acid analogue of TPI (TPAD) was synthesized, and its structure and activity were determined. TPAD has comparable antibacterial activity to TPI on 14 bacterial strains, including four drug-resistant bacteria. Importantly, TPAD has significantly improved stability against enzymatic degradation and decreased hemolytic activity compared to TPI, indicating that it has better therapeutic potential. The induction of bacterial resistance using low concentrations of TPAD resulted in the activation of the QseC/B two-component system. Deletion of this system resulted in at least five-fold improvement of TPAD activity, and the combined use of TPAD with LED209, a QseC/B inhibitor, significantly enhanced the bactericidal effect against three classes of multidrug-resistant bacteria.

Effects of GLP-1 receptor analogue liraglutide and DPP-4 inhibitor vildagliptin on the bone metabolism in ApoE-/- mice.

BACKGROUND: It has been reported that glucagon-like peptide-1 (GLP-1) can alleviate diabetic osteoporosis (DOP). This study was to investigate the effects of GLP-1RA liraglutide and dipeptidyl peptdase-4 (DPP-4) inhibitor vildagliptin on the advanced glycation end products (AGEs)-induced bone injury in ApoE-/- mice with euglycemia. METHODS: The bone markers OC, PINP, PTH, TRACP and CTX, the mRNA and protein expressions of RAGE in the femur, and the femoral morphology index were determined to evaluate whether the osteoporosis was improved by liraglutide or vildagliptin. RESULTS: AGEs adversely affected the bone metabolism, characterized by reduced OC and increased CTX. However, vildagliptin reduced AGEs and increased OC, and liraglutide significantly decreased AGEs and PTH. Both vildagliptin and liraglutide had no effects on the bone metrology and RAGE expression in the femurs of ApoE-/- mice. CONCLUSIONS: The elevated AGEs may exacerbate osteogenesis and increase bone resorption, and vildagliptin/liraglutide may improve bone metabolism.

Antifungal Activity of Gallic Acid In Vitro and In Vivo.

Gallic acid (GA) is a polyphenol natural compound found in many medicinal plant species, including pomegranate rind (Punica granatum L.), and has been shown to have antiinflammatory and antibacterial properties. Pomegranate rind is used to treat bacterial and fungal pathogens in Uyghur and other systems of traditional medicine, but, surprisingly, the effects of GA on antifungal activity have not yet been reported. In this study, we aimed to investigate the inhibitory effects of GA on fungal strains both in vitro and in vivo. The minimal inhibitory concentration (MIC) was determined by the NCCLS (M38-A and M27-A2) standard method in vitro, and GA was found to have a broad spectrum of antifungal activity, with MICs for all the tested dermatophyte strains between 43.75 and 83.33 µg/mL. Gallic acid was also active against three Candida strains, with MICs between 12.5 and 100.0 µg/mL. The most sensitive Candida species was Candida albicans (MIC = 12.5 µg/mL), and the most sensitive filamentous species was Trichophyton rubrum (MIC = 43.75 µg/mL), which was comparable in potency to the control, fluconazole. The mechanism of action was investigated for inhibition of ergosterol biosynthesis using an HPLC-based assay and an enzyme linked immunosorbent assay. Gallic acid reduced the activity of sterol 14α-demethylase P450 (CYP51) and squalene epoxidase in the T. rubrum membrane, respectively. In vivo model demonstrated that intraperitoneal injection administration of GA (80 mg/kg d) significantly enhanced the cure rate in a mice infection model of systemic fungal infection. Overall, our results confirm the antifungal effects of GA and suggest a mechanism of action, suggesting that GA has the potential to be developed further as a natural antifungal agent for clinical use. Copyright © 2017 John Wiley & Sons, Ltd.

Feasibility and safety of functional cholecystectomy by pure NOTES: a pilot animal study.

BACKGROUND: NOTES cholecystectomy has become one of the hottest areas of research. But most of the cases need the assistance of the laparoscope. This study is conducted to evaluate the feasibility and safety of a newly proposed operative method-functional cholecystectomy by pure NOTES. MATERIALS AND METHODS: The functional cholecystectomy was performed on eight female miniature pigs. An incision was made on the vaginal wall, and an endoscope was inserted into the peritoneal cavity to create a pneumoperitoneum to expose the intra-abdominal viscera, gallbladder, and cystic duct. The cystic duct was isolated and closed with a clip. Then, an injection needle was inserted into the gallbladder to suck up the bile. After the gallbladder was washed with saline, an incision was made on the wall of the gallbladder, and the tip of the endoscope was inserted into the gallbladder cavity. After the endoscope was withdrawn, the gallbladder incision was closed with clips in four pigs and was suspended in the other four pigs. The vaginal incision was closed with clips. All the animals were closely monitored and euthanized 28 days after the procedure. Necropsy was performed. RESULTS: The functional cholecystectomy was successfully completed in all eight pigs. No severe intraoperative complications occurred. The animals recovered well postoperatively. At necropsy, no macroscopic signs of intraperitoneal infection or bile leakage in the peritoneal cavity were observed, and the clips were still present on the cystic duct in a good position in all cases. The gallbladder incision healed, with no sign of bile leakage or injury to the adjacent organs. CONCLUSIONS: We successfully performed the functional cholecystectomy by transvaginal approach on pigs, which appears to be feasible, safe, and convenient. Functional cholecystectomy provides a new fitting path to pure NOTES.

[Correlation study on sensory gating P50 and Wisconsin card sorting test in schizophrenics with violent behaviors].

OBJECTIVE: To analyze the variations of sensory gating and executive functions in schizophrenics with violent behaviors, and the relationship between sensory gating and executive functions. METHODS: Thirty-five schizophrenics with violent behaviors (Group V) and 32 healthy control persons (Group C) were recorded by P50 with EGI 256 high density EEG. They were also examined by Wisconsin card sorting test (WCST) and followed up for 3 months. The relationship of sensory gating P50 and WCST was evaluated by Person's analysis. RESULTS: (1) Compared with Group C, Group V showed a higher amplitude of S2-P50 [baseline: (1.8 ± 1.1) vs (0.9 ± 0.6) µV; 3-month treatment: (1.9 ± 1.6) µV]. There was a higher S2/S1 ratio, a lower S1-S2 difference and 100 × (1-S2/S1) at baseline and after 3 months (P < 0.05) [baseline: S2/S1, S1-S2 and 100 × (1-S2/S1) in both groups respectively [(89 ± 49) vs (42 ± 25), (0.7 ± 2.5) vs (1.4 ± 1.2), (11 ± 49) vs (58 ± 25); 3 months treatment: (170 ± 277, -0.0 ± 1.8, -70 ± 277)]; (2) At baseline and after a 3-month treatment, two groups had no difference in learning [baseline (t = -0.585, P = 0.561), 3 months (t = -0.021, P = 0.983)] (P > 0.05) and there were significant differences in other indices (P < 0.05); (3) No relationship was found between S2/S1 ratio, S1-S2 and WCST in two groups at baseline and after a 3-month treatment by Person's analysis. CONCLUSIONS: The schizophrenics with violent behaviors have sensory gating deficits and abnormal executive functions before and after treatment. Sensory gating and executive functions may reflect different brain functions in schizophrenics.

[Sensory gating P50 in schizophrenic patients with and without homicide].

OBJECTIVE: To investigate the change of sensory gating P50 in schizophrenic patients with and without homicide. METHODS: The auditory evoked potentials P50 were recorded from 26 schizophrenic patients with homicide (Sch group), 27 schizophrenic patients without homicide (non-Sch group) and 32 normal controls (NC) using conditioning/testing paradigm presented with auditory double click stimuli by EGI 256 dense array. And the same time, their clinical symptoms were evaluated by positive and negative symptom scale (PANSS). RESULTS: (1) Compared with NC, two Sch groups showed no significant difference in amplitude and latency of S1-P50 [amplitude: NC, Sch group, non-Sch group at Fz: (2.4 + or - 1.6) microV, (2.5 + or - 1.5) microV, (3.4 + or - 2.7) microV; latency: NC, Sch group, non-Sch group at Fz: (68 + or - 19) ms, (67 + or - 20) ms, (61 + or - 19) ms; respectively], but a higher amplitude and delayed latency of S2-P50 [amplitude: NC, Sch group, non-Sch group at Fz: (0.8 + or - 0.7) microV, (2.5 + or - 1.6) microV, (3.3 + or - 2.2) microV; latency: NC, Sch group, non-Sch group at Fz: (50 + or - 26) ms, (75 + or - 19) ms and (70 + or - 24) ms respectively] (P < 0.01), and no significant difference in amplitude and latency of S2-P50 between two Sch groups. (2) Compared with NC, two Sch groups showed a higher S2/S1 ratio [NC, Sch group, non-Sch group at Fz: 35 + or - 26, 153 + or - 137, 125 + or - 85, respectively], lower S2-S1 [NC, Sch group, non Sch group at Fz: 1.69 + or - 1.55, 0.08 + or - 2.41 and 0.17 + or - 2.30, respectively] and 100 (1-S2/S1) [NC, Sch group, non-Sch group at Fz: 65 + or - 26, -53 + or - 137 and -25 + or - 85 respectively] (P < 0.01). And there was no significant difference in S2/S1 ratio, S2-S1 and 100 (1-S2/S1) between two Sch groups. (3) Two Sch groups showed no significant difference in PANSS total, P scale, N scale, and G scale [Sch group: (110 + or - 27), (26 + or - 10), (29 + or - 7), (55 + or - 12); non Sch group: (105 + or - 27), (24 + or - 8), (28 + or - 10) and (53 + or - 12) respectively] (P > 0.05), and no significant correlation with S2/S1 ratio, S2-S1 and 100(1-S2/S1) (P > 0.05). CONCLUSION: Sensory gating deficit exists in schizophrenic patients with and without homicide. And it can be quantified by measuring auditory evoked potential P50, but sensory gating P50 has no difference between schizophrenic patients with and without homicide.