Biodegradation and valorization of feather waste using the keratinase-producing bacteria and their application in environmentally hazardous industrial processes.

Poultry feathers are widely discarded as waste worldwide and are considered an environmental pollutant and a reservoir of pathogenic bacteria. Therefore, developing sustainable and environmentally friendly methods for managing feather waste is one of the important environmental protection requirements. In this study, we investigated a rapid and eco-friendly method for the degradation and valorization of feather waste using keratinase-producing Pseudomonas geniculata H10, and evaluated the applicability of keratinase in environmentally hazardous chemical processes. Strain H10 completely degraded chicken feathers within 48 h by producing keratinase using them as sources of carbon, nitrogen, and sulfur. The culture contained a total of 402.8 µM amino acids, including 8 essential amino acids, which was higher than the chemical treatment. Keratinase was a serine-type metalloprotease with optimal temperature and pH of 30 °C and 9, respectively, and showed relatively high stability at 10-40 °C and pH 3-10. Keratinase was also able to degrade various insoluble keratins such as duck feathers, wool, human hair, and nails. Furthermore, keratinase exhibited more efficient depilation and wool modification than chemical treatment, as well as novel functionalities such as nematicidal and exfoliating activities. This suggests that strain H10 is a promising candidate for the efficient degradation and valorization of feather waste, as well as the improvement of current industrial processes that use hazardous chemicals.

Dysbiosis of Fecal Microbiota in Tg2576 Mice for Alzheimer's Disease during Pathological Constipation.

Tg2576 transgenic mice for Alzheimer's disease (AD) exhibited significant phenotypes for neuropathological constipation, but no research has been conducted on the association of the fecal microbiota with dysbiosis. The correlation between fecal microbiota composition and neuropathological constipation in Tg2576 mice was investigated by examining the profile of fecal microbiota and fecal microbiota transplantation (FMT) in 9-10-month-old Tg2576 mice with the AD phenotypes and constipation. Several constipation phenotypes, including stool parameters, colon length, and histopathological structures, were observed prominently in Tg2576 mice compared to the wild-type (WT) mice. The fecal microbiota of Tg2576 mice showed decreases in Bacteroidetes and increases in the Firmicutes and Proteobacteria populations at the phylum level. The FMT study showed that stool parameters, including weight, water content, and morphology, decreased remarkably in the FMT group transplanted with a fecal suspension of Tg2576 mice (TgFMT) compared to the FMT group transplanted with a fecal suspension of WT mice (WFMT). The distribution of myenteric neurons and the interstitial cells of Cajal (ICC), as well as the enteric nervous system (ENS) function, remained lower in the TgFMT group. These results suggest that the neuropathological constipation phenotypes of Tg2576 mice may be tightly linked to the dysbiosis of the fecal microbiota.

Chrysanthemum boreale Makino Inhibits Oxidative Stress-Induced Neuronal Damage in Human Neuroblastoma SH-SY5Y Cells by Suppressing MAPK-Regulated Apoptosis.

Oxidative stress has been demonstrated to play a pivotal role in the pathological processes of many neurodegenerative diseases. In the present study, we demonstrated that Chrysanthemum boreale Makino extract (CBME) suppresses oxidative stress-induced neurotoxicity in human neuroblastoma SH-SY5Y cells and elucidated the underlying molecular mechanism. Our observations revealed that CBME effectively protected neuronal cells against H2O2-induced cell death by preventing caspase-3 activation, Bax upregulation, Bcl-2 downregulation, activation of three mitogen-activated protein kinases (MAPKs), cAMP response element-binding protein (CREB) and NF-κB phosphorylation, and iNOS induction. These results provide evidence that CBME has remarkable neuroprotective properties in SH-SY5Y cells against oxidative damage, suggesting that the complementary or even alternative role of CBME in preventing and treating neurodegenerative diseases is worth further studies.

Characterization and applicability of novel alkali-tolerant carbonatogenic bacteria as environment-friendly bioconsolidants for management of concrete structures and soil erosion.

Cracking and erosion are critical factors that reduce the mechanical properties and stability of concrete structures and soil, respectively. They are recognized worldwide as severe disasters causing the collapse of many structures including stone heritage and dams, and landslides. Therefore, it is essential to propose effective and environment-friendly management methods to prevent them. Carbonatogenesis has recently received considerable attention as a reliable biological process for remediating cracks in calcareous structures, stabilizing loose soils, and sequestering CO2 in the environment. Isolating and characterizing carbonatogenic bacteria with excellent performance is crucial for applying this process to the field of environmental and civil engineering. The aim of this study was to isolate new CaCO3-precipitating bacteria and investigate various properties for their use as bioconsolidants. Furthermore, the possibility of restoring damaged structures and stabilizing loose sandy soil using isolated strain was investigated. Strain LC13 with urease and CaCO3-precipitating activity was isolated from limestone cave soil in Korea and identified as Arthrobacter sulfureus by phenotypic characterization and 16S rRNA gene analysis. Although cell growth was observed after an adaptation period at pH 11, strain LC13 grew well at pH 7-11, indicating alkali tolerance. The optimal conditions for CaCO3 precipitation were 1.0% yeast extract, 2.5% urea, 0.35% NaHCO3, and 400 mM CaCl2, with an initial pH of 6.5 at 30 °C. Under optimized conditions, maximal CaCO3 (22.92 ± 0.14 g/l) precipitated after 3 days, which was 10.8-fold higher than the value in a urea-CaCl2 medium. CaCO3 precipitation by strain LC13 was associated with an increased pH due to ureolysis and protein deamination. Using an optimized medium as a cementation solution, strain LC13 completely remediated 340-760 µm wide cracks over 3 days, and also restored the spalling of concrete surfaces. Furthermore, the sand treated with LC13 solidified with a surface strength of 14.9 kPa. Instrumental analysis confirmed that the crystals precipitated were a mixture of CaCO3 polymorphs composed of rhombohedral calcite and spherical vaterite. These results suggest that A. sulfureus LC13 may be useful for implementing sustainable biorestoration and environmental management technologies such as the in situ remediation of structural cracks and in situ prevention of soil erosion.

Overexpression of the PanaxginsengCYP703 Alters Cutin Composition of Reproductive Tissues in Arabidopsis.

Cytochrome P450 (CYP) catalyzes a wide variety of monooxygenation reactions in plant primary and secondary metabolisms. Land plants contain CYP703, belonging to the CYP71 clan, which catalyzes the biochemical pathway of fatty acid hydroxylation, especially in male reproductive tissues. Korean/Asian ginseng (Panax ginseng Meyer) has been regarded as one of important medicinal plant for a long time, however the molecular mechanism is less known on its development. In this study, we identified and characterized a CYP703A gene in P. ginseng (PgCYP703A4), regarding reproductive development. PgCYP703A4 shared a high-sequence identity (81-83%) with predicted amino acid as CYP703 in Dancus carota, Pistacia vera, and Camellia sinensis as well as 76% of amino acid sequence identity with reported CYP703 in Arabidopsis thaliana and 75% with Oryza sativa. Amino acid alignment and phylogenetic comparison of P. ginseng with higher plants and known A. thaliana members clearly distinguish the CYP703 members, each containing the AATDTS oxygen binding motif and PERH as a clade signature. The expression of PgCYP704B1 was only detected in P. ginseng flower buds, particularly in meiotic cells and the tapetum layer of developing anther, indicating the conserved role on male reproduction with At- and Os- CYP703. To acquire the clue of function, we transformed the PgCYP703A4 in A. thaliana. Independent overexpressing lines (PgCYP703A4ox) increased silique size and seed number, and altered the contents of fatty acids composition of cutin monomer in the siliques. Our results indicate that PgCYP703A4 is involved in fatty acid hydroxylation which affects cutin production and fruit size.

Enzyme-mediated biocalcification by a novel alkaliphilic Bacillus psychrodurans LC40 and its eco-friendly application as a biosealant for crack healing.

Biocalcification is a natural biochemical process, which has been regarded as a promising method for sequestering heavy metals or carbon dioxide in the environment, healing cracks in concrete structures, and stabilizing soil. One of the key factors in this process is calcium carbonate-producing bacteria. The purpose of this study was to maximize the production of calcium carbonate by alkaliphilic Bacillus psychrodurans LC40 isolated from a limestone cave, by manipulating the medium composition for fast and non-detrimental crack healing, and to investigate the mechanism of its production. Strain LC40 could grow well in the strongly alkaline region (pH 9.5-11), indicating its alkaliphilic nature. The optimal medium for calcium carbonate production contained 2% tryptone, 1.5% urea, 0.15% NaHCO3, and 150 mM calcium formate (pH 6). Using this medium, the yield of calcium carbonate at 72 h was approximately 8.6-fold higher than that obtained through Urea-CaCl2 medium. In this culture, the urease and carbonic anhydrase activities were observed simultaneously, and the pH of the medium was found to have increased to 9.4, leading to maximum calcium carbonate production. This suggests that this pH value is achieved by the synergistic action of the two enzymes, resulting in a high calcium carbonate yield. The crystals were characterized by FESEM, EDS and XRD, which confirmed the production of rhombohedral and spherical calcium carbonate crystals containing vaterite and calcite. Strain LC40 completely healed a 0.75 mm wide crack in a very short time of 3 days using the optimized medium as a cementation solution. Our findings indicate that B. psychrodurans LC40 could be a promising candidate for the development of eco-friendly biosealant applicable to environmentally stressed concrete structures.

Dysbiosis of Fecal Microbiota From Complement 3 Knockout Mice With Constipation Phenotypes Contributes to Development of Defecation Delay.

Significant phenotypes for constipation were detected in complement 3 (C3) knockout (KO) mice, although no research has been conducted on an association with alteration of gut microbiota. To investigate the effects of dysbiosis on fecal microbiota from C3 KO mice with constipation, the composition of fecal microbiota was characterized in mid-colons of 16-week-old C3 KO mice, and their function for defecation delay development was examined after fecal microbiota transplantation (FMT) of C3 KO mice. Some significant alterations in constipation phenotypes, including stool parameters and histopathological structure, were detected in 16-week-old C3 KO mice compared to those of wild-type (WT) mice. Fecal microbiota of C3 KO mice exhibited decreases in Anaerocolumna, Caecibacterium, Christensenella, Kineothrix, and Oscillibacter populations and increases in Prevotellamassilia, Reuthenibacterium, Prevotella, Eubacterium, Culturomica, Bacteroides, and Muribaculum populations. In FMT study, key stool parameters, including weight and water content, were remarkably declined in a transplanted KO (KFMT) group of antibiotics-induced depletion of microbiota (AiDM)-WT and AiDM-KO mice, and a similar change was observed in fecal morphology. However, intestine length decreased in only the KFMT group of AiDM-WT mice compared with that of AiDM-KO mice. The mucosal layer and muscle thickness were commonly decreased in the KFMT group of AiDM-WT and AiDM-KO mice, and significant alterations in the crypt structure of Lieberkuhn and molecular regulators, including AQP8, C-kit, and 5-HT, were observed in the same group. Taken together, results of the present study indicate that dysbiosis of fecal microbiota from C3 KO mice with constipation phenotypes has a key role in the induction and regulation of defecation delay.

Characterization of a Novel CaCO3-Forming Alkali-Tolerant Rhodococcus erythreus S26 as a Filling Agent for Repairing Concrete Cracks.

Biomineralization, a well-known natural phenomenon associated with various microbial species, is being studied to protect and strengthen building materials such as concrete. We characterized Rhodococcus erythreus S26, a novel urease-producing bacterium exhibiting CaCO3-forming activity, and investigated its ability in repairing concrete cracks for the development of environment-friendly sealants. Strain S26 grown in solid medium formed spherical and polygonal CaCO3 crystals. The S26 cells grown in a urea-containing liquid medium caused culture fluid alkalinization and increased CaCO3 levels, indicating that ureolysis was responsible for CaCO3 formation. Urease activity and CaCO3 formation increased with incubation time, reaching a maximum of 2054 U/min/mL and 3.83 g/L, respectively, at day four. The maximum CaCO3 formation was achieved when calcium lactate was used as the calcium source, followed by calcium gluconate. Although cell growth was observed after the induction period at pH 10.5, strain S26 could grow at a wide range of pH 4-10.5, showing its high alkali tolerance. FESEM showed rhombohedral crystals of 20-60 µm in size. EDX analysis indicated the presence of calcium, carbon, and oxygen in the crystals. XRD confirmed these crystals as CaCO3 containing calcite and vaterite. Furthermore, R. erythreus S26 successfully repaired the artificially induced large cracks of 0.4-0.6 mm width.

Therapeutic Applications of Type 2 Diabetes Mellitus Drug Metformin in Patients with Osteoarthritis.

Type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) are common chronic diseases that frequently co-exist. The link between OA and T2DM is attributed to common risk factors, including age and obesity. Several reports suggest that hyperglycemia and accumulated advanced glycosylation end-products might regulate cartilage homeostasis and contribute to the development and progression of OA. Metformin is used widely as the first-line treatment for T2DM. The drug acts by regulating glucose levels and improving insulin sensitivity. The anti-diabetic effects of metformin are mediated mainly via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is an energy sensing enzyme activated directly by an increase in the AMP/ATP ratio under conditions of metabolic stress. Dysregulation of AMPK is strongly associated with development of T2DM and metabolic syndrome. In this review, we discuss common risk factors, the association between OA and T2DM, and the role of AMPK. We also address the adaptive use of metformin, a known AMPK activator, as a new drug for treatment of patients with OA and T2DM.

Effects of mycosubtilin homolog algicides from a marine bacterium, Bacillus sp. SY-1, against the harmful algal bloom species Cochlodinium polykrikoides.

The marine bacterium, Bacillus sp. SY-1, produced algicidal compounds that are notably active against the bloom-forming alga Cochlodinium polykrikoides. We isolated three algicidal compounds and identified these as mycosubtilins with molecular weights of 1056, 1070, and 1084 (designated MS 1056, 1070, and 1084, respectively), based on amino acid analyses and 1H, 13C, and two-dimensional nuclear magnetic resonance spectroscopy, including 1H-15N heteronuclear multiple bond correlation analysis. MS 1056 contains a ß-amino acid residue with an alkyl side chain of C15, which has not previously been seen in known mycosubtilin families. MS 1056, 1070, and 1084 showed algicidal activities against C. polykrikoides with 6-h LC50 values of 2.3 ± 0.4, 0.8 ± 0.2, and 0.6 ± 0.1 µg/ml, respectively. These compounds also showed significant algicidal activities against other harmful algal bloom species. In contrast, MS 1084 showed no significant growth inhibitory effects against other organisms, including bacteria and microalgae, although does inhibit the growth of some fungi and yeasts. These observations imply that the algicidal bacterium Bacillus sp. SY-1 and its algicidal compounds could play an important role in regulating the onset and development of harmful algal blooms in natural environments.

Nitric Oxide-Releasing Bacterial Cellulose/Chitosan Crosslinked Hydrogels for the Treatment of Polymicrobial Wound Infections.

Polymicrobial wound infections are a major cause of infectious disease-related morbidity and mortality worldwide. In this study, we prepared a nitric oxide (NO)-releasing oxidized bacterial cellulose/chitosan (BCTO/CHI) crosslinked hydrogel to effectively treat polymicrobial wound infections. Linear polyethyleneimine diazeniumdiolate (PEI/NO) was used as the NO donor. The aldehyde group of BCTO and the amine of CHI were used as crosslinked hydrogel-based materials; their high NO loading capacity and antibacterial activity on the treatment of polymicrobial-infected wounds were investigated. The blank and NO-loaded crosslinked hydrogels, namely BCTO-CHI and BCTO-CHI-PEI/NO, were characterized according to their morphologies, chemical properties, and drug loading. BCTO-CHI-PEI/NO exhibited sustained drug release over four days. The high NO loading of BCTO-CHI-PEI/NO enhanced the bactericidal efficacy against multiple bacteria compared with BCTO-CHI. Furthermore, compared with blank hydrogels, BCTO-CHI-PEI/NO has a favorable rheological property due to the addition of a polymer-based NO donor. Moreover, BCTO-CHI-PEI/NO significantly accelerated wound healing and re-epithelialization in a mouse model of polymicrobial-infected wounds. We also found that both crosslinked hydrogels were nontoxic to healthy mammalian fibroblast cells. Therefore, our data suggest that the BCTO-CHI-PEI/NO developed in this study improves the efficacy of NO in the treatment of polymicrobial wound infections.

Constipation in Tg2576 mice model for Alzheimer's disease associated with dysregulation of mechanism involving the mAChR signaling pathway and ER stress response.

BACKGROUND: Although constipation has been researched in various neurological disorders, including Parkinson's disease (PD) and spinal cord injury (SCI), the pathological mechanism of this symptom has not been investigated in Alzheimer's disease (AD) associated with loss of nerve cells in the brain. This study was undertaken to gain scientific evidences for a molecular correlation between constipation and AD. METHODS: To understand the etiology, we measured alterations in various constipation parameters, muscarinic acetylcholine receptors (mAChRs) and endoplasmic reticulum (ER) stress response, in 11-month-old Tg2576 transgenic (Tg) mice showing AD-like phenotypes. RESULTS: A high accumulation of amyloid beta (Aß) peptides, a key marker of AD pathology, were detected in the cortex and hippocampus of Tg mice. Furthermore, significant alterations were observed in various constipation parameters including stool weight, histological structure, cytological structure and mucin secretion in Tg2576 mice. Moreover, M2 and M3 expression and the downstream signaling pathways of mAChRs were decreased in the Tg group, as compared with non-Tg (NT) group. Furthermore, activation of ER stress proteins and alteration of ER structure were also detected in the same group. CONCLUSIONS: The results of the present study provide strong novel evidence that the neuropathological constipation detected in Tg2576 mice is linked to dysregulation of the mAChR signaling pathways and ER stress response.

Therapeutic effects of a liquid bandage prepared with cellulose powders from Styela clava tunics and Broussonetia kazinoki bark: Healing of surgical wounds on the skin of Sprague Dawley rats.

Cellulose in different forms has extensively been applied in biomedical treatments, including scaffolding, tissue engineering and tissue formation. To evaluate the therapeutic effects of a liquid bandage (LB) prepared with cellulose powders from Styela clava tunics (SCT) and Broussonetia kazinoki bark (BSLB) for healing cutaneous wounds, the remedial effects of a low concentration (LoBSLB) and a high concentration (HiBSLB) of BSLB on skin regeneration and toxicity in Sprague Dawley rats. Results indicated that the total area of skin involved in the surgical wound was lower in the BSLB­treated group compared with the Vehicle­treated group at days 4­12, although some variations were observed in the HiBSLB­treated group. In addition, the BSLB­treated group showed significantly enhanced width of the re­epithelialization region and epidermal thickness when compared with the Vehicle­treated group. Furthermore, significant stimulation in the expression level of collagen­1 and the signaling pathway of VEGF after topical application of BSLB was indicated. No liver or kidney toxicities were detected for either doses of BSLB. Overall, the results of the present study suggest that BSLB accelerates the process of wound healing in surgical skin wounds of Sprague Dawley rats through stimulation of re­epithelialization and connective tissue formation, without any accompanying significant toxicity.

Bacterial indoleacetic acid-induced synthesis of colloidal Ag2O nanocrystals and their biological activities.

The biosynthesis and biological activity of colloidal Ag2O nanocrystals have not been well studied, although they have potential applications in many fields. For the first time, we developed a reducing agent free, cost-effective technique for Ag2O biosynthesis using Xanthomonas sp. P5. The optimal conditions for Ag2O synthesis were 50 °C, pH 8, and 2.5 mM AgNO3. Using these conditions the yield of Ag2O obtained at 10 h was about five times higher than that obtained at 12 h under unoptimized conditions. Ag2O was characterized by FESEM-EDS, TEM, dynamic light scattering, XRD, and UV-Visible spectroscopy. Indoleacetic acid produced by the strain P2 was involved in the synthesis of Ag2O. Ag2O exhibited a broad antimicrobial spectrum against several human pathogens. Furthermore, Ag2O exhibited 1,1-diphenyl-2-picrylhydrazyl (IC50 = 25.1 µg/ml) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (IC50 = 16.8 µg/ml) radical scavenging activities, and inhibited collagenase (IC50 = 27.9 mg/ml). Cytotoxicity of Ag2O was tested in fibroblast cells and found to be non-toxic, demonstrating biocompatibility.

The Anti-Inflammatory Effects of Fermented Herbal Roots of Asparagus cochinchinensis in an Ovalbumin-Induced Asthma Model.

Introduction: Roots of Asparagus cochinchinensis, which have pharmacologically active ingredients, have received great attention because they show good therapeutic effects for various inflammatory diseases without specific toxicity. This study investigated the anti-asthmatic effects of a butanol extract of Asparagus cochinchinensis roots that had been fermented with Weissella cibaria (BAW) and its possible underlying cholinergic regulation. Methods: Alterations of the anti-asthmatic markers and the molecular response factors were measured in an ovalbumin (OVA)-induced asthma model after treatment with BAW. Results: Treatment with BAW decreased the intracellular reactive oxygen species (ROS) production in lipopolysaccharides (LPS) activated RAW264.7 cells. The results of the animal experiments revealed lower infiltration of inflammatory cells and bronchial thickness, and a significant reduction in the number of macrophages and eosinophils, concentration of OVA-specific IgE, and expression of Th2 cytokines in the OVA + BAW treated group. In addition, a significant recovery of goblet cell hyperplasia, MMP-9 expression, and the VEGF signaling pathway was observed upon airway remodeling in the OVA + BAW treated group. Furthermore, these responses of BAW were linked to recovery of acetylcholine esterase (AChE) activity and muscarinic acetylcholine receptor (mAChR) M3 downstream signaling pathway in epithelial cells, smooth muscle cells, and afferent sensory nerves of OVA + BAW-treated mice. Conclusion: Overall, these findings are the first to provide evidence that the therapeutic effects of BAW can prevent airway inflammation and remodeling through the recovery of cholinergic regulation in structural cells and inflammatory cells of the chronic asthma model.

Biological synthesis and characterisation of silver nanoparticles using Pseudomonas geniculata H10 for pharmaceutical activity.

In the present study, silver nanoparticles (SNPs) were synthesised for the first time using Pseudomonas geniculata H10 as reducing and stabilising agents. The synthesis of SNPs was the maximum when the culture supernatant was treated with 2.5 mM AgNO3 at pH 7 and 40°C for 10 h. The SNPs were characterised by field emission scanning electron microscopy-energy-dispersive spectroscopy, transmission electron microscopy, dynamic light scattering, X-ray diffraction and UV-vis spectroscopy. Fourier transform infrared spectroscopy indicated the presence of proteins, suggesting they may have been responsible for the reduction and acted as capping agents. The SNPs displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 28.301 µg/ml) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate (IC50 = 27.076 µg/ml) radical scavenging activities. The SNPs exhibited a broad antimicrobial spectrum against several human pathogenic Gram-positive and Gram-negative bacteria and Candida albicans. The antimicrobial action of SNPs was due to cell deformation resulting in cytoplasmic leakage and subsequent lysis. The authors' results indicate P. geniculata H10 could be used to produce antimicrobial SNPs in a facile, non-toxic, cost-effective manner, and that these SNPs can be used as effective growth inhibitors in various microorganisms, making them applicable to various biomedical and environmental systems. As far as the authors are aware, this study is the first to describe the potential biomedical applications of SNPs synthesised using P. geniculata.

Quercetin promotes gastrointestinal motility and mucin secretion in loperamide-induced constipation of SD rats through regulation of the mAChRs downstream signal.

CONTEXT: Quercetin (QCT) has been known as a potential therapeutic strategy for gastrointestinal diseases because it contributes to the stabilization of mast cells, the prevention of histamine release and modulation of CaCC chloride channel. OBJECTIVE: We investigated the laxative effect and action mechanism of QCT in Lop-induced constipation model. MATERIALS AND METHODS: Constipation of SD rats was induced by subcutaneous injection of loperamide (Lop) (4 mg/kg weight) in 0.5% Tween 20 twice a day for three days. After 24 h, the constipation group was further treated with 1× PBS (Lop + Vehicle treated group), 10 mg/kg of QCT (Lop + LQCT treated group), 20 mg/kg of QCT (Lop + MQCT treated group) or 40 mg/kg QCT (Lop + HQCT treated group) at once. At 24 h after QCT treatment, the constipation phenotypes were measured and the transverse colon was collected from SD rats. RESULTS: The gastrointestinal motility, the number of stools and histological structures were significantly recovered in Lop + QCT treated group compared with the Lop + Vehicle treated group. Also, above activity of epithelial cells and smooth muscle cells were regulated by the mRNA expression of the muscarinic acetylcholine receptors M2 and M3 (mAChR M2 and M3) and some mediators of their downstream signalling pathway. Finally, laxative effects of QCT on mAChR signalling pathway were significantly inhibited by the treatment of mAChR antagonist in primary smooth muscle of rat intestine cells (pRISMCs). CONCLUSIONS: This study provides the first strong evidence that QCT can be considered an important candidate for improving chronic constipation induced by Lop treatment in animal models.

Improved biosynthesis of silver nanoparticles using keratinase from Stenotrophomonas maltophilia R13: reaction optimization, structural characterization, and biomedical activity.

In the present study, keratinase from Stenotrophomonas maltophilia R13 was used for the first time as a reducing agent for the eco-friendly synthesis of AgNPs. The keratinase produced by strain R13 was responsible for the reduction of silver ions and the subsequent formation of AgNPs. Maximum AgNP synthesis was achieved using 2 mM AgNO3 at pH 9 and 40 °C. Electron microscopy and dynamic light scattering analysis showed AgNPs were spherical and of average diameter ~ 8.4 nm. X-ray diffraction revealed that AgNPs were crystalline. FTIR indicated AgNPs were stabilized by proteins present in the crude enzyme solution of strain R13. AgNPs exhibited a broad antimicrobial spectrum against several pathogenic microorganisms, and the antimicrobial mechanism appeared to involve structural deformation of cells resulting in membrane leakage and subsequent lysis. AgNPs also displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 0.0112 mg/ml), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate radical scavenging (IC50 = 0.0243 mg/ml), and anti-collagenase (IC50 = 23.5 mg/ml) activities.

Ethyl acetate extract from Asparagus cochinchinensis exerts anti­inflammatory effects in LPS­stimulated RAW264.7 macrophage cells by regulating COX­2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity.

Asparagus cochinchinesis (A. cochinchinesis) is a medicine traditionally used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although numerous studies of the anti­inflammatory effects of A. cochinchinesis have been conducted, the underlying mechanisms of such effects in macrophages remain to be demonstrated. To investigate the mechanism of suppressive effects on the inflammatory response in macrophages, alterations of the nitric oxide (NO) level, the cell viability, inducible nitric oxide synthase (iNOS) and cyclooxygenase­2 (COX­2) expression levels, inflammatory cytokine expression, the mitogen-activated protein kinase (MAPK) signaling pathway, cell cycle arrest and reactive oxygen species (ROS) levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells following treatment with ethyl acetate extract from A. cochinchinesis root (EaEAC). RAW264.7 cells pretreated two different concentrations of EaEAC prior to LPS treatment exhibited no significant toxicity. The concentration of NO was significantly decreased in the EaEAC + LPS treated group compared with the vehicle + LPS treated group. A similar decrease in mRNA transcript level of COX­2, iNOS, pro-inflammatory cytokines [tumor necrosis factor­α and interleukin (IL)­1ß] and anti­inflammatory cytokines (IL­6 and IL­10) was detected in the EaEAC + LPS treated group compared with the vehicle + LPS treated group, although the decrease rate varied. Enhancement of the phosphorylation of MAPK family members following LPS treatment was partially rescued in the EaEAC pretreated group, and the cell cycle was arrested at the G2/M phase. Furthermore, the EaEAC pretreated group exhibited a reduced level of ROS generation compared with the vehicle + LPS treated group. Taken together, these results suggest that EaEAC suppresses inflammatory responses through inhibition of NO production, COX­2 expression and ROS production, as well as differential regulation of inflammatory cytokines and cell cycle in RAW264.7 cells. In addition, these results provide strong evidence to suggest that EaEAC may be considered as an important candidate for the treatment of particular inflammatory diseases.

Effects of different cellulose membranes regenerated from Styela clava tunics on wound healing.

The aim of this study was to investigate the therapeutic effects of three different cellulose membranes (CMs) manufactured from Styela clava tunics (SCTs) on the healing of cutaneous wounds. We examined the physical properties and therapeutic effects of three CMs regenerated from SCTs (referred to as SCT­ CMs), including normal CM (SCT­CM), freeze-dried SCT­CM (FSCT­CM) and sodium alginate-supplemented SCT­CM (ASCT­CM) on skin regeneration and angiogenesis using Sprague-Dawley (SD) rats. FSCT­CM exhibited an outstanding interlayered structure, a high tensile strength (1.64 MPa), low elongation (28.59%) and a low water vapor transmission rate (WVTR) compared with the other SCT-CMs, although the fluid uptake rate was maintained at a medium level. In the SD rats with surgically wounded skin, the wound area and score of wound edge were lower in the FSCT­CM-treated group than in the gauze (GZ)-treated group on days 3-6 and 12-14. In addition, a significant attenuation in the histopathological changes was observed in the FSCT­CM-treated group. Furthermore, the expression level of collagen-1 and the signaling pathway of transforming growth factor (TGF)-ß1 were significantly stimulated by the topical application of FSCT­CM. However, no signs of toxicity were detected in the livers or kidneys of the three SCT­CM-treated groups. Overall, our data indicate that the FSCT­CM may accelerate the process of wound healing in the surgically wounded skin of SD rats through the regulation of angiogenesis and connective tissue formation without inducing any specific toxicity.