Skin Barrier-Enhancing Effects of Dermabiotics HDB with Regulation of Skin Microbiota.

In the regulation of inflammatory responses and skin homeostasis, the skin and its microbiota are closely related. Studies have reported that lactic acid bacteria extracts can improve the skin condition and microbiota. In our previous study, we developed probiotic lysates, which are efficacious in improvement of human skin cells and the skin barrier. The skin-moisturizing effect of Dermabiotics HDB (HDB) prepared with Lactiplantibacillus plantarum, and the correlation between changes in the skin microbiota and moisture contents, were evaluated and analyzed in clinical trials. The clinical parameters on the cheeks of 21 female participants were measured using biophysical tools before and after (2 weeks) using HDB or control. The skin microbes were collected and identified using 16s rRNA gene sequencing. HDB significantly improved moisture intensity, transepidermal water loss (TEWL), and hot flush level on the cheek. The beta-diversity of the skin microbiota was different from that of the control in the unweighted UniFrac principal coordinate analysis after using HDB. The genus Lawsonella demonstrated a positive correlation with TEWL and a negative correlation with the moisture contents of the keratin layer, regardless of the use of HDB and control. Conversely, after HDB use, the genus Staphylococcus was increased and associated with a lower hot flush level, while the genera of the phylum Proteobacteria tended to decrease, which is associated with an improved skin condition. Overall, HDB showed clinically proven effects, including skin moisturization with regulation of the skin microbiota.

Engineering of a bacterial outer membrane vesicle to a nano-scale reactor for the biodegradation of ß-lactam antibiotics.

Bacterial outer membrane vesicles (OMVs) are small unilamellar proteoliposomes, which are involved in various functions including cell to cell signaling and protein excretion. Here, we have engineered the OMVs of Escherichia coli to nano-scaled bioreactors for the degradation of ß-lactam antibiotics. This was exploited by targeting a ß-lactamase (i.e., CMY-10) into the OMVs of a hyper-vesiculating E. coli BL21(DE3) mutant. The CMY-10-containing OMVs, prepared from the E. coli mutant cultures, were able to hydrolyze ß-lactam ring of nitrocefin and meropenem to a specific rate of 6.6 × 10-8 and 3.9 × 10-12 µmol/min/µm3 of OMV, which is approximately 100 and 600-fold greater than those of E. coli-based whole-cell biocatalsyts. Furthermore, CMY-10, which was encapsulated in the engineered OMVs, was much more stable against temperature and acid stresses, as compared to free enzymes in aqueous phase. The OMV-based nano-scaled reaction system would be useful for the remediation of a variety of antibiotics pollution for food and agricultural industry.

Gut Microbiota-Mediated Immunomodulatory Effects of Lactobacillus rhamnosus HDB1258 Cultured in the Lava Seawater in the Colitis Mouse Model.

The immunomodulatory effects of Lactobacillus rhamnosus HDB1258 were evaluated in mice with colitis induced by Klebsiella oxytoca (KO). L. rhamnosus HDB1258 was cultured in the lava seawater (LS) to improve its probiotic properties. It increased adhesive ability to mucin with mRNA expression levels of chaperone proteins (such as GroEL/ES, DnaKJ, and HtrA). In the in vivo experiments, administration of KO caused an inflammation on the colon with gut dysbiosis. LH group (oral gavage of HDB1258 1.0 × 109 colony forming units/day) showed that inflammatory biomarkers, including IL-1ß, TNF-α, IL-6, and PGE2, were significantly decreased to less than half of the KO group, and Th1 cells were decreased in the spleen, but Treg cells were not affected. In contrast, the expression levels of secretory IgA and IL-10 were significantly increased, and the composition of gut microbiota in the LH group tended to recover similar to normal mice without any effect on the α-diversity. In conclusion, L. rhamnosus HDB1258 cultured in the LS could regulate competitively pathogenic bacteria in imbalanced flora with its improved mucin adhesive ability and was an effective immunomodulatory adjuvant for treating colitis by its regulatory function on intestinal inflammation.

TXNIP/VDUP1 attenuates steatohepatitis via autophagy and fatty acid oxidation.

Impaired macroautophagy/autophagy has been implicated in experimental and human nonalcoholic steatohepatitis (NASH). However, the mechanism underlying autophagy dysregulation in NASH is largely unknown. Here, we investigated the role and mechanism of TXNIP/VDUP1 (thioredoxin interacting protein), a key mediator of cellular stress responses, in the pathogenesis of NASH. Hepatic TXNIP expression was upregulated in nonalcoholic fatty liver disease (NAFLD) patients and in methionine choline-deficient (MCD) diet-fed mice, as well as in palmitic acid (PA)-treated hepatocytes. Upregulation of hepatic TXNIP was positively correlated with impaired autophagy, as evidenced by a decreased number of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) puncta and increased SQSTM1/p62 (sequestosome 1) expression. Deletion of the Txnip gene enhanced hepatic steatosis, inflammation, and fibrosis, accompanied by impaired autophagy and fatty acid oxidation (FAO) in MCD diet-fed mice. Mechanistically, TXNIP directly interacted with and positively regulated p-PRKAA, leading to inactivation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) and nuclear translocation of TFEB (transcription factor EB), which in turn promoted autophagy. Inhibition of MTORC1 by rapamycin induced autophagy and increased the expression levels of FAO-related genes and concomitantly attenuated lipid accumulation in PA-treated txnip-knockout (KO) hepatocytes, which was further abolished by silencing of Atg7. Rapamycin treatment also attenuated MCD diet-induced steatosis, inflammation, and fibrosis with increased TFEB nuclear translocation and restored FAO in txnip-KO mice. Our findings suggest that elevated TXNIP ameliorates steatohepatitis by interacting with PRKAA and thereby inducing autophagy and FAO. Targeting TXNIP may be a potential therapeutic approach for NASH.Abbreviations: ACOX1: acyl-Coenzyme A oxidase 1, palmitoyl; ACSL1: acyl-CoA synthetase long-chain family member 1; ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BafA1: bafilomycin A1; COL1A1/Col1α1: collagen, type I, alpha 1; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; DGAT1: diacylglycerol O-acyltransferase 1; DGAT2: diacylglycerol O-acyltransferase 2; ECI2/Peci: enoyl-Coenzyme A isomerase 2; EHHADH: enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase; FAO: fatty acid oxidation; FASN: fatty acid synthase; FFA: free fatty acids; GFP: green fluorescent protein; GK/GYK: glycerol kinase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPAM: glycerol-3-phosphate acyltransferase, mitochondrial; GPT/ALT: glutamic pyruvic transaminase, soluble; H&E: hematoxylin and eosin; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; IOD: integral optical density; KO: knockout; Leu: leupeptin; LPIN1: lipin 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MCD: methionine choline-deficient; MMP9: matrix metallopeptidase 9; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver diseases; NASH: nonalcoholic steatohepatitis; PA: palmitic acid; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; qRT-PCR: quantitative real-time PCR; RPS6KB1/p70S6K1: ribosomal protein S6 kinase, polypeptide 1; RPTOR: regulatory associated protein of MTOR complex 1; SCD1: stearoyl-Coenzyme A desaturase 1; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TGFB/TGF-ß: transforming growth factor, beta; TIMP1: tissue inhibitor of metalloproteinase 1; TNF/TNF-α: tumor necrosis factor; TXNIP/VDUP1: thioredoxin interacting protein; WT: wild-type.

Intracellular transformation rates of fatty acids are influenced by expression of the fatty acid transporter FadL in Escherichia coli cell membrane.

Fatty acids have a low permeability through the cell membrane. Therefore, the intracellular biotransformation of fatty acids can be slow due to supply limitations. The effects of expression level of the fatty acid transporter FadL in Escherichia coli on the biotransformations were investigated. The enhanced expression of FadL led to 5.5-fold increase of the maximum reaction rate Vmax (i.e., 200 µmol/min per g dry cells (200 U/g dry cells)) of the recombinant E. coli expressing a hydratase of Stenotrophomonas maltophilia in the periplasm with respect to hydration of oleic acid. The FadL expression level was also critical for oxidation of 12- and 10- hydroxyoctadecanoic acid by the recombinant E. coli expressing an alcohol dehydrogenase (ADH) of Micrococcus luteus. In addition, the multistep biotransformation of ricinoleic acid into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) by the recombinant E. coli expressing the ADH of M. luteus and a Baeyer-Villiger monooxygenase of Pseudomonas putida KT2440 was 2-fold increased to 40 U/g dry cells with expression of FadL to an appropriate level. The FadL expression level is one of the critical factors to determine whole-cell biotransformation rates of not only long chain fatty acids but also hydroxy fatty acids. This study may contribute to whole-cell biocatalyst engineering for biotransformation of hydrophobic substances.

A large intrauterine vascular lesion developing after the successful treatment of a cesarean scar pregnancy with methotrexate injection.

A cesarean scar pregnancy is a rare type of ectopic pregnancy. Induced abortion by local methotrexate (MTX) injection is an effective management approach. We describe a case in which a large intrauterine vascular lesion appeared after the sonographic-guided local injection of MTX, which successfully induced the abortion of the cesarean scar pregnancy. Although a cesarean scar pregnancy may be safely treated with a local MTX injection, close follow-up, including serum ß-human chorionic gonadotropin level measurement and Doppler sonography, is needed because an intrauterine vascular lesion could develop even after a successfully induced abortion. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 46:222-226, 2018.

Quisqualis indica Improves Benign Prostatic Hyperplasia by Regulating Prostate Cell Proliferation and Apoptosis.

Quisqualis indica (QI) has been used for treating disorders such as stomach pain, constipation, and digestion problem. This study was aimed to evaluate the therapeutic efficacy of QI extract on treating benign prostatic hyperplasia (BPH) in LNCaP human prostate cancer cell line and a testosterone-induced BPH rat model. LNCaP cells were treated with QI plus testosterone propionate (TP), and androgen receptor (AR) and prostate specific antigen (PSA) expression levels were assessed by Western blotting. To induce BPH, the rats were subjected to a daily subcutaneous injection of TP (3 mg/kg) for 4 weeks. The rats in treatment group were orally gavaged with QI (150 mg/kg) together with the TP injection. In-vitro studies showed that TP-induced increases in AR and PSA expression in LNCaP cells were reduced by QI treatment. In BPH-model rats, the prostate weight, testosterone in serum, dihydrotestosterone (DHT) concentration and 5α-reductase type 2 mRNA expression in prostate tissue were significantly reduced following the treatment with QI. TP-induced prostatic hyperplasia and the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 were significantly attenuated in QI-treated rats. In addition, QI induced apoptosis by up-regulating caspase-3 and -9 activity and decreasing the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio in prostate tissues of BPH rats. Further investigation showed that TP-induced activation of AKT and glycogen synthase kinase 3ß (GSK3ß) was reduced by QI administration. Therefore, our findings suggest that QI attenuates the BPH state in rats through anti-proliferative and pro-apoptotic activities and might be useful in the clinical treatment of BPH.

Isoimperatorin attenuates airway inflammation and mucus hypersecretion in an ovalbumin-induced murine model of asthma.

Isoimperatorin (IMP), an active natural furocoumarin, has numerous pharmacologic effects, including anti-inflammatory, analgesic, antispasmodic, and anticancer activities. This study aimed to evaluate the preventive activity of IMP in an ovalbumin (OVA)-induced murine model of asthma and to investigate its possible molecular mechanisms. Female BALB/c mice were sensitized on days 0 and 14 via intraperitoneal injection of 20µg OVA. On days 21-23 after the initial sensitization, the mice received an airway challenge with OVA (1% w/v in PBS) for 1h; meanwhile, IMP (10 or 30mg/kg once daily) was administered by gavage on days 18-23. Our results revealed that IMP significantly lowered the productions of interleukin (IL)-4, IL-5, IL-13, eotaxin, and immunoglobulin (Ig)E in bronchoalveolar lavage fluid (BALF), plasma, or lung tissues. Histological studies showed that IMP inhibited OVA-induced inflammatory cell infiltration and mucus production in the respiratory tract. In addition, pretreatment with IMP suppressed the activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular-signal-regulated kinases 1/2 (ERK1/2), and nuclear factor-κB (NF-κB). Together, these results suggest that IMP effectively inhibits airway inflammation and mucus hypersecretion by downregulating the levels of Th2 cytokines and inhibiting NF-κB and MAPK pathways.

Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

3'-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli.

3'-Untranslated region (3'UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3'UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3'UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3'UTR. In summary, 3'UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells.

Impaction of an intrathoracic kidney acted as a shield against herniation of the abdominal viscera in a case of right congenital diaphragmatic hernia.

We describe a case of an intrathoracic kidney combined with right congenital diaphragmatic hernia (CDH) that was diagnosed at 32 weeks of gestation. Although it has been well established that a right CDH shows a poorer outcome than a left CDH, our present case showed a good outcome because there was no herniation of other abdominal viscera, except for the right kidney. Our findings in this case indicate that impaction of the intrathoracic kidney may act as a 'shield' against further herniation of other abdominal viscera into the thoracic cavity.

meso-Dihydroguaiaretic acid attenuates airway inflammation and mucus hypersecretion in an ovalbumin-induced murine model of asthma.

meso-Dihydroguaiaretic acid (MDGA), which is a dibenzylbutane lignin isolated from the ethyl acetate fraction of Saururus chinensis, has various biological activities, including anti-oxidative, anti-inflammatory, anti-bacterial, and neuroprotective effects. However, no report has examined the potential anti-asthmatic activity of MDGA. In this study, we evaluated the protective effects of MDGA on asthmatic responses, particularly airway inflammation and mucus hypersecretion in an ovalbumin (OVA)-induced murine model of asthma. Intragastric administration of MDGA significantly lowered the productions of interleukin (IL)-4, IL-5, IL-13, tumor necrosis-α (TNF-α), eotaxin, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and immunoglobulin (Ig)E in bronchoalveolar lavage fluid (BALF), plasma, or lung tissues. Histological studies showed that MDGA inhibited OVA-induced inflammatory cell infiltration and mucus production in the respiratory tract. Moreover, MDGA markedly attenuated the OVA-induced activations of nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinases 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK). Together, these results suggest that MDGA effectively inhibits airway inflammation and mucus hypersecretion by downregulating the levels of T helper 2 (Th2) cytokines, chemokines, and adhesion molecules, and inhibiting the activations of NF-κB and MAPKs.

Protective Effects of Manassantin A against Ethanol-Induced Gastric Injury in Rats.

Manassantin A, a neolignan isolated from Saururus chinensis, is a major phytochemical compound that has various biological activities, including anti-inflammatory, neuroleptic, and human acyl-CoA : cholesterol acyltransferase (ACAT) inhibitory activities. In this study, we investigated the protective effects of manassantin A against ethanol-induced acute gastric injury in rats. Gastric injury was induced by intragastric administration of 5 mL/kg body weight of absolute ethanol to each rat. The positive control group and the manassantin A group were given oral doses of omeprazole (20 mg/kg) or manassantin A (15 mg/kg), respectively, 1 h prior to the administration of absolute ethanol. Our examinations revealed that manassantin A pretreatment reduced ethanol-induced hemorrhage, hyperemia, and epithelial cell loss in the gastric mucosa. Manassantin A pretreatment also attenuated the increased lipid peroxidation associated with ethanol-induced acute gastric lesions, increased the mucosal glutathione (GSH) content, and enhanced the activities of antioxidant enzymes. The levels of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1ß were clearly decreased in the manassantin A-pretreated group. In addition, manassantin A pretreatment enhanced the levels of cyclooxygenase (COX)-1, COX-2, and prostaglandin E2 (PGE2) and reduced the inducible nitric oxide synthase (iNOS) overproduction and nuclear factor kappa B (NF-κB) phosphorylation. Collectively, these results indicate that manassantin A protects the gastric mucosa from ethanol-induced acute gastric injury, and suggest that these protective effects might be associated with COX/PGE2 stimulation, inhibition of iNOS production and NF-κB activation, and improvements in the antioxidant and anti-inflammatory status.

Genetically obese (ob/ob) mice are resistant to the lethal effects of thioacetamide hepatotoxicity.

Obesity increases the risk of chronic liver diseases, including viral hepatitis, alcohol-induced liver disease, and non-alcoholic steatohepatitis. In this study, we investigated the effects of obesity in acute hepatic failure using a murine model of thioacetamide (TA)-induced liver injury. Genetically obese ob/ob mice, together with non-obese ob/+ littermates, were subjected to a single intraperitoneal injection of TA, and examined for signs of hepatic injury. ob/ob mice showed a significantly higher survival rate, lower levels of serum alanine aminotransferase and aspartate aminotransferase, and less hepatic necrosis and apoptosis, compared with ob/+ mice. In addition, ob/ob mice exhibited significantly lower levels of malondialdehyde and significantly higher levels of glutathione and antioxidant enzyme activities compared with their ob/+ counterparts. Bioactivation analyses revealed reduced plasma clearance of TA and covalent binding of [(14)C]TA to liver macromolecules in ob/ob mice. Together, these data demonstrate that genetically obese mice are resistant to TA-induced acute liver injury through diminished bioactivation of TA and antioxidant effects.

Genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium Thermus thermophilus HB27.

BACKGROUND: Thermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions. RESULTS: We reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media. CONCLUSIONS: The reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Multistep enzymatic synthesis of long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils.

A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid.

Mercury induced the Accumulation of Amyloid Beta (Aß) in PC12 Cells: The Role of Production and Degradation of Aß.

Extracellular accumulation of amyloid beta protein (Aß) plays a central role in Alzheimer's disease (AD). Some metals, such as copper, lead, and aluminum can affect the Aß accumulation in the brain. However, the effect of mercury on Aß accumulation in the brain is not clear. Thus, this study was proposed to estimate whether mercury concentration affects Aß accumulation in PC12 cells. We treated 10, 100, and 1000 nM HgCl2 (Hg) or CH3HgCl2 (MeHg) for 48 hr in PC12 cells. After treatment, Aß40 in culture medium increased in a dose- and time-dependent manner. Hg and MeHg increased amyloid precursor protein (APP), which is related to Aß production. Neprilysin (NEP) levels in PC12 cells were decreased by Hg and MeHg treatment. These results suggested that Hg induced Aß accumulation through APP overproduction and reduction of NEP.