Fucosylation and galactosylation in N-glycans of bovine intestinal alkaline phosphatase and their role in its enzymatic activity.

Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-ß-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-ß-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-ß-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially ß-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1).

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

Structural identification and quantification of unreported sialylated N-glycans in bovine testicular hyaluronidase by LC-ESI-HCD-MS/MS.

Bovine testicular hyaluronidase (BTH), which accelerates the absorption and dispersion of drugs by decomposing hyaluronan in subcutaneous tissues, has been used in medical applications, including local anesthesia, ophthalmology, and dermatosurgery. The requirement of N-glycans for the activity of human hyaluronidase has been reported, and BTH has greater activity than human hyaluronidase. However, the N-glycan characteristics of BTH are unclear. From a commercial BTH source containing additional proteins, purified BTH (pBTH) was obtained using size exclusion chromatography, and the structures and quantities of its N-glycans were analyzed using liquid chromatography (LC)-electrospray ionization-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS). In pBTH, 32 N-glycans were identified, with 12 sialylations (39.0% of total N-glycan content), nine core-fucosylations (31.5%), six terminal galactosylations (14.6%), five high-mannosylations (13.7%), and four bisecting N-acetylglucosamine structures (7.8%). The presence of sialylated glycopeptides in pBTH was confirmed by nano-LC-HCD-MS/MS analysis. The absolute quantity of all N-glycans was calculated as 1.4 pmol (0.6 pmol for sialylation) in pBTH (1.0 pmol). The sialylation level (related to half-life, thermal stability, resistance to proteolysis, and solubility) was 24.4 times higher than that of human hyaluronidase. The hyaluronan degradation activity of de-sialylated pBTH decreased to 41.2 ± 4.2%, showing that sialylated N-glycans were required for pBTH activity as well. This is the first study to identify and quantify 32 N-glycans of pBTH and investigate their structural roles in its activity. The presence of larger amounts of sialylated N-glycans in pBTH than in human hyaluronidase suggests a greater utilization of pBTH.

Immersive virtual reality simulation training for cesarean section: a randomized controlled trial.

BACKGROUND: Caesarean section (CS) is a complex surgical procedure that involves many steps and requires careful precision. Virtual reality (VR) simulation has emerged as a promising tool for medical education and training, providing a realistic and immersive environment for learners to practice clinical skills and decision-making. This study aimed to evaluate the educational effectiveness of a VR simulation program in training the management of patients with premature rupture of membranes (PROM) and CS. MATERIALS AND METHODS: A two-arm parallel randomized controlled trial was conducted with 105 eligible participants randomly assigned to the VR group ( n =53) or the control group ( n =52) in a 1:1 ratio. The VR group received VR simulation training focused on PROM management and CS practice, while the control group watched a video presentation with narrative of clinical scenario and recording of CS. Both groups completed questionnaires assessing their prior experiences with VR, experience in managing patients with PROM and performing CS, as well as their confidence levels. These questionnaires were administered before and after the intervention, along with a mini-test quiz. RESULTS: Baseline characteristics and previous experiences were comparable between the two groups. After the intervention, the VR group had higher confidence scores in all four aspects, including managing patients with PROM, performing CS as an operator, and understanding the indications and complications of CS, compared to the control group. The VR group also achieved significantly higher scores on the mini-test quiz [median (interquartile range), 42 (37-48) in the VR group; 36 (32-40) in the control group, P <0.001]. CONCLUSION: VR simulation program can be an effective educational tool for improving participants' knowledge and confidence in managing patients with PROM and performing CS.

Identification, quantification, and structural role of N-glycans in two highly purified isoforms of sheep testicular hyaluronidase.

Animal-derived hyaluronidase, which hydrolyzes the polysaccharide hyaluronic acid, has been used in medical applications despite its limited purity. Additionally, the N-glycan characterization of sheep testicular hyaluronidase (STH) and its structural role remain poorly understood. In this study, STH was purified from the commercially available STH preparation (containing at least 14 impurity proteins) using heparin-affinity chromatography followed by size exclusion chromatography. The structure and quantity of N-glycans of STH were investigated using liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. Two isoforms, H3S1 and H3S2, of STH were obtained (purity >98 %) with a yield of 3.4 % and 5.1 %, respectively. Fourteen N-glycans, including nine core-fucosylated N-glycans (important for the stability and function of glycoproteins), were identified in both H3S1 and H3S2, with similar quantities of each N-glycan. The amino acid sequences of the proteolytic peptides of H3S1 and H3S2 were compared with those reported in STH. The hyaluronic acid-degrading activity of deglycosylated H3S1 and H3S2 was reduced to 70.8 % and 71.1 % compared to that (100 %) of H3S1 and H3S2, respectively. This is the first report of N-glycan characterization of two highly purified isoforms of STH. These H3S1 and H3S2 will be useful for medical use without unwanted effects of partially purified STH.

Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins.

Sialylated N-glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese hamster ovary cell lines; however, their linkage isomers have not been reported. In this study, N-glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated N-glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the N-acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective m/z value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total N-glycans (100%) for all observed ionization states. Twenty sialylated N-glycan isomers with only α2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated N-glycan isomers (58.8%) in mono- (3 N-glycans; 0.9%), bi- (18; 48.3%), tri- (14; 8.9%), and tetra- (4; 0.7%) antennary structures of mutant were obtained, which comprised mono- (15 N-glycans; 25.4%), di- (15; 28.4%), tri- (8; 4.8%), and tetra- (1; 0.2%) sialylation, respectively, with only α2-3 (10 N-glycans; 4.8%), both α2-3 and α2-6 (14; 18.4%), and only α2-6 (15; 35.6%) linkage(s). These results are consistent with those for α2-3 neuraminidase-treated N-glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.

Peptide-N-glycosidase F or A treatment and procainamide-labeling for identification and quantification of N-glycans in two types of mammalian glycoproteins using UPLC and LC-MS/MS.

BACKGROUND: N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods. OBJECTIVE: To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins. METHODS: N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)]. The structure and quantity of each N-glycan were determined using UPLC and LC-MS/MS. RESULTS: The 21 N-glycans in fetuin and another 21 N-glycans in IgG by either PF-ProA or PA-ProA were identified using LC-MS/MS. The N-glycans in fetuin (8-13 N-glycans were previously reported) and in IgG (19 N-glycans were previously reported), which could not be identified by using the widely used PF-AB, were all identified by using PF-ProA or PA-ProA. The quantities (%) of the N-glycans (>0.1 %) relative to the total amount of N-glycans (100 %) obtained by AB- and ProA-labeling using LC-MS/MS had a similar tendency. However, the absolute quantities (pmol) of the N-glycans estimated using UPLC and LC-MS/MS were more efficiently determined with ProA-labeling than with AB-labeling. Thus, PF-ProA or PA-ProA allows for more effective identification and quantification of N-glycans than PF-AB in glycoprotein, particularly bovine fetuin. This study is the first comparative analysis for the identification and relative and absolute quantification of N-glycans in glycoproteins with PF-ProA and PA-ProA using UPLC and LC-MS/MS.

Novel strategy for dental caries by physiologic dentin regeneration with CPNE7 peptide.

OBJECTIVE: CPNE7-derived functional peptide (CPNE7-DP) has been introduced as a bioactive therapeutics for dentin diseases. CPNE7-DP regenerates tubular dentin on the pulpal side and occlude dentinal tubules. CPNE7-DP was capable to treat dentin hypersensitivity typically associated with dentinal wear at the neck of the tooth. However, the role of CPNE7-DP in another common dentin disease, dental caries, remains uninvestigated. In this study, we evaluated the potential application of CPNE7-DP in dentin caries using an experimental dentin caries model in rats. DESIGN: The stability of CPNE7-DP in caries-like environments including pathologic bacteria of caries or low pH was tested. We established a nutrition-time/hyposalivation-based dental caries rat model by inoculating caries-inducing bacteria and diet for sufficient time. Glycopyrrolate has been treated to induce reversible hyposalivation for accelerating caries progression. Then the tubular dentin regeneration was investigated with histologic methods. Also, modulation of inflammation or autophagy by CPNE7-DP was investigated with marker gene expression in human dental pulp cells (hDPCs) and immunohistochemistry. RESULTS: CPNE7-DP was stable with caries-inducing bacteria and low pH. Establishment of dentin caries was confirmed with radiographic and histologic evaluation. CPNE7-DP regenerated a substantial amount of tubular tertiary dentin and alleviated the pulp inflammation of dentin caries. Under inflammatory conditions, CPNE7-DP reduced the expression of inflammatory cytokines. These phenomena could be the consequence of the modulation of autophagy by CPNE7-DP, which reactivates inflamed odontoblasts. CONCLUSIONS: Overall, CPNE7-DP, which repairs caries through physiological dentin regeneration, might help overcoming the limitations of current restorative caries treatments.

Neuroprotective Effect of 1,3-dipalmitoyl-2-oleoylglycerol Derived from Rice Bran Oil against Cerebral Ischemia-Reperfusion Injury in Rats.

1,3-Dipalmitoyl-2-oleoylglycerol (POP) is a triacylglyceride found in oils from various natural sources, including palm kernels, sunflower seeds, and rice bran. In the current study, the neuroprotective effects and the specific mechanism of POP derived from rice bran oil were investigated for the first time using the middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats. Orally administered POP at 1, 3, or 5 mg/kg (three times: 0.5 h before MCAO, after 1 h of MCAO, and after 1 h of reperfusion) markedly reduced the MCAO/R-induced infarct/edema volume and neurobehavioral deficits. Glutathione depletion and the oxidative degradation of lipids in the rat brain induced by MCAO/R were prevented by POP administration. The upregulation of phosphorylated p38 MAPKs, inflammatory factors (inducible nitric oxide synthase (i-NOS) and cyclooxygenase-2 (COX-2)), and pro-apoptotic proteins (B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) and cleaved caspase-3) and the downregulation of the anti-apoptotic protein (Bcl-2) in the ischemic brain were significantly inhibited by POP administration. In addition, downregulation of phosphatidylinositol 3'-kinase (PI3K), phosphorylated protein kinase B (Akt), and phosphorylated cyclic (adenosine monophosphate) AMP responsive element-binding protein (CREB) expression in the ischemic brain was inhibited by POP administration. These results suggest that POP might exert neuroprotective effects by inhibition of p38 MAPK and activation of PI3K/Akt/CREB pathway, which is associated with anti-oxidant, anti-apoptotic, and anti-inflammatory action. From the above results, the present study provides evidence that POP might be effectively applied for the management of cerebral ischemia-related diseases.

Co3O4/Au Hybrid Nanostructures as Efficient Peroxidase Mimics for Colorimetric Biosensing.

Nanomaterials with enzyme-like characteristics (nanozymes) have emerged as potential replacements for natural enzymes due to their potential to overcome several critical limitations of natural enzymes, including low stability as well as high costs in preparation and purification. Herein, we have developed hybrid nanostructures that incorporate cobalt oxide nanoparticles (Co3O4 NPs) and gold nanoclusters (AuNCs) through electrostatic attraction induced by simple incubation in an aqueous buffer for 2 hours. Owing to the synergistic effect of Co3O4 NPs and AuNCs, the constructed Co3O4/Au hybrid nanostructures yielded highly enhanced peroxidase-like activity and enabled rapid catalytic oxidation of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), producing a blue colored solution depending on the amount of H2O2. Moreover, we observed catalytic activity of the Co3O4/Au hybrid over a broad pH range, especially at physiologically relevant pH in the range of 5.0-7.4, which is advantageous for applications in biological systems. Using the hybrid as peroxidase mimic, we successfully determined the level of target H2O2 or glucose by coupling with glucose oxidase with excellent specificity and sensitivity. Based on this study, we expect that Co3O4/Au hybrid nanostructures can serve as potent peroxidase mimics for the detection of clinically important target molecules.

Delamanid, linezolid, levofloxacin, and pyrazinamide for the treatment of patients with fluoroquinolone-sensitive multidrug-resistant tuberculosis (Treatment Shortening of MDR-TB Using Existing and New Drugs, MDR-END): study protocol for a phase II/III, multicenter, randomized, open-label clinical trial.

BACKGROUND: Treatment success rates of multidrug-resistant tuberculosis (MDR-TB) remain unsatisfactory, and long-term use of second-line anti-TB drugs is accompanied by the frequent occurrence of adverse events, low treatment compliance, and high costs. The development of new efficient regimens with shorter treatment durations for MDR-TB will solve these issues and improve treatment outcomes. METHODS: This study is a phase II/III, multicenter, randomized, open-label clinical trial of non-inferiority design comparing a new regimen to the World Health Organization-endorsed conventional regimen for fluoroquinolone-sensitive MDR-TB. The control arm uses a conventional treatment regimen with second-line drugs including injectables for 20-24 months. The investigational arm uses a new shorter regimen including delamanid, linezolid, levofloxacin, and pyrazinamide for 9 or 12 months depending on time to sputum culture conversion. The primary outcome is the treatment success rate at 24 months after treatment initiation. Secondary outcomes include time to sputum culture conversion on liquid and solid media, proportions of sputum culture conversion on liquid media after 2 and 6 months of treatment, treatment success rate according to pyrazinamide resistance, and occurrence of adverse events grade 3 and above as evaluated by the Common Terminology Criteria for Adverse Events. Based on an α = 0.025 level of significance (one-sided test), a power of 80%, and a < 10% difference in treatment success rate between the control and investigational arms (80% vs. 70%) when the anticipated actual success rate in the treatment group is assumed to be 90%, the number of participants needed per arm to show non-inferiority of the investigational regimen was calculated as 48. Additionally, assuming the proportion of fluoroquinolone-susceptible MDR-TB among participants as 50%, and 5% loss to follow-up, the number of participants is calculated as N/( 0.50 × 0.95), resulting in 102 persons per group (204 in total). DISCUSSION: This trial will reveal the effectiveness and safety of a new shorter regimen comprising four oral drugs, including delamanid, linezolid, levofloxacin, and pyrazinamide, for the treatment of fluoroquinolone-sensitive MDR-TB. Results from this trial will provide evidence for adopting a shorter and more convenient treatment regimen for MDR-TB. TRIAL REGISTRATION: ClincalTrials.gov, NCT02619994 . Registered on 2 December 2015.

Convenient Colorimetric Detection of Thrombin via Aptamer-Mediated Inhibition and Restoration of the Oxidase Activity of Nanoceria.

Cerium oxide nanoparticles, also called nanoceria, have recently gained much attention as oxidase-mimicking nanozymes that catalyze the oxidation of chromogenic substrates for color generation without the addition of H2O2. Herein, we have developed a unique colorimetric biosensor for thrombin in human blood plasma, which relies on thrombin-binding aptamer (TBA)-mediated inhibition of the oxidase activity of nanoceria and its restoration by very selective interactions of TBA with target thrombin. In this system, nanoceria were first incubated with TBA, resulting in quick reduction of the oxidase activity of nanoceria via the adsorption of single-stranded (ss)DNA-type TBA on nanoceria. By the addition of sample solutions containing target thrombin, TBA bound on the nanoceria would strongly interact with free thrombin and be detached from the nanoceria, thereby increasing the available surface area of the nanoceria and consequently enhancing the oxidase activity. Using this strategy, target thrombin was successfully detected at concentrations as low as 100 pM over a wide linear range from 0.1 to 10 nM. The diagnostic capability of this method has been demonstrated by detecting thrombin in human blood plasma, showing its great potential in the practical applications.

Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica.

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the ß-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.

Dilute sulfuric acid fractionation of Korean food waste for ethanol and lactic acid production by yeast.

Fermentation of food waste biomass can be used to produce biochemicals such as lactic acid and ethanol in a cost-effective manner. Korean food waste (KFW) dewatered by a screw press contains 23.1% glucan on a dry basis and is a potential raw material for the production of ethanol and lactic acid through fermentation. This study was conducted to optimize the dilute acid fractionation conditions for KFW fermentation with respect to the H2SO4 concentration (0-0.8% w/v), temperature (130-190 °C), and residence time (1-128 min) using response surface methodology. Dilute sulfuric acid fractionation was carried out using a 30-mL stainless steel reactor under conditions, and then the dilute acid fractionation was scaled-up in 1-L and 7-L stainless steel reactors under the optimal conditions. The hydrolysate was concentrated, liquid-liquid extracted and neutralized for lactic acid and ethanol production. The highest concentration of glucose obtained from the KFW was 26.4 g/L using fractionation with 0.37% w/v H2SO4 at 156 °C for 123.6 min. Using recombinant Saccharomyces cerevisiae containing a codon-optimized lactate dehydrogenase, the yield of lactic acid and ethanol was 77% of the theoretical yield for 17.4 g/L of fermentable sugar at pH 5.5. Additionally, the yield of ethanol produced by Issatchenkia orientalis was 89% of the theoretical yield for 25 g/L of fermentable sugar at pH 3.

Phytoceramide ameliorates ß-amyloid protein-induced memory impairment and neuronal death in mice.

The present study was performed to investigate the protective effect of phytoceramide against ß-amyloid protein (Aß) (25-35)-induced memory impairment and its underlying mechanisms in mice. Memory impairment in mice was induced by intracerebroventricular injection of 15 nmol Aß (25-35) and measured by the passive avoidance test and Morris water maze test. Chronic administration of phytoceramide (10, 25 and 50 mg/kg, p.o.) resulted in significantly less Aß (25-35)-induced memory loss and hippocampal neuronal death in treated mice compared to controls. The decrease of glutathione level and increase of lipid peroxidation in brain tissue following injection of Aß (25-35) was reduced by phytoceramide. Alteration of apoptosis-related proteins, increase of inflammatory factors, and phosphorylation of mitogen activated proteins kinases (MAPKs) in Aß (25-35)-administered mice hippocampus were inhibited by phytoceramide. Phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and phosphorylation of cyclic AMP response element-binding protein (CREB) were suppressed, while phosphorylation of tau (p-tau) was increased in Aß (25-35)-treated mice brain; these effects were significantly inhibited by administration of phytoceramide. These results suggest that phytoceramide has a possible therapeutic role in managing cognitive impairment associated with Alzheimer's disease. The underlying mechanism might involve inhibition of p-tau formation via anti-apoptosis and anti-inflammation activity and promotion of PI3K/Akt/CREB signaling process.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Neuroprotective effect of phytoceramide against transient focal ischemia-induced brain damage in rats.

The present study was conducted to investigate the protective effect of phytoceramide against focal transient ischemic brain damage and the underlying mechanisms. Focal transient ischemic brain damage was produced in rats by occlusion of the middle cerebral artery for 2 h followed by 24 h of reperfusion (MCAO/reperfusion). Orally administered phytoceramide (10, 25, and 50 mg/kg) significantly reduced MCAO/reperfusion-induced brain infarction and edema as well as the development of behavioral disabilities in the animals. Depletion of glutathione levels and lipid peroxidation in brain tissue following MCAO/reperfusion was reduced by administration of phytoceramide. The expressions of phosphorylated extracellular signaling-regulating kinases/mitogen-activated protein kinase (p-ERK1/2 MAPK), inflammatory factors such as cyclooxygenase-2 and inducible nitric oxide synthase, and pro-apoptotic proteins Bax and caspase-3 were increased while the anti-apoptotic protein Bcl-2 was decreased in ischemic brain; these effects were significantly inhibited by treatment with phytoceramide. Furthermore, phytoceramide activated the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway to prevent ischemic brain damage. These results suggest that phytoceramide may help prevent neurodegeneration caused by ischemic stroke due to its anti-oxidant, anti-apoptotic, and anti-inflammatory properties.

Ilex latifolia Prevents Amyloid ß Protein (25-35)-Induced Memory Impairment by Inhibiting Apoptosis and Tau Phosphorylation in Mice.

Ilex latifolia Thunb. (Aquifoliaceae), a Chinese bitter tea called "kudingcha," has been widely consumed as a health beverage and found to possess antioxidant, antidiabetic, antihypertensive, anti-inflammatory, and anti-ischemic activities. The aim of the present study was to investigate the neuroprotective effects of an ethanol extract of I. latifolia against amyloid ß protein (Aß)-induced memory impairment in mice and neurotoxicity in cultured rat cortical neurons. Memory impairment in mice was induced by intracerebroventricular injection of 15 nmol Aß (25-35) and measured by the passive avoidance test and Morris water maze test. Chronic administration of I. latifolia (25-100 mg/kg, p.o.) significantly prevented Aß (25-35)-induced memory loss. I. latifolia also prevented the decrease of glutathione concentrations, increased lipid peroxidation, expression of phosphorylated tau (p-tau), and changes in apoptosis-associated proteins in the memory-impaired mouse brain. Exposure of cultured cortical neurons to 10 µM Aß (25-35) for 36 h induced neuronal apoptotic death. The neuronal cell death, elevation of intracellular Ca(2+) concentration, generation of reactive oxygen species, and expression of proapoptotic proteins caused by Aß (25-35) in the cultured neurons were inhibited by treatment with I. latifolia (1-50 µg/mL). These results suggest that I. latifolia may have a possible therapeutic role in managing cognitive impairment associated with Alzheimer's disease. The underlying mechanism might involve the antiapoptotic effects mediated by antioxidant activity and inhibition of p-tau formation.

A single fraction from Uncaria sinensis exerts neuroprotective effects against glutamate-induced neurotoxicity in primary cultured cortical neurons.

We identified a neuroprotective single fraction among 62 ones of hexane extract from Uncaria sinensis (JGH43IA) and investigated its effects and mechanisms in primary cortical neurons. Pretreatment with JGH43IA showed a significantly increase cell viability in a dose-dependent manner with a decrease in the lactate dehydrogenase release. When we performed morphological assay and flow cytometry to determination of the type of cell death, pretreatment with JGH43IA showed a significant reduction of glutamate-induced apoptotic cell death. Then we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation to elucidate possible pathways of neuroprotection by JGH43IA. Pretreatment with JGH43IA exhibited a significant attenuation of NMDAR GluN2B subunit activation and a decrease in active form of calpain 1 leading to subsequent cleavage of striatal-enriched protein tyrosine phosphatase (STEP). In addition, pretreatment with JGH43IA showed a marked increase of cAMP responsive element binding protein. These results suggest that JGH43IA may have neuroprotective effects through down-regulation of NMDAR GluN2B subunit and calpain 1 activation, and subsequent alleviation of STEP cleavage. This single fraction from U. sinensis might be a useful therapeutic agent for brain disorder associated with glutamate injury.

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.