Chao-Lin Liu

N-Acetylglucosamine: Production and Applications

N-Acetylglucosamine (GlcNAc) is a monosaccharide that usually polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to serving as a component of this homogeneous polysaccharide, GlcNAc is also a basic component of hyaluronic acid and keratin sulfate on the cell surface. In this review, we discuss the industrial production of GlcNAc, using chitin as a substrate, by chemical, enzymatic and biotransformation methods. Also, newly developed methods to obtain GlcNAc using glucose as a substrate in genetically modified microorganisms are introduced. Moreover, GlcNAc has generated interest not only as an underutilized resource but also as a new functional material with high potential in various fields. Here we also take a closer look at the current applications of GlcNAc, and several new and cutting edge approaches in this fascinating area are thoroughly discussed.

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×109 vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.

Inhibition of Acidic Mammalian Chitinase by RNA Interference Suppresses Ovalbumin-Sensitized Allergic Asthma

Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.

Colloid Chitin Azure Is a Dispersible, Low-Cost Substrate for Chitinase Measurements in a Sensitive, Fast, Reproducible Assay

Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.

The validity of clinical feature profiles for cytomegaloviral anterior segment infection

BackgroundAnterior segment cytomegalovirus (CMV) infection, which can be presented as anterior uveitis and corneal endotheliitis, has recently been reported in immunocompetent patients. We would like to access the validity of two presumed characteristic clinical profiles: profile 1, non-herpes simplex virus (HSV)/varicella zoster virus (VZV) corticosteroid-recalcitrant inflammatory ocular hypertensive syndrome (IOHS), and profile 2, corneal endotheliitis with specific coin-shaped keratic precipitates (KPs), that could be helpful in identifying CMV anterior segment intraocular infection.MethodsPatients with either profile 1 or profile 2 or both were enrolled consecutively from the uveitis service in Chang Gung Memorial Hospital, Taoyuan, between January 1, 2006 and May 31, 2010. Diagnostic anterior chamber tapping was performed and followed by real-time quantitative polymerase chain reaction (PCR) to detect herpesviridae DNA including HSV I and II, VZV, CMV, and Epstein–Barr virus.ResultsThirty-one eyes of 30 patients (21 males and nine females) were enrolled in this study. CMV DNA PCR was positive in 29 eyes of 28 patients (20 males and eight females). Nineteen of 20 eyes (19 patients) in profile 1 had positive CMV PCR. Ten of 11 eyes (11 patients) in profile 2 had positive CMV PCR. The positive predictive value of profile 1 and profile 2 was 94.7% and 90.9%, respectively. The positive predictive value of combining the two profiles was 93.3%.ConclusionsNon-HSV/ZVZ corticosteroid-recalcitrant IOHS and corneal endotheliitis with specific coin-shaped KPs could be used as the screening tool for CMV anterior segment intraocular infection.

Isolation and identification of two novel SDS‐resistant secreted chitinases from Aeromonas schubertii

Two SDS‐resistant endochitinases, designated as ASCHI53 and ASCHI61, were isolated from Aeromonas schubertii in a soil sample from southern Taiwan. MALDI‐TOF mass measurement indicates the molecular weights of 53,527 for ASCHI53 and 61,202 for ASCHI61. N‐terminal and internal amino acid sequences were obtained, and BLAST analysis of the sequences and MS/MS peptide sequencing showed that they were novel proteins. Degradation of chitin by these two endochitinases gave rise to hexameric chitin oligosaccharide, a compound known to have several potent biomedical functions. ASCHI53 and ASCHI61 retained, respectively, 65% and 75%, of their chitinase activity in the presence of 5% SDS and 100% of their activity in the presence of 10% β‐mercaptoethanol. These results demonstrate that they are SDS‐resistant endochitinases and probably have a rigid structure.

Preparation and Characterization of Ferrofluid Stabilized with Biocompatible Chitosan and Dextran Sulfate Hybrid Biopolymer as a Potential Magnetic Resonance Imaging (MRI) T2 Contrast Agent

Chitosan is the deacetylated form of chitin and used in numerous applications. Because it is a good dispersant for metal and/or oxide nanoparticle synthesis, chitosan and its derivatives have been utilized as coating agents for magnetic nanoparticles synthesis, including superparamagnetic iron oxide nanoparticles (SPIONs). Herein, we demonstrate the water-soluble SPIONs encapsulated with a hybrid polymer composed of polyelectrolyte complexes (PECs) from chitosan, the positively charged polymer, and dextran sulfate, the negatively charged polymer. The as-prepared hybrid ferrofluid, in which iron chloride salts (Fe3+ and Fe2+) were directly coprecipitated inside the hybrid polymeric matrices, was physic-chemically characterized. Its features include the z-average diameter of 114.3 nm, polydispersity index of 0.174, zeta potential of −41.5 mV and iron concentration of 8.44 mg Fe/mL. Moreover, based on the polymer chain persistence lengths, the anionic surface of the nanoparticles as well as the high R2/R1 ratio of 13.5, we depict the morphology of SPIONs as a cluster because chitosan chains are chemisorbed onto the anionic magnetite surfaces by tangling of the dextran sulfate. Finally, the cellular uptake and biocompatibility assays indicate that the hybrid polymer encapsulating the SPIONs exhibited great potential as a magnetic resonance imaging T2 contrast agent for cell tracking.

N-acetyl glucosamine obtained from chitin by chitin degrading factors in Chitinbacter tainanesis.

A novel chitin-degrading aerobe, Chitinibacter tainanensis, was isolated from a soil sample from southern Taiwan, and was proved to produce N-acetyl glucosamine (NAG). Chitin degrading factors (CDFs) were proposed to be the critical factors to degrade chitin in this work. When C. tainanensis was incubated with chitin, CDFs were induced and chitin was converted to NAG. CDFs were found to be located on the surface of C. tainanensis. N-Acetylglucosaminidase (NAGase) and endochitinase activities were found in the debris, and the activity of NAGase was much higher than that of endochitinase. The optimum pH of the enzymatic activity was about 7.0, while that of NAG production by the debris was 5.3. These results suggested that some factors in the debris, in addition to NAGase and endochitinase, were crucial for chitin degradation.

Beneficial effects of Chlorella-11 peptide on blocking LPS-induced macrophage activation and alleviating thermal injury-induced inflammation in rats.

Chlorella possesses various remarkable biological activities. One component, Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe (Chlorella-11 peptide) was found to be able to suppress LPS-induced NO production and inflammation. However, the molecular mechanism behind these findings and the consistency between in vitro and in vivo data have not been investigated. LPS-activated RAW 264.7 macrophages were used to study in vitro molecular anti-inflammatory effects of Chlorella-11 peptide. After activation, NO production and the expression of iNOS and NF-κB proteins as well as iNOS mRNA were measured using Griess colorimetric assay, Western blotting and RT-PCR, respectively. Alterations in PGE2 and TNF-α contents were also monitored by ELISA. For in vivo studies, thermal injury Wistar rats were used and inflammatory indications e.g. serum malondialdehyde (MDA), TNF-α levels and skin erythema were evaluated 48 h after injury implementation. In vitro results showed that Chlorella-11 peptide produced a dose- and time-dependent inhibition on NO production. The effective inhibition could remain for at least 6 h after LPS activation. It was also found that the expression of LPS-induced iNOS mRNA, iNOS and NF-κB proteins were diminished by the peptide treatment. Concurrently, the levels on TNF-α and PGE2 production after LPS activation were also inhibited. These findings are in agreement with the in vivo data that animal serum MDA and TNF-α levels and skin erythema in rats were considerably reduced compared to the control group (saline-treated). The significance of this study sheds light on the effectiveness of Chlorella-11 peptide in preventing inflammation progression in vitro and in vivo and its potential for clinical applications.

Structural alterations, pore generation, and deacetylation of α- and β-chitin submitted to steam explosion.

The purpose of this study was to use an environmentally friendly steam explosion method to achieve α- and β-chitin structural alterations, pore generation, and deacetylation, enhancing the degree of deacetylation (DD) in chitin and extending its applications. The samples of α- and β-chitin possessing various moisture contents that were exploded at 9 kg/cm(2) exhibited higher DDs, lower densities, lower crystallinity and more porous structures compared to unexploded chitin. After explosion, β-chitin exhibited a larger expansion ratio, lower crystallinity and contained a larger proportion of small-sized particles compared to α-chitin. The highest DD values of exploded α- and β-chitin with 75% moisture content were 42.9% and 43.7%, respectively. The exploded chitin samples with lower moisture content exhibited lower DDs, densities, crystallinity indices, smaller particle sizes, and higher expansion ratios than the chitin samples with higher moisture content. The chitin samples with lower moisture content also contained larger and more numerous pores.