CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis.

Multiple sclerosis (MS) is characterized by heterogeneity in disease course and prediction of long-term outcome remains a major challenge. Here, we investigate five myeloid markers - CHIT1, CHI3L1, sTREM2, GPNMB and CCL18 - in the cerebrospinal fluid (CSF) at diagnostic lumbar puncture in a longitudinal cohort of 192 MS patients. Through mixed-effects and machine learning models, we show that CHIT1 is a robust predictor for faster disability progression. Integrative analysis of 11 CSF and 26 central nervous system (CNS) parenchyma single-cell/nucleus RNA sequencing samples reveals CHIT1 to be predominantly expressed by microglia located in active MS lesions and enriched for lipid metabolism pathways. Furthermore, we find CHIT1 expression to accompany the transition from a homeostatic towards a more activated, MS-associated cell state in microglia. Neuropathological evaluation in post-mortem tissue from 12 MS patients confirms CHIT1 production by lipid-laden phagocytes in actively demyelinating lesions, already in early disease stages. Altogether, we provide a rationale for CHIT1 as an early biomarker for faster disability progression in MS.

Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions.

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency.

The Exploitation of the Glycosylation Pattern in Asthma: How We Alter Ancestral Pathways to Develop New Treatments.

Asthma has reached epidemic levels, yet progress in developing specific therapies is slow. One of the main reasons for this is the fact that asthma is an umbrella term for various distinct subsets. Due to its high heterogeneity, it is difficult to establish biomarkers for each subset of asthma and to propose endotype-specific treatments. This review focuses on protein glycosylation as a process activated in asthma and ways to utilize it to develop novel biomarkers and treatments. We discuss known and relevant glycoproteins whose functions control disease development. The key role of glycoproteins in processes integral to asthma, such as inflammation, tissue remodeling, and repair, justifies our interest and research in the field of glycobiology. Altering the glycosylation states of proteins contributing to asthma can change the pathological processes that we previously failed to inhibit. Special emphasis is placed on chitotriosidase 1 (CHIT1), an enzyme capable of modifying LacNAc- and LacdiNAc-containing glycans. The expression and activity of CHIT1 are induced in human diseased lungs, and its pathological role has been demonstrated by both genetic and pharmacological approaches. We propose that studying the glycosylation pattern and enzymes involved in glycosylation in asthma can help in patient stratification and in developing personalized treatment.

Evolutionary insights into sequence modifications governing chitin recognition and chitinase inactivity in YKL-40 (HC-gp39, CHI3L1).

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.

X-ray structure and mutagenesis analyses of Clostridioides difficile endolysin Ecd09610 glucosaminidase domain.

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove.

N-acetyl-ß-hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic Gram-negative bacillus that can cause the disease melioidosis. Although B. pseudomallei is a recognised member of terrestrial soil microbiomes, little is known about its contribution to the saprophytic degradation of polysaccharides within its niche. For example, while chitin is predicted to be abundant within terrestrial soils the chitinolytic capacity of B. pseudomallei is yet to be defined. This study identifies and characterises a putative glycoside hydrolase, bpsl0500, which is expressed by B. pseudomallei K96243. Recombinant BPSL0500 was found to exhibit activity against substrate analogues and GlcNAc disaccharides relevant to chitinolytic N-acetyl-ß-d-hexosaminidases. In B. pseudomallei, bpsl0500 was found to be essential for both N-acetyl-ß-d-hexosaminidase activity and chitooligosaccharide metabolism. Furthermore, bpsl0500 was also observed to significantly affect biofilm deposition. These observations led to the identification of BPSL0500 activity against model disaccharide linkages that are present in biofilm exopolysaccharides, a feature that has not yet been described for chitinolytic enzymes. The results in this study indicate that chitinolytic N-acetyl-ß-d-hexosaminidases like bpsl0500 may facilitate biofilm disruption as well as chitin assimilation, providing dual functionality for saprophytic bacteria such as B. pseudomallei within the competitive soil microbiome.

The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation.

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at KD 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD.

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059.

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Efficient chito-oligosaccharide utilization requires two TonB-dependent transporters and one hexosaminidase in Cellvibrio japonicus.

Chitin utilization by microbes plays a significant role in biosphere carbon and nitrogen cycling, and studying the microbial approaches used to degrade chitin will facilitate our understanding of bacterial strategies to degrade a broad range of recalcitrant polysaccharides. The early stages of chitin depolymerization by the bacterium Cellvibrio japonicus have been characterized and are dependent on one chitin-specific lytic polysaccharide monooxygenase and nonredundant glycoside hydrolases from the family GH18 to generate chito-oligosaccharides for entry into metabolism. Here, we describe the mechanisms for the latter stages of chitin utilization by C. japonicus with an emphasis on the fate of chito-oligosaccharides. Our systems biology approach combined transcriptomics and bacterial genetics using ecologically relevant substrates to determine the essential mechanisms for chito-oligosaccharide transport and catabolism in C. japonicus. Using RNAseq analysis we found a coordinated expression of genes that encode polysaccharide-degrading enzymes. Mutational analysis determined that the hex20B gene product, predicted to encode a hexosaminidase, was required for efficient utilization of chito-oligosaccharides. Furthermore, two gene loci (CJA_0353 and CJA_1157), which encode putative TonB-dependent transporters, were also essential for chito-oligosaccharides utilization. This study further develops our model of C. japonicus chitin metabolism and may be predictive for other environmentally or industrially important bacteria.

Chitotriosidase as biomarker for early stage amyotrophic lateral sclerosis: a multicenter study.

Objective: Levels of chitotriosidase (CHIT1) are increased in the cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis (ALS) patients reflecting microglial activation. Here, we determine the diagnostic and prognostic potential of CHIT1 for early symptomatic ALS. Methods: Overall, 275 patients from 8 European neurological centers were examined. We included ALS with <6 and >6 months from symptom onset, other motoneuron diseases (oMND), ALS mimics (DCon) and non-neurodegenerative controls (Con). CSF CHIT1 levels were analyzed for diagnostic power and association with progression and survival in comparison to the benchmark neurofilament. The 24-bp duplication polymorphism of CHIT1 was analyzed in a subset of patients (N = 65). Results: Homozygous CHIT1 duplication mutation carriers (9%) invariably had undetectable CSF CHIT1 levels, while heterozygous carriers had similar levels as patients with wildtype CHIT1 (p = 0.414). In both early and late symptomatic ALS CHIT1 levels was increased, did not correlate with patients' progression rates, and was higher in patients diagnosed with higher diagnostic certainty. Neurofilament levels correlated with CHIT1 levels and prevailed over CHIT1 regarding diagnostic performance. Both CHIT1 and neurofilaments were identified as independent predictors of survival in late but not early symptomatic ALS. Evidence is provided that CHIT1 predicts progression in El Escorial diagnostic category in the group of ALS cases with a short duration. Conclusions: CSF CHIT1 level may have additional value in the prognostication of ALS patients with a short history of symptoms classified in diagnostic categories of lower clinical certainty. To fully interpret apparently low CHIT1 levels knowledge of CHIT1 genotype is needed.

Chitotriosidase gene polymorphisms and mutations limit the determination of chitotriosidase expression in sarcoidosis.

Serum chitotriosidase (CTO) activity was proposed as a biomarker in sarcoidosis being potentially useful in diagnostics. Nevertheless, a common duplication polymorphism (c.1049_1072dup24, Dup24) of the CTO gene influences CTO activity and thereby compromises its use in sarcoidosis. Here we aimed to substitute CTO activity with CTO concentration to prevent the confounding effect of Dup24. CTO activity, concentration and genetic backgrounds were determined in 80 histopathology proven sarcoidosis patients and 133 healthy individuals. CTO activities were lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (472 ± 367 mU/L, n = 49; 2300 ± 2105 mU/L, n = 29) than in homozygous wild types (838 ± 856 mU/L, n = 81; 5125 ± 4802 mU/L, n = 48; p < 0.001, respectively). Sera of Dup24 homozygous individuals had no CTO activity. CTO concentrations were also lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (7.2 ± 1.9 µg/L, n = 11; 63.16 ± 56.5 µg/L, n = 29) than in homozygous wild types (18.9 ± 13.0 µg/L, n = 36; 157.1 ± 132.4 µg/L, n = 47, p < 0.001, respectively) suggestive for an interaction between Dup24 mutation and CTO concentration determinations. We also identified a healthy Hungarian male subject without CTO activity carrying a rare mutation (c.(965_993)del), which mutation has been considered unique for Cypriot population to date. Taken together, CTO concentration determination does not add to the CTO activity measurement when CTO is used as a biomarker in sarcoidosis. Therefore, genotyping of CTO gene should be involved in the interpretation of laboratory findings.

New insights in the coordinated amidase and glucosaminidase activity of the major autolysin (Atl) in Staphylococcus aureus.

After bacterial cell division, the daughter cells are still covalently interlinked by the peptidoglycan network which is resolved by specific hydrolases (autolysins) to release the daughter cells. In staphylococci, the major autolysin (Atl) with its two domain enzymes, N-acetylmuramyl-L-alanine amidase (AmiA) and ß-N-acetylglucosaminidase (GlcA), resolves the peptidoglycan to release the daughter cells. Internal deletions in each of the enzyme domains revealed defined morphological alterations such as cell cluster formation in ΔamiA, ΔglcA and Δatl, and asymmetric cell division in the ΔglcA. A most important finding was that GlcA activity requires the prior removal of the stem peptide by AmiA for its activity thus the naked glycan strand is its substrate. Furthermore, GlcA is not an endo-ß-N-acetylglucosaminidase but an exo-enzyme that cuts the glycan backbone to disaccharides independent of its O-acetylation modification. Our results shed new light into the sequential peptidoglycan hydrolysis by AmiA and GlcA during cell division in staphylococci.

Comparative functional analysis between human and mouse chitotriosidase: Substitution at amino acid 218 modulates the chitinolytic and transglycosylation activity.

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been attracting research interest due to their involvement in various pathological conditions such as Gaucher's disease and asthma, respectively. Both enzymes are highly expressed in mice, while the level of AMCase mRNA was low in human tissues. In addition, the chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. Here, we revealed a substantially higher chitinolytic and transglycosylation activity of human Chit1 against artificial and natural chitin substrates as compared to the mouse enzyme. We found that the substitution of leucine (L) by tryptophan (W) at position 218 markedly reduced both activities in human Chit1. Conversely, the L218W substitution in mouse Chit1 increased the activity of the enzyme. These results suggest that Chit1 may compensate for the low of AMCase activity in humans, while in mice, highly active AMCase may supplements low Chit1 activity.

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.

High risk screening for Gaucher disease in patients with splenomegaly and/or thrombocytopenia in China: 55 cases identified.

Gaucher disease (GD) is a common lysosomal storage disorder caused by deficiency of glucocerebrosidase (GCase) due to the pathogenic variants in the GBA gene. The aim of this study was to evaluate the performance of high risk screening program for GD by measuring the enzyme activities of GCase and chitotriosidease in dried blood spots of patients with splenomegaly and/or thrombocytopenia. A total of 787 subjects (364 females and 423 males) with unexplained splenomegaly and/or thrombocytopenia were enrolled in this study from May 2016 to Aug 2019. The cutoff value of GCase activity was set as less than 3.0 pmol/punch/h for screening positive. The diagnosis of GD was confirmed by Sanger sequencing of the GBA gene. Among 131 screening positive cases, 49 patients were confirmed GD. The positive predictive value was 37.4%.Three patients with boundary values (GCase 3-4 pmol/punch/h) and other three splenectomic patients with normal GCase activity were confirmed GD by GBA genetic analysis because of increased chitotriosidase or Gaucher cells in bone marrow. A total of 55 GD cases were identified. The sensitivity and specificity of the high risk screening were 98.2% and 89.5%, respectively. These 55 GD patients presented splenomegaly (100%), hepatomegaly (70.9%), thrombocytopenia (83.6%). The level of GCase in GD patients was (1.7 ± 1.6) pmol/punch/h. The increased chitotriosidase (383.8 ± 130.2 pmol/punch/h) was found in 42 (76.4%) patients with GD. Molecular genetic analysis identified 44 variants in the GBA gene, including 11 novel variants. The results showed the high risk screening for GD is accurate, rapid and cost-effective.

LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid ß-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

Dramatically changed immune-related molecules as early diagnostic biomarkers of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, with a low 5-year survival rate because of the absence of effective clinical biomarkers for early diagnosis. Based on the immunosurveillance theory, we proposed that changes in the immune system are more pronounced than tumour-associated antigens during the early stage of cancer. Therefore, a new strategy was designed to screen early diagnostic biomarkers from peripheral leukocytes in early-stage NSCLCs with transcriptome sequencing. A total of 358 immune-related differentially expressed genes were identified between early-NSCLC patients and healthy individuals. Orosomucoid-1 (ORM1, a acute phase protein), the total ORM and chitotriosidase-1 (involved in degradation of chitobiose) were selected for further verification in 210 serum samples by western blotting, ELISA and nephelometry immunoassay (based on immuno-scatter turbidmetry). Receiver operating characteristic curve analysis show that ORM1 and total ORM have excellent diagnostic efficacies, with area under the curve of 0.862 and 0.920, respectively, which significantly distinguished very early-NSCLC (IA) from healthy samples. Flow cytometry results showed that CD15+ neutrophils made up 73% of ORM1+ peripheral leukocytes. In mouse lung cancer model, serum ORM1, but not liver ORM1, changed significantly in the early stage of NSCLC. ORM1 expression in peripheral leukocytes was regulated by TGF-ß and mediated by the TGF-ß/Smad signalling pathway. Our results indicated that combined ORM and TGF-ß could be a promising clinical biomarker in the diagnosis of early NSCLC.

Molecular cloning and characterization of genes encoding chitinase and N-acetyl-ß-glucosaminidase in Dolerocypris sinensis.

Chitinases and N-acetyl-ß-glucosaminidase (NAG) are important in molting and growth of crustaceans. In ostracods, the genes encoding these enzymes have not been characterized. The aim of the present study was to clone the genes encoding chitinase (DsChi) and NAG (DsNAG) from the ostracod, Dolerocypris sinensis, elucidate the phylogenetic relationships between the cloned genes and known chitinolytic enzymes, and determine the expression patterns of these genes at different stages of growth in the presence of an environmental pollutant. The genes were amplified from the genomic DNA of the organism using polymerase chain reaction (PCR). The products from PCR were cloned and characterized with bioinformatics tools, and their expression patterns at different growth stages were determined using real-time quantitative PCR (qRT-PCR). Nine and five introns were identified in DsChi and DsNAG genes, respectively. When compared with protein sequences available in GenBank, chitinase from D. sinensis was most closely related to that of Macrobrachium nipponense (61 % homology). The NAG of D. sinensis was most closely related to that of Limulus polyphemus (55.6 % homology). Based on phylogenetic analysis of known chitinases from crustaceans and insects, the D. sinensis chitinase tightly clustered in the same branch with chitinases from species within the Malacostraca class. In contrast, NAG of D. sinensis was clustered with NAG from F. candida.The level of expression of DsChi mRNA was significantly higher than that of DsNAG throughout the period of growth (p < 0.05). Treatment of D. sinensis cells with fenoxycarb significantly downregulated the expressions of DsChi and DsNAG throughout the period of growth (p < 0.05). These results show that the protein products of DsChi and DsNAG possess remarkable biochemical properties characteristic of a chitinase and NAG, respectively.

Lacto-N-tetraose synthesis by wild-type and glycosynthase variants of the ß-N-hexosaminidase from Bifidobacterium bifidum.

Lacto-N-biose 1,2-oxazoline was prepared chemo-enzymatically and shown to be a donor substrate for ß-1,3-glycosylation of lactose by the wild-type and glycosynthase variants (D320E, D320A, Y419F) of Bifidobacterium bifidum ß-N-hexosaminidase. Lacto-N-tetraose, a core structure of human milk oligosaccharides, was formed in 20-60% yield of donor substrate (up to 8 mM product titre), depending on the degree of selectivity control by the enzyme used.