N-acetylglucosaminyl Transferase Research Articles

In silico design and mechanistic study of niosome-encapsulated curcumin against multidrug-resistant Staphylococcus aureus biofilms

Curcumin, an important natural component of turmeric, has been known for a long time for its antimicrobial properties. This study aimed to investigate the anti-biofilm action of the niosome-encapsulated curcumin and explore the involved anti-biofilm mechanism. In silico investigations of ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) were first performed to predict the suitability of curcumin for pharmaceutical application. Curcumin showed low toxicity but at the same time, low solubility and low stability, which, in turn, might reduce its antimicrobial activity. To overcome these intrinsic limitations, curcumin was encapsulated using a biocompatible niosome system, and an encapsulation efficiency of 97% was achieved. The synthesized curcumin-containing niosomes had a spherical morphology with an average diameter of 178 nm. The niosomal curcumin was capable of reducing multi-drug resistant (MDR) Staphylococcus aureus biofilm 2–4-fold compared with the free curcumin. The encapsulated curcumin also demonstrated no significant cytotoxicity on the human foreskin fibroblasts. To understand the interaction between curcumin and S. aureus biofilm, several biofilm-related genes were analyzed for their expression. N-acetylglucosaminyl transferase (IcaD), a protein involved in the production of polysaccharide intercellular adhesion and known to play a function in biofilm development, was found to be downregulated by niosomal curcumin and showed high binding affinity (-8.3 kcal/mol) with curcumin based on molecular docking analysis. Our study suggests that the niosome-encapsulated curcumin is a promising approach for the treatment of MDR S. aureus biofilm and can be extended to biofilms caused by other pathogens.

Loss of N-acetylglucosaminyl transferase V is involved in the impaired osteogenic differentiation of bone marrow mesenchymal stem cells.

The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of β-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the β-catenin, BMP type 2 (BMP2) and TGF-β signals and involved in the process of OP.

Review on Molecular Cross-talk of Biofilm Producing Mechanisms of Staphylococcus aureus

Here the review converses the `molecular cross-talk` of biofuel production mechanisms for Staphylococcus aureus. Staphylococcus aureus is a leading cause of bacterial infections globally in both healthcare and community settings. The succes of this bacterium is the of an expansive repertoire of virulence factors in combination with acquired antibiotic resistance and propensity for biofilm formation. S. aureus leverages these factors to adapt to and subvert the host immune response. With the burgeoning fiels of immunometabolism, is has become clear that the metabolic program of leukocytes dictates their inflammatory status and overall effectiveness is clearing an infection. The treatment of S. aureus infections become complicated due to the capacity of S. aureus �multidrug-resistant� occurs because of biofilm formationon the surfaces depending on biotic and abiotic factors, genetic factors, and numerous environmental, which vary from species to species. A broad range of molecular phenomenon contributes a high range of recalcitrance that is insisting on the biofilm formation. The previous published literature illustrated that all strains of Staphylococcal sp. contain the �ica locus� and several can form biofilms in vitro condition. Absences of �ica locus� results diminish of capability to produce biofuels, along with `PIA gene`, or mediate `N-acetyl glucosaminyl transferase activity� in vitro condition.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.

APEX1 regulates alternative splicing of key tumorigenesis genes in non-small-cell lung cancer

BackgroundAberrant alternative splicing (AS) contributes to tumor progression. Previous studies have shown that apurinic-apyrimidinic endonuclease-1 (APEX1) is involved in tumor progression. It is unknown whether APEX1 functions in tumor progression by regulation of AS. It is also unknown whether APEX1 can regulate non-small-cell lung cancer (NSCLC) proliferation and apoptosis. We analyzed APEX1 expression levels in 517 lung NSCLC samples from the TCGA (Cancer Genome Atlas) database. The impact of APEX1 over expression on A549 cell proliferation and apoptosis was detected by the methyl thiazolyl tetrazolium assay and by flow cytometry. The transcriptome of A549 cells with and without APEX1 over expression was determined by Illumina sequencing, followed by analysis of AS. RT-qPCR validated expression of APEX1-related genes in A549 cells. We have successfully applied RNA-seq technology to demonstrate APEX1 regulation of AS.ResultsAPEX1 expression was shown to be upregulated in NSCLC samples and to reduce cell proliferation and induce apoptosis of A549 cells. In addition, APEX1 regulated AS of key tumorigenesis genes involved in cancer proliferation and apoptosis within MAPK and Wnt signaling pathways. Each of these pathways are involved in lung cancer progression. Furthermore, validated AS events regulated by APEX1 were in key tumorigenesis genes; AXIN1 (axis inhibition protein 1), GCNT2 (N-acetyl glucosaminyl transferase 2), and SMAD3 (SMAD Family Member 3). These genes encode signaling pathway transcription regulatory factors.ConclusionsWe found that increased expression of APEX1 was an independent prognostic factor related to NSCLC progression. Therefore, APEX1 regulation of AS may serve as a molecular marker or therapeutic target for NSCLC treatment.

First report of soft rot of onion caused by Pectobacterium wasabiae in Japan.

Onion (Allium cepa L.) is one of the important vegetables in Japan. In the summer of 2019, onions with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. Diseased onion showed chlorosis, maceration of leaves, and rotted bulbs. We sampled some diseased onions and isolated three independent isolations (NAONI191, NAONI192 and NAONI193) from infected bulbs on LB medium. These strains were identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All the strains exhibited pectolytic activity on potato slices. DNA from these strains yielded a single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR. However, DNA from these strains did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. The sequence analysis of 16S rDNA (LC597917- LC597919) showed more than 98% identities to P. wasabiae strains (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597925- LC597927), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597972-LC597974), isocitrate dehydrogenase (icdA, LC597998- LC597998LC598000), malate dehydrogenase(mdh, LC598024- LC598026), mannitol-1-phosphate dehydrogenase (mtlD, LC598050- LC598052), glucose-6-phosphate isomerase (pgi, LC598076- LC598078) and gamma-glutamyl phospate reductase (proA, LC598099- LC598101)), and all clustered into a clade containing other confirmed strains of P. wasabiae. As a result, these three strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these three strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against onion (cv. 'Hayate') was confirmed by the field experiments with 5 weeks growth plants sprayed with bacterial suspension (1×107cfu/ml) resulting in soft rot on the plants about four weeks after inoculation, whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of onion in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of onion in Japan.

First report of soft rot of cabbage caused by Pectobacterium wasabiae in Japan.

Cabbage (Brassica oleracea var. capitata) is one of the important vegetables in Japan. In the summer of 2019, some cabbages with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. All diseased plants showed grey to brown discoloration and expanding water-soaked lesions on leaves. We obtained two independent strains (NACAB191 and NACAB192) from diseased leaves. DNA from these strains yielded an expected single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR, but did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. These two strains grew at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative and rod-shaped. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All strains exhibited pectolytic activity on potato slices. The sequence analysis of 16S rDNA (LC597897 and LC597898) showed more than 98% identities to P. wasabiae strain (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA10 (Kumer et al. 2018) using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597923 and LC597924), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597970 and LC597971), isocitrate dehydrogenase (icdA, LC597996 and LC597997), malate dehydrogenase(mdh, LC598022 and LC598023), mannitol-1-phosphate dehydrogenase (mtlD, LC598048 and LC598049), glucose-6-phosphate isomerase (pgi, LC598074 and LC598075) and gamma-glutamyl phospate reductase (proA, LC598079 and LC598080)), and all clustered NACAB191 and NACAB192 into a clade containing other confirmed strains of P. wasabiae. As a result, these two strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these two strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against cabbage (cv. 'Rakuen') was confirmed by the field experiments with five weeks growth plants sprayed with bacterial suspension (1×107cfu/ml). Thirty cabbages per strain were used in this study, 12 plants treated the suspension of NACAB191 and 16 plants treated the suspension of NACAB192 which died with the same soft rot symptoms about four weeks after inoculation. Whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of cabbage in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of cabbage in Japan.

N-Glycan Branching Is Required for Development of Mature B Cells.

Galectins have been implicated in inhibiting BCR signaling in mature B cells but promoting pre-BCR signaling during early development. Galectins bind to branched N-glycans attached to cell surface glycoproteins to control the distribution, clustering, endocytosis, and signaling of surface glycoproteins. During T cell development, N-glycan branching is required for positive selection of thymocytes, inhibiting both death by neglect and negative selection via enhanced surface retention of the CD4/CD8 coreceptors and limiting TCR clustering/signaling, respectively. The role of N-glycan branching in B cell development is unknown. In this study, we report that N-glycan branching is absolutely required for development of mature B cells in mice. Elimination of branched N-glycans in developing B cells via targeted deletion of N-acetylglucosaminyl transferase I (Mgat1) markedly reduced cellularity in the bone marrow and/or spleen and inhibited maturation of pre-, immature, and transitional stage 2 B cells. Branching deficiency markedly reduced surface expression of the pre-BCR/BCR coreceptor CD19 and promoted spontaneous death of pre-B cells and immature B cells in vitro. Death was rescued by low-dose pre-BCR/BCR stimulation but exacerbated by high-dose pre-BCR/BCR stimulation as well as antiapoptotic BclxL overexpression in pre-B cells. Branching deficiency also enhanced Nur77 induction, a marker of negative selection. Together, these data suggest that, as in T cells, N-glycan branching promotes positive selection of B cells by augmenting pre-BCR/BCR signaling via CD19 surface retention, whereas limiting negative selection from excessive BCR engagement.

First Report of Pectobacterium parmentieri Causing Stem Rot Disease of Potato in Russia

Bacterial (aerial) stem rot of potato (Solanum tuberosum) in Russia is caused by the pectolytic bacteria Pectobacterium carotovorum subsp. carotovorum, P. atrosepticum, or Dickeya spp. (De Boer and Kelman 2001). In 2011 to 2014, over 100 pectinolytic enterobacterial isolates were obtained from green potato plants with blackleg symptoms found in four districts (Odintsovo, Dmitrov, Kashira, and Ruza) of Moscow region in severely infected commercial fields planted with cultivars Nevskii, Gala, and Lady Clear. Disease incidence was from 1 to 10% of infected plants in the field in July to August. From 5 to 15 samples were assayed for each of eight evaluated fields. Six bacterial strains isolated during the survey exhibited pectolytic ability on crystal violet pectate (CVP) agar and potato slices, and failed to grow at 37°C, but physiological tests did not conclusively distinguish the bacterium as P. atrosepticum (Khayi et al. 2016). They were identified as Pectobacterium parmentieri (formerly P. wasabiae group isolated from potato plants) (Kornev et al. 2012) based on their inability to elicit hypersensitive reaction on Nicotiana tabacum and their ability to utilize raffinose and lactose. These bacterial strains were gram negative, rod shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and ONPG-positive and positive for acid production from d-galactose, lactose, melibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-d-glucoside, and d-arabitol. All the strains exhibited pectolytic activity on potato slices. Reference strains of P. carotovorum NCPPB 312, P. atrosepticum NCPPB 549, and P. parmentieri LMG 29774 obtained from international collections were used as controls. Molecular identification of the bacterium was performed with 16S rRNA and recombinase A (recA) coding sequences as previously described (Waleron et al. 2002). BLAST analysis of the 16S rRNA DNA sequence for the strain F148 (syn. PB20) (GenBank accession no. MG493237.1) revealed 99% identity to the 16S rRNA of P. parmentieri WPP163 (Kornev et al. 2012). The other five strains had sequence of 16S rRNA identical to F148. Phylogenetic reconstruction using DNA sequences of recA gene from P. parmentieri F009, F034, F035, F127, F148 (PB20), and F149 (GenBank accession nos. MG518488, MG518489, MG518490, MG518491, and MG518492) and other related taxa clustered these bacteria with strain SCRI488 and other P. parmentieri strains, readily distinguishing them from other closely related species of Pectobacterium. Pathogenicity assays were conducted on stems of four young potato plants for each strain. Five-week-old ‘Docker’ potato plants were wound inoculated by injecting 10 μl of a bacterial suspension (10⁸ CFU/ml) into each stem. Plants were exposed to a 72-h incubation period with 90 to 100% relative humidity at 30°C, and lesions were measured. All inoculated plants exhibited stem rot/blackleg symptoms similar to those observed in the field, including brown water-soaked lesions. Symptoms were not observed on water-inoculated controls. The bacteria reisolated from all inoculated stems caused pitting on CVP and exhibited the same morphology as the original culture and, thus, were confirmed as P. parmentieri using 16S rRNA and recA sequences, fulfilling Koch’s postulates. To our knowledge, this is the first report of bacterial stem rot/blackleg of potato caused by P. parmentieri in the Russian Federation. According to results of several independent diagnostic laboratories, P. parmentieri had second place in occurrence on seed potatoes after P. carotovorum subsp. brasiliense (about 20% of the tested seed stocks) in field season 2017.

TGF-β1 Causes EMT by Regulating N-Acetyl Glucosaminyl Transferases via Downregulation of Non Muscle Myosin II-A through JNK/P38/PI3K Pathway in Lung Cancer.

Epithelial to mesenchymal transition (EMT) is a major determinant of cancer metastasis and is closely linked with TGF-β1. Intracellular proteins, including E. Cadherin, N. Cadherin and Vimentin are directly related to EMT that affect cell migration and adhesion; on the other hand, non muscle myosin (NM) has a central role in cytokinesis, migration and adhesion. We aimed to explore the association of EMT and metastasis with TGF-β1 through regulation of non-muscle myosin II-A (NMII-A) and its interaction with Hexosamine Biosynthesis Pathway (HBP). Protein expression changes were assessed by western blotting and immunofluorescent staining while transcription level changes were assessed by qRT-PCR. EMT was assessed by phenotypic analysis, wound healing, proliferation and transwell migration assay in vitro while in vivo studies were conducted in BALB/c nude mice for lung orthotopic and tail vein metastasis models. We demonstrated that regulation of JNK/ P38/PI3K by TGF-β1 led to down expression of NMII-A which promoted EMT and lung cancer metastasis. This down expression of NMII-A conversely upregulated the expression of Core 2 N-acetyl Glucosaminyl Transferase mucin type (C2GnT-M) and further facilitated up-regulation and down-regulation of N-acetylglucosaminyltransferase (GnT) -V and -III respectively; moreover, NMII-A K.D cells showed 3 times more tendency to migrate towards brain in vivo. The study reports a novel pathway through which NMII-A negatively regulates EMT and metastasis via up regulation of C2GnT-M, GnT-V and down expression of GnT-III. These findings of lung cancer may further be required to study other cancer types.

N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-L-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I.

Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-L-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man5 (cgl-IDUA) or Man6-8 (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K m) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases.

The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans

BackgroundThe pathogenic potential of Candida albicans depends on adhesion to the host cells mediated by highly glycosylated adhesins, hyphae formation and growth of biofilm. These factors require effective N-glycosylation of proteins.Here, we present consequences of up- and down-regulation of the newly identified ALG13 gene encoding N-acetylglucosaminyl transferase, a potential member of the Alg7p/Alg13p/Alg14p complex catalyzing the first two initial reactions in the N-glycosylation process. MethodsWe constructed C. albicans strain alg13∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. Gene expression and enzyme activity were measured using RT-qPCR and radioactive substrate. Cell wall composition was estimated by HPLC DIONEX. Protein glycosylation status was analyzed by electrophoresis of HexNAcase, a model N-glycosylated protein in C. albicans. ResultsBoth decreased and elevated expression of ALG13 changed expression of all members of the complex and resulted in a decreased activity of Alg7p and Alg13p and under-glycosylation of HexNAcase. The alg13 strain was also defective in hyphae formation and growth of biofilm. These defects could result from altered expression of genes encoding adhesins and from changes in the carbohydrate content of the cell wall of the mutant. General significanceThis work confirms the important role of protein N-glycosylation in the pathogenic potential of C. albicans.

Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients.

Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting GlcNAc and demonstrate the importance of N-glycosylation in cancer progression.

Burkholderia sp. induces functional nodules on the South African invasive legume Dipogon lignosus (Phaseoleae) in New Zealand soils.

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678(T) which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

First Report of Pectobacterium wasabiae Causing Soft Rot of Cabbage in Malaysia.

Soft rot of cabbage (Brassica rapa) occurs sporadically in Malaysia, causing economic damage under the hot and wet Malaysian weather conditions that are suitable for disease development. In June 2011, 27 soft rotting bacteria were isolated from cabbage plants growing in the Cameron Highlands and Johor State in Malaysia where the economic losses exceeded 50% in severely infected fields and greenhouses. Five independent strains were initially identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and elicit hypersensitive reaction (HR) on Nicotiana tabaccum and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG-positive and positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-D-glucoside, and D-arabitol. All the strains exhibited pectolytic activity on potato slices. PCR assays were conducted to distinguish P. wasabiae from P. carotovorum subsp. brasiliensis, P. atrosepticum, and other Pectobacterium species using primers Br1f/L1r (2), Eca1f/Eca2r (1), and EXPCCF/EXPCCR, respectively. DNA from strains did not yield the expected amplicon with the Br1f/L1r and Eca1f/Eca2r, whereas a 550-bp amplicon typical of DNA from P. wasabiae was produced with primers EXPCCF/EXPCCR. ITS-RFLP using the restriction enzyme, Rsa I, produced similar patterns for the Malaysian strains and the P. wasabiae type strain (SCRI488), but differentiated it from P. carotovora subsp. carotovora, P. atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya chrysanthemi type strains. BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. KC445633) showed 99% identity to the 16S rRNA of Pw WPP163. Phylogenetic reconstruction using concatenated DNA sequences of mdh and gapA from P. wasabiae Cc6 (KC484657) and other related taxa (4) clustered Malaysian P. wasabiae strains with P. wasabiae SCRI488, readily distinguishing it from other closely related species of Pectobacterium. Pathogenicity assays were conducted on leaves and stems of four mature cabbage plants for each strain (var. oleifera) by injecting 10 μl of a bacterial suspension (108 CFU/ml) into either stems or leaves, and incubating them in a moist chamber at 80 to 90% relative humidity at 30°C. Water-soaked lesions similar to those observed in the fields and greenhouses were observed 72 h after injection and bacteria with similar characteristics were consistently reisolated. Symptoms were not observed on water-inoculated controls. The pathogenicity test was repeated with similar results. P. wasabiae was previously reported to cause soft rot of horseradish in Japan (3). However, to our knowledge, this is the first report of P. wasabiae infecting cabbage in Malaysia.

Characterization and downstream mannose phosphorylation of human recombinant α‐L‐iduronidase produced in Arabidopsis complex glycan‐deficient (cgl) seeds

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme™). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells—UDP-GlcNAc:lysosomal enzyme N−acetylglucosamine (GlcNAc)−1−phosphotransferase—and GlcNAc−1−phosphodiester α−N−acetylglucosaminidase (the ‘uncovering enzyme’). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

Knockdown of N-Acetylglucosaminyl Transferase V Ameliorates Hepatotoxin-Induced Liver Fibrosis in Mice

Aberrant N-glycosylation caused by altered N-acetyl glucosaminyltransferase V (GnT-V) expression is known to regulate tumor invasion and metastasis by modulating multiple cytokine signaling pathways. However, the exact role of GnT-V in the development of liver fibrosis has not been clearly defined. Here, we induced mouse liver fibrosis by ip injections of carbon tetrachloride (CCl4) or thioacetamide (TAA) and observed significant increase of hepatic GnT-V during the processes of liver fibrogenesis. Meanwhile, upregulations of GnT-V were detected in the activated hepatic stellate cells (HSCs) and injured hepatocytes. To knock down hepatic GnT-V expression, adenovirus that expressed the GnT-V siRNA was injected via the tail vein. Adenovirus-mediated delivery of GnT-V siRNA dramatically reduced the GnT-V expression in fibrotic liver and activated HSC in vivo and consequently alleviated CCl4- or TAA-induced liver fibrosis as assessed through collagen deposition and profiles of profibrogenic markers. Furthermore, knockdown of GnT-V in HSCs reduced transforming growth factor beta (TGF-β)/Smad signaling and blunted the activated HSC phenotype. The suppression of TGF-β/Smad signaling in HSCs correlated with the decrease of GnT-V-modified β1,6-branched N-glycan on TGF-β receptors. Knockdown of GnT-V also suppressed platelet-derived growth factor (PDGF)-induced HSC proliferation and migration through inhibiting PDGF/Erk signaling. Finally, we demonstrated that knockdown of GnT-V profoundly suppressed TGF-β1-induced epithelial-mesenchymal transition (EMT) in hepatocytes by morphological assessment and reversal of EMT markers. In conclusion, this study demonstrates that GnT-V is implicated in hepatotoxin-induced liver fibrosis, and targeting GnT-V may be a feasible and promising approach for treating liver fibrosis.

First Report of Soft Rot Disease Caused by Pectobacterium wasabiae on Sweet Potato, Tomato, and Eggplant in Malaysia.

In August 2011, sweet potato (Ipomoea batatas), tomato (Solanum lycopersicum), and eggplant (S. melongena) crops from major growing areas of the Cameron highlands and Johor state in Malaysia were affected by a soft rot disease. Disease incidence exceeded 80, 75, and 65% in severely infected fields and greenhouses of sweet potato, tomato, and eggplant, respectively. The disease was characterized by dark and small water-soaked lesions or soft rot symptoms on sweet potato tubers, tomato stems, and eggplant fruits. In addition, extensive discoloration of vascular tissues, stem hollowness, and water-soaked, soft, dark green lesions that turned brown with age were observed on the stem of tomato and eggplant. A survey was performed in these growing areas and 22 isolates of the pathogen were obtained from sweet potato (12 isolates), tomato (6 isolates), and eggplant (4 isolates) on nutrient agar (NA) and eosin methylene blue (EMB) (4). The cultures were incubated at 27°C for 2 days and colonies that were emerald green on EMB or white to gray on NA were selected for further studies. All bacterial cultures isolated from the survey exhibited pectolytic ability on potato slices. These bacterial isolates were gram negative; rod shaped; N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG positive; and were also positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. They were negative for indol production, phosphatase activity, reducing substances from sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-mathyl-D-glocoside, and D-arabitol. The bacteria did not grow on NA at 37°C. Based on these biochemical and morphological assays, the pathogen was identified as Pectobacterium wasabiae (2). In addition, DNA was extracted and PCR assay with two primers (16SF1 and 16SR1) was performed (4). Partial sequences of 16S rRNA (GenBank Accession Nos. JQ665714, JX494234, and JX513960) of sweet potato, tomato, and eggplant, respectively, exhibited a 99% identity with P. wasabiae strain SR91 (NR_026047 and NR_026047.1). A pathogenicity assay was carried out on sweet potato tubers (cv. Oren), tomato stems (cv. 152177-A), and eggplant fruits (cv. 125066x) with 4 randomly representative isolates obtained from each crop. Sweet potato tubers, tomato stems, and eggplant fruits (4 replications) were sanitized in 70% ethyl alcohol for 30 s, washed and rinsed in sterile distilled water, and needle punctured with a bacterial suspension at a concentration of 108 CFU/ml. Inoculated tubers, stems, and fruits were incubated in a moist chamber at 90 to 100% RH for 72 h at 25°C when lesions were measured. All inoculated tubers, stems, and fruits exhibited soft rot symptoms after 72 h similar to those observed in the fields and greenhouses and the same bacteria were consistently reisolated. Symptoms were not observed on controls. The pathogenicty test was repeated with similar results. P. wasabiae have been previously reported to cause soft rot on Japanese horseradish (3), and aerial stem rot on potato in New Zealand (4), the U.S. (2), and Iran (1). To our knowledge, this is the first report of sweet potato, tomato, and eggplant soft rot caused by P. wasabiae in Malaysia.

Preparation and application of a ‘clickable’ acceptor for enzymatic synthesis of heparin oligosaccharides

A ‘clickable’ disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.

First Report of Pectobacterium wasabiae Causing Aerial Stem Rot of Potato in Washington State.

Aerial stem rot of potato (Solanum tuberosum), also known as bacterial stem rot, is often caused by the pectolytic bacteria Pectobacterium (Erwinia) carotovorum subsp. carotovorum, P. atrosepticum, or Dickeya spp. (3). A survey was carried out in August 2008 in 'Russet Burbank' potato fields exhibiting aerial stem rot symptoms in the Columbia Basin of Washington State. One bacterial strain isolated during the survey, PwO405, exhibited pectolytic ability on crystal violet pectate (CVP) agar and potato slices and failed to grow at 37°C, but physiological tests did not conclusively distinguish the bacterium as P. atrosepticum (1). The bacterium was positive for ONPG, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. Molecular identification of the bacterium was performed with 16S rRNA, aconitase (acnA), and malate dehydrogenase (mdh) coding sequences as previously described (2,4). Partial sequences of 16S rRNA (1,408 bp) and acnA (412 bp) genes (GenBank Accession Nos. JQ723958 and JQ723959, respectively) exhibited 99% shared identities with P. wasabiae strain WPP163, while the mdh sequence (435 bp) (GenBank Accession No. JQ723960) exhibited 100% shared identity with mdh sequences from three P. wasabiae strains (NZEC9, NZEC10, and NZEC8974). Maximum parsimony analysis using concatenated acnA and mdh sequences from this study and Pectobacterium sequences previously deposited in GenBank (2,4) clustered strain PwO405 with other P. wasabiae strains. Three 7-week-old 'Russet Norkotah' potato plants were wound-inoculated by inserting a sterile 23 gauge needle just above a central leaf axil at a depth of 1 mm. A 10-μl drop of inoculum (104 CFU) was placed on the wound. Plants were exposed to a 24-h leaf wetness period (90 to 100% RH in a mist chamber) and lesions were measured. All three inoculated plants exhibited aerial stem rot symptoms similar to those observed in the field, including brown water-soaked lesions that spread acropetally and basipetally. Upon drying, the lesions became shriveled and turned dark brown to black. Some plants exhibited hollowing of the stems and unilateral wilt on the side of the lesion. Symptoms were not observed on water-inoculated controls. The bacteria that were reisolated into pure culture from all three inoculated stems caused pitting on CVP and exhibited the same morphology as the original culture and were confirmed as P. wasabiae using 16S rRNA, acnA, and mdh coding sequences, fulfilling Koch's postulates. Stem rot ability of the bacterium was also confirmed on four potato cultivars: 'Ranger Russet,' 'Russet Burbank,' 'Russet Norkotah,' and 'Umatilla Russet' by wound-inoculating six single-stem plants of each cultivar as described above. To our knowledge, this is the first report of aerial stem rot of potato caused by P. wasabiae in Washington State.