Surface Carbohydrate Research Articles

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Galactose-functionalized polyHIPE scaffolds for use in routine three dimensional culture of mammalian hepatocytes.

Three-dimensional (3D) cell culture is regarded as a more physiologically relevant method of growing cells in the laboratory compared to traditional monolayer cultures. Recently, the application of polystyrene-based scaffolds produced using polyHIPE technology (porous polymers derived from high internal phase emulsions) for routine 3D cell culture applications has generated very promising results in terms of improved replication of native cellular function in the laboratory. These materials, which are now available as commercial scaffolds, are superior to many other 3D cell substrates due to their high porosity, controllable morphology, and suitable mechanical strength. However, until now there have been no reports describing the surface-modification of these materials for enhanced cell adhesion and function. This study, therefore, describes the surface functionalization of these materials with galactose, a carbohydrate known to specifically bind to hepatocytes via the asialoglycoprotein receptor (ASGPR), to further improve hepatocyte adhesion and function when growing on the scaffold. We first modify a typical polystyrene-based polyHIPE to produce a cell culture scaffold carrying pendent activated-ester functionality. This was achieved via the incorporation of pentafluorophenyl acrylate (PFPA) into the initial styrene (STY) emulsion, which upon polymerization formed a polyHIPE with a porosity of 92% and an average void diameter of 33 μm. Histological analysis showed that this polyHIPE was a suitable 3D scaffold for hepatocyte cell culture. Galactose-functionalized scaffolds were then prepared by attaching 2′-aminoethyl-β-d-galactopyranoside to this PFPA functionalized polyHIPE via displacement of the labile pentafluorophenyl group, to yield scaffolds with approximately ca. 7–9% surface carbohydrate. Experiments with primary rat hepatocytes showed that cellular albumin synthesis was greatly enhanced during the initial adhesion/settlement period of cells on the galactose-functionalized material, suggesting that the surface carbohydrates are accessible and selective to cells entering the scaffold. This porous polymer scaffold could, therefore, have important application as a 3D scaffold that offers enhanced hepatocyte adhesion and functionality.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galβ1-3 or Galα1-4Galβ1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.

Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: Potential role of surface carbohydrates in successful parasitism

In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts.

Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In this assay, various S. aureus bacteria or culture supernatant dilutions are exposed to human pooled serum. S. aureus specific IgGs from the serum are captured to the IgG-accessible staphylococcal antigens present on the bacterial surface, or in supernatant, in a dose dependent manner. The remaining non-captured IgGs are then quantified by incubation with Luminex beads to which the individual recombinant S. aureus surface proteins and carbohydrates are covalently linked. The signals obtained are inversely proportional to the antigen concentration measured, and binding patterns were dependent on the particular S. aureus isolate used, growth conditions and growth phase. For example, the detection of cell wall teichoic acid (WTA) and peptidoglycan (PG) carbohydrate structures increased significantly when bacteria entered the stationary growth phase, whereas the amounts of clumping factors (ClfA and ClfB) decreased significantly. Using this technique, the appearance of several S. aureus gene knockout strains were studied. We demonstrated that in an S. aureus srtA gene knockout strain, srtA-dependent cell-surface-associated proteins were detectable yet not covalently linked to the PG layer. In conclusion, we show that this multiplex competitive Luminex assay is a suitable method for the rapid and simultaneous determination of S. aureus IgG-accessible cell-surface-associated proteins and carbohydrates in various growth phases and growth conditions in different S. aureus strains.

Differences in Selection Behaviors and Chemical Cues of adult Asian Citrus Psyllids, Diaphorina citri, on Healthy and Huanglongbing-Infected Young Shoots of Citrus Plants

The pathogen Candidatus Liberibacter asiaticus (Las) causes devastating citrus huanglongbing in many parts of Asia. The host selection behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psyllidae), the only known Las vector, is critical to its epidemics. Las-infected citrus plants attract adult psyllids by altering their color and chemical cues. However, it is unclear whether any changes in selection behavior or chemical cues occur in young shoots. Selection behavior bioassays showed the number of adults and eggs on Las-infected shoots was significantly higher than on healthy shoots. Significantly more adults responded to the odor of Las-infected shoots than to that of uninfected shoots. GC-MS analysis of volatile constituents showed that the quantitatively dominant constituents were β-elemene and β-phellandrene in both samples, (+)-epi-bicyclosesquiphellandrene was only detected in Las-infected shoots, but ocimene, germacrene D, δ-cadinene, d-longifolene, and α-sinensal were detected only in healthy shoots. Analysis of the concentrations of surface-soluble carbohydrates indicated that the levels of sucrose, fructose, and glucose were significantly higher on the surfaces of Las-infected shoots than on the surfaces of healthy shoots. Alteration of volatile compounds and soluble surface carbohydrates of young shoots after Las infection could be attributed to the preference of Asian citrus psyllids for Las-infected young shoots. The knowledge obtained from this investigation may contribute to novel control measures for plant diseases transmitted by insect vectors.

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejunid-altro- and l-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-D-manno- and GDP-6-deoxy-D-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex L-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-D-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaGNCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG81176 (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces L-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaGNCTC is not necessary for L-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG81176, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.

Possible role of a cell surface carbohydrate in evolution of resistance to viral infections in old world primates.

Due to inactivation of the α1,3-galactosyltransferase gene (GGTA1, or the α1,3GT gene) approximately 28 million years ago, the carbohydrate αGal (Galα1,3Galβ1,4GlcNAc) is not expressed on the cells of Old World monkeys and apes (including humans) but is expressed in all other mammals. The proposed selective advantage of this mutation for these primates is the ability to produce anti-Gal antibodies, which may be an effective immune component in neutralizing αGal-expressing pathogens. However, loss of α1,3GT expression may have been advantageous by providing natural resistance against viral pathogens that exploited the α1,3GT pathway or cell surface αGal for infection. Infections of paired cell lines with differential expression of α1,3GT showed that Sindbis viruses (SINV) preferentially replicate in α1,3GT-positive cells, whereas herpes simplex viruses type 1 and type 2 (HSV-1 and HSV-2) preferentially grow in cells lacking α1,3GT. Viral growth and spread correlated with the ability of the different viruses to successfully initiate infection in the presence or absence of α1,3GT expression. GT knockout (KO) suckling mice infected with SINV strains (AR339 and S.A.AR86) experienced significant delay in onset of disease symptoms and mortality compared to wild-type (WT) B6 suckling mice. In contrast, HSV-2-infected GT KO mice had higher viral titers in spleen and liver and exhibited significantly more focal hepatic necrosis than WT B6 mice. This study demonstrates that α1,3GT activity plays a role in the course of infections for certain viruses. Furthermore, this study has implications for the evolution of resistance to viral infections in primates.

A disposable electrochemiluminescence device for ultrasensitive monitoring of K562 leukemia cells based on aptamers and ZnO@carbon quantum dots

We developed a new electrochemiluminescence (ECL) platform for ultrasensitive and selective detection of leukemia cells. In order to construct the platform, the nonporous gold with controllable three-dimensional porosity and good conductivity was used to modify the screen-printed carbon electrode. The carbon quantum dots (CQDs) coated ZnO nanosphere (ZnO@CQDs) were used as good ECL label with low cytotoxicity and good biocompatibility. Structure characterization was obtained by means of transmission electron microscopy and scanning electron microscopy images. The aptamer was used for cell capture and the concanavalin A conjugated ZnO@CQDs was used for selective recognition of the cell surface carbohydrate. The proposed method showed a good analytical performance for the detection of K562 cells ranging from 1.0×102 to 2.0×107cellsmL−1 with a detection limit of 46cellsmL−1. The as-proposed device has the advantages of high sensitivity, nice specificity and good stability and could offer great promise for sensitive detection of leukemia cells in response to therapy.

Carbohydrate-Decorated PCL Fibers for Specific Protein Adhesion

Ultrafine biocompatible fibers decorated with carbohydrates were prepared by electrospinning. Both bulk- and surface-modification approaches have been investigated and compared in terms of practicability and grafting density along the fibrous mats. On one hand, bulk-functionalized fibers were prepared by electrospinning of native and galactose-modified PCL polymers. The size and morphology of the resulting fibers was strongly influenced by the galactose-PCL content as observed by electron microscopy. Successful surface modification was evidenced by water contact angle measurements, but a rather low carbohydrate density was attained, as indicated by a colorimetric quantification. On the other hand, efficient and versatile surface-glycosylation was achieved after modification of azido-functionalized electrospun fibers by CuAAC click-chemistry. Homogeneous ultrafine PCL fibers, decorated with azide functions, have been made completely hydrophilic upon coupling with propargyl-α-d-mannoside and propargyl-β-d-galactoside. Specific adhesion of lectins further attested good bioavailability of the surface carbohydrate residues, suggesting interesting perspectives of the latter approach in the development of bioactive materials for tissue engineering.

Interaction between the genomes of Lactococcus lactis and phages of the P335 species

Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (Φ KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome.

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate–protein interaction profiling and cell surface carbohydrate expression evaluation

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 10(10). The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.

Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species

Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain–Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.

Phosphatidylserine Exposure and Surface Sugars in Two Leishmania (Viannia) braziliensis Strains Involved in Cutaneous and Mucocutaneous Leishmaniasis

Phosphatidylserine (PS) and surface carbohydrates (SC) are known as virulence factors that may contribute to the different clinical symptoms ranging from self-healing cutaneous leishmaniasis lesions to fatal visceral disease. Leishmania (Viannia) braziliensis causes localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). We analyzed PS exposure and SC expression associated with 2 primary L. braziliensis isolates from patients with LCL or MCL. The role of PS exposure was also addressed during promastigotes phagocytosis by macrophages. We observed higher PS exposure on the surface of late stationary growth phase promastigotes from patients with LCL, compared with those from patients with MCL, and both strains were alive during PS display. Reduction in the infectivity index was observed during macrophage interaction with late stationary growth phase promastigotes in which PS was blocked by annexin V. The major surface carbohydrates detected on LCL and MCL promastigotes were α-Man, α-Glc, and α-Gal. However, α-β-GalNAc, although observed on the surface of the LCL strain during the late stationary growth phase was highly expressed on the surface of early stationary growth phase promastigotes. Our results suggest that PS and SC can modulate interactions between Leishmania organisms and host cells and may be important for the outcome of the clinical course of diseases caused by L. braziliensis.

Cultivation and Characterization of Cornea Limbal Epithelial Stem Cells on Lens Capsule in Animal Material-Free Medium

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.

Structural Basis of Rotavirus Strain Preference toward N -Acetyl- or N -Glycolylneuraminic Acid-Containing Receptors

The rotavirus spike protein domain VP8* is essential for recognition of cell surface carbohydrate receptors, notably those incorporating N-acylneuraminic acids (members of the sialic acid family). N-Acetylneuraminic acids occur naturally in both animals and humans, whereas N-glycolylneuraminic acids are acquired only through dietary uptake in normal human tissues. The preference of animal rotaviruses for these natural N-acylneuraminic acids has not been comprehensively established, and detailed structural information regarding the interactions of different rotaviruses with N-glycolylneuraminic acids is lacking. In this study, distinct specificities of VP8* for N-acetyl- and N-glycolylneuraminic acids were revealed using biophysical techniques. VP8* protein from the porcine rotavirus CRW-8 and the bovine rotavirus Nebraska calf diarrhea virus (NCDV) showed a preference for N-glycolyl- over N-acetylneuraminic acids, in contrast to results obtained with rhesus rotavirus (RRV). Crystallographic structures of VP8* from CRW-8 and RRV with bound methyl-N-glycolylneuraminide revealed the atomic details of their interactions. We examined the influence of amino acid type at position 157, which is proximal to the ligand's N-acetyl or N-glycolyl moiety and can mutate upon cell culture adaptation. A structure-based hypothesis derived from these results could account for rotavirus discrimination between the N-acylneuraminic acid forms. Infectivity blockade experiments demonstrated that the determined carbohydrate specificities of these VP8* domains directly correlate with those of the corresponding infectious virus. This includes an association between CRW-8 adaption to cell culture, decreased competition by N-glycolylneuraminic acid for CRW-8 infectivity, and a Pro157-to-Ser157 mutation in VP8* that reduces binding affinity for N-glycolylneuraminic acid.

Distinct contributions of Galgt1 and Galgt2 to carbohydrate expression and function at the mouse neuromuscular junction

At the mammalian neuromuscular junction (NMJ), the CT (cytotoxic T cell) carbohydrate antigen [GalNAcβ1,4[Neu5Ac/Gcα2,3]Galβ1,4GlcNAc-] is a unique synaptic cell surface carbohydrate present in both the presynaptic and postsynaptic membranes. Here we show that Galgt1, which synthesizes the β1,4GalNAc linkage of the CT carbohydrate on gangliosides, is required for presynaptic expression of the CT carbohydrate at the NMJ, while Galgt2, which can synthesize the β1,4GalNAc of the CT carbohydrate on glycoproteins, is required for postsynaptic expression. Proper postsynaptic localization of the CT carbohydrate also required muscle expression of dystroglycan, a known muscle substrate for Galgt2. Transgenic overexpression of Galgt2 in skeletal myofibers altered the expression of synaptic muscle proteins and altered neuromuscular topography, which was partially NCAM-dependent, while an increase in postsynaptic AChR-rich domains was observed in both neuron- and skeletal muscle-specific Galgt2 transgenic mice. By contrast, overexpression of Galgt1 in muscle did not allow for increased expression of CT carbohydrate on the sarcolemmal membrane and instead caused muscle pathology. Loss of Galgt2 increased intracellular accumulation of acetylcholine receptors and acetylcholinesterase within skeletal myofibers, suggesting an additional role for Galgt2 in neuromuscular stability. These experiments demonstrate that Galgt1 and Galgt2 contribute in distinct ways to the expression and function of synaptic βGalNAc-containing carbohydrates at the NMJ.

The effect of human immunodeficiency virus-1 on monocyte-derived dendritic cell maturation and function.

Dendritic cells (DC) are mediators of the adaptive immune response responsible for antigen presentation to naive T cells in secondary lymph organs. Human immunodeficiency virus (HIV-1) has been reported to inhibit the maturation of DC, but a clear link between maturation and function has not been elucidated. To understand further the effects of HIV-1 on DC maturation and function, we expanded upon previous investigations and assessed the effects of HIV-1 infection on the expression of surface molecules, carbohydrate endocytosis, antigen presentation and lipopolysaccharide (LPS) responsiveness over the course of maturation. In vitro infection with HIV-1 resulted in an increase in the expression of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as well as decreases in maturation-induced CCR7 and major histocompatibility complex (MHC)-II expression. Retention of endocytosis that normally occurs with DC maturation as well as inhibition of antigen presentation to CD8(+) T cells was also observed. Mitogen-activated protein kinase (MAPK) responsiveness to LPS as measured by phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK)1/2 was not affected by HIV-1 infection. In summary, in-vitro HIV-1 impairs DC maturation, as defined by cell surface protein expression, with selective alterations in mature DC function. Understanding the mechanisms of DC dysfunction in HIV infection will provide further insight into HIV immune pathogenesis.

Poly- N -Acetylglucosamine Expression by Wild-Type Yersinia pestis Is Maximal at Mammalian, Not Flea, Temperatures

ABSTRACTNumerous bacteria, including Yersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis by Y. pestis have been reported to be maximal at 21 to 28°C or “flea temperatures,” facilitating the regurgitation of Y. pestis into a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation by Y. pestis have used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WT Y. pestis. Extracts of WT sylvatic Y. pestis strains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WT Y. pestis is maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection.

Cultivation and characterization of corneal limbal epithelial stem cells on lens capsule in animal material‐free medium

AbstractPurpose To develop simple, reproducible and animal‐materials free method for cultivating and differentiating human limbal stem cells (LSCs) into corneal epithelium on human lens capsule (LC) for clinical transplantation.Methods Limbal tissues (2x2x0,25 mm) were harvested from cadavers (44 males and 33 females, age 70,5+9,3 years) within 12 hours of biologic death. All tissue collection complied with the guidelines of the Helsinki Declaration and was approved by the Regional Ethical Committee. Cell viability was measured with the MTT and annexin‐FITC/propidium iodide assays. Characterization of the growing cells was performed by RT‐qPCR and immunfluorescence stainings accordingly.Results Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (&gt;97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were tested for positivity for markers of stemness (P63, ABCG2, CK19, ITGA9, VIM), proliferation (Ki‐67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non‐hematopoietic, ‐endothelial and ‐mesenchymal stem cell phenotype, while the cell adhesion molecules, integrins and lectin‐based surface carbohydrate profiling showed a specific pattern on the LESCs.Conclusion We report a method for isolating and expanding cornea LESCs from cadavers or alternatively from autologous donors capable of producing viable cell outgrowth on LC for possible treatment of LESC deficiency.