A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation

Alkyl glycosides are surfactants with good biodegradability and low toxicity, attractive to produce by an enzymatic method to get a well-defined product. In this paper, we report a novel thermostable variant of a family 3 beta-glucosidase to be an efficient catalyst in alkyl-glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule. The enzyme has an apparent optimum for hydrolysis at 90 degrees C, which coincides with its unfolding temperature. The total activity is lower at lower temperature (60 degrees C), but the ratio of alcoholysis/hydrolysis is slightly more favourable. This ratio is however more heavily influenced by the water content and the pH. Optimal reaction conditions for hexyl glucoside synthesis from p-nitrophenyl-beta-glucopyranoside were a water/hexanol two-phase system containing 16% (v/v) water, pH 5.8, and a temperature of 60 degrees C. Under these conditions, the total initial reaction rate was 153 micromol min(-1)mg(-1) and the alcoholysis/hydrolysis ratio was 5.1. Comparing with alcoholysis/hydrolysis ratios of other beta-glycosidases, TnBgl3B can be considered to be a very promising catalyst for alkyl glucoside production.     

The M4 muscarinic acetylcholine receptor plays a key role in the control of murine hair follicle cycling and pigmentation

Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in M4R KO mice, compared to age-matched wild type controls. On day 17, when mice enter the first hair growth cycle, the KO mice still showed a slightly retarded catagen phase. Subsequently, hair follicles of the KO mice stayed in a highly significantly prolonged telogen phase, while wild type mice had already far progressed in the hair cycle by entry into anagen. Most strikingly, the M4R KO mice did not engage in follicular melanogenesis and failed to produce pigmented hair shafts. The current pilot study suggests that the M4R plays a fundamental role in the control of the murine hair follicle cycling and is an essential signaling element in the control of hair follicle pigmentation.     

Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis

A pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range—more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250 pmol in 20 μl using a Tris–HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS–PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points.     

Initiation of Antiretroviral Therapy (ART) at Different Stages of HIV-1 Disease Is Not Associated with the Proportion of Exhausted CD8+ T Cells

CD8⁺ T cell-restricted immunity is important in the control of HIV-1 infection, but continued immune activation results in CD8⁺ T cell dysfunction. Early initiation of antiretroviral treatment (ART) and the duration of ART have been associated with immune reconstitution. Here, we evaluated whether restoration of CD8⁺ T cell function in HIV-1-infected individuals was dependent on early initiation of ART. HIV-specific CD107a, IFNγ, IL-2, TNFα and MIP-1β expression by CD8⁺ T cells and the frequency of CD8⁺ T cells expressing PD-1, 2B4 and CD160 were measured by flow cytometry. The frequency of CD8⁺ T cells expressing the inhibitory markers PD-1, 2B4 and CD160 was lower in ART-treated individuals compared with ART-naïve individuals and similar to the frequency in HIV-uninfected controls. The expression of the three markers was similarly independent of when therapy was initiated. Individuals treated before seroconversion displayed an HIV-specific CD8⁺ T cell response that included all five functional markers; this was not observed in individuals treated after seroconversion or in ART-naïve individuals. In summary, ART appears to restore the total CD8⁺ T cell population to a less exhausted phenotype, independent of the time point of initiation. However, to preserve multifunctional, HIV-1-specific CD8⁺ T cells, ART might have to be initiated before seroconversion.     

Bilingualism and Performance on Two Widely Used Developmental Neuropsychological Test Batteries

The present study investigated the effect of bilingualism on the two widely used developmental neuropsychological test batteries Wechsler Intelligence Scale for Children – Fourth Edition (WISC-IV) and A Developmental Neuropsychological Assessment, Second Edition (NEPSY-II) in children. The sample consisted of 100 Finland-Swedish children in two age groups. About half (n = 52) of the participants were early simultaneous bilinguals, and the other half (n = 48) were monolinguals. As no Finland-Swedish versions of the tests are available at the moment, both tests were translated and adapted to suit this population. The results revealed no difference in the performance between bilingual and monolingual children. This speaks against a cognitive advantage in bilingual children and indicates that development of separate norms for monolingual and bilingual children is not needed for clinical use.     

The Effect of a Mutation in the Thyroid Stimulating Hormone Receptor (TSHR) on Development,Behaviour and TH Levels in Domesticated Chickens

The thyroid stimulating hormone receptor (TSHR) has been suggested to be a “domestication locus” in the chicken, due to a strong selective sweep over the gene found in domesticated chickens, differentiating them from their wild ancestor the Red Junglefowl (RJF). We investigated the effect of the mutation on development (incubation time), behaviour and thyroid hormone levels in intercross chickens homozygous for the mutation (d/d), wild type homozygotes (w/w) or heterozygotes (d/w). This allowed an assessment of the effect of genotype at this locus against a random mix of RJF and WL genotypes throughout the rest of the genome, controlling for family effects. The d/d genotype showed a longer incubation time, less fearful behaviours, lower number of aggressive behaviours and decreased levels of the thyroid hormone T4, in comparison to the w/w genotype. The difference between TSHR genotypes (d/d vs. w/w) in these respects mirrors the differences in development and behaviour between pure domesticated White Leghorns and pure RJF chickens. Higher individual T3 and T4 levels were associated with more aggression. Our study indicates that the TSHR mutation affects typical domestication traits, possibly through modifying plasma levels of thyroid hormones, and may therefore have been important during the evolution of the domestic chicken.     

Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity

The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1–2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sd^a-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.Campylobactor jejuni infection is widespread in industrialized countries and is the greatest source of food poisoning worldwide. Infection and the resulting diarrhea are the most common cause of death among young children in developing countries, with an estimated 2.1–2.5 million cases per year in the USA. C. jejuni leads to severe gastroenteritis and is also linked to Guillain-Barré and irritable bowel disease (1, 2). Contaminated poultry meat is the most common source of infection, with up to 88% of poultry products being infected (3). The bacteria are commensal in chickens and exist throughout their intestinal tract. However, they do not invade the epithelium of the gut. Purified mucin glycoproteins from the caecum and small and large intestine have been shown to exhibit inhibitory properties against the bacteria in vitro (4). The commensal relationship between C. jejuni occurrence in chickens and the inability to invade the intestinal tissues is poorly understood. Enhanced understanding of how the properties of the chicken mucosal barrier differ from humans; as well as changes in mucin composition along the chicken intestinal tract, have the potential to explain the commensal behavior of C. jejuni in this species. Such information could also provide leads for the development of agents to limit infection.Intestinal mucus in vertebrates is a hydrated gel comprised mainly of very high molecular weight and polymeric secreted mucin glycoproteins that are heavily O-glycosylated. O-linked glycans constitute up to 80% of the mucin by weight and represent an abundant potential carbon source for the resident microflora. They also present targets for bacterial adhesion and chemotaxis that may be exploited by both commensal and pathogenic organisms. Intestinal mucus is a dynamic barrier layer that is mainly secreted by goblet cells. It forms a supramucosal layer to protect the gastrointestinal epithelial cells against infection. A density gradient of viscoelastic and highly hydrated mucins polymers exists from the inner to the outer mucus layer. This stratification is most clearly demonstrable in the colon, where a relatively tightly attached mucus layer proximal to the epithelium is normally devoid of bacteria, but a less cross-linked more superficial layer is heavily colonized (5). In humans, it has been shown that the abundance of bacteria is highest in the large intestine, where colonization of the mucosal epithelium is contested by a thick mucus layer (∼700 μm) and more rapid mucus turnover (6). In the small intestine, however, where bacteria are much less abundant, the mucus layer is between 100–400 μm thick. O-glycosylation confers the mucus component of the mucosal barrier with much of the protective properties required to separate the abundant gut microflora from the immune system of its host. Defects in mucin glycosylation lead to severe inflammation and susceptibility to infection, and the glycans themselves have been shown to be ligands that can block the binding of microorganisms (7–14). Thus, secreted gel-forming mucins are key components of intestinal defense, beyond which bacterial pathogens must normally penetrate to cause pathology through interaction with epithelial cells.Several C. jejuni adhesion proteins required for colonization of chickens have been identified (15), but the mechanisms by which the bacteria initiate interactions with carbohydrates on mucosal surfaces remain unclear. However, many hypotheses can be made from previous studies where bacterial-carbohydrate interactions have been investigated (7). Additionally, bovine mucins and l-fucose are known chemoattractants for C. jejuni (8), and l-fucose is a substrate for C. jejuni growth (13). Glycans from human breast milk bearing the H(O) blood group antigen (which presents α1–2-linked fucose) also inhibits infection (12). d-glucose, d-mannose, and d-fucose, but not the l-sugar equivalents, inhibited the binding of C. jejuni to human colonic Caco-2 cells (16). Muc1, a membrane-bound mucin, is up-regulated during infection in mice, and knockouts are highly prone to C. jejuni infection with transepithelial translocation (11). These studies have provided considerable insight into C. jejuni behavior, but the specific relationship between the bacteria and mucin glycans in vivo remains unidentified.

Table I

Oligosaccharide-specific C. jejuni interactions

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10^-7 and 2.7×10^-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca²⁺-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.