Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid

VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV‐1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β‐elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4‐derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5‐N‐formyl‐legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid‐like sugar, in an archaeal‐derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N‐acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC‐ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4‐derived peptides bearing the N‐glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N‐glycosylation apparatus, as determined by LC‐ESI/MS of heterologously expressed VP4. Here, however, the N‐linked pentasaccharide was the same as shown to decorate the S‐layer glycoprotein in this species. Hence, N‐glycosylation of the haloarchaeal viral protein, VP4, is host‐specific. These results thus present additional examples of archaeal N‐glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.     

CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.     

Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection

TLR7 is an innate signaling receptor that recognizes single-stranded viral RNA and is activated by viruses that cause persistent infections. We show that TLR7 signaling dictates either clearance or establishment of life-long chronic infection by lymphocytic choriomeningitis virus (LCMV) Cl 13 but does not affect clearance of the acute LCMV Armstrong 53b strain. TLR7(-/-) mice infected with LCMV Cl 13 remained viremic throughout life from defects in the adaptive antiviral immune response-notably, diminished T?cell function, exacerbated T?cell exhaustion, decreased plasma cell maturation, and negligible antiviral antibody production. Adoptive transfer of TLR7(+/+) LCMV immune memory cells that enhanced clearance of persistent LCMV Cl 13 infection in TLR7(+/+) mice failed to purge LCMV Cl 13 infection in TLR7(-/-) mice, demonstrating that a TLR7-deficient environment renders antiviral responses ineffective. Therefore, methods that promote TLR7 signaling are promising treatment strategies for chronic viral infections.     

Enzymatic pretreatment for preparing starch nanocrystals

Starch nanocrystals (SNCs) are crystalline platelets resulting from the acid hydrolysis of starch. A limiting factor for their more widespread use is their preparation duration. Therefore, this study investigates the possibility of developing an enzymatic pretreatment of starch to reduce the acid hydrolysis duration. A screening of three types of enzymes, namely, α-amylase, β-amylase, and glucoamylase, is proposed, and the latter was selected for a pretreatment. Compared with the regular kinetics of hydrolysis for preparing SNC, that of pretreated starch was much faster. The extent of hydrolysis normally reached in 24 h was obtained after only 6 h, and the regular final yield (15% after 5 days) was reached in 45 h. AFM and X-ray diffraction measurements confirmed that the obtained nanoparticles were indeed SNC.     

Do allopatric male Calopteryx virgo damselflies learn species recognition?

There is a growing amount of empirical evidence that premating reproductive isolation of two closely related species can be reinforced by natural selection arising from avoidance of maladaptive hybridization. However, as an alternative for this popular reinforcement theory, it has been suggested that learning to prefer conspecifics or to discriminate heterospecifics could cause a similar pattern of reinforced premating isolation, but this possibility is much less studied. Here, we report results of a field experiment in which we examined (i) whether allopatric Calopteryx virgo damselfly males that have not encountered heterospecific females of the congener C. splendens initially show discrimination, and (ii) whether C. virgo males learn to discriminate heterospecifics or learn to associate with conspecifics during repeated experimental presentation of females. Our experiment revealed that there was a statistically nonsignificant tendency for C. virgo males to show initial discrimination against heterospecific females but because we did not use sexually naïve individuals in our experiment, we were not able to separate the effect of innate or associative learning. More importantly, however, our study revealed that species discrimination might be further strengthened by learning, especially so that C. virgo males increase their association with conspecific females during repeated presentation trials. The role of learning to discriminate C. splendens females was less clear. We conclude that learning might play a role in species recognition also when individuals are not naïve but have already encountered potential conspecific mates.     

Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4⁺ T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4⁺ T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Further evidence for the role of ENPP1 in obesity: association with morbid obesity in Finns

The aim of this study was to investigate a series of single-nucleotide polymorphisms (SNPs) in the genes MC2R, MC3R, MC4R, MC5R, POMC, and ENPP1 for association with obesity. Twenty-five SNPs (2-7 SNPs/gene) were genotyped in 246 Finns with extreme obesity (BMI > or = 40 kg/m2) and in 481 lean subjects (BMI 20-25 kg/m2). Of the obese subjects, 23% had concomitant type 2 diabetes. SNPs and SNP haplotypes were tested for association with obesity and type 2 diabetes. Allele frequencies differed between obese and lean subjects for two SNPs in the ENPP1 gene, rs1800949 (P = 0.006) and rs943003 (P = 0.0009). These SNPs are part of a haplotype (rs1800949 C-rs943003 A), which was observed more frequently in lean subjects compared to obese subjects (P = 0.0007). Weaker associations were detected between the SNPs rs1541276 in the MC5R, rs1926065 in the MC3R genes and obesity (P = 0.04 and P = 0.03, respectively), and between SNPs rs2236700 in the MC5R, rs2118404 in the POMC, rs943003 in the ENPP1 genes and type 2 diabetes (P = 0.03, P = 0.02 and P = 0.02, respectively); these associations did not, however, remain significant after correction for multiple testing. In conclusion, a previously unexplored ENPP1 haplotype composed of SNPs rs1800949 and rs943003 showed suggestive evidence for association with adult-onset morbid obesity in Finns. In this study, we did not find association between the frequently studied ENPP1 K121Q variant, nor SNPs in the MCR or POMC genes and obesity or type 2 diabetes.     

Cholesterol substitution increases the structural heterogeneity of caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.