Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.     

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO₂ fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation.     

Effect of viscoelastic relaxation on moisture transport in foods. Part I: Solution of general transport equation

Within the framework of continuum mechanics, Singh et al. [1] developed an integro-differential equation, which applies to both Darcian (Fickian) and non-Darcian (non-Fickian) modes of fluid transport in swelling biological systems. A dimensionless form of the equation was obtained and transformed from moving Eulerian to the stationary Lagrangian coordinates. Here a solution scheme for the transport equation is developed to predict moisture transport and viscoelastic stresses in spheroidal biopolymeric materials. The equation was solved numerically and results used for predicting drying and sorption curves, moisture profiles, and viscoelastic stresses in soybeans. The Lagrangian solution was obtained by assembling together several schemes: the finite element method was used to discretize the equation in space; non-linearity was addressed using the Newton-Raphson method; the Volterra term was handled via a time integration scheme of Patlashenko et al. [2] and the Galerkin rule was used to solve the time-differential term. The solution obtained in Lagrangian coordinates was transformed back to the Eulerian coordinates. In part II of this sequence we present the numerical results.Revised version: 5 October 2003     

Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.     

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape,banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops,including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including d Cas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumerfriendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.     

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected α-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.Pathogens are detected by the host when antigenic molecules directly contact immune receptors during the early stages of infection. The strategy of intracellular infection allows viruses, certain bacteria and protozoa to partially cloak themselves from the immune response by physically encapsulating their antigens within host cells. Intracellular residence also takes advantage of immune tolerance mechanisms that prevent autoimmune destruction of self. T cells play a central role in immunity to intracellular pathogens because they can respond to antigens that are generated inside cells and then transported to the surface of infected cells after binding to antigen-presenting molecules. The antigen-presenting molecules encoded in the major histocompatibility complex are widely known for presenting peptide fragments of proteins (1). More recently, human and mouse members of the CD1 (cluster of differentiation 1) system have been shown to present small amphipathic molecules, including a variety of membrane lipids, glycolipids, and lipopeptides, greatly expanding the molecular structures recognized by the cellular immune system (2, 3).Among human CD1 proteins (CD1a, CD1b, CD1c, CD1d, and CD1e), each CD1 isoform is expressed on a different spectrum of antigen-presenting cells. Human CD1a proteins are distinguished from other CD1 proteins by high expression levels on the surface of intradermal Langerhans cells, which play a role in barrier immune function (4). Human T cell clones have been shown to directly recognize CD1a proteins in the presence of exogenous foreign antigens (5) or in the presence of sulfatide and other self lipids (6, 7), suggesting a role for CD1a in T cell activation. In addition, mycobacteria and other intracellular pathogens have been shown to increase CD1a expression in lesions found in leprosy and tuberculosis patients, implying a possible role for CD1a in the response to infection, especially at mucosal or skin sites (8–10). Analysis of the molecular target recognized by CD1a-restricted T cell clone (CD8-2) allowed the identification of a foreign antigen presented by CD1a as dideoxymycobactin (DDM) (11).²Mycobactin binds iron to promote Mycobacterium tuberculosis survival. DDM was initially isolated (11) from antigenic lipid extracts of M. tuberculosis, a pathogen that kills ∼1.7 million humans annually on a worldwide basis (12). The determination of DDM structure was based on mass spectrometric and NMR studies of limiting amounts of natural material derived from the pathogenic organisms, so that not all elements of its chemical structure could be formally determined. Instead, its assigned structure was facilitated by obvious parallels of dideoxymycobactin with mycobactin, a lipopeptide siderophore (13, 14). Iron is required for reduction-oxidation reactions involving respiration and other basic metabolic pathways in bacterial pathogens (13). Environmental mycobacteria have at least two iron uptake pathways, but mycobactin and the related molecule carboxymycobactin represent the only known dedicated iron uptake pathway for pathogenic species like M. tuberculosis (15, 16). Highlighting the physiological importance of the mycobactin pathway, deletion of mycobactin synthase B limits M. tuberculosis survival in cells (13, 14). Also, mammalian innate immune systems produce siderocalin, a 20-kDa lipocalin that binds both ferric and apo siderophores, preventing their uptake and subsequent iron delivery to microbes (17–20). The small available yields of natural material highlighted the need for a straightforward method to synthesize DDM for studies of its role in mycobacterial iron acquisition and testing T cell responses in human populations, as well as to provide authentic standards to investigate unknown aspects of natural DDM stereochemistry. Here we report two syntheses for production of DDM in solution phase and solid phase. Comparison of synthetic and natural DDMs gives unexpected insight into the stereochemical structures of the methylserine, oxazoline, and butyrate moieties of DDM and provides direct evidence that the T cell response is highly specific for a unique aspect of DDM structure that protrudes from the surface of the CD1a-DDM complexes.     

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.