Sugar-quantum dot conjugates for a selective and sensitive detection of lectins

One-pot synthesized neoglycoconjugates with a reactive thiol group are introduced here for functionalization with carbohydrates for solubilization and stabilization of CdSe-ZnS quantum dots in aqueous solution. Three different sizes of quantum dots (QDs) with lactose, melibiose, and maltotriose on their surface have been utilized, for the first time, for lectin detection through agglutination assay. The sugar-QDs thus synthesized were characterized by transmission election microscopy (TEM), fluorescence, and absorption spectroscopy. Agglutination of sugar-QDs by three different lectins occurred through specific multivalent carbohydrate-lectin interactions and was studied extensively by monitoring the scattered light at 600 nm. This assay was very selective, which has been demonstrated by a more selective binding of soybean agglutinin (SBA) with melibiose-QD, as compared to lactose-QD, and specific deagglutination caused by alpha-d-galactose, while alpha-d-mannose did not show any effect. The detection sensitivity of the maltotriose-QD was tested with Concanavalin A (ConA), and as little as 100 nM of the lectin was detected using light scattering. The detection sensitivity of this protocol has been enhanced considerably by the fluorescence properties of QDs. This agglutination process seems to occur through formation of smaller soluble aggregates, which further associate to form larger aggregates.     

A geminivirus betasatellite encoded βC1 protein interacts with PsbP and subverts PsbP-mediated antiviral defence in plants

Geminivirus disease complexes potentially interfere with plants physiology and cause disastrous effects on a wide range of economically important crops throughout the world. Diverse geminivirus betasatellite associations exacerbate the epidemic threat for global food security. Our previous study showed that βC1, the pathogenicity determinant of geminivirus betasatellites induce symptom development by disrupting the ultrastructure and function of chloroplasts. Here we explored the betasatellite-virus-chloroplast interaction in the scope of viral pathogenesis as well as plant defence responses, using Nicotiana benthamiana—Radish leaf curl betasatellite (RaLCB) as the model system. We have shown an interaction between RaLCB-encoded βC1 and one of the extrinsic subunit proteins of oxygen-evolving complex of photosystem II both in vitro and in vivo. Further, we demonstrate a novel function of the Nicotiana benthamiana oxygen-evolving enhancer protein 2 (PsbP), in that it binds DNA, including geminivirus DNA. Transient silencing of PsbP in N. benthamiana plants enhances pathogenicity and viral DNA accumulation. Overexpression of PsbP impedes disease development during the early phase of infection, suggesting that PsbP is involved in generation of defence response during geminivirus infection. In addition, βC1-PsbP interaction hampers non-specific binding of PsbP to the geminivirus DNA. Our findings suggest that betasatellite-encoded βC1 protein accomplishes counter-defence by physical interaction with PsbP reducing the ability of PsbP to bind geminivirus DNA to establish infection.     

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

Background  

Heat stress (HS) and related illnesses are a major concern in military, sports, and fire brigadiers. HS results in physiologic responses of increased temperature, heart rate and sweating. In heat stroke, inflammatory response plays an important role and it is evidenced that turpentine (T) induced circulating inflammatory cytokines reduced survival rate and duration at 42°C. Here we report the alteration in the protein expression in liver cells upon HS with and without T treatment using two dimensional gel electrophoresis (2-DE), tryptic in-gel digestion and MALDI-TOF-MS/MS approaches     

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n = 12), cemetery urns (n = 23), and miscellaneous containers that included two tree holes (n = 19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) resulting in the accumulation of Ac(1)PIM(1) and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed 'Cg-LM-A') and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed 'Cg-LM-B'. Further chemical analyses established the lipoglycan possessed an alpha-D: -glucopyranosyluronic acid-(1 --> 3)-glycerol (GlcAGroAc(2)) based anchor which was then further glycosylated by 8-22 mannose residues, with Man(12-20)GlcAGroAC(2) molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-alpha by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity.     

Liposomal packaging generates Wnt protein with in vivo biological activity

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.     

Preprocessing of tandem mass spectrometric data to support automatic protein identification

Liquid chromatography tandem mass spectrometry is a major tool for identifying proteins. The fragment spectra of peptides can be interpreted automatically in conjunction with a sequence database search. With the development of powerful automatic search engines, research now focuses on optimizing the result returned from database searches. We present a series of preprocessing steps for fragment spectra to increase the accuracy and specificity of automatic database searches. After processing, the correct amino acid sequences from the database can be related better to the fragment spectra. This increases the sensitivity and reliability of protein identifications, especially with very large genomic databanks, and can be important for the systematic characterization of post-translational modifications.     

Rapid shortening of telomere length in response to ceramide involves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase

Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.     

Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin

Introduction: Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin.

Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as ‘enoxaparin’, ‘mass spectrometry’, ‘low molecular weight heparin’, ‘structural characterization’, etc.

Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.