Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.     

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.     

Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.     

Automatic particle selection: results of a comparative study

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.     

Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.In 2002 renal cell cancer accounted for more than 200,000 cases and 100,000 deaths worldwide (33). Unfortunately, chemotherapy, radiotherapy, and immunotherapy yield low response rates (9, 17) in this cancer type. Thus, prognosis for patients is poor, especially when the disease is metastatic, as median survival is only 8 months (19). Although recently approved drugs, such as sorafenib, sunitinib, temsirolimus, and bevacizumab, have provided additional tools for treatment of renal cell cancer (7), they are usually not curative, and thus new treatment approaches are needed.Oncolytic vaccinia viruses are promising agents for cancer treatment and have shown compelling results in preclinical tumor models (40, 42, 45). Moreover, good safety and preliminary evidence of antitumor efficacy were seen in phase 1 clinical trials (22, 26, 32). Vaccinia virus has a strong oncolytic effect due to its fast replication cycle (45) and a high innate tropism to cancer tissue (34). Tumor targeting can be further improved by deleting vaccinia virus genes that are necessary for replication in normal cells but not in cancer cells. For example, deletions of either thymidine kinase (TK) or vaccinia virus growth factor (VGF) or both have been shown to reduce pathogenicity compared to wild-type virus (3, 5, 27). To enhance antitumor potency, oncolytic vaccinia viruses can be armed with therapeutic transgenes, such as immunostimulatory factors (26) or suicide genes (14, 16, 35). With regard to kidney cancer, an arming approach with antiangiogenenic molecules seems logical, considering the high vascularization characteristic of renal tumors (20).Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis and is highly expressed in renal cell cancers (29). VEGF binds to the fms-like-tyrosine kinase receptor (flt-1 or VEGFR-1) and kinase domain region receptor (KDR or VEGFR-2) with high affinity (13). The soluble vascular endothelial growth factor receptor 1-Ig fusion protein (VEGFR-1-Ig) used in this study is derived from the membrane-bound VEGFR-1 and binds human and murine VEGF without inducing vascular endothelial cell mitogenesis (31). Blocking VEGF with this or closely related molecules has been shown to inhibit tumor growth in several cancer models (18, 21, 25, 39).Although tumor cell selective replication can be enhanced by deletion of TK and/or VGF to reduce pathogenicity (3, 5, 27), high doses of attenuated vaccinia virus may increase serum cytokine concentrations which parallel the onset of toxic events, as seen with other viral vectors (2, 38). The potential “early” toxicity associated with oncolytic vaccinia viruses has not been completely elucidated heretofore (36, 46).Given the high vascularization of renal cell cancers and the pressing need to generate new antitumor agents with increased safety and efficacy, we hypothesized that an oncolytic vaccinia virus targeted by TK and VGF deletions and armed with VEGFR-1-Ig would exhibit enhanced antitumor efficacy due to its antiangiogenic properties in renal cell cancer models compared to a nonarmed control virus, allowing reduction of the treatment dose.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

DPPC Bilayers in Solutions of High Sucrose Content

The properties of lipid bilayers in sucrose solutions have been intensely scrutinized over recent decades because of the importance of sugars in the field of biopreservation. However, a consensus has not yet been formed on the mechanisms of sugar-lipid interaction. Here, we present a study on the effect of sucrose on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers that combines calorimetry, spectral fluorimetry, and optical microscopy. Intriguingly, our results show a significant decrease in the transition enthalpy but only a minor shift in the transition temperature. Our observations can be quantitatively accounted for by a thermodynamic model that assumes partial delayed melting induced by sucrose adsorption at the membrane interface.