New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus

Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.     

Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage‐derived EVs of the proliferative response of CD4⁺ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin‐specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF‐1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF‐ and EF‐1α‐deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF‐1α alone or in combination with HRF conferred a long‐lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei‐derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV‐mediated immune suppression in malaria‐infected individuals.     

The adaptation of a two-compartment cell renewal system to external demands

Summary The inner enamel epithelium (IEE) covers the labial tooth aspect as a one cell layer which, when cut sagittally, appears as a longitudinal cell column extending from the tooth origin toward the periphery. Following sudden tooth shortening, the IEE responds by an increased cell production which later declines below normal values. The perturbation affects all cell kinetic parameters; the progenitor compartment, which initially increases, diminishes in size toward end of the experiment. The cell cycle transition times, which initially decline, rise toward the end of the experiment. The mean normal daily cell production rate of 70 cell % (i.e. 70 cells are produced by 100 progenitors) increases to 111 cell % and then declines to a low of 51 cell %. The IEE response typifies the behavior of other cell renewal systems such as intestinal epithelium and epidermis.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Highly conserved and cis-acting lncRNAs produced from paralogous regions in the center of HOXA and HOXB clusters in the endoderm lineage

Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar orthologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5–7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.     

Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.     

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Passive mechanical properties of the lumbar multifidus muscle support its role as a stabilizer

The purpose of this study was to compare the passive mechanical properties and titin isoform sizes of the multifidus, longissimus, and iliocostalis muscles. Given our knowledge of each muscle's architecture and the multifidus’ operating range, we hypothesized that multifidus would have higher elastic modulus with corresponding smaller titin isoforms compared to longissimus or iliocostalis muscles. Single-fiber and fiber-bundle material properties were derived from passive stress–strain tests of excised biopsies (n=47). Titin isoform sizes were quantified via sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) analysis. We found that, at the single-fiber level, all muscles had similar material properties and titin isoform sizes. At the fiber-bundle level, however, we observed significantly increased stiffness (～45%) in multifidus compared to longissimus and iliocostalis muscles. These data demonstrate that each muscle may have a different scaling relationship between single-fiber and fiber-bundle levels, suggesting that the structures responsible for higher order passive mechanical properties may be muscle specific. Our results suggest that divergent passive material properties are observed at size scales larger than the single cell level, highlighting the importance of the extracellular matrix in these muscles. In addition to architectural data previously reported, these data further support the unique stabilizing function of the multifidus muscle. These data will provide key input variables for biomechanical modeling of normal and pathologic lumbar spine function and direct future work in biomechanical testing in these important muscles.     

Fungal apoptosis: function, genes and gene function

Cells of all living organisms are programmed to self-destruct under certain conditions. The most well known form of programmed cell death is apoptosis, which is essential for proper development in higher eukaryotes. In fungi, apoptotic-like cell death occurs naturally during aging and reproduction, and can be induced by environmental stresses and exposure to toxic metabolites. The core apoptotic machinery in fungi is similar to that in mammals, but the apoptotic network is less complex and of more ancient origin. Only some of the mammalian apoptosis-regulating proteins have fungal homologs, and the number of protein families is drastically reduced. Expression in fungi of animal proteins that do not have fungal homologs often affects apoptosis, suggesting functional conservation of these components despite the absence of protein-sequence similarity. Functional analysis of Saccharomyces cerevisiae apoptotic genes, and more recently of those in some filamentous species, has revealed partial conservation, along with substantial differences in function and mode of action between fungal and human proteins. It has been suggested that apoptotic proteins might be suitable targets for novel antifungal treatments. However, implementation of this approach requires a better understanding of fungal apoptotic networks and identification of the key proteins regulating apoptotic-like cell death in fungi.