Dissociation between mature phenotype and impaired transmigration in dendritic cells from heparanase-deficient mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking.     

Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli

A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.     

Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK)

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK), a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK) may trigger apoptosis.For efficient overexpression, Bcl-x(AK) was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK) appeared as an essential and initial step. Bcl-x(AK) was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L). A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.     

The Colony Predation Algorithm

This paper proposes a new stochastic optimizer called the Colony Predation Algorithm(CPA)based on the corporate predation of animals in nature.CPA utilizes a ma...     

Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157:H7

Chloroplast genetic engineering offers an opportunity for high level expression and cost-effective recombinant protein production. Escherichia coli O157:H7 is one of the most important zoonotic pathogens causing hemorrhagic colitis (HC) and the life-threatening hemolytic-uremic syndrome in humans worldwide. The occurrence of zoonotic E. coli O157:H7 outbreaks in recent years has led to increased efforts in the development of safe and cost-effective immunogenic antigens against E. coli O157:H7. EspA and Tir/Intimin proteins are the important virulence factors which are encoded by the LEE locus of enterohemorrhagic E. coli. In this study, we hypothesized that the high level expression of the chimeric form of these effectors in chloroplasts and using tobacco transplastomic plants as an oral delivery system for the development of an edible-base vaccine would induce an immune response for the prevention of E. coli 0157:H7 attachment and colonization in animal model mice. The prokaryotic codon-optimized EIT protein was expressed in plastid genome via chloroplast transformation. Putative transplastomic plants were analyzed by PCR, and Southern blot analysis confirming chloroplast integration and homoplasmy in the T1 progeny. Immunoblotting and ELISA assays demonstrated that the EIT protein was expressed in chloroplasts and accumulated up to 1.4 % of total soluble protein in leaf tissue. In mice orally immunized with transplastomic tobacco plant leaves, high immunological responses (IgG and IgA specific antibodies) were detected in serum and feces. Finally, the challenging assay with E. coli O157:H7 in immunized mice showed reduced bacterial shedding.     

Individual Pause-and-Go Motion Is Instrumental to the Formation and Maintenance of Swarms of Marching Locust Nymphs

The principal interactions leading to the emergence of order in swarms of marching locust nymphs was studied both experimentally, using small groups of marching locusts in the lab, and using computer simulations. We utilized a custom tracking algorithm to reveal fundamental animal-animal interactions leading to collective motion. Uncovering this behavior introduced a new agent-based modeling approach in which pause-and-go motion is pivotal. The behavioral and modeling findings are largely based on motion-related visual sensory inputs obtained by the individual locust. Results suggest a generic principle, in which intermittent animal motion can be considered as a sequence of individual decisions as animals repeatedly reassess their situation and decide whether or not to swarm. This interpretation implies, among other things, some generic characteristics regarding the build-up and emergence of collective order in swarms: in particular, that order and disorder are generic meta-stable states of the system, suggesting that the emergence of order is kinetic and does not necessarily require external environmental changes. This work calls for further experimental as well as theoretical investigation of the neural mechanisms underlying locust coordinative behavior.     

Neural Control of Gas Exchange Patterns in Insects: Locust Density-Dependent Phases as a Test Case

The adaptive significance of discontinuous gas exchange cycles (DGC) in insects is contentious. Based on observations of DGC occurrence in insects of typically large brain size and often socially-complex life history, and spontaneous DGC in decapitated insects, the neural hypothesis for the evolution of DGC was recently proposed. It posits that DGC is a non-adaptive consequence of adaptive down-regulation of brain activity at rest, reverting ventilatory control to pattern-generating circuits in the thoracic ganglia. In line with the predictions of this new hypothesis, we expected a higher likelihood of DGC in the gregarious phase of the desert locust (Schistocerca gregaria, Orthoptera), which is characterized by a larger brain size and increased sensory sensitivity compared with the solitary phase. Furthermore, surgical severing of the neural connections between head and thoracic ganglia was expected to increase DGC prevalence in both phases, and to eliminate phase-dependent variation in gas exchange patterns. Using flow-through respirometry, we measured metabolic rates and gas exchange patterns in locusts at 30°C. In contrast to the predictions of the neural hypothesis, we found no phase-dependent differences in DGC expression. Likewise, surgically severing the descending regulation of thoracic ventilatory control did not increase DGC prevalence in either phase. Moreover, connective-cut solitary locusts abandoned DGC altogether, and employed a typical continuous gas exchange pattern despite maintaining metabolic rate levels of controls. These results are not consistent with the predictions of the neural hypothesis for the evolution of DGC in insects, and instead suggest neural plasticity of ventilatory control.     

Endothelins and their receptors in embryo implantation

As a critical stage of pregnancy, the implantation of blastocysts into the endometrium is a progressive, excessively regulated local tissue remodeling step involving a complex sequence of genetic and cellular interplay executed within an optimal time frame. For better understanding the causes of infertility and, more importantly, for developing powerful strategies for successful implantations and combating infertility, an increasing number of recent studies have been focused on the identification and study of newly described substances in the reproductive tree. The endothelins (ET), a 21-aminoacidic family of genes, have been reported to be responsible for the contraction of vascular and nonvascular smooth muscles, including the smooth muscles of the uterus. Therefore, this review aims to comprehensively discuss the physiological role of endothelins and signaling through their receptors, as well as their probable involvement in the implantation process.