A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.     

Cross-talk between transforming growth factor-β and Wingless/Int pathways in lung development and disease

Lung development depends on accurate and precise patterning of a pulmonary anlagen, consisting of both endodermally and mesodermally derived progenitor cells. In this process, the need to establish communication and control among individual cells is paramount. Transforming growth factor-β (TGFβ) and Wingless/int (Wnt) signaling pathways serve this need. The individual functional repertoire of the two pathways is further expanded by cross-talk and integration of signaling at multiple levels taking advantage of their hard-wired multi-component signal transduction platforms. Cross-talk creates the possibility for both specificity and versatility in signaling during development and during repair of injured tissue. Understanding the mechanics and the physiological implications of this cross-talk is necessary for therapeutic or preventive targeting of either TGFβ or Wnt signaling pathways.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Impact of postharvest exogenous γ-aminobutyric acid treatment on cucumber fruit in response to chilling tolerance

Low-temperature storage is generally used to extend postharvest lifetime and to inhibit decay of cucumber fruit, but it also enhances the intensity of chilling injury. The capability of γ-aminobutyric acid to enhance antioxidant enzyme activities and reduce chilling injury was studied in cucumber (Cucumis sativus L.) fruit stored at 1 °C for 5 weeks. The purpose of this study was to define if the GABA-induced modification in antioxidant system and phospholipase activity is linked to the reduced chilling injury in cold-stored cucumber fruit. Alleviation of chilling injury by GABA treatment was related to increased content of proline, endogenous GABA and enhanced activities of CAT and SOD, together with reduced activities of PLC, PLD and LOX. We suggest that PLC, LOX and PLD are associated with chilling injury initiation by involvement in a signaling pathway and membrane deterioration. Therefore the results obtained in this study suggest GABA’s potential for postharvest applications for reducing chilling injury symptom in cucumber fruit.     

Molecular identification and phylogenetic relationships of Coleoidea (Mollusca: Cephalopoda) from the Persian Gulf and Oman Sea reveals a case of cryptic diversity

The Persian Gulf and Oman Sea constitute one of the most important marine ecosystems and have many economically important aquatic species, including several coleoid cephalopods. Some coleoids are difficult to identify using traditional morphological characteristics. In this study, two mitochondrial fragments, cytochrome oxidase I (COI) and the large ribosomal subunit (16S rRNA), were used for identification of coleoid species in four regions in the northern Persian Gulf and Oman Sea. The study led to the identification of potential cryptic species of Sepia, Amphioctopus and Uroteuthis. Furthermore, Euprymna hyllebergi was reported for the first time from the Persian Gulf. A high diversity of Coeloidea was found in the study area. Mean intraspecific and interspecific nucleotide distances for COI were 0%–2% and 2%–7%, respectively, while these values for 16S rRNA sequences were 0%–1% and 1%–4%. Given the uncertainty about species identity and the high levels of intraspecific genetic diversity reported for some species in GenBank, a comprehensive global study will be needed to resolve the taxonomic status of several coleoid species.     

Citrinin detection by intensified fluorescence signal of a FRET-based immunosensor using magnetic/silica core–shell

The specific immune-reaction between the anti-citrinin antibody immobilized on the surface of magnetic/silica core–shell (MSCS) and the citrinin–Rho123–BSA conjugate brings the Rho123 fluorophore as an acceptor and the QDs as a donor in close spatial proximity and causes FRET for occurring upon photo-excitation of the QDs. The novelties of this study include: (1) immobilization of the MSCS; (2) large amount of the immobilized QDs, and (3) immobilization of a large amount of Rho123 on the BSA macromolecule. Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride. Magnetic nanoparticles were synthesized using FeSO₄ and FeCl₃. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Transmission electron microscopy (TEM) analysis was used for investigating shape and monodispersity of the nanoparticles. EDC/NHS was used as a cross linking agent for immobilization of the QDs, conjugation of citrinin to amino groups of BSA, labeling of BSA with Rho123 and also for immobilization of the amino-functionalized MSCS on the immobilized QDs. Immobilization of the anti-citrinin antibody on the surface of the amino-functionalized MSCS was performed by Schiff-base mechanism. By using these three effective strategies, sensitivity of the designed nanobiosensor was incredibly enhanced as a very low limit of detection (up to 0.1 pM). The feasibility of this technique was tested by the detection of citrinin in the spiked human serum. Results showed that there was a linear correlation between the decreased fluorescence intensity of the Rho123 and increased fluorescence intensity of the QDs with increasing concentration of citrinin in the spiked samples in the range of 1–6 pM. According to obtained results, we conclude that this highly sensitive detection scheme is a easy, quick and impressive method that can be used in optical-based nanosensors.