Distribution of Pathogenic Genes aatA, aap, aggR, among Uropathogenic Escherichia coli (UPEC) and Their Linkage with StbA Gene

Urinary tract infection (UTI) with E. coli (UPEC) is one of the most common bacterial infections among human beings. In addition to the host predisposing factors, genes are also proposed to have an important role in the occurrence of UTIs. This study investigated the distribution of three pathogenic genes including aggR, aap and aatA among UPEC infected samples and their linkage with stbA, the essential gene for maintaining of pAA plasmid. A total of 244 samples were collected from patients with UTIs through clinical laboratories located in western side of Tehran (Iran) during years 2008–2009. E. coli isolation was performed according to standard laboratory methods. DNAs were extracted from samples using Boiling method, and the presence of aap, aggR, aatA and stbA genes were investigated by PCR. No pathogenic genes (aap, aggR, aatA) were found in 104 out of 244 UPEC samples, while 14 of them were carrying stbA gene. Out of 140 UPEC samples with pathogenic genes, 94 (46.6%) were carrying aap gene, 52 (23%) aggR gene, and 80 (35.4%) aatA gene. A total of 18 samples were also carrying all pathogenic genes together. Moreover, 44 out of 144 samples were carrying stbA gene. The results obtained by this study showed that the aggR, aap and aatA pathogenic genes have different existence patterns in different E. coli strains that infect different organs. Our study also showed that these three plasmid genes in EAEC strains are able to transpose in the genome and change their level of linkage with pAA plasmid essential gene stbA. Meanwhile, this study confirmed that aggR, aap and aatA genes are not specific to only EAEC strains.     

Significant enhancement of lignan accumulation in hairy root cultures of Linum album using biotic elicitors

Linum album has been shown to accumulate some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, we examined the effects of fungal elicitors on the production of lignans in L. album hairy root cultures. The biosynthesis of lignans was differentially affected by fungal elicitors. Fusarium graminearum extract induced the highest increase of PTOX, 190 μg g^?1 dry weight (DW), and lariciresinol, 260 μg g^?1 DW, which was two-fold and three-fold greater than the untreated control, respectively, while Trichoderma viride extract enhanced the accumulation of MPTOX, instead of PTOX, up to 160 µg g^?1 DW, which was 2.4-fold greater than the control. The enhancing effects of fungal elicitors on lignans production was correlated with the increased expression of some key genes involved in the biosynthesis of these compounds, phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl-alcohol dehydrogenase and pinoresinol-lariciresinol reductase.     

Inducible transient expression of Smpd3 prevents early lethality in fro/fro mice

Sphingomyelin phosphodiesterase 3 (SMPD3) is a pleiotropic lipid metabolizing enzyme involved in multiple physiological processes. A deletion mutation in the murine Smpd3 gene called fragilitas ossium (fro) leads to severe skeletal abnormalities in the developing fro/fro embryos. Although fro/fro mice can be useful to study many different aspects of SMPD3 functions, their perinatal lethality makes it difficult to generate a sufficient number of mice for controlled studies. In fact, on the C57BL/6 genetic background, none of the fro/fro mice survive beyond the perinatal stage. In this study, we used the “Tet‐On” inducible gene expression system to express Smpd3 transiently in fro/fro;ROSA‐rtTA;TRE‐Smpd3 embryos on the C57BL/6 background. This induced Smpd3 expression corrected all the skeletal abnormalities in these embryos and prevented their early death. However, induction of Smpd3 expression in the adolescent fro/fro;ROSA‐rtTA;TRE‐Smpd3 mice was not sufficient to correct the defects in trabecular bone mineralization and the impaired growth of the long bones. This novel mouse model will be a useful tool to study SMPD3 biology in vivo. genesis 52:408–416, 2014. © 2014 Wiley Periodicals, Inc.     

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.     

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na⁺ transport, effects at least partially involving regulation of the epithelial Na⁺ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na⁺ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, ^V617FJAK2 or ^K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I _amil) in dual electrode voltage clamp. Moreover, I _amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I _amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I _amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I _amil. In colonic epithelium, I _amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.     

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Effects of manipulation of water and nitrogen regime on the water relations of the desert shrub Larrea tridentata

Summary Water and nitrogen regimes of Larrea tridentata shrubs growing in the field were manipulated during an annual cycle. Patterns of leaf water status, leaf water relations characteristics, and stomatal behavior were followed concurrently. Large variations in leaf water status in both irrigated and nonirrigated individuals were observed. Predawn and midday leaf water potentials of nonirrigated shrubs were lowest except when measurements had been preceded by significant rainfall. Despite the large seasonal variation in leaf water status, reasonably constant, high levels of turgor were maintained. Pressure-volume curve analysis suggested that changes in the bulk leaf osmotic potential at full turgor were small and that nearly all of the turgor adjustment was due to tissue elastic adjustment. The increase in tissue elasticity with increasing water deficit manifested itself as a decrease in the relative water content at zero turgor and as a decrease in the tissue bulk elastic modulus. Because of large hydration-induced displacement in the osmotic potential and relative water content at zero turgor, it was necessary to use shoots in their natural state of hydration for pressure-volume curve determinations. Large diurnal and seasonal differences in maximum stomatal conductance were observed, but could not easily be attributed to variations in leaf water potential or leaf water relations characteristics such as the turgor loss point. The single factor which seemed to account for most of the diurnal and seasonal differences in maximum stomatal conductance between individual shrubs was an index of soil/root/ shoot hydraulic resistance. Daily maximum stomatal conductance was found to decrease with increasing soil/root/ shoot hydraulic resistance. This pattern was most consistent if the hydraulic resistance calculation was based on an estimate of total canopy transpiration rather than the more commonly used transpiration per unit leaf area. The reasons for this are discussed. It is suggested that while stomatal aperture necessarily represents a major physical resistance controlling transpiration, plant hydraulic resistance may represent the functional resistance through its effects on stomatal aperture.     

Evaluation of the Trophic Diatom Index for assessing water quality in River Gharasou,western Iran

Water quality, diatom species composition and biomass estimates were performed in the Gharasou River in western Iran. Five sites along the River Gharasou were sampled every month from April to September 2005. Physical and chemical factors (pH, NO₃–N, PO₄–P, dissolved oxygen, total suspend solids, total dissolved solids, conductivity, turbidity, chemical oxygen demand and biological oxygen demand) were measured along with biological properties of the periphyton including biomass, ash-free dry mass, chlorophyll-a concentration and the taxonomic composition diatom assemblages. Information from the diatom assemblage was used to calculate the Trophic Diatom Index and biovolume. The TDI was significantly correlated with measures of human disturbance at the sites (e.g. PO₄–P, NO₃–N and dissolved oxygen) as well as to biomass measures (chlorophyll a, ash-free dry mass and biovolume). The sensitivity of the TDI and its component metrics to environmental stressors supports the use of this index for monitoring ecological conditions in streams in Iran and to aid diagnosis of the cause of their impairment. Handling editor: L. Naselli-Flores