The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O-2(center dot-)) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O-2(center dot-) (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed

Biodiversity, Species Protection, and Animal Welfare Under International Law

The chapter explores the influence of the concept of animal welfare on international biodiversity law. A close examination of the recent evolution of this branch of international law shows that animal welfare has an ambivalent place in biodiversity-related agreements. Indeed, while welfare is only a faint consideration in the development of international regimes dealing with biodiversity as a whole, the concept has become an essential element for agreements dealing with the conservation of specific endangered species. Despite its role in these agreements, the place of animal welfare in international biodiversity law highlights that this corpus of rules is currently insufficient to be an effective tool for the protection of wildlife welfare. The last section of this study suggests that the adoption of international rules aiming at ensuring the protection of wild animals’ welfare could serve the double purpose of strengthening the conservation purpose of biodiversity regimes while also filling the welfare gap of international biodiversity law

Preclinical animal research on therapy dosimetry with dual isotopes

Preclinical research into radionuclide therapies based on radiation dosimetry will enable the use of any LET-equivalent radionuclide. Radiation dose and dose rate have significant influence on dose effects in the tumour depending on its radiation sensitivity, possibilities for repair of sublethal damage, and repopulation during or after the therapy. Models for radiation response of preclinical tumour models after peptide receptor radionuclide therapy based on the linear quadratic model are presented. The accuracy of the radiation dose is very important for observation of dose-effects. Uncertainties in the radiation dose estimation arise from incomplete assay of the kinetics, low accuracy in volume measurements and absorbed dose S-values for stylized models instead of the actual animal geometry. Normal dose uncertainties in the order of 20% might easily make the difference between seeing a dose-effect or missing it altogether. This is true for the theoretical case of a homogeneous tumour type behaving in vivo in the same way as its cells do in vitro. Heterogeneity of tumours induces variations in clonogenic cell density, radiation sensitivity, repopulation capacity and repair kinetics. The influence of these aspects are analysed within the linear quadratic model for tumour response to radionuclide therapy. Preclinical tumour models tend to be less heterogenic than the clinical conditions they should represent. The results of various preclinical radionuclide therapy experiments for peptide receptor radionuclide therapy are compared to the outcome of theoretical models and the influence of increased heterogeneity is analysed when the results of preclinical research is transferred to the clinic. When the radiation dose and radiobiology of the tumour response is known well enough it may be possible to leave the current phenomenological approach in preclinical radionuclide therapy and start basing these experiments on radiation dose. Then the use of a gamma ray-emitting radionuclides for a chemically comparable beta-particle-emitting paired isotope for therapy evaluation would be feasible

DPEP1 Inhibits Tumor Cell Invasiveness, Enhances Chemosensitivity and Predicts Clinical Outcome in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. To identify biologically relevant genes with prognostic and therapeutic significance in PDAC, we first performed the microarray gene-expression profiling in 45 matching pairs of tumor and adjacent non-tumor tissues from resected PDAC cases. We identified 36 genes that were associated with patient outcome and also differentially expressed in tumors as compared with adjacent non-tumor tissues in microarray analysis. Further evaluation in an independent validation cohort (N = 27) confirmed that DPEP1 (dipeptidase 1) expression was decreased (T: N ratio ∼0.1, P<0.01) in tumors as compared with non-tumor tissues. DPEP1 gene expression was negatively correlated with histological grade (Spearman correlation coefficient = −0.35, P = 0.004). Lower expression of DPEP1 in tumors was associated with poor survival (Kaplan Meier log rank) in both test cohort (P = 0.035) and validation cohort (P = 0.016). DPEP1 expression was independently associated with cancer-specific mortality when adjusted for tumor stage and resection margin status in both univariate (hazard ratio = 0.43, 95%CI = 0.24–0.76, P = 0.004) and multivariate analyses (hazard ratio = 0.51, 95%CI = 0.27–0.94, P = 0.032). We further demonstrated that overexpression of DPEP1 suppressed tumor cells invasiveness and increased sensitivity to chemotherapeutic agent Gemcitabine. Our data also showed that growth factor EGF treatment decreased DPEP1 expression and MEK1/2 inhibitor AZD6244 increased DPEP1 expression in vitro, indicating a potential mechanism for DPEP1 gene regulation. Therefore, we provide evidence that DPEP1 plays a role in pancreatic cancer aggressiveness and predicts outcome in patients with resected PDAC. In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments

Specific and Sensitive Detection of H. pylori in Biological Specimens by Real-Time RT-PCR and In Situ Hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens

NOA1 Functions in a Temperature-Dependent Manner to Regulate Chlorophyll Biosynthesis and Rubisco Formation in Rice

NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-supressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice

Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilumbinding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection