Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?

Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.

     

Inhibitory effects of acetylsalicylic acid on exocrine pancreatic carcinogenesis

Abstract

We investigated short (6 months) and long (12 months) term inhibitory effects of low (200 ppm) and high (400 ppm) dosages of acetylsalicylic acid (aspirin) on exocrine pancreatic carcinogenesis. It is known that exocrine pancreatic carcinogenesis can be detected by the presence of atypical acinar cell foci (AACF) in pancreas. We investigated possible inhibitory effects of acetylsalicylic acid in an azaserine-treated rat model. AACF were produced in rats by injection with azaserine according to previous studies. Our findings showed that the number, volume and diameter of pancreatic AACF were reduced after acetylsalicylic acid application. These observations suggest that acetylsalicylic acid may exert a protective effect against neoplastic development of pancreatic acinar cells in azaserine injected rats. Our findings corroborate reports in the literature concerning the effects of aspirin in reducing neoplastic development.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.     

Triterpenoids from the Stem Bark of Melia toosendan and Determination of Their Absolute Configurations at C(24)

Six new triterpenoids, meliasenins S–X ( 1 – 6 , resp.), were isolated from the stem bark of Melia toosendan. Their structures were elucidated by mass spectrometry, NMR experiments, and comparison with the known compounds. Particularly, the absolute configuration at C(24) in new compounds was determined through their CD spectra of the [Pr(FOD)₃] complex (fod=1,1,1,2,2,3,3,7,7,7‐decafluoroheptane‐4,6‐dione) in CCl₄, as well as by using Mosher's method.     

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1β expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.     

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin

The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.     

The Effects of Catalyst Layer Deposition Methodology on Electrode Performance

The catalyst layer of the cathode is arguably the most critical component of low‐temperature fuel cells and carbon dioxide (CO₂) electrolysis cells because their performance is typically limited by slow oxygen (O₂) and CO₂ reduction kinetics. While significant efforts have focused on developing cathode catalysts with improved activity and stability, fewer efforts have focused on engineering the catalyst layer structure to maximize catalyst utilization and overall electrode and system performance. Here, we study the performance of cathodes for O₂ reduction and CO₂ reduction as a function of three common catalyst layer preparation methods: hand‐painting, air‐brushing, and screen‐printing. We employed ex‐situ X‐ray micro‐computed tomography (MicroCT) to visualize the catalyst layer structure and established data processing procedures to quantify catalyst uniformity. By coupling structural analysis with in‐situ electrochemical characterization, we directly correlate variation in catalyst layer morphology to electrode performance. MicroCT and SEM analyses indicate that, as expected, more uniform catalyst distribution and less particle agglomeration, lead to better performance. Most importantly, the analyses reported here allow for the observed differences over a large geometric volume as a function of preparation methods to be quantified and explained for the first time. Depositing catalyst layers via a fully‐automated air‐brushing method led to a 56% improvement in fuel cell performance and a significant reduction in electrode‐to‐electrode variability. Furthermore, air‐brushing catalyst layers for CO₂ reduction led to a 3‐fold increase in partial CO current density and enhanced product selectivity (94% CO) at similar cathode potential but a 10‐fold decrease in catalyst loading as compared to previous reports.     

Genome-wide association analysis for lethal brachycephalic-like facial dysmorphia in Labrador Retrievers

A GWAS was performed for inborn X-linked facial dysmorphia with severe growth retardation in Labrador Retrievers. This lethal condition was mapped on the X chromosome at 17–21 Mb and supported by eight SNPs in complete LD. Dams of affected male puppies were heterozygous for the significantly associated SNPs and male affected puppies carried the associated alleles hemizygously. In the near vicinity to the associated region, RPS6KA3 was identified as a candidate gene causing facial dysmorphia in humans and mice known as Coffin–Lowry syndrome. Haplotype analysis showed significant association with the phenotypes of all 18 animals under study. This haplotype was validated through normal male progeny from a dam with the not-associated haplotype on both X chromosomes but male affected full-sibs with the associated haplotype.