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.

     

Lyciumamide A,a dimer of phenolic amide,protects against NMDA-induced neurotoxicity and potential mechanisms in vitro

Journal of Molecular Histology - Currently, the excessive activation of N-methyl-D-aspartate receptors (NMDARs) is considered to be a crucial mechanism of brain injury. Lycium barbarum A (LyA) is a...     

Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7 Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.     

Hemodynamic investigation of a patient-specific abdominal aortic aneurysm with iliac artery tortuosity

Abstract

This paper describes a systematic investigation on the hemodynamic environment in a patient-specific AAA with tortuous common iliac artery(CIA) and external iliac artery (EIA). 3D reconstructions from CT scans and subsequent computational simulation are carried out. It is found out that the Newtonian and non-Newtonian models have very similar flow field and WSS distribution. More importantly, it is revealed that the torturous CIA maintained its helical flow. It is concluded that the assumption of Newtonian blood is adequate in capturing the intra-aneurysmal hemodynamics. Moreover, it is speculated that the physiological spiral flow protects the twisted CIA from the thrombosis formation.     

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.     

Study on the Interaction between the Inclusion Complex of Hematoxylin with β-Cyclodextrin and DNA

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n_hematoxylin:n_{β-cyclodextrin} = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n_{β-cyclodextrin -hematoxylin}:n_DNA = 2:1, binding constant was K^? _298.15K = 5.29 × 10⁴ L·mol^?1, and entropy worked as driven force in this action.     

A Fast Workflow for Identification and Quantification of Proteomes

The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.The performance of mass spectrometry has been improved tremendously over the last few years (1–3), making mass spectrometry-based proteomics a viable approach for large-scale protein analysis in biological research. Scientists around the world are striving to fulfill the promise of identifying and quantifying almost all gene products expressed in a cell line or tissue. This would make mass spectrometry-based protein analysis an approach that is compatible to the second-generation mRNA deep-seq technique (4, 5).Two liquid chromatography (LC)-MS strategies have been employed to achieve deep proteome coverage. One is a single run with a long chromatography column and gradient to take advantage of the resolving power of HPLC to reduce the complexity of peptide mixtures; the other is a sequential run with two-dimensional separation (typically ion-exchange and reverse phase) to reduce peptide complexity. It was reported by two laboratories that 2761 and 4500 proteins were identified with a 10 h chromatography gradient on a dual pressure linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap Velos)(6–8). Similarly, 3734 proteins were identified using a 8 h gradient on a 2 m long column with a hybrid triple quadrupole - time of flight (Q-TOF, AB sciex 5600 Q-TOF)(9) mass spectrometer. The two-dimensional approach has yielded more identification with longer time. For example, 10,006 proteins (representing over 9000 gene products, GPs)¹ were identified in U2OS cell (10), and 10,255 proteins (representing 9207 GPs) from HeLa cells (11). It took weeks (for example, 2–3 weeks) of machine running time to achieve such proteome coverage, pushing proteome analysis to the level that is comparable to mRNA-seq. With the introduction of faster machines, human proteome coverage now has reached the level of 7000–8500 proteins (representing 7000–8000 GPs) in 3 days (12). Notwithstanding the impressive improvement, the current approach using long column and long gradient suffers from inherent limitations: it takes long machine running time and it is challenging to keep reproducibility among repeated runs. Thus, current throughput and reproducibility have hindered the application of in-depth proteomics to traditional biological researches. A timesaving approach is in urgent need.In this study, we used the first-dimension (1D) short pH 10 RP prefractionation to reduce the complexity of the proteome (13), followed by sequential 30 min second-dimension (2D) short pH 3 reverse phase-(RP)-LC-MS/MS runs for protein identification (14). The results demonstrated that it is possible to identify 8000 gene products from mammalian cells within 12 h of total MS measurement time by applying this dual-short 2D-RPLC-MS/MS strategy (Fast sequencing, Fast-seq). The robustness of the strategy was revealed by parallel testing on different MS systems including quadrupole orbitrap mass spectrometer (Q-Exactive), hybrid Q-TOF (Triple-TOF 5600), and dual pressure linear ion-trap orbitrap mass spectrometer (LTQ-Orbitrap Velos), indicating the inherent strength of the approach as to merely taking advantage of the better MS instruments. This strategy increases the efficiency of MS sequencing in unit time for the identification of proteins. We achieved identification of 2200 proteins/30 mins on LTQ-Orbitrap Velos, 2800 proteins/30 mins on Q-Exactive and Triple-TOF 5600 respectively. We further optimized Fast-seq and worked out a quantitative-version of the Fast-seq workflow: Fast-quantification (Fast-quan) and applied it for protein abundance quantification in HUVEC cell that was treated with a drug candidate MLN4924 (a drug in phase III clinical trial). We were able to quantify > 6700 GPs in 1 day of MS running time and found 99 proteins were up-regulated with high confidence. We expect this efficient alternative approach for in-depth proteome analysis will make the application of MS-based proteomics more accessible to biological applications.     

Cell-based high-throughput proliferation and cytotoxicity assays for screening traditional Chinese herbal medicines

A simple, reliable, high-throughput screening method was developed and used to assess the pharmaceutical effects of extracts of traditional Chinese herbal medicines (TCHMs). This method is based on 3-dimensional (3-D) cultures of mouse embryonic stem (mES) and human colon cancer and breast cancer cells expressing enhanced green fluorescent protein (EGFP) in polyethylene terephthalate (PET) fibrous scaffolds on modified 384-well plates with online monitoring of culture fluorescence for dynamic responses of cells to drugs present in culture media. Cell responses to deoxycholic acid and the extracts of 3 TCHMs (Ganoderma lucidum spores, Ginkgo biloba, and Epimedium brevicornum) at various concentrations were investigated for their effects on proliferation and cytotoxicity. The screening results, i.e., the growth responses of cancer cells to those drugs, were consistent with what have been reported in the literature, confirming the reliability of the new screening approach. Different from previous screening methods for both TCHMs and western medicines that used animal models or 2-D cell-based assays with single cell lines, this 3-D cell-based screening method employs both cancer and normal cells and thereby provides a way for quick, direct evaluation of the anticancer effects of TCHMs. This method also offers assessment on the side effects of TCHMs.     

Interferon Induced IFIT Family Genes in Host Antiviral Defense

Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IFN-stimulated genes. This family contains a cluster of duplicated loci. Most mammals have IFIT1, IFIT2, IFIT3 and IFIT5; however, bird, marsupial, frog and fish have only IFIT5. Regardless of species, IFIT5 is always adjacent to SLC16A12. IFIT family genes are predominantly induced by type I and type III interferons and are regulated by the pattern recognition and the JAK-STAT signaling pathway. IFIT family proteins are involved in many processes in response to viral infection. However, some viruses can escape the antiviral functions of the IFIT family by suppressing IFIT family genes expression or methylation of 5'' cap of viral molecules. In addition, the variants of IFIT family genes could significantly influence the outcome of hepatitis C virus (HCV) therapy. We believe that our current review provides a comprehensive picture for the community to understand the structure and function of IFIT family genes in response to pathogens in human, as well as in animals.