Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Properties of STAT1 and IRF1 enhancers and the influence of SNPs

Background

STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.

Results

We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.

Conclusions

These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.

     

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

Search for critical loading condition of the spine–A meta analysis of a nonlinear viscoelastic finite element model

The relative vulnerability of spinal motion segments to different loading combinations remains unknown. The meta-analysis described here using the results of a validated L2–L3 nonlinear viscoelastic finite element model was designed to investigate the critical loading and its effect on the internal mechanics of the human lumbar spine. A Box-Behnken experimental design was used to design the magnitude of seven independent variables associated with loads, rotations and velocity of motion. Subsequently, an optimization method was used to find the primary and secondary variables that influence spine mechanical output related to facet forces, disc pressure, ligament forces, annulus matrix compressive/shear stresses and anulus fibers strain. The mechanical responses with respect to the two most-relevant variables were then regressed linearly using the response surface quadratic model. Axial force and sagittal rotation were identified as the most-relevant variables for mechanical responses. The procedure developed can be used to find the critical loading for finite element models with multi input variables. The derived meta-models can be used to predict the risk associated with various loading parameters and in setting safer load limits.     

Database and tools for metabolic network analysis

Metabolic network analysis has attracted much attention in the area of systems biology. It has a profound role in understanding the key features of organism metabolic networks and has been successfully applied in several fields of systems biology, including in silico gene knockouts, production yield improvement using engineered microbial strains, drug target identification, and phenotype prediction. A variety of metabolic network databases and tools have been developed in order to assist research in these fields. Databases that comprise biochemical data are normally integrated with the use of metabolic network analysis tools in order to give a more comprehensive result. This paper reviews and compares eight databases as well as twenty one recent tools. The aim of this review is to study the different types of tools in terms of the features and usability, as well as the databases in terms of the scope and data provided. These tools can be categorised into three main types: standalone tools; toolbox-based tools; and web-based tools. Furthermore, comparisons of the databases as well as the tools are also provided to help software developers and users gain a clearer insight and a better understanding of metabolic network analysis. Additionally, this review also helps to provide useful information that can be used as guidance in choosing tools and databases for a particular research interest.     

Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells

Molecular Biology Reports - Despite the advancements in primary brain tumour diagnoses and treatments, the mortality rate remains high, particularly in glioblastoma (GBM). Chemoresistance,...     

Biodynamics model for operator head injury in stand-up lift trucks

Biodynamics and injury potential of operators in stand-up rider lift truck accidents have been investigated with a special focus on head injury. An anthropomorphic test device (ATD) model was used as an operator surrogate in computer simulations of off-the-dock (OTD) and tip-over (TO) accidents. The biomechanical model representing the ATD was developed based on rigid body segments, and then combined with a rigid body truck model in the accident simulations. The operator compartment of the truck model was enclosed with a rear door. The computed kinematics are in agreement with the results of previous experimental testing. A 2D finite element model of the head was created to compute head impact decelerations in the sagittal plane. Values of the head injury criterion for the TO cases were computed from the model and shown to compare favourably with experimental values. The results advance the state of knowledge concerning injury potential in TO and OTD accidents and simulation models for such accidents.     

Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.