Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered-charge peptide adsorbents

Ion-exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion-exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein-free diffusion, hindered diffusion, and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered-charge adsorbent and other surface interactions at the molecular scale.     

Control of pollen-mediated gene flow in transgenic trees

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.     

Impact of DAG stimulation on mineral synthesis, mineral structure and osteogenic differentiation of human cord blood stem cells

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy.Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the + DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca-P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.     

HLA-A*02:07 is a protective allele for EBV negative and a susceptibility allele for EBV positive classical Hodgkin lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10⁻⁵). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.     

Utilization of ELISA Using Thioredoxin Peroxidase-1 and Tandem Repeat Proteins for Diagnosis of Schistosoma japonicum Infection among Water Buffaloes

Background

The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests.

Methodology/Principal Findings

Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%).

Conclusions/Significance

These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.     

Bral1: "Superglue" for the extracellular matrix in the brain white matter

Bral1 is a link protein that stabilizes the binding between lecticans and hyaluronic acid and thus maintains the extracellular matrix assembly in the CNS. Bral1 is specifically located in the white matter around the nodes of Ranvier. Recent studies suggest its function in promoting saltatory neural conduction. This article reviews the current knowledge about the structure, expression and function of this link protein.     

Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB₁ cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB₁R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB₁R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB₁R-DOR heteromer-specific antibody, we found increased levels of CB₁R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB₁R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB₁R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB₁R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.