Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

Multi-layered Graphene Silica-Based Tunable Absorber for Infrared Wavelength Based on Circuit Theory Approach

Graphene can be utilized as a tunable material for a wide range of infrared wavelength regions due to its tunable conductivity property. In this paper, we use Y-shaped silver material resonator placed over the top of multiple graphene silica-layered structures to realize the perfect absorption over the infrared wavelength region. We propose four different designs by placing the graphene sheet over silica. The absorption and reflectance performance of the structures have been explored for 1500- to 1600-nm wavelength range. The proposed design also explores the absorption tunability of the structure for the different values of graphene chemical potential. We have reported the negative impedance for the perfect absorption for proposed metamaterial absorber structures. All the metamaterial absorbers have reported 99% of its absorption peaks in the infrared wavelength region. These designs can be used as a tunable absorber for narrowband and wideband applications. The proposed designs will become the basic building block of large photonics design which will be applicable for polariser, sensor, and solar applications.

     

Role for the Epidermal Growth Factor Receptor in Chemotherapy-Induced Alopecia

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.     

Isolation and characterization of microsatellite markers from the marine isopods Serolis paradoxa and Septemserolis septemcarinata (Crustacea: Peracarida)

This study reports the successful isolation of highly informative microsatellite marker sets for two marine serolid isopod species. For Serolis paradoxa (Fabricius, 1775), 13, and for Septemserolis septemcarinata (Miers, 1875), eight polymorphic microsatellite markers were isolated using the reporter genome enrichment protocol. The number of alleles per locus (N(A) ) and the observed heterozygosity (H(O) ) encompass a wide range of variation within S. paradoxa (N(A) 3-31, H(O) 6-89%) and S. septemcarinata (N(A) 2-18, H(O) 9-94%). The suitability of the newly isolated markers for population genetic studies is evaluated.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

An improved method for scoring protein-protein interactions using semantic similarity within the gene ontology

Background  

Semantic similarity measures are useful to assess the physiological relevance of protein-protein interactions (PPIs). They quantify similarity between proteins based on their function using annotation systems like the Gene Ontology (GO). Proteins that interact in the cell are likely to be in similar locations or involved in similar biological processes compared to proteins that do not interact. Thus the more semantically similar the gene function annotations are among the interacting proteins, more likely the interaction is physiologically relevant. However, most semantic similarity measures used for PPI confidence assessment do not consider the unequal depth of term hierarchies in different classes of cellular location, molecular function, and biological process ontologies of GO and thus may over-or under-estimate similarity.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

Hypoxanthine guanine phosphoribosyltransferase distorts the purine ring of nucleotide substrates and perturbs the pKa of bound xanthosine monophosphate

Enzymatic efficiency and structural discrimination of substrates from nonsubstrate analogues are attributed to the precise assembly of binding pockets. Many enzymes have the additional remarkable ability to recognize several substrates. These apparently paradoxical attributes are ascribed to the structural plasticity of proteins. A partially defined active site acquires complementarity upon encountering the substrate and completing the assembly. Human hypoxanthine guanine phosphoribosyltransferase (hHGPRT) catalyzes the phosphoribosylation of guanine and hypoxanthine, while the Plasmodium falciparum HGPRT (PfHGPRT) acts on xanthine as well. Reasons for the observed differences in substrate specificities of the two proteins are not clear. We used ultraviolet resonance Raman spectroscopy to study the complexes of HGPRT with products (IMP, GMP, and XMP), in both organisms, in resonance with the purine nucleobase electronic absorption. This led to selective enhancement of vibrations of the purine ring over those of the sugar-phosphate backbone and protein. Spectra of bound nucleotides show that HGPRT distorts the structure of the nucleotides. The distorted structure resembles that of the deprotonated nucleotide. We find that the two proteins assemble similar active sites for their common substrates. While hHGPRT does not bind XMP, PfHGPRT perturbs the pK(a) of bound XMP. The results were compared with the mutant form of hHGPRT that catalyzed xanthine but failed to perturb the pK(a) of XMP.