A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases

© 2015 Macmillan Publishers Limited. All rights reserved. Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene

Host-Based Th2 Cell Therapy for Prolongation of Cardiac Allograft Viability

Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use “direct” host T cell therapy for prolongation of allograft viability as an alternative to “indirect” therapy mediated by donor T cell infusion

An Attempt to Understand Kidney's Protein Handling Function by Comparing Plasma and Urine Proteomes

With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form “reference profiles” of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology.After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched.The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma) and output (urine), it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It can also be anticipated that there will be more applications for proteomics in organ function research

Prevention of catheter lumen occlusion with rT-PA versus heparin (Pre-CLOT): study protocol of a randomized trial [ISRCTN35253449]

BACKGROUND: Many patients with end-stage renal disease use a central venous catheter for hemodialysis access. A large majority of these catheters malfunction within one year of insertion, with up to two-thirds due to thrombosis. The optimal solution for locking the catheter between hemodialysis sessions, to decrease the risk of thrombosis and catheter malfunction, is unknown. The Prevention of Catheter Lumen Occlusion with rt-PA versus Heparin (PreCLOT) study will determine if use of weekly rt-PA, compared to regular heparin, as a catheter locking solution, will decrease the risk of catheter malfunction. METHODS/DESIGN: The study population will consist of patients requiring chronic hemodialysis thrice weekly who are dialyzed with a newly inserted permanent dual-lumen central venous catheter. Patients randomized to the treatment arm will receive rt-PA 1 mg per lumen once per week, with heparin 5,000 units per ml as a catheter locking solution for the remaining two sessions. Patients randomized to the control arm will receive heparin 5,000 units per ml as a catheter locking solution after each dialysis session. The study treatment period will be six months, with 340 patients to be recruited from 14 sites across Canada. The primary outcome will be catheter malfunction, based on mean blood flow parameters while on hemodialysis, with a secondary outcome of catheter-related bacteremia. A cost-effectiveness analysis will be undertaken to assess the cost of maintaining a catheter using rt-PA as a locking solution, compared to the use of heparin. DISCUSSION: Results from this study will determine if use of weekly rt-PA, compared to heparin, will decrease catheter malfunction, as well as assess the cost-effectiveness of these locking solutions

Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice

Earlier diagnosis and treatment of Alzheimer's disease would greatly benefit from the identification of biomarkers at the prodromal stage. Using a prominent animal model of aspects of the disease, we here show using clinically relevant methodologies that very young, pre-pathological PDAPP mice, which overexpress mutant human amyloid precursor protein in the brain, exhibit two cryptic deficits that are normally undetected using standard methods of assessment. Despite learning a spatial memory task normally and displaying normal brain glucose uptake, they display faster forgetting after a long delay following performance to a criterion, together with a strong impairment of brain glucose uptake at the time of attempted memory retrieval. Preliminary observations suggest that these deficits, likely caused by an impairment in systems consolidation, could be rescued by immunotherapy with an anti-β-amyloid antibody. Our data suggest a biomarker strategy for the early detection of β-amyloid-related abnormalities

Bone Marrow Support of the Heart in Pressure Overload Is Lost with Aging

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response.To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging.Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice.BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging

Trace Levels of Innate Immune Response Modulating Impurities (IIRMIs) Synergize to Break Tolerance to Therapeutic Proteins

Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1st dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins

Vancomycin Activates σB in Vancomycin-Resistant Staphylococcus aureus Resulting in the Enhancement of Cytotoxicity

The alternative transcription factor σB is responsible for transcription in Staphylococcus aureus during the stress response. Many virulence-associated genes are directly or indirectly regulated by σB. We hypothesized that treatment with antibiotics may act as an environmental stressor that induces σB activity in antibiotic-resistant strains. Several antibiotics with distinct modes of action, including ampicillin (12 µg/ml), vancomycin (16 or 32 µg/ml), chloramphenicol (15 µg/ml), ciprofloxacin (0.25 µg/ml), and sulfamethoxazole/trimethoprim (SXT, 0.8 µg/ml), were investigated for their ability to activate this transcription factor. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. The transcription levels of selected genes associated with virulence were also measured. Real-time quantitative reverse transcription PCR was employed to evaluate gene transcription levels. Contact hemolytic and cytotoxicity assays were used to evaluate cell damage following antibiotic treatment. Antibiotics that target the cell wall (vancomycin and ampicillin) and SXT induced σB activity in VRSA strains. Expression of σB-regulated virulence genes, including hla and fnbA, was associated with the vancomycin-induced σB activity in VRSA strains and the increase in cytotoxicity upon vancomycin treatment. These effects were not observed in the sigB-deficient strain but were observed in the complemented strain. We demonstrate that sub-minimum inhibitory concentration (sub-MIC) levels of antibiotics act as environmental stressors and activate the stress response sigma factor, σB. The improper use of antibiotics may alter the expression of virulence factors through the activation of σB in drug-resistant strains of S. aureus and lead to worse clinical outcomes