The thermodynamic consequences of interactions of native bovine serum albumin (BSA) with two smaller solutes (glycine betaine or urea) in aqueous solution are characterized by a novel application of vapor pressure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are either accumulated or excluded near the surface of a protein. From VPO measurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dependence of the solute molarity (C3) on that of BSA (C2) at fixed temperature (37 °C), pressure (∼1 atm), and osmolality (over the range 0−1.6 molal). After some thermodynamic transformations, these results yield values of m ≡ limm2→0(∂m3/∂m2)T,P,μ3, which characterizes the interdependence of solute molalities when temperature, pressure, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in terms of the molecular exclusion or accumulation of the solute (relative to water) near the protein surface. Within experimental uncertainty, m is proportional to m3 both for glycine betaine (0−0.9 m) and for urea (0−1.6 m). For glycine betaine ∂m /∂m3 = −49 ± 4, a value consistent with the interpretation that this solute is completely excluded from the hydrated surface of BSA, whereas for urea ∂m /∂m3 = 6 ± 1, which indicates a moderate extent of accumulation at the surface of native BSA. The preferential accumulation of solutes (e.g., urea) that have some binding affinity for a protein can be quantified and interpreted using the two-domain model if the extent of hydration of the protein has been determined using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if general, justifies the use of glycine betaine as a thermodynamic probe of the changes in hydration that accompany protein folding, protein association, and protein−ligand binding interactions.
Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1cD2 allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L578→P578 and S1027→P1027 polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.
How is cancer information exchanged among laypersons, clinical professionals, and medical researchers? High hopes for the role of computers in medical information exchange have been reflected in science fiction for decades. After at least two information technology paradigm shifts (personal computers and the Internet) and countless successful implementations of, for example, shared electronic records, knowledge bases, decision support systems, speech-to-text tools, natural language processing tools, or remote monitoring devices in a variety of medical and nonmedical settings, we are in the process of realizing these high hopes.
Abstract Background: Neuroblastoma is an aggressive solid tumor with poor prognosis. Novel therapies are needed to help increase the survival and quality of life for affected children. It has been previously shown that bortezomib results in apoptosis in neuroblastoma both in vitro and in vivo. PCI-24781 is a novel pan-HDAC inhibitor which has been shown to be synergistic with bortezomib in lymphoma models. We hypothesize the combination of bortezomib with PCI-24781 would increase cytotoxicity to neuroblastoma. Methods: Several established neuroblastoma cell lines as well as patient-derived primary neuroblastoma cultures were grown in 96-well plates and treated with bortezomib and PCI-24781, both separately and in combination for 48 hours. Cell viability was assessed by calcein AM assays and IC50's and drug synergy was determined at each cell line tested. mRNA was collected from drug treated cell lines and effects on cell signaling was evaluated using U133 mRNA expression arrays and Ingenuity analysis. Cell lysates prepared from drug treated cells were evaluated by western blot for caspase-3 and PARP cleavage. Nude mice were injected with 107 SMS-KCNR cells subcutaneously in the left flank. Mice were treated with daily doses of either 0.5 mg/kg bortezomib, 12.5 mg/kg PCI-24781, or the combination. Mice tumors were imaged using the IVIS lumina imager twice per week and caliper measurements were also obtained weekly until tumor max was reached. Results: All neuroblastoma cell lines and patient cells tested showed sensitivity to bortezomib and PCI-24781 treatment in calcien AM cell viability assays with IC50's for bortezomib less than 50nM and IC50's for PCI-24781 less than 500nM. The combination of bortezomib and PCI-24781 was more cytotoxic than either drug alone. Cells treated with combined treatment showed an increase in caspase-3 and PARP activity confirming apoptosis. Expression analysis showed differential expression in the Notch and Wnt pathways between drug treated and vehicle treated cells. The xenograft models showed a significant decrease in tumor volume in mice treated with both bortezomib and PCI-24781 when compared to the single agent treatment groups or to the control group. Conclusions: Bortezomib and PCI-24781 inhibit neuroblastoma growth both in vitro and in vivo to a greater extent than either drug alone. The mechanism of action is currently being investigated further but certainly involves caspase-3 mediated apoptosis. This combination therapy appears to be effective and well tolerated in the mouse model and would be a novel therapy for neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5260.
Abstract Background: Neuroblastoma is the most common extracranial solid tumor in children, and treatment options for recurrent neuroblastoma are limited. Using molecular profiling to target the molecular vulnerabilities of individual patient tumors with existing therapeutic agents may result in a rational, data-driven approach to treatment with potential to improve clinical outcomes. Methods: This was a multicenter study through the Neuroblastoma and Medulloblastoma Translational Research Consortium. The primary objective of this study was to evaluate the feasibility of supporting real-time treatment decisions through predictive modeling of genome-wide mRNA gene expression data from neuroblastoma tumor biopsies. Feasibility was defined as completion of tumor biopsy, histopathological evaluation, RNA extraction and quality control, gene expression profiling within a CLIA-certified laboratory, bioinformatic analysis, generation of a drug prediction report, molecular tumor board review yielding a formulated treatment plan, independent medical monitor review, treatment initiation within a 2-week period and completion of one cycle of therapy. The secondary objectives included a reproducibility study of patient biopsies, safety and response to therapy. Research validation in cell culture models of patient tumors was performed. Results: Fourteen patients with multiply relapsed neuroblastoma were enrolled between July 2011 and November 2012. All biopsies passed histopathology and RNA quality control. Generation of gene expression data and its analysis (3-8 days), reports which linked this data into medically actionable drug candidates (0-3 days), molecular tumor board (1-6 days) and independent medical monitor review (1 -4 days) were all completed in real-time. The average time was 12.4 days from biopsy to initiation of treatment. There were no unexpected serious adverse events on study and patients tolerated therapy well. Clinical benefit was seen 50% of patients. Triplicate biopsies showed reproducible sets of drugs and the comparatively large differences in drug lists between patients show that the drug lists are tailored to patients. Comparison of RNA expression profiles with RNA sequencing from each patient showed strong correlation. Conclusion: This study shows that it is feasible to create therapeutic treatment plans based on genomic profiling in real time and that patients are able to be treated safely on a tumor board derived molecular guided therapy using existing medications. Incorporation of further genomic studies to evaluate additional molecular profiling techniques in order to make more informed individual therapeutic treatment plans will be evaluated in future studies. Citation Format: Giselle L. Sholler, Genevieve Bergendahl, Alyssa VanderWerff, William Ferguson, William Roberts, Don Eslin, Jacqueline Kraveka, Joel Kaplan, Deanna Mitchell, Nehal Parikh, Kathleen Neville, Takamaru Ashikaga, Jeffrey Bond, Gina Hanna, Melinda Merchant, Matt Huentelman, Jason Corneveaux, Jeffrey Trent. A feasibility trial using molecular-guided therapy for the treatment of patients with refractory or recurrent neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-160. doi:10.1158/1538-7445.AM2013-LB-160
