Pathways of unfolding a protein depend in principle on the perturbation-whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40-45 degrees C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat beta(1-4) spectrin-an alpha-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches T(m), however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.

Background Numerous pre-clinical studies using bone marrow derived cells for the treatment of traumatic brain injury and stroke have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and other functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe TBI demonstrated safety and potentially a CNS structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Methods Adults (aged 18-55) with severe traumatic brain injury (GCS 5-8) and without signs of serious other injury or irreversible brain injury (see Table 1) were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6,9,12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest/infusion related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included MRI based measurements of supratentorial and corpus callosal volumes as well as DTI based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months post-injury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pre-treatment, post-treatment, and at 1 and 6 month follow-up. Results There were no serious adverse events related to harvest/infusion. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were down-regulated after BMMNC infusion. Conclusions Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by CNS structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are down-regulated after cell infusion. A Phase 2, prospective, randomized trial excluding the highest dose is warranted and can be powered based upon structural outcome variables.

Abbreviations used: CHO -Chinese hamster ovary; DTT -dithiothreitol; EDTAethylenediaminotetraacetic acid; EIA -enzyme immunoassay; LAD -left anterior descending; SDS -sodium dodecyl sulphate Abstract: Apelin interacts with the APJ receptor to enhance inotropy. In heart failure, apelin-APJ coupling may provide a means of enhancing myocardial function. The alterations in apelin and APJ receptor concentrations with ischemic cardiomyopathy are poorly understood. We investigated the compensatory changes in endogenous apelin and APJ levels in the setting of ischemic cardiomyopathy. Male, Lewis rats underwent LAD ligation and progressed into heart failure over 6 weeks. Corresponding animals underwent sham thoracotomy as control. Six weeks after initial surgery, the animals underwent hemodynamic functional analysis in the presence of exogenous apelin-13 infusion and the hearts were explanted for western blot and enzyme immunoassay analysis. Western blot analysis of myocardial APJ concentration demonstrated increased APJ receptor protein levels with heart failure (1890750±133500 vs. 901600±143120 intensity units, n=8, p=0.00001). Total apelin protein levels increased with ischemic heart failure as demonstrated by enzyme immunoassay (12.0±4.6 vs. 1.0±1.2 ng/ml, n=5, p=0.006) and western blot (1579400±477733 vs. 943000±157600 intensity units, n=10, p=0.008). Infusion of apelin-13 significantly enhanced myocardial function in sham and failing hearts. We conclude that total myocardial apelin and APJ receptor levels increase in compensation for ischemic cardiomyopathy. ISCHEMIC HEART FAILURE ENHANCES ENDOGENOUS MYOCARDIAL APELIN AND APJ RECEPTOR EXPRESSION

Background-Heart failure is a global health concern. As a novel therapeutic strategy, the induction of endogenous myocardial regeneration was investigated by initiating cardiomyocyte mitosis by expressing the cell cycle regulator cyclin A2. Methods and Results-Lewis rats underwent left anterior descending coronary artery ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing cyclin A2 (n ϭ32) or empty adeno-null (n ϭ32). Cyclin A2 expression was characterized by Western Blot and immunohistochemistry. Six weeks after surgery, in vivo myocardial function was analyzed using an ascending aortic flow probe and pressure-volume catheter. DNA synthesis was analyzed by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis was analyzed by phosphohistone-H3 expression. Myofilament density and ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and tapered off by 4 weeks.

Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E2 (PGE2) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement.

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The direct macrocycle synthesis of α-isocyano-ω-carboxylic acids via an Ugi multicomponent reaction is introduced. This multicomponent reaction (MCR) protocol differs by being especially short, convergent, and versatile, giving access to 12-22 membered rings.

This novel revascularization strategy of bone marrow stimulation and intramyocardial delivery of the endothelial progenitor cell chemokine stromal cell-derived factor yielded significantly enhanced myocardial endothelial progenitor cell density, vasculogenesis, geometric preservation, and contractility in a model of ischemic cardiomyopathy.