Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery.

The objective of this study was to discover and to validate novel noninvasive biomarkers that distinguish between benign prostate hyperplasia (BPH) and localized prostate cancer (PCa), thereby helping to solve the diagnostic dilemma confronting clinicians who treat these patients.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

Identifying a molecular target is essential for tumor-targeted nanomedicine. Current cancer nanomedicines commonly suffer from poor tumor specificity, "off-target" toxicity, and limited clinical efficacy. Here, we report a method to screen and identify new molecular targets for tumor-targeted nanomedicine based on a quantitative analysis. In our proof-of-principle study, we used comparative flow cytometric screening to identify ICAM-1 as a potential target for metastatic melanoma (MM). We further evaluated ICAM-1 as a MM targeting moiety by characterizing its (1) tumor specificity, (2) expression level, (3) cellular internalization, (4) therapeutic function, and (5) potential clinical impact. Quantitation of ICAM-1 protein expression on cells and validation by immunohistochemistry on human tissue specimens justified the synthesis of antibody-functionalized drug delivery vehicles, which were benchmarked against appropriate controls. We engineered ICAM-1 antibody conjugated, doxorubicin encapsulating immunoliposomes (ICAM-Dox-LPs) to selectively recognize and deliver doxorubicin to MM cells and simultaneously neutralize ICAM-1 signaling via an antibody blockade, demonstrating significant and simultaneous inhibitory effects on MM cell proliferation and migration. This paper describes a novel, quantitative metric system that identifies and evaluates new cancer targets for tumor-targeting nanomedicine.

Honokiol (HNK) is a small molecule with potent anti-inflammatory and anti-tumorigenic properties; yet the molecular targets of HNK are not well studied. Hyperactivation of the receptor tyrosine kinase c-Met and overexpression of the cytoprotective enzyme heme oxygenase-1 (HO-1) play a critical role in the growth and progression of renal cell carcinoma (RCC). Interestingly, the calcineurin inhibitor (CNI) cyclosporine A (CsA), an immunosuppressant used to prevent allograft rejection, can also increase the risk of RCC in transplant patients. We studied the potential role of c-Met signaling axis on CNI-induced renal tumor growth and tested the anti-tumor efficacy of HNK. Importantly, CNI treatment promoted c-Met induction and enhanced c-Met-induced Ras activation. We found that HNK treatment effectively down-regulated both c-Met phosphorylation and Ras activation in renal cancer cells. It inhibited the expression of both c-Met- and CNI-induced HO-1, and promoted cancer cell apoptosis. In vivo, HNK markedly inhibited CNI-induced renal tumor growth; and it decreased the expression of phospho-c-Met and HO-1 and reduced blood vessel density in tumor tissues. Our results suggest a novel mechanism(s) by which HNK exerts its anti-tumor activity through the inhibition of c-Met-Ras-HO-1 axis; and it can have significant therapeutic potential to prevent post-transplantation cancer in immunosuppressed patients.

Tissue ischemia adversely affects the function of mitochondria, which results in impairment of oxidative phosphorylation and compromised recovery of the affected organ. The impact of ischemia on mitochondrial function has been extensively studied in the heart because of the morbidity and mortality associated with injury to this organ. As conventional methods to preserve cardiac cell viability and contractile function following ischemia are limited in their efficacy, we developed a unique approach to protect the heart by transplanting respiration-competent mitochondria to the injured region. Our previous animal experiments showed that transplantation of isolated mitochondria to ischemic heart tissue leads to decreases in cell death, increases in energy production, and improvements in contractile function. We also discovered that exogenously-derived mitochondria injected or perfused into ischemic hearts were rapidly internalised by cardiac cells. Here, we used three-dimensional super-resolution microscopy and transmission electron microscopy to determine the intracellular fate of endocytosed exogenous mitochondria in human iPS-derived cardiomyocytes and primary cardiac fibroblasts. We found isolated mitochondria are incorporated into cardiac cells within minutes and then transported to endosomes and lysosomes. The majority of exogenous mitochondria escape from these compartments and fuse with the endogenous mitochondrial network, while some of these organelles are degraded through hydrolysis.

Urinary biomarkers can diagnose and monitor pathophysiologic conditions in the central nervous system (CNS). However, focus is often on single diseases, with limited data on discriminatory capability of this approach in a general setting. Here, we demonstrate that different classes of CNS disease exhibit distinct biomarker patterns, evidence of disease-specific "fingerprinting." Urine from 218 patients with pathology-confirmed tumors or cerebrovascular disease, controls (n = 33) were collected. ELISA and/or bead-based multiplexing quantified levels of 21 putative urinary biomarkers. Analysis identified biomarkers capable of distinguishing each disease from controls and other diseases. Mann-Whitney U tests identified biomarkers with differential expression between disease types and controls (P ≤ 0.001). Subsequent receiver-operating characteristic (ROC) analyses revealed distinguishing biomarkers with high sensitivity and specificity. Areas under the curve (AUCs) ranged 0.8563-1.000 (P values ≤ 0.0003), sensitivities ranged 80.00-100.00%, and specificities ranged 80.95-100.00%. These data demonstrate proof-of-principle evidence that disease-specific urinary biomarker signatures exist. In contrast to non-specific responses to ischemia or injury, these results suggest that urinary biomarkers accurately reflect unique biological processes distinct to different diseases. This work can be used to generate disease-specific panels for enhancing diagnosis, assisting less-invasive follow-up and herald utility by revealing putative disease-specific therapeutic targets.

Objective: To determine brain magnetic resonance imaging (MRI) measures of cerebrospinal fluid (CSF) and whole brain volume of full-term and premature infants following surgical treatment for thoracic non-cardiac congenital anomalies requiring critical care. Methods: Full-term (n = 13) and pre-term (n = 13) patients with long-gap esophageal atresia, and full-term naïve controls (n = 19) < 1 year corrected age, underwent non-sedated brain MRI following completion of thoracic non-cardiac surgery and critical care treatment. Qualitative MRI findings were reviewed and reported by a pediatric neuroradiologist and neurologist. Several linear brain metrics were measured using structural T1-weighted images, while T2-weighted images were required for segmentation of total CSF and whole brain tissue using the Morphologically Adaptive Neonatal Tissue Segmentation (MANTiS) tool. Group differences in absolute (mm, cm3) and normalized (%) data were analyzed using a univariate general linear model with age at scan as a covariate. Mean normalized values were assessed using one-way ANOVA. Results: Qualitative brain findings suggest brain atrophy in both full-term and pre-term patients. Both linear and volumetric MRI analyses confirmed significantly greater total CSF and extra-axial space, and decreased whole brain size in both full-term and pre-term patients compared to naïve controls. Although linear analysis suggests greater ventricular volumes in all patients, volumetric analysis showed that normalized ventricular volumes were higher only in premature patients compared to controls. Discussion: Linear brain metrics paralleled volumetric MRI analysis of total CSF and extra-axial space, but not ventricular size. Full-term infants appear to demonstrate similar brain vulnerability in the context of life-saving thoracic non-cardiac surgery requiring critical care as premature infants.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing, with concomitant high incidence of lipoprotein abnormalities. Cardiovascular disease (CVD) is the main cause of death in subjects with NAFLD and management of dyslipidaemia is pivotal for prevention. We aimed to determine cardiovascular risk and indication for statin therapy in subjects with NAFLD.

Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAFV600E-positive thyroid cancer cells to the BRAFV600E-inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC.

Any imbalance between reactive oxygen species (ROS) generation and the anti-oxidant capacity lead to cellular oxidative stress. Many chemotherapeutic agents mediate their cytotoxic functions through the generation of ROS. c-Met, a receptor tyrosine kinase, is over-expressed in renal cancer and plays very crucial role(s) in its growth and survival. Here, we show that c-Met activation protected renal cancer cells from ROS, oxidative stress and cytotoxicity induced by the anti-cancer agent sorafenib (used for renal cancer treatment); and it markedly attenuated sorafenib-induced DNA damage. Activated c-Met promoted the anti-apoptotic proteins (Bcl-2 and Bcl-xL) and inhibited apoptotic cleaved caspase-3. We found that the cytoprotective function of c-Met against sorafenib-induced ROS generation and apoptosis was mediated primarily through the activation of anti-oxidant Nrf2-HO-1. c-Met promoted the nuclear localization of Nrf2 and hindered its binding with the inhibitory protein Keap1. Silencing of Nrf2 attenuated the protective action of c-Met against sorafenib-induced oxidative stress. To evaluate the physiological significance of our findings, in a tumor xenograft model, we observed that a combination treatment with pharmacological inhibitors of c-Met and it's anti-oxidant downstream effecter HO-1 markedly reduced the growth of renal tumor in vivo; it increased the oxidative stress, DNA damage and apoptotic markers in the tumor xenografts, along with reduced tumor vessel density. Our observations indicate that the c-Met-Nrf2-HO-1 pathway plays a vital role in relieving ROS-mediated oxidative stress of renal tumors. Targeting this pathway can significantly increase the oxidative stress to promote apoptotic death of cancer cells.

Cryolipolysis is a noninvasive, skin cooling treatment for local fat reduction that causes prolonged hypoesthesia over the treated area. We tested the hypothesis that cryolipolysis can attenuate nociception of a range of sensory stimuli, including stimuli that evoke itch. The effects of cryolipolysis on sensory phenomena were evaluated by quantitative sensory testing (QST) in 11 healthy subjects over a period of 56 days. Mechanical and thermal pain thresholds were measured on treated and contralateral untreated (control) flanks. Itch duration was evaluated following histamine iontophoresis. Unmyelinated epidermal nerve fiber and myelinated dermal nerve fiber densities were quantified in skin biopsies from six subjects. Cryolipolysis produced a marked decrease in mechanical and thermal pain sensitivity. Hyposensitivity started between two to seven days after cryolipolysis and persisted for at least thirty-five days post treatment. Skin biopsies revealed that cryolipolysis decreased epidermal nerve fiber density, as well as dermal myelinated nerve fiber density, which persisted throughout the study. In conclusion, cryolipolysis causes significant and prolonged decreases in cutaneous sensitivity. Our data suggest that controlled skin cooling to specifically target cutaneous nerve fibers has the potential to be useful for prolonged relief of cutaneous pain and might have a use as a research tool to isolate and study cutaneous itch-sensing nerves in human skin.

Insulin-like growth factor 2 (Igf2), a member of the insulin gene family, is important for brain development and has known neurotrophic properties. Though Igf2, its receptors, and binding proteins, are expressed in the adult CNS, their role in the adult brain is less well-understood. Here we studied how Igf2 deficiency affects brains of adult Igf2 knockout (Igf2(-/-)) mice following neurotoxic insult produced by the glutamate analog kainic acid (KA). Igf2(-/-) mice exhibited attenuated epileptiform activity in response to KA and were less susceptible to hippocampal neurodegeneration compared with Igf2(+/+) mice. Other brain areas protected by the lack of Igf2 included the amygdala complex, septal nuclei, and thalamic region. Apoptosis, as determined by TUNEL and Hoechst 33342 staining, was accordingly less for Igf2(-/-) mice. Hippocampal slices from Igf2(-/-) mice also were protected against the effects epileptogenic effects of KA compared to Igf2(+/+) mice suggesting that neuroprotection afforded by a lack of Igf2 may be developmental in origin and experiments demonstrating enhanced synaptic inhibition in slices taken from Igf2(-/-) mice support this hypothesis. Taken together, these results suggest that Igf2 may be important for mechanisms and circuits that contribute to neurodegeneration and epilepsy.

Extracellular matrix proteins and enzymes involved in degradation have been found to be associated with tissue fibrosis and ureteropelvic junction obstruction (UPJO). In this study we developed a promising urinary biomarker model which can identify reduced renal function in UPJ obstruction patients. This can potentially serve as a non-invasive way to enhance surgical decision making for patients and urologists.

Catheter radiofrequency (RF) ablation for cardiac arrhythmias is a painful procedure. Prior work using functional near-infrared spectroscopy (fNIRS) in patients under general anesthesia has indicated that ablation results in activity in pain-related cortical regions, presumably due to inadequate blockade of afferent nociceptors originating within the cardiac system. Having an objective brain-based measure for nociception and analgesia may in the future allow for enhanced analgesic control during surgical procedures. Hence, the primary aim of this study is to demonstrate that the administration of remifentanil, an opioid widely used during surgery, can attenuate the fNIRS cortical responses to cardiac ablation.

Previous studies in preterm infants report white matter abnormalities of the corpus callosum (CC) as an important predictor of neurodevelopmental outcomes. Our cross-sectional study aimed to describe qualitative and quantitative CC size in critically ill infants following surgical and critical care for long-gap esophageal atresia (LGEA) - in comparison to healthy infants - using MRI. Non-sedated brain MRI was acquired for full-term (n = 13) and premature (n = 13) patients following treatment for LGEA, and controls (n = 20) <1 year corrected age. A neuroradiologist performed qualitative evaluation of T1-weighted images. ITK-SNAP was used for linear, 2-D and 3-D manual CC measures and segmentations as part of CC size quantification. Qualitative MRI analysis indicated underdeveloped CC in both patient groups in comparison to controls. We show no group differences in mid-sagittal CC length. Although 2-D results were inconclusive, volumetric analysis showed smaller absolute (F(2,42) = 20.40, p < 0.001) and normalized (F(2,42) = 16.61, p < 0.001) CC volumes following complex perioperative treatment for LGEA in both full-term and premature patients, suggesting delayed or diminished CC growth in comparison to controls, with no difference between patient groups. Future research should look into etiology of described differences, neurodevelopmental outcomes, and role of the CC as an early marker of neurodevelopment in this unique infant population.

The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Currently available local anesthetics have analgesic durations in humans generally less than 12 hours. Prolonged-duration local anesthetics will be useful for postoperative analgesia. Previous studies showed that in rats, combinations of tetrodotoxin (TTX) with bupivacaine had supra-additive effects on sciatic block durations. In those studies, epinephrine combined with TTX prolonged blocks more than 10-fold, while reducing systemic toxicity. TTX, formulated as Tectin, is in phase III clinical trials as an injectable systemic analgesic for chronic cancer pain. Here, we examine dose-duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine 0.25% (2.5 mg/mL) solutions, with or without epinephrine 5 µg/mL (1:200,000) in rats. Percutaneous sciatic blockade was performed in Sprague-Dawley rats, and intensity and duration of sensory blockade was tested blindly with different Tectin-bupivacaine-epinephrine combinations. Between-group comparisons were analyzed using ANOVA and post-hoc Sidak tests. Nerves were examined blindly for signs of injury. Blocks containing bupivacaine 0.25% with Tectin 10 µM and epinephrine 5 µg/mL were prolonged by roughly 3-fold compared to blocks with bupivacaine 0.25% plain (P < 0.001) or bupivacaine 0.25% with epinephrine 5 µg/mL (P < 0.001). Nerve histology was benign for all groups. Combinations of Tectin in bupivacaine 0.25% with epinephrine 5 µg/mL appear promising for prolonged duration of local anesthesia.

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas(-/-) murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.