Umit Kaldirim

The role of preconditioning and N-acetylcysteine on oxidative stress resulting from tourniquet-induced ischemia-reperfusion in arthroscopic knee surgery.

BACKGROUND The aim of this study was to investigate the effects of ischemic preconditioning (IPC) and N-acetylcysteine (NAC) on oxidative stress resulting from tourniquet-induced ischemia-reperfusion (IR) period in arthroscopic knee surgery. METHODS Forty-five patients who had arthroscopic knee surgery for meniscal and chondral lesions and for pathologic medial plica were included in this study. They were assigned to the following treatment groups: control (group C; n=15), IPC (group P; n=15), and NAC (group N; n=15). Subjects in the control group underwent routine surgical procedures. Subjects in the preconditioning group were subjected to temporary ischemia, with tourniquet performed by three compression cycles of 5 minutes followed by 5 minutes of reperfusion just before the application of tourniquet inflation. Subjects in the NAC group received 10 mg/kg NAC dissolved in 100 mL 0.9% normal saline intravenously 30 minutes before tourniquet inflation. An hour before the tourniquet was applied (preischemia) and 2 hours after tourniquet was removed (reperfusion), blood samples (to test for metabolites) were obtained. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (TAC), and total oxidant status (TOS) were measured in all serum samples. Results were compared between preischemia and reperfusion in three groups. RESULTS MDA in the control group was found to be increased significantly compared with preischemia, whereas MDA in IPC and NAC groups did not change insignificantly. SOD and GSH activities in the control group were found to be increased significantly, whereas SOD and GSH activities in IPC and NAC groups did not change significantly after reperfusion. TAC in the control group was found to be decreased and TOS was found to be increased significantly, but TAC and TOS in IPC and NAC groups were not significantly different after reperfusion. Mean serum MDA, TOS, SOD, and GSH-Px levels were lower in group P than group C at reperfusion period (p<0.05). Mean serum SOD levels were lower in group P than group N at reperfusion period (p<0.05). CONCLUSIONS Tourniquet-induced IR period in routine arthroscopic knee surgery resulted in oxidative stress by increasing MDA, SOD, GSH-Px, TOS and decreasing TAC. NAC and IPC had protective effect on occurrence of oxidative stress resulting from IR period by preventing MDA, SOD, GSH-Px, TAC, and TOS changes in routine arthroscopic knee surgery.

A comparison of suprascapular nerve block and procedural sedation analgesia in shoulder dislocation reduction

OBJECTIVES Dislocation of the shoulder joint is one of the most common dislocations. The reduction procedure is a painful procedure. In this study, 2 different treatment groups were compared for pain control during shoulder dislocation reduction. It was aimed to evaluate the differences between the groups in reduction, success, length of hospital stay, complications, side effects, patient-physician satisfaction, and ease of application. METHODS The study was planned to be prospective and randomized. As procedural sedation analgesia (SA), titration of ketamine 1 to 2 mg/kg was administered intravenously to group 1. Suprascapular nerve block (SNB) was applied under ultrasound guidance (USG) to group 2. Conformity to normal distribution of variables was examined with the Kolmogorov-Smirnov test. The χ2 test and Fisher test were used to evaluate differences between the groups in categorical variables and the Mann-Whitney U test, and a value of P<.05 was accepted as statistically significant. RESULTS The study comprised a total of 41 patients; 20 in the group 1 and 21 in the group 2. No statistically significant difference was determined between the groups in terms of age (P=.916), sex (P=.972), reduction success (P=.540), and patient-physician satisfaction (P=.198). The time spent in the emergency department (ED) by patients in the SA group was signficantly longer compared with the SNB group. No side effects were observed in the SNB group. CONCLUSIONS Suprascapular nerve block, which can be easily applied under USG in the ED, can be evaluated as a good alternative to SA in the reduction of shoulder dislocations.

Effect of Preconditioned Hyperbaric Oxygen and Ozone on Ischemia-Reperfusion Induced Tourniquet in Skeletal Bone of Rats

BACKGROUND The aim of the study was to investigate effect of I/R injury on bone tissue and protective role of hyperbaric oxygen precondition (HBO-PC) and ozone precondition (O(3)-PC) on I/R injury by using biochemical analysis. MATERIALS AND METHODS Thirty-two rats were included in study. The animals were divided into four equal groups: sham operation, I/R, I/R+HBO and I/R+O(3). One session of 60 min, 3 ATA, 3-4 L/min, 100% oxygenation was defined as one dose of HBO. First dose of HBO was administrated 72 h before ischemia. Subsequent, one-dose of HBO administrated per 12 hours until ischemia time (total seven doses); 0.7 mg/kg ozone/oxygen mixture intraperitoneally was defined as one dose of ozone. First dose of O(3) was administered 72 h before ischemia (total four doses). I/R model was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 22 h of reperfusion. The right tibia and were harvested. Tissue was assayed for levels of malondialdehyde (MDA) and protein carbonyl (PCO), activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). RESULTS MDA and PCO levels were increased in I/R group. SOD activity was increased; GSH-Px activity was decreased in I/R group. MDA and PCO levels were decreased, SOD and GSH-Px activities were increased in both HBO+I/R and O(3)+I/R groups. CONCLUSION It has been shown that levels of MDA and PCO in bone were increased followed by 2 h of ischemia and 22 h of reperfusion period. Ozone-PC and HBO-PC has protective effect against skeletal bone I/R injury by decreasing levels of MDA and PCO, increasing activities of SOD and GSH-Px in rats.

The protective effects of ozone therapy in a rat model of acetaminophen-induced liver injury

OBJECTIVES Acetaminophen (APAP) overdose may cause acute liver injury. Ozone therapy (OT) is shown to reduce inflammation and necrosis in several entities. Thus, we have designed this study to evaluate the efficacy of OT in a rat model of APAP-induced liver injury. METHODS Twenty-seven Sprague-Dawley rats were divided into three groups: sham, APAP and APAP+OT groups. In the APAP and the APAP+OT groups, liver injury was induced by oral administration of 1 g/kg APAP. The APAP+OT group received a single dose ozone/oxygen mixture (0.7 mg/kg) intraperitoneally 1h after APAP administration. All animals were killed at 24 hour after APAP administration. Blood samples and liver tissues were harvested to determine liver injury and oxidative stress parameters. Liver tissues and blood samples were obtained for biochemical and histopathological analyses. RESULTS APAP administration caused necrosis in the liver after 24h. The degrees of liver necrosis of the APAP group were higher than the other groups (in both p<0.05, respectively). In the APAP+OT group, liver antioxidant enzymes activities were significantly higher than the APAP group (p<0.05), but were lower than the sham group (p<0.05). In the sham group, serum neopterin, a marker of cell-mediated immunity, concentrations (4.8±1.2 nmol/L) were lower than the APAP (14.7±1.4 nmol/L) and APAP+OT groups (7.5±2.4 nmol/L) (in both p<0.05, respectively). CONCLUSION Our results showed that OT prevented liver necrosis in rats and reduced neopterin levels. These findings suggest that the use of OT as an adjuvant therapy which might improve the outcome in APAP induced liver injury.

Evaluation and Comparison of the Effects of Hyperbaric Oxygen and Ozonized Oxygen as Adjuvant Treatments in an Experimental Osteomyelitis Model

BACKGROUND We evaluated and compared the efficacy of ozone (O(3)) and hyperbaric oxygen (HBO) therapies in an experimental rat model of osteomyelitis. MATERIALS AND METHODS Forty-eight male Sprague-Dawley rats were divided into sham, osteomyelitis (control), vancomycin (V), vancomycin + HBO (VHB), vancomycin + O(3) (VO), and vancomycin + HBO + O(3) (VOHB) groups. Osteomyelitis was induced by a bone injection of 10(8) CFU/mL methicillin-resistant Staphylococcus aureus. HBO was administered daily at 2.8-atm pressure for 90 min; O(3) therapy was provided as intraperitoneal injections of 0.7 mg/kg O(3)/O(2) gas mixture once daily. Treatments were continued from d 7 to 21 after induction of osteomyelitis. Bone tissues and blood samples were harvested for biochemical, histopathologic, and microbiologic analyses. RESULTS Rats in the sham, VO, and VOHB groups gained weight but those in the control, V, and VHB groups did not. Levels of malondialdehyde, superoxide dismutase, and glutathione peroxidase were lower in the VHB, VO, and VOHB groups than in V and control groups. Levels of interleukin-10 and -1β and tumor necrosis factor-α were decreased in the VHB, VO, and VOHB groups; transforming growth factor-β was increased in these groups compared with V and control groups (P ≤ 0.001). Bacteria counts in VOHB were significantly lower than those in group of V (P = 0.012). Histopathologic scores in group VO were significantly lower than those in group V (P = 0.046). CONCLUSIONS O(3) was as effective as HBO in decreasing oxidative parameters and inflammatory cytokines. Rats in the VO and VOHB groups gained more weight than did the other groups. Bacteria counts were significantly decreased in group VOHB compared with the other groups. Histopathologic scores in group VO were significantly decreased compared with the other groups.

Effects of medical ozone therapy on acetaminophen-induced nephrotoxicity in rats

Acetaminophen (APAP), also known as paracetamol, is the commonest cause of toxic ingestion in the world. Because overdose of APAP has life-threatening effects on kidney, treatment of APAP-induced nephrotoxicity has life-saving importance. Aim of the study was to evaluate the efficacy of medical ozone therapy in experimental model of APAP toxication. Twenty-one male Wistar rats (200–250 g) were randomly assigned into three groups containing seven rats each: Sham, control (only APAP treated), and APAP + ozone therapy groups. Rats were killed 48 hours after administration of APAP. Urea, creatinine levels in the blood, and malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity in renal tissue were measured. Kidney tissues were stained with hematoxylin and eosin for histological assessment. APAP administration deteriorated the renal functions and significantly elevated renal MDA levels and depleted SOD and GSH-Px activities. Ozone therapy significantly reduced the MDA level, increased the SOD and GSH-Px activities, and normalized the renal histology. In conclusion, our study results are consistent with encouraging data for ozone therapy on APAP-induced nephrotoxicity in rats by improving antioxidant mechanism and oxidative stress.

Poly(ADP-ribose) polymerase inhibition modulates experimental acute necrotizing pancreatitis-induced oxidative stress, bacterial translocation and neopterin concentrations in rats

Various studies have been performed to find out novel treatment strategies for acute necrotizing pancreatitis (ANP). Inhibition of poly(ADP-ribose) polymerase (PARP) is shown to reduce inflammation in several pathological conditions. We aimed to evaluate the efficacy of benzamide, a PARP inhibitor, in an experimental model of ANP. Thirty Sprague–Dawley rats were divided into three groups: sham-operated, ANP and ANP + benzamide groups. All groups except the sham-operated group were subjected to the ANP procedure, induced by infusing of 1 mL/kg of 3% sodium taurocholate into the common biliopancreatic duct. The ANP + benzamide group received 100 mg/kg/day benzamide intraperitoneally for a total of three days after induction of pancreatitis. The surviving animals were killed at the fourth day and the pancreas was harvested for biochemical, microbiological and histological analysis. Blood samples were also obtained from the animals. In the ANP group, a significant increase was observed in concentrations of serum amylase and neopterin and tissue oxidative stress indices (malondialdehyde, superoxide dismutase and glutathione peroxidase). Almost all of these changes were found to be reversed to near their normal values in the ANP + benzamide group. Histological injury scores were significantly higher in the ANP group than in the sham group (P < 0.05, ANP versus sham), and were significantly lower in the ANP + benzamide group than in the ANP group (P < 0.05, ANP + benzamide versus ANP). Evaluation of bacterial translocation identified significantly fewer infected sites in the ANP + benzamide group than in the ANP animals (P < 0.01). We observed that inhibition of PARP with benzamide reduced the severity, the mortality, the bacterial translocation rates and the neopterin concentrations in an experimental ANP model in rats. These findings suggest that it may be possible to improve the outcome of ANP by using PARP inhibitors.

The protective effects of taurine on experimental acute pancreatitis in a rat model

The aim of this study was to investigate the protective effects of taurine (Tau) on experimental acute pancreatitis (AP) in a rat model by measuring cytokines and oxidant stress markers. Forty rats were randomly divided into four groups: sham, AP, Tau and AP + Tau. AP was induced with sodium taurocholate. No treatment was given to the AP. All rats were killed 5 days later. Pancreatic tissues of rats and blood samples were obtained. Tau treatment significantly decreased serum amylase activity (p < 0.001), total injury score (p < 0.001), malondialdehyde levels (p < 0.001) and myeloperoxidase (MPO) activity (p < 0.001). There was no significant difference between the Tau and AP + Tau groups in serum and pancreatic tumor necrosis factor-α, interleukin (IL)-1β and IL-6 levels (p = 1.000). Histopathologic scores in the AP + Tau and Tau groups were significantly lower compared with the AP group (both p < 0.001). These results showed that Tau reduces lipid peroxidation, amylase and MPO activities and the concentrations of proinflammatory cytokines secondary to AP and also increases superoxide dismutase and glutathione peroxidase activities in rats with sodium taurocholate-induced AP. It also has a marked ameliorative effect at histopathologic lesions. With these effects, Tau protects the cells from oxidative damage, reduces inflammation and promotes regression of pancreatic damage.

Hyperbaric oxygen treatment and N-acetylcysteine ameliorate acetaminophen-induced liver injury in a rat model.

An overdose of acetaminophen (APAP) produces centrilobular hepatocellular necrosis. We aimed to investigate the hepatoprotective effects of N-acetylcysteine (NAC) only and hyperbaric oxygen (O2) treatment (HBOT) combined with NAC, and their anti-inflammatory properties in liver tissue. In the current study, a total of 32 male Sprague Dawley rats were divided into 4 groups: sham, APAP, NAC, and NAC + HBOT. In the APAP, NAC, and NAC + HBOT groups, liver injury was induced by oral administration of 1 g/kg APAP. The NAC group received 100 mg/kg NAC per day. NAC + HBOT group received intraperitoneal injection of 100 mg/kg/day NAC and were given HBOT at 2.8 ATA pressure with 100% O2 inhalation for 90 min every 12 h for 5 days. Rats in the sham group received distilled water only by gastric tube. All animals were killed on day 6 after APAP or distilled water administration. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic neopterin, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) levels were measured. There was a significant increase in serum AST and ALT activities in the APAP group compared with the sham group (in both p = 0.001). NAC and NAC + HBOT groups had significant decreases in hepatic neopterin, TNF-α, and IL-6 levels compared with the APAP group. APAP administration caused extensive hepatic necrosis. NAC and NAC + HBO treatments significantly reduced APAP-induced liver injury. Our results showed that the liver damage in APAP toxicity was attenuated by NAC and NAC + HBO treatments. NAC + HBOT exhibit hepatoprotective activity against APAP-induced liver injury in rats.

Serum neuron-specific enolase and S-100β levels as prognostic follow-up markers for oxygen administered carbon monoxide intoxication cases.

O of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, predominantly drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3’ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate posttranscriptional regulation of RRM2 and chemosensitivity by let-7a and that the accumulation of let-7a precursors as a favorable biomarker for judging chemoresistance in pancreatic cancer.W the use of screening mammography, the diagnosis of ductal carcinoma in situ (DCIS) is increasing worldwide. Patients diagnosed with DCIS either have not yet developed or may never develop invasive breast cancer (IBC). Presently, an indolent tumor can not be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients, a major clinical dilemma. We are interested in developing tissue biomarkers (BM) to determine the likelihood that initial breast tumor identified on diagnostic biopsy remains contained in situ, as opposed to becoming invasive. Molecular studies predicted that the transition from in situ to invasive disease was associated with quantitative rather than qualitative differences in gene and protein expression. We developed imaging-based method to measure protein expression as a continuous variable in fixed tissue. We assumed that DCIS accompanying microinvasive carcinoma (T1mic) represented an early progressive DCIS, because invasion increases the likelihood of metastasis. We theorized that molecular BMs of progressive DCIS are the measurable parameters that distinguish cells in in situ component of T1mic from cells in normal/benign epithelium. We performed quantitative protein profiling on 210 archived tissues: 42 histologically normal, 19 benign, 54 cancer in situ, and 95 IBC. Our measurements in DCIS revealed previously unidentified quantitative differences in the insulin-like growth factor I receptor, Ras oncogene like protein 1, and Rho GTPase guanine nucleotide exchange factor VAV2, the proteins implicated in the regulation of invasion in preclinical models. These new findings may open doors to molecular-based predictions of individualized risk for developing invasion in DCIS.High quality biospecimens with appropriate clinical annotation are critical in the era of biomarker discovery in personalized medicine. Several pre-analytical variables affect human biospecimen integrity for biomarker research in cancer. This situation is applicable to a variety of biospecimens including plasma/serum and fixed cancer tissues used for biomarker analysis. The U.S. National Cancer Institute (NCI) Biorepositories and Biospecimen Research Branch (BBRB) was established in 2005 to coordinate NCI’s biospecimen resource activities and address those issues that affect access to the high quality specimens for biomarker research. A Biospecimen Research Network (BRN) was established to fund research to develop additional evidence-based practices used to develop serum and tissue biomarkers for human biospecimen integrity. We describe the development of assays and identification of biomarkers that may be used as sentinel markers of plasma/tissue stability in biobanks using mass-spectroscopy proteomics, circulating miRNA and immunostaining of FFPE tissues (AQUA technology). The first NCI/BBRB-funded project involves the identification of protein biomarkers using mass-spectrometry and illumina arrays in serum obtained from breast cancer and matched normal subjects, to develop guidelines for blood collection and storage. A second project studied effects of pre-analytical variables on circulating miRNA and identification and validation of new and improved housekeeping miRNA and biomarkers associated with breast cancer. In another study, a series of biomarkers have been validated by construction of tissue microarray (TMA) from 93 breast cancer specimens with known time to fixation as a pre-analytical variable. A tissue quality index (TQI) model was generated to predict the time to fixation and tissue quality by studying a subset of biomarker proteins in breast cancer tissues using AQUA scores. This presentation will outline the progressive efforts taken by BRN, investigator-led projects to identify and validate biomarkers for human biospecimen integrity.F of bioactive lipids by oxygenases is known to play both a protective and pro-thrombotic role in circulation. 12-lipoxygenase (12-LOX) and its oxidized products play an important but unresolved role in regulation of platelet function. 12-LOX oxidation of the fatty acid, dihomo-γ-linolenic acid (DGLA), produces the novel bioactive metabolite 12-hydroxyeicosatetrienoic acid (12(S)-HETrE). Recent work suggests that while 12(S)-HETE (produced from 12-LOX oxidation of AA) is pro-thrombotic to the platelet, 12(S)-HETrE acts in a protective manner in platelets to limit activation. Therefore, we sought to identify the mechanism by which DGLA inhibits platelet activation through 12(S)-HETrE. Delineating the mechanism by which this previously unknown metabolite regulates cellular activity is essential to begin to understand how 12-LOX oxidation of DGLA can potentially lead to regulation of a number of physiological processes including thrombosis. To address these questions, we have employed pharmacological and whole animal studies. Pharmacological intervention has confirmed an integral role of 12-HETrE production through 12-LOX to negatively regulate activity in the platelet. These observations have been confirmed in mice lacking the 12-LOX gene. Further, dietary supplementation of DGLA-enriched chow supports our ex vivo studies exogenously adding DGLA and/or 12-HETrE to attenuate agonist-induced platelet activation. Hence, these studies are the first to begin to elucidate the underlying mechanisms by which omega-3 and 6 fatty acids are protective against cardiovascular disease and stroke.P genetics has been unable to replicate studies which link genotype with treatment outcome. In large measure the difficulty in producing reliable markers has been a consequence of the heterogeneity of depression. Almost all depression trials or genetic studies use “response” as the surrogate outcome measure. By definition, response is defined as a 50% reduction on analytical metric when comparing entry and exit from the trial. This implies that subjects remain symptomatic for their presenting symptoms. We have analyzed The Sequenced Treatment Alternatives for Relieving Depression (STAR*D) NIMH multicenter clinical. An endophenotype was defined by baseline depression severity and minimal co-morbidity. The outcome metric we utilized compared non-responders with true remitters. No subjects defined as responders were included in our genotypic analysis. Polymorphic markers were chosen before genotypic analysis. We identified 255 subjects of European/Caucasian ancestry who met our inclusion criteria with 66% non-responders and 33% true remitters. To our knowledge this is the first genetic analysis of depression subjects where the relevant surrogate clinical outcome of symptom relief was used. Our analysis identified 27 SNPs in 12 genes within muscarinic, nicotinic, adrenergic, serotonergic, dopaminergic, reward and signal transduction pathways. A haplotype analysis indicates that certain alleles can predict true remission. Our findings demonstrate using an endophenotypic analysis can identify biomarkers predicting relevant clinical outcomes in depression. Further work is underway to validate the original findings in additional data sets and novel depressed patients.Biological samples from patients are invaluable. Ideally the samples should be preserved for the same period of time as the duration of their corresponding medical records. Urine is a body fluid that can be non-invasively acquired and contains important biological information about the patient. Simple and inexpensive urinary protein sample preservation can be the starting point for comprehensive biological sample storage just like medical record of patients. Here, we propose a method to adsorb urinary proteins to a membrane named Urimem that can then be dried and stored. This method is very simple and inexpensive and requires minimal sample handling. It does not use organic solvents. The proteins on the membrane are dry and are prevented from degradation. The membrane may even be able to be stored at room temperature at least for weeks. The quantity of eluted proteins from a membrane is sufficient for biomarker validation experiments. Comprehensive historical biological information can also be used in retrospective studies to understand the pathophysiology of disease and the relationships among diseases as well as to monitor the long-term efficacy and side effects of treatments. With this information, medical research can be conducted more easily, considerably faster, and more economically, ultimately benefiting the patients who provided the samples. Thus, we believe that it is possible to preserve urinary protein samples from each stage of disease development for every consenting patient in a hospital. This can potentially change the landscape of medical research and medical practice in the future.Phosphatidylserine (PS) exposure is one of the most prominent and ubiquitous fingerprints of dying cells, making it an attractive biomarker for molecular imaging. Synthetic bis-zinc(II)-dipicolylamine (Zn-DPA) derivatives have high selectivity for biological membranes enriched with PS. Our study aims to apply PET/NIRF imaging with a novel DPA-containing probe ( 18 F-MTTI-170) to visualize and evaluate cell death induced by Paclitaxel in a U87MG tumor xenograft model. In vitro toxicity of Paclitaxel to U87MG cells was determined by a colorimetric assay. The response of U87MG cells to Paclitaxel treatment was determined by flow cytometry, fluorescence staining, and cell uptake study. Established U87MG tumors in nude mice were daily treated with a combination of All-Trans Retinoic Acid (ATRC) (1.5 μg/kg) and Paclitaxel (45 μg/kg). Longitudinal PET imaging was performed with 18 F-MTTI-170 before treatment and at day 3, 6, and 9 after treatment. NIRF imaging was carried out with 19 F-MTTI-170 before treatment and at day 4, 7, and 11 after treatment. Our data demonstrated that U87MG human glioma cells are sensitive to Paclitaxel treatment. After being treated with Paclitaxel for 15 h, U87MG cells were stained with PSVue643 (Cy5-Zn-DPA). The strong red fluorescence signal was identified in the cytosol of the treated cells but not on the untreated cells. Besides, the fluorescent signal was effectively blocked by co-incubation with excess amount of unlabeled Zn-DPA. For cell uptake study, about 1.5% of 18 F-MTTI-170 uptake in Paclitaxel-treated U87MG cells was determined after 1 hr incubation, which is significantly higher than 0.69% and 0.39% observed for 18 F-FP-DPA (single modality compound) and F-FP-Dye (negative control), suggesting that the Zn-DPA moiety is indeed the component binding to PS, and the cell uptake of 18 F-MTTI-170 is significantly higher than that of 18 F-FP-DPA. Daily treatment with ATRC and Paclitaxel effectively inhibited the growth of U87MG tumors by inducing cell death. The cell death was clearly visualized by 18 F-MTTI-170 PET. The tumor uptake, which was observed at day 9 after treatment, was significantly higher than that in the untreated tumors with a ratio of 6.70. The NIRF imaging results are consistent with the findings by PET. In conclusion, PET/NIRF imaging with MTTI-170 is sensitive enough to allow visualization of Paclitaxel induced cell death in U87MG tumor xenograft model. Fully quantitative imaging of tumor response to therapy with MTTI-170 offers the potential to provide early assessment of cancer treatment efficacy leading to individually tailored therapeutic plans with improved outcomes.Serum neuron-specific enolase (NSE) and S-100β levels are considered novel biochemical markers of neuronal cell injury. In this study, the initial and post-treatment levels of NSE and S-100β were compared in carbon monoxide (CO) poisoning patients, who received normorbaric oxygen (NBO) or hyperbaric oxygen (HBO) therapy. Forty consecutive patients with acute CO poisoning were enrolled in this prospective, observational study. According to their clinical symptoms and observations, twenty patients were treated with NBO, and the other twenty with HBO. Serum S-100β and NSE levels were measured both at time of admission and 6 h later (post-treatment). Serum NSE and S-100β values decreased significantly in both of the therapeutic modalities. The initial and post-treatment values of NSE and S-100β in NBO or HBO patients were comparable. A clear negative correlation was observed between the decrease of NSE and S-100β levels and initial blood carboxyhemoglobin levels. In conclusion, the present results suggested the use of serum S-100β and NSE levels as indicators for brain injury. Due to the significant increase of their values with oxygen therapy, they may also be useful as prognostic follow-up markers. However, the current findings reflected no difference between the efficacy of NBO or HBO therapy.T activation of NFκB, a central coordinator of immune responses, is tightly regulated in order to achieve its normal transient activation in response to stress. In cancer, NFκB is activated abnormally, contributing to oncogenesis and progression. The regulation of transient activation is complex, and we are still learning the details of how the essential fine control is achieved. Here, we uncover a novel regulatory pathway for NFκB that is driven by cycles of lysine methylation and demethylation. Using a novel lentiviral Validation-Based Insertional Mutagenesis (VBIM) method, we identified the F-box leucine repeat rich protein 11 (FBXL11), a known histone H3 lysine 36 (H3K36) demethylase, as a potent negative regulator of NFκB. Deletion of the demethylase domain of FBXL11 abolishes this activity. Knocking the expression of FBXL11 down activates NFκB, as does over-expression of the corresponding histone H3K36 methylase, NSD1. The p65 subunit of NFκB binds to NSD1 and FBXL11, and significant methylation of K218 and K221 of p65 was detected in cells with constitutively active NFκB or upon cytokine stimulation. Importantly, FBXL11 is transcribed in response to NFκB activation and thus, like the well known inhibitor IκB, FBXL11 participates in an auto-regulatory negative feedback loop. We show that lysine methylation is an important regulatory post-translational modification of NFκB that is mediated by the FBXL11-NSD1 enzyme pair. Furthermore, we demonstrate that the VBIM technique is a powerful tool for gene discovery that has broad applications in many different systems.H we report a core-shell plasmonic nanoparticle (NP) design based on silver nanodecahedrons (Ag NDs). We have developed a new photo-assisted synthesis process to prepared the Ag NDs and a self-assembly procedure to coat the Ag NDs with a uniform layer of silica in which Raman tags may be incorporated. This core-shell Ag nano-composite exhibits strong surface enhanced Raman scattering (SERS) because of its localized surface plasmon resonance (LSPR). A unique property offered by the Ag NDs is their widely tunable LSPR peak between 420 660 nm. Until now, there are very few reported cases of Ag nanoparticles that exhibit LSPR in the RED region since the natural plasmonic resonance of Ag occurs in the UV-blue region. This has led to problems with most Raman-active tags as they only produce transition-enhanced Raman signal in the REDGREEN region. The core-shell design also ensures that the Ag nano-particles are completely protected against degradation due to oxidation. As a result of the reported core-shell Ag NDs, the use of Raman tags for labeling biomolecules now has a much better prospect. Raman-based labeling is known to be a better choice than the common fluorescence approach because of the wide spectral diversity offered by Raman tags. In addition, field enhancement due to LSPR in the core-shell Ag NDs may lead to a variety of applications including non-linear harmonics generation, photo-induced reaction with long wavelength radiation and florescence enhancement.Personalized and molecular-based cancer treatment depends on biomarkers. Biomarker research and assay development is becoming increasingly important because it drives drug discovery, clinical diagnostics, prognostics and treatment decisions. Immunohistochemistry (IHC), which examines protein localization with preserved tissue morphology, is one of the most important assays to detect protein biomarkers. This technique has been applied in many facets of biological science, especially in assays required using archival formalin-fixed, paraffin embedded (FFPE) tissues. The major challenges for IHC are sensitivity and quantification. AQUA (Automatic Quantitative Analysis) is a fluorescence IHC imaging analysis technology that could automatically quantify protein expression in tissues with high sensitivity and accuracy. We have applied AQUA technology for both basic and clinical research. Using this technology, we identified biomarkers that related to cetuximab treatment in a mouse xenograft model. We determined the expression of certain proteins in cancer patient cohorts and correlated the protein expression levels with pathologic and clinical outcomes. We found several proteins that could serve as prognostic or predictive biomarkers. With the emergence of new biomarkers from recent genetic discoveries, numerous novel targeted therapies for cancer are already in clinical trials, with many more to be developed in the coming decades. Personalized medicine is about the identification of patients that are most likely to benefit from treatment and assess treatment response. AQUA technology can dramatically speed up the progress of such challenges and therefore represents the future for biomarker research. Biography Yang is an Assistant Professor at Fox Chase Cancer Center, where she takes charge of the Tissue Research Service at the Biosample Repository Core Facility. She is a leading expert in the field of immunohistochemistry (IHC) and quantitative analysis. She has applied IHC to study the molecular alterations of known and novel signaling pathways that regulate embryogenesis, organ morphogenesis and tumorigenesis. Her research results can be found in prestigious journals such as Development, Am. J. Path., JBC et. al. Recently Dr. Yang extended the automatic quantitative analysis (AQUA) IHC assay system for tumor biomarker research. Dr. Yang serves in the editorial board of the Journal of Cancer Research Updates and the Journal of Analytical Oncology. She is ad hoc reviewers in a number of journals including Biomarkers, Biochemical Pharmacology, Current Pharmaceutical Biotechnology, Current Proteomics, Experimental Neurology et.al.M technologies have recently come to the fore in the context of biomarker discovery. Metabolic dysfunction is a noted hallmark in numerous disease states, and thus multivariate measurement and analysis of metabolite parameters provides a potential window into both diagnostic as well as prognostic realms. In this presentation, the advantages and disadvantages of biomarker discovery by NMR metabolomics methods will be discussed. In particular, the use of quantitative methods for metabolite analysis will be contextulized against other tools such as mass spectrometry based metabolite discovery. A biological context will be provided in our clinical oncology studies in the areas of pancreatic, colorectal, lung and brain cancer.O of the HER2/neu receptor occurs in 15 to 30 percent of breast tumors and is linked to poorer prognosis. Currently HER2/neu expression status determines whether patient will receive trastuzumabbased treatment. In clinical practice, overexpression of HER2/neu is identified using IHC or FISH, both of which are invasive approaches requiring tissue samples. Serum assays for HER2/neu receptor have been reported but the use is very limited due to serum interference factors (e.g. human anti-animal antibodies) that lead to false test results and inconsistency with tissue Her2 status. We have developed an ELISA based approach implementing an MBB buffer to eliminate false results and to obtain more accurate assessment of HER2/neu levels. Using this refined assay we retroactively measured HER2/neu levels from 56 patients. Pre-treatment (e.g. surgery, radiation, or chemotherapy) samples were available from 12 patients, of which 6 patients were tissue HER2/neu positive and the other six were negative. All the HER2/ neu positive samples had higher serum levels than negative ones (p < 0.05). We observed a decrease in serum HER-2/neu values after surgery in two out of 5 patients. In 5 patients experienced recurrence or metastasis, HER2/neu level significantly increased from the treatment-established baseline, accompanying recurrence and before metastasis. Our results indicate that we can monitor HER2 ECD as a biomarker over the course of disease progression and treatment.N antibodies against viruses represent a major mechanism of host protection against viral infections. Most if not all marketed vaccines elicit neutralizing antibodies. Currently, neutralizing anti-viral therapeutic monoclonal antibodies are being developed for the treatment and/or the prevention of viral diseases. Standard neutralization assays to assess the viral neutralization activities of antibodies have historically been functional plaque assays. Plaque assays are time-consuming, laborintensive and challenging to implement in clinical studies especially those involving a large number of patients. Here we described the development and implementation of fast and robust Fluorescent Foci (FFA)-based microneutralization (MN) assays. These assays, using viruses expressing enhanced green fluorescence protein (EGFP), allow for higher throughput, better precision, and shorter assay turn-around time making them suitable for use in large clinical studies. We will first describe the establishment of a novel FFA-based MN assay to detect and quantify neutralizing antibodies against EBV. This assay uses EBV-GFP and an engineered epithelial cell line. All assay conditions impacting assay performance were optimized and the assay was automated using liquid handling and high-content imaging systems. The robustness and precision of the optimized assay were demonstrated using serum samples from mice, rabbits and humans (n > 600). In addition, a companion EBV-specific IgG ELISA assay was developed. Significant correlation (r2 = 0.89) between both assays was demonstrated using 358 rabbit serum samples. A MN assay was similarly developed for Respiratory Syncytial virus (RSV) to support the development of MedImmune’s anti-RSV therapeutic mAbs and RSV vaccine candidates. The 2-year assay control trending of RSV MN assay will be presented to demonstrate the robustness and precision of this assay.