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The activation of the α1-adrenoceptor-(α1-AR) by norepinephrine results in the G-protein (Gqα) mediated increase in the phosphoinositide-specific phospholipase C (PLC) activity. The byproducts of PLC hydrolytic activity, namely, 1,2-diacylglycerol and inositol-1,4,5-trisphosphate, are important downstream signal transducers for increased protein synthesis in the cardiomyocyte and the subsequent hypertrophic response. In this article, evidence was outlined to demonstrate the role of cardiomyocyte PLC isozymes in the catecholamine-induced increase in protein synthesis by using a blocker of α1-AR and an inhibitor of PLC. The discussion was focused on the α1-AR-Gqα-PLC-mediated hypertrophic signalling pathway from the viewpoint that it may compliment the other β1-AR-Gs protein-adenylyl cyclase signal transduction mechanisms in the early stages of cardiac hypertrophy development, but may become more relevant at the late stage of cardiac hypertrophy. From the information provided here, it is suggested that some specific PLC isozymes may potentially serve as important targets for the attenuation of cardiac hypertrophy in the vulnerable patient population at-risk for heart failure.

Summary Background The shortage of single-use N95 respirator masks (NRMs) during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted consideration of NRM recycling to extend limited stocks by healthcare providers and facilities. Aim To assess potential reuse via autoclaving of NRMs worn daily in a major urban Canadian hospital. Methods NRM reusability was assessed following collection from volunteer staff after 2–8 h use, sterilization by autoclaving and PortaCount fit testing. A workflow was developed for reprocessing hundreds of NRMs daily. Findings Used NRMs passed fit testing after autoclaving once, with 86% passing a second reuse/autoclave cycle. A separate cohort of used masks pre-warmed before autoclaving passed fit testing. To recycle 200–1000 NRMs daily, procedures for collection, sterilization and re-distribution were developed to minimize particle aerosolization risk during NRM handling, to reject NRM showing obvious wear, and to promote adoption by staff. NRM recovery ranged from 49% to 80% across 12 collection cycles. Conclusion Reuse of NRMs is feasible in major hospitals and other healthcare facilities. In sharp contrast to studies of unused NRMs passing fit testing after 10 autoclave cycles, we show that daily wear substantially reduces NRM fit, limiting reuse to a single cycle, but still increasing NRM stocks by ∼66%. Such reuse requires development of a comprehensive plan that includes communication across staffing levels, from front-line workers to hospital administration, to increase the collection, acceptance of and adherence to sterilization processes for NRM recovery.

It is now well established that ischemia/reperfusion (I/R) injury is associated with the compromised recovery of cardiac contractile function. Such an adverse effect of I/R injury in the heart is attributed to the development of oxidative stress and intracellular Ca2+-overload, which are known to induce remodeling of subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils. However, repeated episodes of brief periods of ischemia followed by reperfusion or ischemic preconditioning (IP) have been shown to improve cardiac function and exert cardioprotective actions against the adverse effects of prolonged I/R injury. This protective action of IP in attenuating myocardial damage and subcellular remodeling is likely to be due to marked reductions in the occurrence of oxidative stress and intracellular Ca2+-overload in cardiomyocytes. In addition, the beneficial actions of IP have been attributed to the depression of proteolytic activities and inflammatory levels of cytokines as well as the activation of the nuclear factor erythroid factor 2-mediated signal transduction pathway. Accordingly, this review is intended to describe some of the changes in subcellular organelles, which are induced in cardiomyocytes by I/R for the occurrence of oxidative stress and intracellular Ca2+-overload and highlight some of the mechanisms for explaining the cardioprotective effects of IP.

According to the World Health Organization, cancer is a leading cause of death worldwide, representing almost 10 million deaths in 2020 [...]

Ineffective healing and treatment of foot ulcers can lead to an infection and gangrene of the wound area that ultimately results in the loss of the limb. The incidence of foot ulcers is higher in patients with diabetes, peripheral vascular disease and kidney disease. Accordingly, this study was undertaken to assess the ability of foot bathing in CO2-enriched water to heal foot ulcers. The design was a double-blinded, randomized, placebo-controlled study. Patients with at least one foot ulcer were randomized to receive either a treatment with bath therapy at 37 ± 0.5 °C containing either 1000–1200 ppm CO2-enriched tap water (the intervention) or non-carbonated tap water at 37 ± 0.5 °C (the control group). Treatment was conducted three times/week for 15 min per session for up to 16 weeks for a total of 48 treatment sessions. Before and at the end of every treatment month, wound size, wound area oxygenation and the ankle brachial index were measured. In addition, the McGill pain questionnaire was conducted. Blood was also collected at these time points (for a total of five collections) for the measurement of different biomarkers. While no significant differences (p &lt, 0.05) in the group/time interaction effect were observed, a clear separation within the wound area reduction/wound area/oxygenated Hb outcomes was seen between placebo (control) and treatment (CO2) group. This pilot study is suggestive that bathing in CO2-enriched water may accelerate the healing of foot ulcers.

The objective of this research is to use metabolomic techniques to discover and validate plasma metabolite biomarkers for the diagnosis of early-stage non-small cell lung cancer (NSCLC). The study included plasma samples from 156 patients with biopsy-confirmed NSCLC along with age and gender-matched plasma samples from 60 healthy controls. A fully quantitative targeted mass spectrometry (MS) analysis (targeting 138 metabolites) was performed on all samples. The sample set was split into a discovery set and validation set. Metabolite concentration data, clinical data, and smoking history were used to determine optimal sets of biomarkers and optimal regression models for identifying different stages of NSCLC using the discovery sets. The same biomarkers and regression models were used and assessed on the validation models. Univariate and multivariate statistical analysis identified &beta, hydroxybutyric acid, LysoPC 20:3, PC ae C40:6, citric acid, and fumaric acid as being significantly different between healthy controls and stage I/II NSCLC. Robust predictive models with areas under the curve (AUC) &gt, 0.9 were developed and validated using these metabolites and other, easily measured clinical data for detecting different stages of NSCLC. This study successfully identified and validated a simple, high-performing, metabolite-based test for detecting early stage (I/II) NSCLC patients in plasma. While promising, further validation on larger and more diverse cohorts is still required.

The five-year survival rate of lung cancer patients is very low, mainly because most newly diagnosed patients present with locally advanced or metastatic disease. Therefore, early diagnosis is key to the successful treatment and management of lung cancer. Unfortunately, early detection methods of lung cancer are not ideal. In this brief review, we described early detection methods such as chest X-rays followed by bronchoscopy, sputum analysis followed by cytological analysis, and low-dose computed tomography (LDCT). In addition, we discussed the potential of metabolomic fingerprinting, compared to that of other biomarkers, including molecular targets, as a low-cost, high-throughput blood-based test that is both feasible and affordable for early-stage lung cancer screening of at-risk populations. Accordingly, we proposed a paradigm shift to metabolomics as an alternative to molecular and proteomic-based markers in lung cancer screening, which will enable blood-based routine testing and be accessible to those patients at the highest risk for lung cancer.

Aim: Spermidine/spermine N1-acetyltransferase (SSAT-1) regulates cell growth, proliferation and death. Amantadine is converted by SSAT-1 to acetylamantadine (AA). In our earlier studies, although SSAT-1 was activated in patients with cancer, a number of ostensibly healthy adult volunteers had higher than expected AA concentration. This study was therefore undertaken to examine the outlier group. Materials & methods: A follow up of urine analysis for AA by liquid chromatography-tandem mass spectrometry as well as clinical assessments and additional blood analyses were conducted. Results: In some of the outlier controls, higher than expected AA concentration was linked to increased serum carcinoembryonic antigen. Clinical and radiographic assessments revealed underlying abnormalities in other cases that could represent premalignant conditions. Hematology tests revealed elevations in white blood cells and platelets, which are markers of inflammation. Conclusion: High urine concentration of AA could be used as a simple and useful test for screening of cancer in high-risk populations., Graphical abstract, Lay abstract This study describes the potential of a novel noninvasive urine test for screening of cancer using a safe and approved drug, amantadine. We have previously measured high urinary concentration of the acetylated form of amantadine in patients diagnosed with cancer. However, higher than expected acetylated amantadine concentration was also measured in some of the healthy adult volunteers. Subsequent clinical assessments revealed that these healthy individuals could have early clinical signs of cancer. This is a simple test, which may serve as a useful tool for routine screening in populations considered at high risk for cancer.

Background: Lung cancer is the most common cause of cancer-related deaths worldwide. Early diagnosis is crucial to increase the curability chance of the patients. Low dose CT screening can reduce lung cancer mortality, but it is associated with several limitations. Metabolomics is a promising technique for cancer diagnosis due to its ability to provide chemical phenotyping data. The intent of our study was to explore metabolomic effects and profiles of lung cancer patients to determine if metabolic perturbations in the SSAT-1/polyamine pathway can distinguish between healthy participants and lung cancer patients as a diagnostic and treatment monitoring tool. Patients and Methods: Plasma samples were collected as part of the SSAT1 Amantadine Cancer Study. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and quantify metabolite concentrations in lung cancer patient and control samples. Standard statistical analyses were performed to determine whether metabolite concentrations could differentiate between healthy subjects and lung cancer patients, as well as risk prediction modeling applied to determine whether metabolic profiles could provide an indication of cancer progression in later stage patients. Results: A panel consisting of 14 metabolites, which included 6 metabolites in the polyamine pathway, was identified that correctly discriminated lung cancer patients from controls with an area under the curve of 0.97 (95% CI: 0.875-1.0). Conclusion: When used in conjunction with the SSAT-1/polyamine pathway, these metabolites may provide the specificity required for diagnosing lung cancer from other cancer types and could be used as a diagnostic and treatment monitoring tool.

Poor quality and quantity maternal nutrition during pregnancy exerts permanent and damaging effects on the heart of the developing fetus. The developmental origin of adult heart disease is considered an important and critical factor in the pathogenesis of myocardial abnormalities in later life. Low birth mass, a marker of intrauterine stress, has been linked to a predisposition to heart disease. In this article, our work on the impact of exposure to a low-protein diet, in utero, on the developing heart and its long-term consequences are discussed. Other studies providing some supportive evidence are also described. It is proposed that normal fetal nutrition, growth, and development through efficient maternal nutrition (as well as other predisposing factors) before and during pregnancy may serve as a strategy for the primary prevention of heart disease.

Aim: Spermidine/spermine N1-acetyltransferase (SSAT-1) plays a critical role in cell growth, proliferation and death, and is known to be activated in human cancer cells. Amantadine, a US FDA-approved antiviral drug, is a substrate for SSAT-1 and can be used to indirectly measure SSAT-1 activity because of its conversion to acetylamantadine (AA). This study was undertaken to further validate SSAT-1 activity in breast and lung cancer patients. Results: An increase in the urinary concentration of AA in lung and breast cancer patients was observed. The 0–2 h collection time point was determined to be optimal in revealing significant differences in urinary AA concentration between healthy controls and cancer patients. Conclusion: The high urine concentration of AA could be used as a simple and useful test for the detection of breast and lung cancer., Lay abstract This study describes a novel noninvasive urine test for detecting and screening of breast and lung cancer using a safe and approved drug called amantadine. Higher concentration of the acetylated form of amantadine in the urine are detectable in the urine of both breast and lung cancer patients as compared with healthy adult volunteers. This test is simple and may serve as a useful tool for determining the presence of breast and lung cancer.

Aim: SSAT-1 is an enzyme that plays a critical role in cell growth. Amantadine, a FDA-approved antiviral drug, is a substrate for SSAT-1. The utility of amantadine as an agent to demonstrate elevated SSAT-1 activity linked to cancer was conducted. Results: High levels of SSAT-1 expression were measured in tumor human cell lines, and in breast, prostate and lung tumor tissue. An increase in the urinary levels of acetylated amantadine in cancer patients was observed. Conclusion: Increases in SSAT-1 contents in tumor tissue could be of value in targeting cancers with high SSAT-1 expression for confirmation/quantification. The high levels of acetylated amantadine could be used as a simple and useful screening test for the presence of cancer., Lay abstract In response to cancer, cells tend to overproduce specific enzymes as a self-defense mechanism. By using a safe and reliable method to capture and measure the excess enzyme spermidine/spermine N1-acetyltransferase-1, the presence of cancer can be established. This study describes a novel approach of detecting and screening cancer noninvasively in the urine of cancer patients using a safe and approved drug called amantadine that acts as a smart-tracking agent. Higher levels of the acetylated form of amantadine are detectable in the urine of cancer patients, which may serve as a detection tool. In addition, increases in the amount of spermidine/spermine N1-acetyltransferase-1 in tumor tissue may provide a tool for determining the presence of cancer during pathology assessment.

e14536 Background: In the evolving role of biomarkers, proteinomic signatures related to up-regulation of polyamine synthesis including spermine/spermidine acetyltransferase-1 (SSAT-1) appear to be promising markers of malignancy. Acetylamantadine (AA) excretion, a measure of SSAT-1 up-regulation, has been shown to be a marker for malignant proliferation. In preliminary analyses of ostensibly normal individuals from Canada (Winnipeg) and Bangladesh (Dhaka), a proportion were identified to have evidence of SSAT-1 up-regulation above the expected non-malignant range (outliers). These outliers were assessed and followed for development of identifiable medical conditions. Methods: SSAT-1 up regulation was assessed in 60 ostensibly normal individuals by analysis of urinary excretion of AA after ingestion of amantadine 200 mg x 1 dose. AA was assayed using HPLC-mass spec techniques as previously described. Outliers who consented were followed-up by clinical examinations, routine biochemical and hematological tests and radiographs, where indicated. Results: Total AA average (median) excretion at 6 hrs in the Winnipeg cohort was 579+/-252 vs 1699+/-633 ng in the Dhaka cohort. Average urinary concentration at 6 hrs was 3.75+/-0.75 vs 21+/-20 ng/ml. In outliers consenting to follow-up, 1 case of invasive malignancy was identified, 6 cases of pre-malignant neoplastic change, chronic liver disease in 7 cases and chronic inflammation in 2 cases. In others, no malignant or inflammatory conditions have yet been identified. The range of SSAT-1 activity in individuals living in Bangladesh was significantly higher than in the Canadian cohort. Bangladesh volunteers live in an area of known to have high natural contamination with arsenic, a recognized carcinogen and they are exposed to high levels of air pollution. Conclusions: By this assay, high SSAT-1 activity has been confirmed in patients harbouring malignancy; however, pre-malignant and inflammatory conditions may result in a positive test. Follow-up of individuals with elevated SSAT-1 activity may reveal unrecognized malignancies, pre-malignant neoplasms, liver disease, inflammatory conditions and possibly false-positivity in individuals exposed to carcinogens such as arsenic. In further work, SSAT-1 up-regulation is being correlated with 5 other putative polyamine metabolite markers for accurate diagnosis of lung cancer.

Flaxseed contains ω-3 fatty acids, lignans, and fiber that together may provide benefits to patients with cardiovascular disease. Animal work identified that patients with peripheral artery disease may particularly benefit from dietary supplementation with flaxseed. Hypertension is commonly associated with peripheral artery disease. The purpose of the study was to examine the effects of daily ingestion of flaxseed on systolic (SBP) and diastolic blood pressure (DBP) in peripheral artery disease patients. In this prospective, double-blinded, placebo-controlled, randomized trial, patients (110 in total) ingested a variety of foods that contained 30 g of milled flaxseed or placebo each day over 6 months. Plasma levels of the ω-3 fatty acid α-linolenic acid and enterolignans increased 2- to 50-fold in the flaxseed-fed group but did not increase significantly in the placebo group. Patient body weights were not significantly different between the 2 groups at any time. SBP was ≈10 mm Hg lower, and DBP was ≈7 mm Hg lower in the flaxseed group compared with placebo after 6 months. Patients who entered the trial with a SBP ≥140 mm Hg at baseline obtained a significant reduction of 15 mm Hg in SBP and 7 mm Hg in DBP from flaxseed ingestion. The antihypertensive effect was achieved selectively in hypertensive patients. Circulating α-linolenic acid levels correlated with SBP and DBP, and lignan levels correlated with changes in DBP. In summary, flaxseed induced one of the most potent antihypertensive effects achieved by a dietary intervention.

BACKGROUND: Foot ulceration usually precedes more serious foot complications such as infection, gangrene or amputation. The risk of developing foot ulcers has been estimated to be higher in individuals with peripheral arterial disease, diabetes and kidney disease. OBJECTIVE: To evaluate the healing potential of CO2-enriched water in patients with foot ulceration. METHODS: This is a multi-centered; placebo controlled and randomized clinical trial. Patients will have at least 1 distal extremity ulcer and will be randomized to receive a treatment with bath therapy at 37oC containing either 1,000-1200 ppm CO2-enriched tap water (the intervention), or non- carbonated tap water (the control group). The treatment will be carried out 3 times/week for 15 minutes per session for 4 months for a total of 48 treatments. At baseline and at the end of every treatment month, wound assessment, measurement of limb oxygenation, ankle brachial pressures as well as McGill pain questionnaire will be carried out. Blood will also be collected at baseline and at the end of every month of the treatment period (for a total of 5 collections) for the measurement of different biomarkers. BASELINE DATA: At baseline all 14 participants have at least one distal extremity ulcer and 93% (13/14) are diabetic. Although 6 of the participan`ts have peripheral arterial disease (left and/or right limb) as evidenced by an ankle brachial index (ABI)

An important part of training the next generation of physicians is ensuring that they are exposed to the integral role that research plays in improving medical treatment. However, medical students often do not have sufficient time to be trained to carry out any projects in biomedical and clinical research. Many medical students also fail to understand and grasp translational research as an important concept today. In addition, since medical training is often an international affair whereby a medical student/resident/fellow will likely train in many different countries during his/her early training years, it is important to provide a learning environment whereby a young medical student experiences the unique challenges and value of an international educational experience. This article describes a program that bridges the gap between the basic and clinical research concepts in a unique international educational experience. After completing two semester curricula at Alfaisal University in Riyadh, Kingdom of Saudi Arabia, six medical students undertook a summer program at St. Boniface Hospital Research Centre, in Winnipeg, MB, Canada. The program lasted for 2 mo and addressed advanced training in basic science research topics in medicine such as cell isolation, functional assessment, and molecular techniques of analysis and manipulation as well as sessions on the conduct of clinical research trials, ethics, and intellectual property management. Programs such as these are essential to provide a base from which medical students can decide if research is an attractive career choice for them during their clinical practice in subsequent years. An innovative international summer research course for medical students is necessary to cater to the needs of the medical students in the 21st century.

Background Flaxseed is an important source of alpha-linolenic acid an essential omega-3 fatty acid. The possibility that a supplementation of the diet with foods rich in alpha-linolenic acid, antioxidants and fiber (like flaxseed) has not been investigated. Methods/design The primary objective is to determine whether consumption of a diet rich in FLAX seed over a one year period has any beneficial cardiovascular effects in patients with P eripheral A rterial D isease (FLAX-PAD study). This is a single center, prospective, double blinded, randomized controlled clinical trial aimed at in 110 patients over 40 years old and with peripheral arterial disease. Patients will receive 30 g of milled flaxseed (or placebo) per day. Primary endpoints are incidence of myocardial infarction and stroke. Secondary measures include: requirement for surgical interventions, exercise and cardiopulmonary performance, cardiac arrhythmias, serum lipid profile, arterial sufficiency, blood pressure, inflammatory profile, platelet function, changes in drug dosage levels, as well as nutrigenomic and biomarker profiles in the blood. Recruitment and baseline examinations started in October 2008. Baseline data of the 110 patients is shown. Conclusions FLAX-PAD will generate data on the safety, tolerability, cardiovascular efficacy and genomic response to a diet rich in flaxseed. It will determine the effects on primary and secondary events (stroke, myocardial infarctions, angina pectoris, cardiac arrhythmias) as well as in secondary endpoints (exercise performance, blood pressure and circulating lipid levels) in patients with PAD.

It has long been recognized that certain indigenous populations, such as the traditional living Inuit, have been relatively spared from ischemic heart disease despite their high fat diet. There is evidence to suggest that elevated serum levels of oxidated cholesterol may be a strong risk factor for atherogenesis. This study was performed to examine the plasma levels of lipid, triglyceride, lipoprotein cholesterol and oxidated cholesterol derivatives in the Inuit of the Keewatin district of Canada. Lipoprotein isolation from plasma was performed by serial centrifugation and apolipoprotein concentrations and native and esterified cholesterol content of the lipoprotein isolated were determined. Analysis of cholesterol and its oxidation products was performed using high pressure liquid chromatography. The Inuit showed a decreased concentration of both VLDL apolipoprotein and LDL apolipoprotein. The incidence of oxidatived cholesterol in Inuit VLDL and LDL was higher than control except for the 25 α-hydroxy derivative in VLDL. These data provide insight into the relative contributions of genetic and environmental influences on the development of ischemic heart disease.

BACKGROUND A single chain antibody fragment, NovoMAb-G2-scFv, derived from a human anti-tumor monoclonal antibody recognizes tumor antigen molecules expressed on a wide variety of human cancers including melanoma, breast carcinoma, colon adenocarcinoma, squamous cell carcinoma, lung carcinoma, and prostate carcinoma. This study was designed to evaluate the use of a NovoMab-G2-scFv/cyanine fluorochrome (Cy5.5.18) conjugate as diagnostic tool for in vivo imaging of tumors. METHODS The NovoMab-G2-scFv-Cy5 complex was administered to athymic mice injected subcutaneously with human melanoma tumor cells, and the distribution of fluorescence was imaged noninvasively using a charge-coupled device camera. Images were acquired 2, 6, 12, 24, 48, and 72 hours after injection. RESULTS Fluorescence was detected at the tumor site after injection of NovoMab-G2-scFv-Cy5 but not after injection of a labeled irrelevant control antibody fragment. Fluorescence from the tumor site peaked 2 hours after injection and gradually declined, reaching a minimum 72 hours after injection. Fluorescence was also apparent in the kidneys, indicating clearance of the complex through the kidneys. Results suggest that 16% and 73% of the antibody is located in the tumor and kidneys, respectively. Imaging of isolated organs confirmed the presence of the NovoMab-G2-scFv-Cy5 complex in tumors, kidneys, and liver. No fluorescence was observed in other organs. CONCLUSIONS Specific binding of the antibody-dye complex to the tumor was observed, and the kinetics of binding to tumors and kidneys were determined. These results suggest that the NovoMab-G2-scFv-Cy5.5 complex may be used for noninvasive tumor localization. Cancer 2000;89:1134–44. © 2000 American Cancer Society.

When LDL is oxidized by a free radical generating system, both cholesterol and fatty acyl moieties within the LDL are modified. Oxidized low density lipoprotein (oxLDL) can induce alterations in the Ca2+ transients in isolated cardiomyocytes. It is unclear if oxidation of the LDL fatty acyl chains (in phospholipids, triglycerides and cholesteryl esters) or oxidation of the LDL unesterified cholesterol is more important in producing the oxLDL-induced alteration in cellular calcium transients. Therefore, we investigated the possible role of oxidized cholesterol and fatty acyl chain peroxidation in the effects of LDL on Ca2+ transients. Cholesterol oxidase (CO) treatment of LDL produced oxidized cholesterol plus H2O2. The H2O2 peroxidized the LDL fatty acyl chains, as indicated by an increased malondialdehyde (MDA) content. The cell systolic [Ca2+] was significantly increased after incubation with CO-treated LDL. Diastolic [Ca2+] was unchanged. MDA content in the CO-treated LDL correlated with the change in systolic [Ca2+] of treated cells. Catalase, a scavenger of H2O2, inhibited MDA formation in the CO-treated LDL and prevented the increment in systolic [Ca2+] in the treated cells. A similar stimulatory effect on the Ca2+ transient was observed if cells were treated with LDL after exposure to only H2O2 and not CO. Direct exposure of myocytes to H2O2 (without LDL) failed to produce a stimulatory effect on the calcium transient but caused an increment in cellular diastolic Ca2+. Exposure of myocytes to CO alone (without LDL) produced a significant increment in diastolic [Ca2+] and this effect was not prevented by catalase. These results suggest that fatty acid peroxidation in the LDL moiety is more important than oxidized cholesterol in the generation of ox-LDL-induced increases in Ca2+ transients in isolated cardiomyocytes. Further, oxidation of in situ cell membrane cholesterol will destroy cell integrity. Our data also underline the importance of adding extracellular lipid in any study of the effects of oxygen free radicals on cellular function. Under conditions of radiologic or chemically-induced generation of oxygen-derived free radicals, cardiac dysfunction and damage may be induced by this process.

Clinical trials constitute one of the final stages along the testing continuum that is so needed for the introduction of new products, methods, and ground-­breaking technologies. Currently, most economies have recognized the importance clinical trials play as part of the entire value chain from idea to product. While most jurisdictions have developed systems of safeguards for the protection of human subjects involved in clinical trials, there are huge disconnects among jurisdictions, institutions and investigators. This article provides some generally accepted international standards and guidelines in relation to subject protection as it pertains to recruitment, confidentiality, monitoring, data storage and data transfer. The need for all trial protocols to be reviewed by a qualified and registered ethics committee, as well as that all legal requirements are met to ensure patient protection, is also highlighted. Since governments and industry see clinical trials as a critical and necessary step in the product development process, there is a need for more translational research, which will increase the demand for more participation in clinical trials. This article will therefore, also address the importance of patient protection if we are to meet these requirements.

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (approximately 150-200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity.

According to the World Health Organization, counterfeit medicines are medicines that are fraudulently mislabeled, contain wrong ingredients, absent of active ingredients, contain insufficient active or over-active ingredients or have fake packaging. The production and distribution of counterfeit medicines has become a significant problem in both affluent and developing nations. It is now widely believed that approximately 15% of drugs worldwide could be counterfeit, although in some developing countries it may be closer to 50% and rising. Due to consumer-initiated importation of prescription drugs through internet sites and other means of cross border purchasing, the potential risks of exposure to counterfeit products has increased dramatically. In addition, counterfeit medicines have become more difficult to detect as counterfeiters have become more creative and bold in the manufacturing of fake drugs. This poses an important threat to global public health and patient safety. In addition, the financial loss and irreparable damage to the reputation of pharmaceutical companies, health care centers and dispensing pharmacies is substantial. This paper briefly highlights the incidence and global impact of counterfeit medicines from a public health and economic perspective and addresses some of the strategies including identifying the risks and threat from counterfeit medicines as well as the analytical procedures for combating the growth, availability and trafficking of counterfeit drugs.

Intracellular pH and [Na+] in the heart are regulated by the sarcolemmal membrane Na(+)-H+ exchange pathway. No data are currently available regarding the adaptation of this system to pathological conditions in the heart. Because ionic interactions with the heart are altered in cardiomyopathy during chronic experimental diabetes, it was hypothesized that Na(+)-H+ exchange may become abnormal. In addition, the effects of treating diabetic rats with daily injection of L-propionylcarnitine were investigated to determine whether alterations in lipid metabolism may be involved in any potential changes in ion transport. Rats were injected with streptozotocin (65 mg/kg) and killed 8–10 wk later, and sarcolemmal membrane vesicles were isolated from pooled ventricles. Significant depressions in Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity and Na(+)-Ca2+ exchange were observed in the diabetic preparations in comparison to control. L-Propionylcarnitine treatment of the diabetic rats partially normalized these activities. A striking depression in cardiac sarcolemmal Na(+)-H+ exchange was observed in the diabetic animals in comparison to control, and this was not a result of a nonspecific increase in membrane permeability. L-Propionylcarnitine treatment of the diabetic rats did not improve sarcolemmal Na(+)-H+ exchange.

It has long been recognized that certain indigenous populations, such as the traditional living Inuit, have been relatively spared from ischemic heart disease despite their high fat diet. There is evidence to suggest that elevated serum levels of oxidated cholesterol may be a strong risk factor for atherogenesis. This study was performed to examine the plasma levels of lipid, triglyceride, lipoprotein cholesterol and oxidated cholesterol derivatives in the Inuit of the Keewatin district of Canada. Lipoprotein isolation from plasma was performed by serial centrifugation and apolipoprotein concentrations and native and esterified cholesterol content of the lipoprotein isolated were determined. Analysis of cholesterol and its oxidation products was performed using high pressure liquid chromatography. The Inuit showed a decreased concentration of both VLDL apolipoprotein and LDL apolipoprotein. The incidence of oxidatived cholesterol in Inuit VLDL and LDL was higher than control except for the 25 alpha-hydroxy derivative in VLDL. These data provide insight into the relative contributions of genetic and environmental influences on the development of ischemic heart disease.

We have used immunofluorescence imaging to study binding of a monoclonal antibody fragment to subcutaneously implanted human melanoma cells in nude mice. The data acquired using this nontraditional approach was then analyzed using standard pharmacokinetic methods to produce estimates of k/sub e/ (0.06 h/sup -1/), t/sub 1/2/ (16 h), mean residency time (23.4 h) and percent exposure of the antibody to the tumor (40%). To our knowledge this is the first time standard pharmacokinetic analyzes have been conducted on immunofluorescence imaging data. The combination of this novel imaging technique and standard pharmacokinetic analytical methods should prove to be a useful tool for comparing the properties of antibody fragments in animal models.

This study demonstrates the potential of in vivo, whole body fluorescence imaging for pharmacokinetic studies. The distribution of a novel human anti-tumour antibody fragment, NovoMab-G2-scFv, labelled with a fluorescent dye (Cy5.5.18) was monitored in vivo. The NovoMab-G2-scFv–Cy5 complex was injected via the tail vein into nude mice bearing subcutaneous human melanoma tumour cells. The distribution of fluorescence NovoMab-G2-scFv–Cy5 was imaged non-invasively using a charge-coupled device (CCD) camera. Whole body fluorescence images were acquired 2, 6, 12, 24, 48 and 72 h post-injection. Fluorescence was detected at the tumour site following injection of NovoMab-G2-scFv–Cy5 but not following injection of a labelled irrelevant antibody fragment, demonstrating specific binding of the antibody–dye complex to the tumour. Fluorescence from the tumour site peaked 2 h post-injection and gradually declined, reaching a minimum 72 h post-injection. Fitting an exponential decay to fluorescence data extracted from images allowed the half-time of the antibody at the tumour site to be calculated, and a value of 7.7 h was obtained. Fluorescence was also apparent in the kidneys, indicating clearance of the NovoMab-G2-scFv through the kidneys. Again, fluorescence intensity decreased with time, reaching a minimum 72 h post-injection. Imaging of isolated organs (ex vivo) confirmed the presence of the antibody–dye complex in the tumours, kidneys and liver. No fluorescence was observed in the brain, heart, lungs or spleen, suggesting that these organs do not accumulate the NovoMab-G2-scFv–dye complex., Shedding New Light on Disease : Optical Diagnostics for the New Millennium, June 25-30, 2000, Winnipeg, Canada

A number of fluorescence imaging techniques show diagnostic promise. Imaging endogenous fluorescence has been proposed as a method for cancer diagnosis. Unfortunately, tissue autofluorescence is relatively weak and poor contrast between malignant and normal tissue is seen. Contrast may be enhanced with the addition of fluorescent materials that are selectively accumulated by malignant cells, such as fluoroscein or porphyrin derivatives. The limited penetration of light at the emission maxima of these materials restricts the use of fluorescence techniques utilizing these chromophores to superficial phenomena. However, many potential applications still exist. For example, monitoring fluorescence during surgery may allow resection margins to be clearly delineated. Other exogenous chromophores that may have may have diagnostic utility include indocyanine green (ICG). Techniques based upon visualization of the distribution of ICG fluorescence (i.e. choroidal angiography) are already prominent in ophthalmology. ICG fluorescence imaging may also find a useful niche in monitoring of burns and transplant tissue. In addition, monitoring of vascular parameters during cardiac surgery presents exciting opportunities. For example, low oxygen levels (e.g. during bypass surgery) in the heart can result in alterations in microvascular permeability. As ICG is largely bound to serum albumin, it should not be seen in extravascular spaces in normal hearts. However, increased permeability will allow albumin to diffuse into the extravascular spaces, and diffuse fluorescence across the surface of the heart will be seen. In principle, the infusion of polymers (e.g. dextrans) of various molecular weights labeled with dyes that fluoresce at various wavelengths will allow the assessment of the porosity of capillary beds in such systems. Immunofluorescence techniques have the potential to provide unmatched sensitivity and specificity. The unique nature of antibody–antigen interactions ensures specific delivery of the fluorophore to the site of interest. The specific interaction of labeled antibodies with antigens means that the fluorophore persists in the body for a prolonged period of time (days). Following a single injection of labeled antibody, repeated measurements on the same site over the course of hours or days allow kinetic information to be readily obtained. In principle, this means that the effect of therapeutic intervention, i.e. radiation therapy, chemotherapy, etc., can be monitored.

A novel technique is described for the evaluation of membrane integrity in isolated nuclei. Membrane integrity is assessed by measuring nuclear fragility in response to high salt conditions. Salt-induced disruption of the nuclear membrane is followed by spectrophotometric monitoring of released nucleotides. The assay is based on determining the amount of salt necessary to induce release of 50% of the total pool of releasable nucleotides. This allows semiquantitative comparison of relative nuclear membrane strength or integrity of different samples in response to high salt conditions. In this manner, the effects of altered nuclear membrane composition or metabolism on membrane integrity may be monitored.

In vivo models of myocardial infarction following coronary artery ligation in the rat still suffer from high early mortality and a low rate of success of myocardial infarction. This study investigated the possibility of reducing early mortality and increasing the rate of myocardial infarction by modifications of surgical techniques. Eighteen rats were divided into two groups: normal control (3 rats) and ligation (15 rats). The major modifications of surgical techniques used in this study include: (1) no exteriorization of the heart, (2) ligation of the origins of the branches rather than the main trunk of the left coronary artery, (3) removal of air from the chest after closure, (4) supplying oxygen immediately after extubation. Following surgery, the rats recovered uneventfully and 11 rats were alive after 16 weeks. One rat, with a large myocardial infarction, died 2 h after surgery. Early mortality (during surgery and 1 week after surgery) was 6.7% with a success rate of myocardial infarction of 85%. The left ventricle in the ligation group showed significant dilation relative to normal and sham-operated control hearts (317% of control hearts, p < 0.001). However, myocardial mass did not increase. The average infarct size was 33%. These results demonstrate that a reduction in early mortality and an increased success rate of myocardial infarction can be achieved by modifications of surgical techniques. (Mol Cell Biochem 176: 227-233, 1997)

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Calcium is an important signal in key nuclear events, including cell cycle timing, regulation of gene expression, and activation of nuclear kinases and phosphatases. It is therefore important to identify calcium binding proteins in the nucleus which may play roles in these functions, and to determine whether these proteins are located in the nuclear envelope or in the nucleoplasm. Rat hepatic and pig cardiac nuclei were isolated and treated with a high salt solution to separate nucleoplasmic proteins from those associated with the nuclear envelope. The presence of calcium binding proteins was then revealed by Stains-All staining of electrophoretic gels and45Ca2+overlays of Western blots. Four major calcium binding proteins were putatively identified in the pig cardiac nuclei, and another three in the rat hepatic nuclei. Proteins of 110, 93 and 35 kDa were observed in the pig cardiac nuclear envelope fraction, and another of 55 kDa in the pig cardiac high salt fraction. A 93-kDa protein was observed in the rat hepatic nuclear envelope fraction, and others of 120 and 110 kDa in the rat hepatic high salt fraction. A tentative identification has been made of the 93-kDa protein in each tissue type as calnexin, and of the cardiac 55 kDa protein as calsequestrin. This study, therefore, has putatively identified for the first time the presence of several calcium binding proteins which have distinct partitioning within hepatic and cardiac nuclei. This localization may play an important functional role within the nuclei.

Infrared spectroscopy was applied to the investigation of normal and oxidatively modified hepatic nuclei. The hepatic nuclei were oxidized by two different free-radical-generating systems. Infrared spectra of oxidized nuclei were remarkably different from those of normal nuclei; the major alteration found in the spectra of oxidized nuclei was the emergence of a new population of nucleic acids with a hydrogen-bonding pattern different from that of the normal phosphodiester groups, and a redistribution of the hydrogen bonding of the protein amide groups of the histones, indicative of protein-structural rearrangements. The spectral changes in the phosphate bands of the nucleic acid resemble those previously observed in different types of malignant tissue, and suggest that there could be a link between nuclei oxidation and carcinogenesis which may involve a free-radical-mediated process.

A growing number of studies have implicated the Na+/H+ exchanger in ischemic/reperfusion damage to the heart [1–7]. Other studies have now demonstrated accelerated ion flow through this exchanger in hypoxic/ reoxygenation insult to the heart as well [8–10]. While these are important pathological events, they are largely immediate reactions of Na+/H+ exchange activity in response to changes in the intracellular ionic environment in the heart. These reactions occur so quickly that they likely do not involve structural changes in the protein or alterations in protein synthesis. However, during low-flow ischemia of a relatively long duration, there are indications that these pathways are beginning to be activated. Changes in the Na+/H+ exchange mRNA message have been detected in hearts after three hours of low-flow ischemia [11]. These changes do not occur in global ischemia experiments, which employ considerably shorter ischemic periods (≤1 hour) [11,12]. Chronic alterations in the exchanger would be more likely to occur during conditions where an adaptive stimulus is placed upon the heart for a much longer period of time and/or where the tissue is allowed sufficient time to induce its adaptive mechanisms. A stimulus that frequently induces an adaptive response in the heart is myocardial disease.

Cholesterol oxidase (3 beta-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3-beta-hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.

Oxygen free radicals have the ability to oxidize cholesterol. However, nothing is known about the effects of cholesterol oxidation on ion transport in isolated myocardial membranes. The purpose of the present study was to investigate the effects of in situ oxidative modification of sarcolemmal cholesterol on Ca2+ flux. Cholesterol oxidase was used to oxidatively modify membrane cholesterol. After incubation of cardiac sarcolemmal vesicles with cholesterol oxidase, cholest-4-en-3-one (cholestenone) was the predominant species of oxidated cholesterol produced. Cholesterol oxidase inhibited sarcolemmal Na(+)-Ca2+ exchange in a concentration-dependent manner. Both the Vmax and Km of the reaction were altered after cholesterol oxidase treatment. Extensive treatment of the sarcolemmal membranes with cholesterol oxidase increased the passive permeability characteristics of the membrane. Passive Ca2+ efflux from the sarcolemmal vesicles was stimulated by increasing the concentration of cholesterol oxidase. ATP-dependent Ca2+ uptake was also inhibited after cholesterol oxidase treatment, but it was not as sensitive as the Na(+)-Ca2+ exchange. Conversely, passive Ca2+ binding to sarcolemmal vesicles was strikingly stimulated by cholesterol oxidase treatment. The results demonstrate that oxidative modification of sarcolemmal membrane cholesterol can directly affect ionic interactions with the sarcolemmal vesicle and provide potentially important mechanistic information for the molecular basis of the effects of free radicals on ion flux and function in the heart.