This paper describes design and development of a self-management program, delivered by telerehabilitation (TR), to address the problem of chronic lower limb swelling in persons with limited mobility. The 18.6 million persons with limited mobility in the USA are at increased risk for chronic lower limb swelling and related secondary complications, including cellulitis and skin ulcers. Over time, chronic swelling often progresses to lymphedema, an incurable condition requiring lifelong care. Without successful self-management, lymphedema and its related complications inevitably worsen. Access and adherence to appropriate treatment are challenging for persons with limited mobility. Program development involved a structured process to establish content validity , readability, suitability, and selection of a TR platform to deliver the educational program. Our goal was to develop a program that would engage patients in self-management skills. The TR software platform chosen, Versatile and Integrated System for Telerehabilitation (VISYTER) was designed to facilitate face-to-face delivery of an interactive home-based self-management program via the internet in real time. Results demonstrated validity of the educational program and ease of use with TR. Future plans are to evaluate ability of this approach to promote self-management skills, home monitoring, and improved management of persons with lymphedema and limited mobility.  Lymphedema, an incurable health condition, occurs when an impairment of the lymphatic system results in a lymphatic load that is greater than lymphatic transport capacity. The consequence is accumulation of protein-rich fluid in the interstitial space , 2. OverPeople with mobility limitations are at high risk for chronic lower extremity swelling. The return of fluids via the venous and lymphatic system is facilitated by muscle contractions of the legs. This physiologic mechanism is hindered or absent in people who use wheelchairs for mobility. Sitting for long periods of time applies pressure to capillaries and lymphatic vessels that also impedes flow. Poor body trunk alignment negatively affects the normal respiratory pump for the lymphatic system and further reduces lymphatic flow .Complete decongestive therapy (CDT) is the gold standard treatment for management of chronic lymphedema –16. The Access to therapists specializing in CDT is challenging. First, there are few certified lymphedema therapists and those available are primarily located in urban areas. Patients with limited mobility may have difficulty obtaining transportation that accommodates their needs. Public transportation is often difficult to access or unavailable, especially in rural areas . Most whTelerehabilitation (TR) has been defined as “the application of telecommunications, remote sensing and operation, and computing technologies to the delivery of medical rehabilitation services at a distance” , p. 115.TR has the potential to improve access to specialty care and provide services within the home environment, thereby meeting needs expressed by those with mobility limitations. Limitation in mobility impacts an estimated 18.6 million people in the USA . StudiesSelf-management is defined as “an individual's ability to manage the symptoms, treatment, physical and psychosocial consequences and lifestyle changes inherent in living with a chronic disease” , p.563. Self-management theory is grounded in the expectation that increasing patients' belief in their ability to manage their illness will result in positive change and better health care outcomes . This beIn order to address barriers faced by people with limited mobility, an alternative treatment strategy was proposed and designed for remote delivery via TR. The TR-PUMPS educational program was developed as a critical component of a clinical trial to evaluate the effects of a TR for immobile individuals with chronic edema/lymphedema of the lower limbs (NIDRR Grant no. H133E090002). In the clinical trial, TR was chosen as a novel delivery method for a modified lymphedema treatment protocol including advanced pneumatic compression. A review of the literature revealed no previous studies of self-management strategies for swelling in this high-risk population with limited mobility. Based on current best practice for treatment of chronic edema/lymphedema, a draft version of the educational protocol was developed with input from three certified lymphedema therapists. Based on current best practice for treatment of chronic edema/lymphedema, the script identified 10 learning goals central to self-management for chronic edema of the lower extremities see . The nexFlesh Reading Ease Scale, Flesch-Kincaid, Gunning Fog Index, SMOG Formula, and Fry Formula [Studies have shown that patient educational materials often have a higher readability level than recommended by the American Medical Association (5th to 6th grade) –56. Pati Formula . These f Formula . TherefoCorporate Voice, Grammatix IV, Microsoft Word for Windows, and RightWriter. Results showed significant differences between the several formulas, but no significant difference in the means of overall grade levels produced by the Corporate Voice, Grammatrix IV, and RightWriter software programs.Electronic software packages offer another option. Mailloux, et al.  performeRightWriter  was used to evaluate the readability level of the educational scripts. RightWriter employs two readability formulas, the Flesch-Kincaid and the Gunning Fog Index and also evaluates for active versus passive voice and jargon. A second evaluation was completed using the Microsoft Word for Windows readability program.Because of the variability in readability level that could result from these various tools, we followed the recommendations to use several formulas and software programs , 59. TheNext, the final revised scripts were used to develop videos for the TR protocol. A video was developed for each of the 10 steps with the length of each video ranging from 1.5 to 11 minutes. The videos illustrated specific skills such as decongestive exercises and donning and doffing of compression garments. Each video was subdivided into individual skills or tasks to permit ease of locating and viewing during TR sessions. To establish content validity, the videos were viewed by eight additional board-certified lymphedema therapists who anonymously ranked each video on accuracy and completeness of the information as well as the clarity of the presentation. A 5-point Likert scale (5 = strongly agree to 1 = strongly disagree) was used to determine the therapists' level of agreement with descriptive statements about the content of each video See .When the TR program is operational, the videos will be available electronically via a portal that participants can access from their home computer. The videos will also be used during teleconferencing to teach the 10-step program and to reinforce teaching during review sessions.To supplement the videos, an educational booklet was developed using the same 10-step approach. Illustrations and still frames from the videos were used to augment the script. The booklet was evaluated for suitability utilizing the Suitability Assessment of Materials (SAM) , 61. Sui Versatile and Integrated System for Telerehabilitation (VISYTER) software platform was chosen for use in this self-management program and tailored to specific educational needs. VISYTER was developed at the University of Pittsburgh, School of Health and Rehabilitation Sciences, Department of Rehabilitation Science and Technology with funding from the National Institute for Disability and Rehabilitation Research via the Rehabilitation Engineering and Research Center on Telerehabilitation. It was selected because of its demonstrated effectiveness and flexibility in conducting remote face-to-face evaluations [Theluations . VISYTERluations . Minimumluations . VISYTERThe use of the VISYTER system was tailored for use in the delivery of educational materials by loading the videos onto the system and performing laboratory testing of all capabilities prior to actual participant use. The first step when used in the home involves downloading the software onto a participant's home computer. Participants will be provided with a ClearOne CHAT 60 speakerphone. If no built-in web cam is available, a Logitech HD C910 web cam will be used for face-to-face videoconferencing. A second camera, the Logitech Orbit AF, can be connected to the participant's computer for use in skin and skill assessments. The Logitech Orbit AF will provide clinicians with the ability to remotely control the camera's movement and zoom capabilities. The clinician at the remote computer site will use a Logitech HD C910 web cam and a Logitech USB headset. A personal user ID and password will be assigned to each participant to enable them to log on to VISYTER. Each participant will also be assigned his or her own virtual clinic room. VISYTER's virtual clinic rooms are housed on the server and can only be accessed by authorized users. An assigned room administrator will determine the user's access and specific access capabilities which might vary depending upon their role . TraininTo implement the self-management program, the participant and clinician will connect to a VISYTER virtual clinic room via the portal in what is termed the “Lymphedema Venue” for each teleconferencing session. During the initial session, participants will be asked to identify five specific occupational performance goals. A plan of action will be developed in a collaborative effort between the participant and clinician. The participant's comprehension on lymphedema self-management will be evaluated, and any barriers that could negatively affect the participant's ability to perform the 10 steps of the self-management program will be identified See .Real-time interactive education and evaluation sessions will occur a minimum of once per week for six weeks. The frequency of sessions will be determined by mutual agreement based on a joint review of the participant's current knowledge of their condition and self-management skills. Educational videos will be used to train the participants using the media sharing capabilities of the VISYTER software. Comprehension and retention of the educational material will be evaluated through participant verbal recall and performance of return demonstrations. All electronic communications will be encrypted to maintain privacy. TR interventions can be recorded and saved in an archived data base within the server.The final step involved uploading the videos to the Lymphedema venue and the testing of the VISYTER software's performance in the delivery of the educational protocol within a laboratory setting. To conduct this evaluation, we transmitted from our virtual clinic workstation to distant rooms to test transmission in a simulated home setting. Bandwidth was tested to determine the speed necessary for providing the self-management program. RightWriter , Flesh Kincaid results indicated a readability level of a 4.01 grade level. The Gunning Fog Index resulted in readability level of a 5.61-grade level. The average of these two formulas resulted in an overall readability level below the 5th-grade level. A second evaluation using the Microsoft Word for Windows readability program resulted in a Flesh Kincaid readability level at a 3.4-grade level.When evaluated for readability utilizing the computer softwareWhen content validity of the educational videos was determined by eight experienced, board-certified lymphedema therapists, the mean score was 4.5 ± 0.35 with a range of 4.1–4.9 see .The supplemental educational booklet, developed using the same script as the videos, was evaluated for suitability utilizing the Suitability Assessment of Materials . A scoreQuickTime Format, used for the videos was not compatible with the VISYTER software platform. The video file format was therefore changed to the Windows Media Video Format to allow sharing of the videos during video conferencing. The ability for participants to access and download videos from the lymphedema portal was verified.The video file format, Transmission speed was determined using two cameras plus audio upload on the participants' side and one camera and media sharing and audio download on the clinician's side. It was determined that a medium-quality video was sufficient for the face to-face interaction. However, high video quality was required for the remote camera for visualization during skin assessments. Results of the laboratory testing determined that speeds of 1220 kbps upload and download were the minimum requirements and the speed of 1620+ kbps upload and download provided optimal teleconferencing audio and visual quality.The study's researchers' collaborative efforts resulted in the development of a comprehensive self-management program for patients with chronic lymphedema with appropriate readability and suitability scores that was highly rated by external evaluators in regard to its content validity and selection of a software platform with the potential to provide in-home teleconferencing and assessment capabilities.Healthy People 2010 defines health literacy as “the degree to which individuals have the capacity to obtain, process, and understand basic health information and services needed to make appropriate health decisions” . The 200 plain and simple project from the Iowa Department of Public Health is recommended and may be accessed at http://www.idph.state.ia.us/PlainAndSimple/Readability.aspx. This website provides information on commonly used readability formulas and access to readability calculating software at no cost.A variety of readability software and formulas are available to evaluate readability levels. A concern, as shown in our study, is that different readability formulas can produce discrepancies in grade-level results with scores deviating as high as 41% . ConsequSAM may be found in Teaching Patients with Low Literacy Skills (2nd Ed) by Doak et al. [http://www.hsph.harvard.edu/healthliteracy/resources/.Readability level is not the only factor that determines the effectiveness of written material when attempting to address health literacy disparity , 61, 74.With advances in information technology, telecommunications offers health care providers the opportunity to utilize new approaches to health care delivery. The current health care delivery system is focused on acute care. With the aging population and the increase in chronic conditions, this health care delivery system is not meeting the public's needs. Chronic conditions result in seven out of ten deaths in the USA. As of 2005, 133 million people in the USA suffered from at least one chronic condition. This number is projected to increase to 157 million people by 2020 . The incThe evidence-based educational materials developed as part of the self-management program for lower limb chronic swelling/lymphedema in persons with limited mobility were found to be valid, accurate, and complete with high ratings of clarity. The readability and suitability ratings indicated that the materials are appropriate for various levels of health literacy in the study population. The self-management program described may be applicable to address other chronic conditions such as diabetes, heart failure, and chronic obstruction pulmonary disease. We believe TR is a viable method of providing a home-based self-management program on lower limb chronic swelling/lymphedema in people with mobility limitations and can decrease the burden associated with lifelong management of this debilitating condition.


CAH, a chronic condition with essentially life threatening aspects, is a frightening concept for families to come to terms with, even more so in the isolation of country areas. Having adequate knowledge and understanding is important in maintaining a sense of normality for these families and is an essential component to management both medically and within the family.The ultimate goal of this study was to improve patient access to education and support, and improve patient outcomes though increased knowledge and understanding. The principle aim of this study was to extend the education service we provide for our patients locally at specialist centres, to the patients who live in isolated regions of NSW and Australia wide. A comprehensive psychosocial education program (PEP) which was developed for patients and families with Congenital Adrenal Hyperplasia (CAH) and run at our local centre, was developed into an audiovisual format. In doing so, the program could be facilitated by one experienced health professional, therefore eliminating the need for a team of specialists to travel country areas, which is logistically difficult and considerably costly.Outreach services in NSW Australia, often provide only specialist medical care, with country families having only limited access to education, counselling and support services, that is routinely provided in metropolitan centres. In order to meet their needs the development of this innovative education program into an audiovisual format was planned and developed in order to provide an educational resource not only for patients and families, but also for clinicians.The PEP has been run 4 times with the program designed to cover the essential information required for families to know and understand the condition. Four 20 minutes sessions: What is CAH, Adolescent/Adult Issues, Psychosocial Issues and Sick Day Management, are followed by a 4 minute session on how to inject Hydrocortisone.The process of developing an audiovisual educational tool will be discussed and the essential factors which need to be considered, in developing such programs into an audiovisual format will be discussed in detail.This program is used in conjunction with the CAH Knowledge Questionnaire (CAHKAQ)


This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins.Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10 Biotin, also known as vitamin B7 and vitamin H, is a water-soluble micronutrient required by most organisms. In mammals, biotin acts as a coenzyme for four carboxylases: propionyl-coenzyme A (CoA) carboxylase, pyruvate carboxylase, methylcrotonoyl-CoA carboxylase, and acetyl-CoA carboxylase. The first three are located in mitochondria and the fourth in the cytoplasm. These enzymes catalyze critical reactions in the intermediary metabolism of gluconeogenesis, fatty acid synthesis, and amino acid catabolism Biotin deficiency is rare among constitutionally healthy people as this nutrient is widely distributed in foods and also amply synthesized by the intestinal flora, meeting with our low daily requirement (approximately15–70 µg/day) Due to its small size, biotin can be covalently linked to a number of proteins without affecting their biological activity. As biotin binds avidin or streptavidin with extremely high affinity and specificity, biotinylation of proteins and macromolecules is applied widely in biochemical assays In this study, we looked for human serum IgM antibodies reacting with biotin (Bio-IgM) and developed an indirect EIA to quantify their level and assess their seroprevalence. The adverse effects of biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse virus IgM EIAs and one commercial assay. The biotin IgM and streptavidin/avidin were shown to compete for binding to biotin. The affinities of the biotin IgM were also determined.To measure seroreactivities against biotin among the general population, an indirect IgM EIA (Bio-EIA) was set up with biotinylated BSA as antigen. The EIA OD values of 612 serum samples from 459 adults were plotted in a bar chart : 553  had biotin IgM and among 316 senior individuals, 10 (3.2%), as shown in Trichodysplasia-spinulosa polyomavirus (TSV), human bocavirus 1 (HBoV), parvovirus B19 (B19V)] and by one commercial kit (B19V EIA of Biotrin). In To assess whether the biotin IgM affects biotinylation-based antiviral IgM assays, all available Bio-IgM-positive serum samples were examined by four inhouse virus IgM EIAs of µ-capture format [MCV, Serum samples, 9 Bio-IgM positive and 9 negative, were applied in a µ-capture EIA , which were formed by biotin covalently bound to the constant region of IgM or IgG Since biotin IgM was initially found in a serum sample with false positivity in our inhouse IgM assay, we determined the susceptibility of several assays to it. We found that, serum samples with high levels of biotin IgM affected adversely all virus IgM assays, while samples containing low levels of the antibody showed low interference level in most assays. Indeed, biotin IgM was shown to be an important contributor to false positivities in virus IgM assays. Moreover, our results showed that among µ-capture IgM assays for four different viruses, the effects of biotin IgM were different. The MCV IgM EIA was more susceptible than the others. This difference may depend on the conformation of viral particles or their degree of biotinylation. Thus, at least two ways emerge to limit the effect of the biotin IgM: one is to optimize the amount of biotin in VLP trimming, and the other is to employ an appropriate borderline zone upon setting up the cutoffs according to the false reactivities derived from biotin IgM.We did study in detail whether the different effects of biotin antibodies on IgM assays would relate with the conformations of the biotinylated antigens. As MCV VLPs were more susceptible than BSA, it is possible that ordered supramolecular assemblies are more susceptible than isolated polypeptides. Of note, biotin itself in solution never gave a positive signal. To account for this, three possibilities existed: (a) the soluble biotin did not bind to the biotin IgM captured on the plate; (b) the IgM-attachment abolished biotin’s ability to bind to streptavidin; (c) IgM would bind the long spacer arm (LC) of biotinylation reagent (Sulfo-NHS-LC-Biotin) instead of the biotin itself. To find the answer, we performed competition assays with soluble biotin in MCV IgM EIA. The fact that soluble biotin displaced biotinylated MCV VLPs without causing positive results itself, proved that biotin could not bind IgM antibodies and streptavidin simultaneously and that the binding was specific for the biotin itself and not the spacer arm. As avidin is a natural constituent in human’s diet and binds to biotin similarly as streptavidin does −3–10−4 mol/L is similar to that of many natural antibodies with their respective antigens We furthermore assessed the binding strength of biotin IgM by measuring IC50 Since our study cohorts are residents in Finland, it would be worthwhile to investigate biotin antibodies in non-Finnish populations. On the other hand, as our seroprevalence data are not population-based, they do not necessarily disclose the precise prevalence of biotin IgM antibodies even among the Finnish population. Nevertheless, the finding of biotin IgM in human sera opens a new venue for research of vitamin-directed immunity. The potential effects of these antibodies on human health call for investigation. These antibodies might adversely affect the uptake of biotin from circulation, or might help in clearance of excessive biotin and its metabolites in blood.The study protocol was reviewed and approved by the Ethics Committees of Turku and Helsinki University Hospitals. Written informed consent was obtained from all participants. If senior participant was unsure about the consent, or he/she was assessed by a geriatrician to have reduced capacity to consent, a written consent was obtained from the next of kin. If participants were children, consent was obtained from parents or guardians.The occurrence of biotin IgM antibodies was determined in 612 serum samples from two groups of adults and 678 samples from children. The first group, referred to as “Adults” comprised 144 sera from University students and our Institute staff, and the second group, “Seniors”, comprised 468 sera from 316 senior citizens . All senior citizens were in-patients of Turku City Hospital and participated to Virel study which aims to investigate detailed molecular virus etiology in the elderly and its links to illness severity and long-term outcome. The last group, “Children”, comprised sera from 349 children at sampling age of 1–12 years , which were previously studied for MCV and TSV primary infection 2O2 were added, the reaction was stopped after 25 min with 0.5 M H2SO4, and the absorbances at 492 nm were recorded. For background reactivity, a BSA-EIA was set up by coating the plates with unbiotinylated BSA and performed as above.An indirect EIA was set up for detection of biotin IgM in human serum samples Bio-EIA, . For useIn cutoff determination, all sera yielding OD <0.15 units in Bio-EIA were considered negative, and the samples with higher OD were re-examined by both Bio-EIA and BSA-EIA; and the net OD was obtained by subtraction of BSA-EIA background. Samples with net OD ≥0.3 were considered positive, those with net OD <0.2 were considered negative, and those with net OD between 0.2 and 0.3 were considered borderline.For detection of biotin IgM by immunoblot, 1 µg/lane of biotinylated BSA (Bio-BSA) or BSA was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Whatman Protran BA 85), blocked in 5% BSA in Tris buffered saline containing 0.1% Tween 20 (TBST) for 30 min at room temperature (RT). The serum samples diluted 1∶100 or 1∶50 in 5% BSA/TBST were kept on the blots at 4°C overnight. After washing with TBST, a polyclonal rabbit peroxidase-conjugated anti-human IgM  diluted 1∶20,000 in 5% BSA/TBST was applied for 1 hour at RT. After washes, the blots were developed using the SuperSignal West Dura Extended Substrate Detection kit  and exposed on X-ray films (Fuji Super RX).2O2 for 15 min at 37°C. In competition assay, the biotinylated antigen was mixed with a nonbiotinylated one, and added into the plates  e plates .Based on the Bio-EIA, a competition assay was set up to measure biotin IgM affinity


The commercial NMP-22 urine assays for bladder cancer (BCa) detect nuclear mitotic apparatus protein 1 (NUMA1) using monoclonal antibodies. It remains unclear whether these assays are monitoring a tumor antigen or some other phenomenon associated with the disease state. In this study, we investigated the influence of urinary cellular and protein concentration, and hematuria on the performance of the NMP-22 tests in an experimental model.Pooled urine from healthy subjects were spiked with varying concentrations of benign (UROtsa) cells, cancer cells , whole blood or serum, prior to analysis with both NMP22® Bladder Cancer ELISA test and the NMP22® BladderChek® point-of-care test.6) or the cell lysate from these cells (306 μg protein) resulted in a false-positive result. High concentrations of pooled-cancer cells (106) or cell lysate (30.6 μg and above) resulted in a positive NMP-22 assay. Concordance between the NMP-22 ELISA assay and the NMP-22 point of care assay was >90%.Urines from control subjects were negative for NMP-22. The addition of whole blood at 50ul/10 ml, but not serum, resulted in a false-positive result. Furthermore, the addition of a high concentration of benign urothelial cells (10Rather than detecting a specific tumor antigen, urinary NMP-22 assays may be measuring the cellularity or amount of cell turnover that may be introduced into the urine by a variety of conditions, including surface shedding from bladder tumors. The absence of significant urinary cellularity in some cases due to lesion characteristics or the timing of sampling may result in false-negative NMP-2 assays. Currently, voided urinary cytology (VUC) is the most widely used non-invasive urine test, with reported sensitivities ranging from 13–75% and specificities ranging from 85–100% . A couplA assays .bona fide tumor antigen, or some other phenomenon associated with BCa such as hematuria or urothelial turnover. Noting the limitations of the BTA assays, we investigated the influence of urinary cellular concentration, urinary protein concentration and hematuria on the performance of the NMP-22 tests in an experimental model.Another commercial assay that has been used for BCa detection in voided urine samples is the nuclear matrix protein-22 (NMP-22) test, which detects the nuclear mitotic apparatus protein 1 (NUMA1), using monoclonal antibodies . Two ass0C until use. Two hundred ml of urine and 10 ml of whole blood were collected from three healthy subjects. None of the subjects had any current or history of urological disorder. Microscopic examination of the urine samples showed no cells and all samples were tested by urinary dipstick test for abnormalities. Pertinent clinical information for each subject was recorded.Under IRB approval and informed consent, serum samples were collected from 20 men with no previous history of BCa and 20 men with newly diagnosed BCa. Serum was isolated, snap frozen and stored at -802 in air.Human bladder cancer cell lines; RT4 , T24 , UM-UC-14 , and KU-7  were available for analysis. The benign human bladder cell line, UROtsa, was a generous gift from Dr. Donald Sens at the University Of North Dakota School Of Medicine . T24, UM-UC-14 and KU-7 cell lines were maintained in RPMI1640 media. UROtsa and RT4 cells were maintained in low-glucose DMEM and McCoy’s 5A medium , respectively. All media were supplemented with 10% fetal bovine serum, 100 units/ml of penicillin and 100 μg/ml of streptomycin. All cells were incubated at 37°C in a humidified atmosphere of 5% CO4 (low concentration), 1 ×105 (medium concentration) and 1 ×106 (high concentration) cells were each added to triplicate 10 ml pooled urine samples. Equal numbers of RT4, T24, UM-UC-14 and KU-7 were pooled, and 1 ×104 (low concentration), 1 × 105 (medium concentration) and 1 × 106 (high concentration) cells were added to triplicate 10 ml pooled urine aliquots. For cell lysate analyses, 1 × 106 cells of each cell line were lysed by RIPA buffer  and total protein concentration measured. The total proteins extracted from 1 × 106 cells of UROtsa, RT4, T24, UM-UC-14 and KU-7 were 431ug, 369ug, 174ug, 261ug and 293 μg, respectively, with a mean total protein extract of 306 μg. In the following experiments, 3.06 μg, 30.6 μg and 306 μg of cellular proteins corresponding to 1 × 104 (low concentration), 1 × 105 (medium concentration) and 1 ×106 (high concentration) cells, were used. These UROtsa lysates and pooled cancer cell lysates were added to pooled urine samples in triplicate. To monitor hematuria influence, pooled whole blood  from 20 subjects with and 20 subjects without BCa was added analyzed in triplicate in 10 ml urine samples. Serum samples from 20 subjects with and 20 subjects without BCa were pooled. Serum protein concentrations similar to the cellular lysate concentrations, 3.06 μg (low concentration), 30.6 μg (medium concentration) and 306 μg (high concentration) were added to triplicate pooled urine samples. Figure To create the experiment model, 200 ml of freshly voided urine samples from three healthy controls were collected in sterile containers. All subjects’ urines tested negative for NMP-22 using both the NMP22® Bladderchek® test and the NMP22® ELISA kit assay. The urine samples from the three subjects were pooled, mixed and distributed into 10 ml aliquots in 15 ml centrifuge tubes. The human bladder cell lines were washed, trypsinized and counted. For UROtsa, 1 ×10The NMP-22 assays: NMP22®BladderChek Test® and NMP22® Bladder Cancer ELISA Test Kit were purchased from Alere Scarborough, Inc. . The NMP22®Bladderchek Test® was performed by adding 4 drops of urine into the well of a disposable test device for all urine samples. Results were considered to be positive if any band, no matter how faint, appeared in the test zone 30 min after adding the urine to the well. The NMP-22 ELISA assay was conducted according to the manufacturer’s instructions. Briefly, 200 μl of urine was added to the 96-well plate. Calibration curves were prepared using purified standards for each protein assessed. Curve fitting was accomplished by linear regression following manufacturer’s instructions. Based on published literature, a cutoff value of 10 U/mL was used to define a positive NMP-22 ELISA assay ,10. Urint-test was used to compare NMP-22 expression levels in cell lines. Statistical significance in this study was set at p < 0.05 and all reported p values were 2-sided. All analyses were performed with PRISM software version 5.00 .A Student p < 0.05). One bladder cancer cell line, KU-7, did not express higher NMP-22 levels than UROtsa. The NMP-22 level recorded for a pool of the four cancer cell lines was significantly higher than the NMP-22 level of UROtsa .The NMP22® Bladder Cancer ELISA Test was performed on a panel of human bladder cell lines  of UROtsa or pooled cancer cells produced a positive NMP-22 test. Cellular lysates from 1 × 105 and 1 × 106 pooled cancer cells, and from 1 × 106 UROtsa cells produced a positive result. The only discordance seen between the NMP22® Bladder Cancer ELISA Test and the NMP22® Bladdercheck® test was seen when UROtsa lysate (1 × 105 cells) produced a positive NMP22® Bladdercheck® test but a negative NMP-22 ELISA assay.The results from applying both the NMP22® Bladder Cancer ELISA Test and the NMP22® Bladdercheck® Test on samples taken from our experimental model are described in Table The addition of whole blood at 5ul and 20ul per 10 ml of urine did not cause a positive NMP-22 result, but spiking 50 μl/10 ml did produce false-positive NMP-22 assays. The fact that the false-positive effect was cellular was confirmed by the addition of pooled serum samples obtained from subjects with or without BCa. Neither serum sample caused NMP-22 test positivity, even at the highest level of 306 ug of protein. Overall, the concordance rates between the NMP22® Bladdercheck® test and the NMP22® ELISA kit assay was > 90% throughout the analyses.et al. reported that when whole blood was added to a urine sample, positive NMP-22 results increased in parallel with the increase in the amount of red blood cells in the sediment. In addition, the leukocyte count in the urine sediment also had a significant impact, and the investigators concluded that pyuria and hematuria significantly affected urinary NMP-22 [Using a novel experimental model, we evaluated a number of factors that may influence the NMP-22 urine tests. Although the model does not mimic the actual physiological situation exactly, the aim was to test whether the NMP-22 analyte could originate from blood (serum or whole blood) or benign cells of urothelial origin. Previous studies have investigated the influence of blood in the NMP-22 assay. Atsu y NMP-22 . In our http://www.proteinatlas.org). The Atlas provides immunohistochemistry data, and expression is confirmed in the majority of cases using 2 or more antibodies for the same antigen [NMP-22 (nuclear matrix protein-22) is also known as nuclear mitotic apparatus protein (NUMA or NUMA-1) ,13. The  antigen . NUMA-1 We also queried public databases for transcriptome profile data. NUMA-1 expression is reported to be increased in tumors of a variety of tissues, but given the epithelial expression profile of NUMA-1 this can be misleading unless the details of how the cellular composition of solid tissue specimens was corrected for in specific studies are known. However, of note, the profile databases do report that NUMA-1 mRNA is high in whole blood. This is of interest because blood incursion into the bladder can create difficulties in interpretation of urine tests for bladder cancer. We showed in our experimental model that spiking of NMP-22 negative urine with whole blood can cause a false-positive result.If the NMP-22 test is measuring a nuclear protein that is present in the majority of cell types throughout the body, and one that is particularly prevalent in the normal bladder lining, how does the test perform reasonably well for the detection of BCa. The test could be detecting NMP-22 that is introduced into the urine by hematuria, and since hematuria is the most associated symptom of bladder cancer, then the test would perform no better than a test for hemoglobin as long as the threshold and sensitivity of the two assays were comparable. Conversely, measuring NMP-22 introduced by bleeding would impact the sensitivity of the test, calling falsely positive cases that had hematuria through non-malignant causes such as infection or trauma. Alternatively, perhaps the NMP-22 test is a measure of the release and lysis of urothelial cells into the urine. Bladder tumors are often friable and shed large numbers of tumor cells into the urine relative to the normal turnover of healthy urothelia. The total number of urothelia shed per unit of time will be greater if the patient has a bladder lesion. NMP-22 is a nuclear scaffold protein and is not known to be secreted, thus, cell lysis must occur for the NMP-22 test to detect soluble protein. If cell lysis occurs at a constant rate, then the increased number of cells released into the urine in the presence of a lesion may suffice for a test, but it is also possible that tumor cells lyse in urine more readily than normal urothelia, thereby amplifying the test to some extent. A likely scenario is that a combination of these factors  contribute to the NMP-22 test result. As long as the test outperforms VUC, one can argue that it has value, but identifying what any bladder cancer test is actually measuring would seem to be pertinent and should enable improvement of a given test, or provide insight into how best to combine multiple markers.et al. reported from a large prospective screening study that NMP-22 outcomes are affected by gross hematuria, urinary tract infections and concentrated urine (creatinine > 2.5 g/L) [Other investigators have associated false-positive NMP-22 tests with instrumentation of the genitourinary tract and inflammation  and grad2.5 g/L) . Further2.5 g/L) . Similar2.5 g/L) . Based oIn combination, these findings raise the question of whether the NMP-22 tests are detecting a bona fide tumor antigen in urine samples or detecting a surrogate for cellularity/cell turnover or hematuria introduced into the urine by a friable bladder tumor. The absence of significant urinary cellularity in subjects with BCa may result in false-negative NMP-22 assays.NMP-22: Nuclear matrix protein 22; NUMA1: Nuclear mitotic apparatus protein 1; BCa: Bladder cancer; VUC: Voided urinary cytology; BTA: Bladder tumor antigen; ELISA: Enzyme-linked immunosorbent assay.The authors declare that they have no competing interests.MM Study concept and design, acquisition of data, data analysis, drafting of manuscript. SG Study concept and design, drafting of manuscript, supervision. EGG acquisition of data and data analysis. WR acquisition of data and data analysis. SR acquisition of data and data analysis. CJR Study concept and design, drafting of manuscript, supervision. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/12/23/prepub


Synthetic heteroglycoclusters are being subjected to increasing interest due to their potential to serve as selective ligands for carbohydrate-binding proteins. In this paper, we describe an expedient strategy to prepare cyclopeptides displaying well-defined distributions and combinations of carbohydrates. By using both oxime ligation and copper(I)-catalyzed alkyne–azide cycloaddition, two series of compounds bearing binary combinations of αMan, αFuc or βLac in an overall tetravalent presentation, and either 2:2 or 3:1 relative proportions, have been prepared. Multivalent interactions between carbohydrates and proteins play key roles in diverse biological events, including fertilization, cell–cell communication, host–pathogen interactions, immune response and cancer metastasis . SynthetInterestingly, recent reports have highlighted that the association of different sugar units instead of a single motif, namely heteroglycocluster, reflects the presence of sugars found in native biological systems more closely than homoglycocluster does. Although recognition mechanisms are not fully understood, these studies suggest that heteroglycoclusters should interact with proteins through distinct binding sites, which may influence both affinity and selectivity –25. In t1-3  and the utilization of microwave or ultrasonic irradiation are parameters that can influence the reaction kinetics, improve the yields and sometimes prevent side reactions. In a previous study, we observed that a tetravalent glycocluster can be obtained in good yields and as a unique 1,4-regioisomer by using a catalytic amount of copper micropowder in a mixture of isopropanol and ammonium acetate buffer + calcd for C60H96N20O22Na, 1471.7; found, 1471.8. After the addition of acetone (100 µL) to the crude, the reaction mixture was lyophilized, then resuspended with t-BuOH/AcONH4 100 mM pH 7.4 . Compound 1b  and copper micropowder  were next added to the solution and the resulting mixture was left under stirring at room temperature. After 4 h, copper was removed by centrifugation and the supernatant purified by semipreparative HPLC. Compound 6-Fuc/Man was obtained in 91% yield (5.9 mg). Analytical RP-HPLC: tR 7.73 min (gradient: 5 to 100% B in 20 min); 1H NMR  δ 8.07 , 8.06 , 7.81 , 7.80 , 5.62 , 5.60 , 4.99 , 4.86–4.68 , 4.47–4.35 , 4.16–3.63 , 3.36–3.20 , 2.39–2.29 , 2.16–1.27 , 1.22 ; ESI+–MS (m/z): [M + H]+ calcd for C78H124N20O34, 1885.9; found, 1886.0. Compounds 6-Man/Fuc, 6-Man/Lac, 6-Fuc/Lac, 6-Lac/Man and 6-Lac/Fuc were prepared following the same experimental conditions.A solution of 7  and 2a  was stirred at 37 °C in 0.1% TFA in H2O (600 µL). After 3 h, analytical HPLC revealed the total conversion of 7 into the corresponding monovalent intermediate. Analytical HPLC tR 10.57 min (gradient: 5 to 100% B in 20 min); ESI+–MS (m/z): [M + H]+ calcd for C52H84N21O17, 1274.6; found, 1274.8. After the addition of acetone (100 µL) to the crude, the reaction mixture was lyophilized, then resuspended with t-BuOH/AcNH4 100 mM pH 7.4 . Compound 1b  and copper micropowder  were next added to the solution and the resulting mixture was left under stirring at room temperature. After 4 h, copper was removed by centrifugation and the supernatant purified by semipreparative HPLC. Compound 8-Fuc/Man was obtained in 88% yield (10.0 mg). Analytical RP-HPLC: tR 7.66 min (gradient: 5 to 100% B in 20 min); 1H NMR  δ 8.05 , 8.02 , 7.79 , 5.61 , 5.60 , 4.99 , 4.85–4.66 , 4.48–4.32 , 4.15–3.64 , 3.29–3.19 , 2.40–2.30 , 2.15–1.28 , 1.22 ; ESI+–MS (m/z): [M + H]+ calcd for C79H126N21O34, 1912.9; found, 1913.2. Compounds 8-Man/Fuc, 8-Man/Lac, 8-Fuc/Lac, 8-Lac/Man and 8-Lac/Fuc were prepared under the same experimental conditions.A solution of 6-Man/Fuc, 6-Man/Lac, 6-Fuc/Man, 6-Fuc/Lac, 6-Lac/Man and 6-Lac/Fuc) and 3:1  described in this article.The Supporting Information file contains analytical details of all heteroglycoclusters of series 2:2 (File 1Crude RP-HPLC profiles and ESI-MS spectra for the heteroglycoclusters.


Pharmacovigilance is a corrective process originating in pharmaco-epidemiology. The 1997 Erice Declaration, presented at the World Health Organisation, became the basis on which the concept was implemented internationally for conventional systems of medicine. The increasing international acceptance of Ayurveda, led regulators to implement a similar program for Ayurveda, particularly as some medical professionals, scientists and members of the public reported adverse reactions after taking Ayurvedic formulations. The World Health Organisation therefore persuaded the Department of AYUSH, Ministry of Health and Family Welfare, Government of India, to implement a pharmacovigilance program for Ayurveda, as a means to ensuring the safety and efficacy of Ayurvedic medicines. After a year of due diligence, the pharmacovigilance program was launched nationally on 29 September 2008. Since that time, Ayurveda, Siddha and Unani medicines have been monitored according to the provisions of a protocol prepared by the National Pharmacovigilance Resource Centre, IPGTRA, Jamnagar, and approved by Department of AYUSH. The program was reviewed, first, on 21st January 2009 by the National Pharmaco-vigilance Consultative Committee for ASU drugs (NPCC-ASU), and again, on 15 Feburary, 2010, when an evaluation meeting effectively rubber stamped the program. Among the outcomes of these meetings were several suggestions of measures to improve the program’s efficiency. Recent developments include the constitution of pharmacovigilance centers at all Ayurveda Teaching institutes and research centers. The first two, in the 1920’s and 1940’s, failed to sustain on account of the discovery of antibiotics like the sulfa drugs and penicillin, but once again due to lack of cures for chronic disease and side-effects of western drugs, developed countries started looking to Ayurveda for treatments to restore wellness.[The 1980’s were witness to the 20As a medical science, Ayurveda includes considerations of the natural human life-span and life-cycle, awareness of the nature of human life on earth, and reasons for it. True integration of body, mind and spirit, of the psychic and the somatic, are among its unique features. Being a system of preventive medicine and health promotion, its accurate application results in a full life-span enjoyed in healthy well-being.In its origins Ayurveda was carefully and systematically developed. As a result, it is now being confirmed by measures of many scientific parameters. It not only provides well-based medical cures for disease, but its holistic approaches use unique principles of diet, life-style and, particularly, therapeutics, to balance and enrich all aspects of the physiology and psyche. In light of these aspects of Ayurveda past and present, the system’s potential for promotion of health and wellness is well-acknowledged.On the other side of the coin, Ayurveda treatments have come under attack for several reasons. Unethical companies are under scrutiny for the production of adulterated and misbranded medicaments by inaccurate methods, while some of its practitioners have indulged in illegal practice.All these events led the Department of AYUSH, Ministry of Health and Family Welfare, Government of India, to implement a National Pharmacovigilance Program for Ayurveda, Unani, and Siddha systems of medicine, in order to systematically monitor adverse drug reactions (ADR).Pharmacovigilance is defined as ‘the detection, assessment, understanding, and prevention of adverse effects of drugs or any other possible drug related problems’. This definition plainly covers the objectives of the AYUSH program and its coverage area as per the WHO guidelines.Dravyaguna), pharmaceutics , and therapeutics (Chikitsa).[Although a technical term equivalent to “pharmacovigilance” does not feature in Ayurvedic texts, the spirit of pharmacovigilance is vibrant throughout Ayurveda’s classical literature. The Brihattrayi and Laghutrayi repeatedly emphasize the major goals of pharmacovigilance, to improve patient care and safety during treatment, and thus to promote rational use of medications. These are recurrent themes of Ayurvedic pharmacology (hikitsa). It is prAnupan therapeutic method and Shodhan pharmaceutics principles probably also contribute to the prevention of many undesired and unforeseen events. Prevention of this kind is a major goal of pharmacovigilance programs.The Ayurvedic literature gives details of drug-drug and drug-diet incompatibilities based on elaborately described qualitative differences in ingredients or quantitative proportions. These factors undoubtedly prevent the onset of many otherwise unfortunate reactions. Ayurveda’s According to Article 2 of the World Health Organization’s constitution, its mandate from its Member States is “to dIn accordance with these resolutions, WHO has acted very quickly whenever widespread health disorders have arisen. The 1961Thalidomide disaster was the driving stroke which compelled WHO to initiate a program to thoroughly monitor all prescribed drugs. In its sixteenth World Health Assembly in 1963, resolution WHA 16:36 emphasized the need for rapid dissemination of information on ADRs.Pilot Research Project for International Drug Monitoring. The program fulfilled basic purposes.[Erice Declaration, an international agreement eventually signed by all member states to agree on uniform standards for reporting ADR’s.In 1968 as a result of reports of drugs producing adverse reactions, WHO initiated its purposes. Finally,The Erice Declaration represented significant progress. It challenges all players, including:Public Health administratorsHealth professionalsThe Pharmaceutical industryGovernmentsDrug regulatorsMedia, andConsumers“to strive towards the highest ethical, professional, and scientific standards in protecting and promoting the safe use of medicines.” The declaration charges governments and all involved in determining policies relating to the benefit, harm, effectiveness, and risk of medicines, to be accountable for what they communicate to both the public and patients. It calls for honesty when communicating drug safety information, even when such information is incomplete and investigations are still underway. It further stipulates that patients be transparently informed of all facts, assumptions, and uncertainties concerning safety profiles of their medicines. Considerable effort has subsequently been made to achieve its goals.Further in 2002, India agreed to send all its ADRs arising from use of conventional medicine to WHO’s ADR Monitoring Centre in Uppsala, Sweden, where its international ADR database receives reports from National Centers in 65 countries using ‘MedDRA’ and WHO ART terminology: the ‘Medical Dictionary for Drug Regulatory Activities’ and ‘World Health Organization Adverse Reaction Terminology’ for ADR’s.India introduced the program in accordance with obligations from agreements signed in 1997 and 2002. A new national network of pharmacovigilance centers was established in 2004, coordinated by the Central Drugs Standard Control Organisation (CDSCO). In this A common understanding between all the pharmacovigilance program’s conventional medicine stakeholders in India was that, to deal with certain objectives not addressed by the 2004 system, the program should be reorganized. The program was relaunched in April 2010 by CDSCO in association with a new key player, the Department of Pharmacology of the All India Institute of Medical Sciences, New Delhi. Up to now, more than 40 medical colleges have been nominated as pharmacovigilance centers under a policy aiming to cover all medical colleges in the country. For conventional medicine, the program has thus been redesigned, and implemented in the hope that it will provide a fool-proof system of ensuring medicines’ safety.13The idea of a corresponding pharmacovigilance program for traditional medicine began inIn October, 2007, BHU’s Institute of Medical Sciences’ Department of Rasa Shastra organized a seminar-cum-workshop entitled “Safety Profile of Ayurvedic Dosage Forms”. The seminar’s technical report submitted to WHO strongly influenced how the Ayurvedic medicines’ pharmacovigilance safety program was implemented.The next concrete step sponsored by WHO was taken by the Institute of Post Graduate Teaching and Research in Ayurveda (IPGTRA), at Gujarat Ayurveda University, Jamnagar, which organized a workshop on the possibility of implementing pharmacovigilance programs for Ayurvedic medicine in December, 2007. In view of the program’s potential importance, the Dept of AYUSH requested IPGTRA to prepare a protocol and ADR reporting format to implement pharmacovigilance for Ayurveda, Siddha, and Unani (ASU) drugs.The draft protocol was discussed and technically analyzed by a consultative committee of concerned experts consisting of specialists in different fields (i.e. pharmacologists and Vaidyas in different disciplines) in Delhi in August, 2008, sponsored by the WHO national office. Incorporating expert suggestions, the draft was finalized and released by Department of AYUSH Secretary, Ms Anita Das, on 29 September 2008. IPGTRA was subsequently declared National Pharmacovigilance Resource Centre for ASU Drugs. From that date, India’s present ASU systems pharmacovigilance program has been in operation. The struReporting procedures for India’s pharmacovigilance program for ASU drugsIndia’s National Program of Pharmacovigilance for ASU drugs was adopted under the following blueprint in accordance with recommendations of the expert group made at its meeting on 28-29 August, 2008.The National Pharmacovigilance Programme for ASU drugs (NPP ASU) shall encourage reporting of all suspected drug related adverse events, including those suspected to have been caused by interaction with any other drugs or food incompatibilities. Reporting of seemingly insignificant or common adverse reactions may be important, since it could highlight a widespread prescribing problem.The program particularly solicits reports ofall adverse reactions suspected to have been caused by ASU drugs either alone or in conjunction with other drugsall suspected drug interactionsreactions to any other drugs suspected of significantly affecting a patient’s management, including reactions suspected for events in the following categoriesdeathlife threatening hospitalization disability congenital anomalyrequired intervention to prevent permanent impairment or damage.The prescribed ‘Adverse Drug Event Reporting Form for ASU Drugs’ shall be used for the purpose of the National Pharmacovigilance Programme for ASU. The form may be downloaded from the webpages of Gujarat Ayurveda University or WHO’s India Office.20Any health care professional may report suspected adverse drug events. The program does not accept reports from lay members of the public, nor others than health care professionals. Others can report through the physician under whom they have undergone treatment.Reporting should be done in a prescribed format through a local pharmacovigilance center.Information in the forms is to be handled in all confidentiality. Peripheral pharmacovigilance centers forward the form to their regional pharmacovigilance centers where causality analysis is carried out. The information is then forwarded to the National Pharmacovigilance Resource Centre, where it is consolidated, statistically analyzed, and forwarded to the Dept of AYUSH.Departments of government, hospitals, and academic institutions, involved in clinical pharmacology, clinical pharmacy, clinical toxicology, or epidemiology have been identified as the best hosts to set up and house centers. Those applying will be assessed for infrastructure and other resource requirements, since proper planning is essential to establish and run a pharmacovigilance center successfully. Also, government support is necessary at least at the national level.www.ayurveduniversity.com portal, where a complete list of centers may be found. Continuing Medical Education and public meetings are being conducted to raise health professional’s awareness of ASU drugs.At present, besides the National Center in Jamnagar, 8 Regional Centers and 30 Peripheral Centers  use, and promotion of understanding, education, and clinical training in pharmacovigilance for Ayurvedic medicines and its effective communication to the public. Many cases have been reported in the recent past regarding ADRs and drug-drug interactions at various national and international forums.deha dhatus will be antagonistic (virodha) towards them. Such antagonism may result from properties, combination, processing, place, time, dose etc. or natural composition.That Ayurvedic drugs are safe due to their natural origin is a popular conception, but it is true to the extent that many are used as foodstuffs and deserve the classification Generally Recognized as Safe - GRAS. References in the Ayurvedic classics to possible adverse reactions to certain Ayurvedic medicines alert us against accepting this concept as universal, however. This is particularly true if medicines are not prepared properly, or if preconditions for their administration are not respected by both physician and patient, which will increase the possibility of an ADR occurring. Charak Sutra Sthana 26 warns that substances contrary to ahar) is as important for cure as medicine. The texts state that an ailment can only be cured by following proper diet (pathya). In this context, Charaka lists dietary incompatibilities between particular foodstuffs[In Ayurveda, diet . More recently, on 15 February, 2010, the Secretary, Department of AYUSH chaired an evaluation meeting of the Pharmacovigilance Program for ASU Drugs, new initiatives to implement the program more effectively. The evaluation meeting effectively rubber stamped the program. Among the outcomes of these meetings were several suggestions of measures to improve the program’s efficiency. NPRC, Jamnagar, presented details of 103 ADRs reported from all over the country and their causality assessment. This is a matter of some concern. More recent developments include constitution of pharmacovigilance centers at all Ayurveda teaching institutes and research centers.On the basis of the above, we conclude that there is a need for a proper postmarketing surveillance program to observe quality, safety, and efficacy of Ayurvedic drugs, which is now available in the form of National Pharmacovigilance Programme for ASU Drugs.The success of any pharmacovigilance system lies in its ability to prevent further adverse reactions on the basis of information received. This will be possible only when physicians are vitally alert to the onset or offset of any ADRs. They need to prioritize their contributions to make the pharmacovigilance program for Ayurvedic medicines a success.


Dosidicus gigas plays an important role in marine food webs both as predator and prey. We investigated the ontogenetic and spatiotemporal variability of the diet composition of jumbo squid in the northern Humboldt Current system. For that purpose we applied several statistical methods to an extensive dataset of 3,618 jumbo squid non empty stomachs collected off Peru from 2004 to 2011. A total of 55 prey taxa was identified that we aggregated into eleven groups. Our results evidenced a large variability in prey composition as already observed in other systems. However, our data do not support the hypothesis that jumbo squids select the most abundant or energetic taxon in a prey assemblage, neglecting the other available prey. Indeed, multinomial model predictions showed that stomach fullness increased with the number of prey taxa, while most stomachs with low contents contained one or two prey taxa only. Our results therefore question the common hypothesis that predators seek locally dense aggregations of monospecific prey. In addition D. gigas consumes very few anchovy Engraulis ringens in Peru, whereas a tremendous biomass of anchovy is potentially available. It seems that D. gigas cannot reach the oxygen unsaturated waters very close to the coast, where the bulk of anchovy occurs. Indeed, even if jumbo squid can forage in hypoxic deep waters during the day, surface normoxic waters are then required to recover its maintenance respiration (or energy?). Oxygen concentration could thus limit the co-occurrence of both species and then preclude predator-prey interactions. Finally we propose a conceptual model illustrating the opportunistic foraging behaviour of jumbo squid impacted by ontogenetic migration and potentially constrained by oxygen saturation in surface waters.The jumbo squid  Dosidicus gigas is the most abundant nektonic squid in the surface waters of the world ocean D. gigas is a large squid with high fecundity The ommastrephid jumbo squid D. gigas plays an important role in marine food webs both as predator and prey D. gigas is a part-time resident of the OMZ thanks to adapted behavior and specific metabolic characteristics D. gigas. We also decipher one paradox in the jumbo squid diet: why do they hardly forage on the tremendous biomass of anchovy Engraulis ringens distributed off coastal Peru? We show that the shallow OMZ in this area could hamper the co-occurrence of jumbo squids and anchovies, impacting jumbo squid foraging behaviour. We finally propose a conceptual model of jumbo squid trophic ecology including the ontogenetic cycle, oxygen conditions and prey availability.Previous studies showed that the feeding ecology of jumbo squid is highly variable in time and space A total of 5320 stomachs were collected from jumbo squids caught between 2004 and 2011 by the authorized industrial jigging fishery off Peru  . No animD. gigas only. We were therefore not able to precisely estimate the importance of natural cannibalism with our dataset that was still blurred by artificially induced cannibalized conspecifics. We thus removed these 859 stomachs and worked with the remaining 4461 (83.9%), from which 3618 were not empty  . Stomach contents were weighed and the different items constituting a single taxon were sorted, counted and weighed. Jigging vessels use 2 kW lights (no use of bait) to attract jumbo squids. Biases can be associated with fishing gear and tactic but jigging is recommended for diet studies tomachs) . We probIdentifiable fresh remains and diagnostic hard parts were used to determine the number of each prey item. For fish otoliths and cephalopod beaks, the maximum number of left or right otoliths and the greatest number of either upper or lower beaks were used to estimate the number of fish and cephalopods, respectively. Prey items were identified to the most precise possible taxonomic level using keys and descriptions for fish A stomach fullness weight index  was calculated A clear relationship exists between squid size and maturity stages  indicatii)i≥1. The logistic form was chosen to predict the probabilities of observing different prey richness as a function of the covariates of interest.In order to analyse the potential effects of explanatory variables on the number of taxa per stomach, a proportional-odds model for ordinal response The potential effects of explanatory variables  on stomach fullness index and diet of jumbo squid were first investigated using Kruskal-Wallis (KW) non-parametric tests. This preliminary approach allowed us to perform an initial inspection of the dataset. Length, distance to the shelf break and SSTA were then each divided in four ordered categories, according to their ecological interpretation (the number of stomachs is given for each category); for mantle length: less than 40 cm (559), 41–60 cm (1553), 61–80 cm (934), over 80 cm (572); for distance to the shelf: less than 50 km (840), 51–75 km (682), 76–130 km (829), over 130 km (1267); for SSTA: less than −1.5°C (616), −1.49 to −0.5°C (899), −0.49 to 0.5°C (1299), over 0.5°C (804). Stomach numbers for the four seasons were: summer (690 stomachs), fall (1068), winter (997) and spring (863). However this approach did not account for dependence and interactions between explanatory variables, and then did not elucidate the complex relationships between the type of prey and the environmental factors. In addition the sampling scheme was very unbalanced in space and time. To cope with these issues, we ran a classification and regression tree (CART) analysis proposed by MASS package for the proportional odds-model rpart package for the classification tree.Analyses were conducted using the statistical open source R software (R Core Team 2013), with the P<0.01) and increased significantly with distance to the shelf (P<0.01). On the opposite, effect of SSTA was not significant (P>0.05), but slightly higher values of stomach fullness weight index occurred in spring (P<0.01).The size of the 4461 selected squids ranged from 14.3 to 114.2 cm ML . OverallDosidicus gigas and other Cephalopoda) were the dominant food source in %O, %N and %W (Vinciguerria lucetia occurred frequently in the stomach contents (36%), representing an average percentage of nearly 20% by weight and 25% by number. The three Myctophidae taxa  occurred in 1577 samples , and contributed 15% by weight and 18.3% by number. Teleosteii were frequent in the stomachs (21.7%) and represented 12.7% by weight and 11.7% by number.Cephalopoda  and reached 43.2% for large squids with ML>80 cm. The percentage of Euphausiidae also increased significantly (V. lucetia (21% to 5.6%) and Myctophum sp. (7.2% to 1.3%) decreased significantly while jumbo squid increased in size (The diet composition of jumbo squid in weight varied according to size . The maificantly  with siz in size .V. lucetia (32%) in spring and less Cephalopoda (26%), Euphausiidae (2.8%) and Teleosteii (9.3%) (V. lucetia was low (13.7%) and Engraulidae were very rare (0.4%).No clear tendency appeared with the season , except i (9.3%) . In summD. gigas varied significantly with the distance to the shelf break  and a significant decrease in cannibalism (from ∼11 to 6.6%) (Diet changed according to SSTA . Trend fto 6.6%) .Based on the detailed 55 prey taxa, the prey richness in the stomachs was very low. A maximum of seven prey taxa was observed in one stomach only, while a single prey taxon was recovered in 48.0% of the stomachs and 30.7% had two prey taxa . Results were similar with the eleven aggregated taxa: a maximum of seven prey taxa, 48.6% with one prey taxon and 31.1% with two prey taxa . Consequently, analyses were performed with the eleven aggregated taxa .According to the Akaike information criterion (AIC), the proportional-odds model with two covariates  was the most parsimonious . The estimated values of the parameters were used to compute the probabilities of observing 1, 2, or 3+  prey taxa in a stomach as a function of stomach fullness or distance to the shelf. Increasing the stomach fullness led to a sharp increase in the probability of recovering 3+ prey taxa in a stomach and to a marked decrease of the probability to observe only one taxon . After aV. lucetia (predicted probability = 0.44) at a distance to the shelf break higher than 191 km, in winter and spring. The node 5 showed a high probability of cannibalism (predicted probability = 0.32) for medium size (between 60 and 80 cm ML) individuals with stomach fullness higher than 0.2. From the node 6 on, squids had a smaller ML (less than 60 cm). The node 6 also showed a high probability of cannibalism (predicted probability = 0.46) for SSTA <0.425°C, in individuals with fullness greater than 2.08, located at less than 209 km to the shelf break. The node 7, characterised by the teleosteii (predicted probability = 0.60), had the same characteristics than the node 6, except a more offshore location. Nodes 8 to 10 showed a relatively balanced diet and were separated from nodes 6 and 7 by a lower fullness (<2.08). Nodes 11 to 13 corresponded to fullness ≥0.2, size <60 cm and SSTA ≥0.425°C. Node 11 was associated to high SSTA (≥1.09°C), short distance to the shelf break (<197 km), and predicted a dominance of cephalopods (predicted probability = 0.37). In nodes 12 (distance to the shelf break greater than 197 km) and 13 (SSTA<1.09°C), V. lucetia was largely dominant .The pruned classification tree showed 13 nodes . The firThis work is based on an extensive dataset on jumbo squid diet encompassing a large range of spatiotemporal location and sizes. Beyond the usual diet description, our results allowed us to provide new knowledge on jumbo squid trophic ecology, in particular on prey distribution under different environmental conditions and on the role that could be played by the dissolved oxygen.Using the detailed (55 taxa) or aggregated (eleven taxa) databases, prey richness in stomachs was similar with an average of 1.8 taxa per stomach. This unexpected result has several consequences. It first empirically validates the eleven aggregated taxonomic groups . Second,Trachurus murphyi; Identifying cephalopods food is tricky V. lucetia and myctophiids) recovered in the jumbo squid stomachs confirmed the structuring role of spatial matching in the jumbo squid-prey interactions. This prey group contributed mainly during spring and far from the coast, when jumbo squid was more offshore. In addition, small jumbo squids distributed far from the coast consumed more mesopelagic fish than larger individuals located closer to the coast. This pattern was unexpected again, but is in accordance with the distribution pattern of mesopelagic fish that are distributed more offshore than euphausiids Mesopelagic fish  D. gigas consumes very few anchovy in Peru, whereas a tremendous biomass of anchovy is potentially available. Furthermore, off Peru, anchovy is concentrated in schools or dense aggregations within the thin surface oxygenated layer D. gigas succeeds in the OMZ by managing hypoxia via metabolic suppression In the California Current system Dosidicus gigas trophic ecology in the northern Humboldt Current system  according to mantle size.(TIF)Click here for additional data file.Figure S2Distribution of the fullness weight index (FWI) of non-empty jumbo squid stomach.(TIF)Click here for additional data file.Table S1Overall description of the 55 prey taxa observed in jumbo squid stomach sampled off Peru during 2004–2011. Are indicated, the taxonomic information, the mean value (±standard deviation) of the proportion by weight (%Weight) and by number (%Number) as well as the frequency of occurrence (%Occurrence).(DOCX)Click here for additional data file.Table S2Yearly description of the 55 prey taxa observed in jumbo squid stomach sampled off Peru during 2004–2011. Proportion by weight: %W; proportion by number: %N; frequency of occurrence: %O.(DOCX)Click here for additional data file.Table S3Result of the Kruskal-Wallis test performed on the 11 dietary groups according to Size, Distance to the shelf-break, Season and SSTA. Significant differences are in bold.(DOCX)Click here for additional data file.


Teens do not meet guidelines for healthy eating and physical activity. The Internet may be an effective method for delivering programs that help them adopt healthy behaviors.To collect information to design content and structure for a teen-friendly website promoting healthy eating and physical activity behaviors.Qualitative research, encompassing both focus group and interview techniques, were used to design the website. Participants were 12-17 year olds in Houston, Texas, and West Lafayette, Indiana.A total of 133 participants took part in 26 focus groups while 15 participated in one-on-one interviews to provide guidance for the development of teen-friendly content and structure for an online behavior change program promoting healthy eating and physical activity to 12-17 year olds. The youth made suggestions to overcome common barriers to healthy eating and physical activity. Their feedback was used to develop “Teen Choice: Food & Fitness,” a 12-week online behavior change program, populated by 4 cartoon character role models.It is critical that members of the target audience be included in formative research to develop behavior change programs that are relevant, appealing, and address their needs and interests. Youth obesity has reached epidemic proportions  and adolTo be effective, behavior change interventions must meet the expectations of today’s technology-savvy users . Online Although there has been concern regarding youth access to computers and the Internet among the general population, data reveal a somewhat different picture. Nationally representative surveys of media use reveal that home computer access among 8-18 year olds ranges from 89% to 94%, while 74% to 88% have home Internet access . ApproxiFocus groups and one-on-one interviews were conducted to inform the content and structure of the online program, as well as to evaluate its components during development. The study was approved by the institutional review boards of Baylor College of Medicine in Houston, Texas, and Purdue University in West Lafayette, Indiana. Adolescents aged 12-17 years were recruited from youth centers and schools using standard recruitment techniques. Participants provided written parental consent and their own verbal assent prior to participation. Teens could participate in only one activity .Trained moderators and assistant moderators  conducteThe focus groups covered two general areas: (1) what youth thought should be included in an online program about healthy eating or physical activity and (2) suggestions for overcoming common barriers to healthy eating or physical activity. The information obtained on barriers and suggested solutions was used to create short role model video clips to be included in the online behavior change program under development.To spark the discussion, separate lists of commonly reported barriers were created for healthy eating -41 and pAdditional focus groups were conducted to develop an online “calculator” that would be easy to use, in a format acceptable to this age group, and that provided information requested by the adolescents on what and how much to eat and how much physical activity they needed to do. An existing healthy eating calculator developed for adults was used to prompt discussion.Using information obtained from the focus groups, investigators created the online program components. Interviews were then conducted to review the materials prior to finalizing the program.Interviews (conducted in Houston only) followed a semi-structured script. Probes and prompts were used to expand and clarify responses. Print versions of the teen cartoon characters  and components of the behavior change program were used to guide the discussion. Two types of interviews were conducted: one set was to review role model scripts and teen characters; the second set of interviews was to review online behavior change program content and structure prior to finalizing the program. Participants for the first set of interviews were recruited from the same locations as the focus group participants using procedures described above. The second set of interviews was promoted only at the Children’s Nutrition Research Center in Texas and interviews were conducted with children, relatives, and/or neighbors of faculty and staff.In total, 18 initial focus groups—10 on healthy eating (n = 50 participants) and 8 on physical activity (n = 45 participants)—were conducted . ParticiThe top barriers to healthy eating and physical activity suggested by the groups are listed in An additional 8 focus groups (n = 38) were conducted to develop a healthy eating and physical activity “calculator” to provide tailored information on nutritional needs and physical activity. Participants suggested a graphic “pie chart” figure that would provide both textual and visual information. They also suggested that users should be able to click on the various “wedges” of the pie to access additional information about that component. This design would give participants control over how much and what information they accessed.Using information from the focus groups that addressed common barriers to healthy eating and physical activity and how to overcome them , 12 roleUsing information from the focus groups and interviews, program components for the online program were developed. A final set of interviews (n = 5) was conducted to review program components prior to completion. During these one-on-one interviews, adolescents were shown the components and queried about their thoughts regarding relevance, appropriateness, and appeal. The interviews indicated no changes were needed to program content.The name selected by participants for the 12-week online behavior change program was “Teen Choice: Food & Fitness.” The online program contains an initial log-on page where teens enter their unique username and password to log on to the program website that is hosted on a secure server. Once on the website, they can view the 12 role model stories addressing barriers to healthy eating (n = 6) and physical activity (n = 6)  led by 4Control Program Content and StructureFor the randomized controlled trial to test the effectiveness of the online program at improving eating and physical activity behaviors, a control condition was needed. It was constructed by removing the role model stories and the goal setting, planning, self-monitoring, goal review, and problem-solving components from the treatment intervention .This paper reports the results of formative research with adolescents to create an online program promoting healthy eating and physical activity that would appeal to this age group. Their recommendations were used to guide decisions about the online program content and structure. As such, it provides guidance on how to involve the target audience in the development of an online behavior change program.Internet and computer use are high among youth , thus ofOnline programs provide an engaging venue for achieving behavior change through both personal mastery and observational learning. Personal mastery can be promoted in an online program through inclusion of self-regulatory activities, such as goal setting, planning, self-monitoring, and problem-solving activities; thus, it is important for these activities to be developmentally appropriate and utilize a format that appeals to the target audience . AdditioObservational learning occurs by watching others  perform a particular behavior and receive rewards . This prAttracting and maintaining attention is an important first step in observational learning because it initiates learning and behavior change processes . EnhanciRecruitment for the randomized controlled trial to test the online program began in late 2009 and concluded in October, 2011. Data collection is currently underway.Design of online programs promoting behavior change should be a collaborative effort between researchers and members of the target audience, such as teens. Suggestions for achieving this are:1. A realistic timeline is essential. Allow ample time for recruitment, data collection, analysis, interpretation, and application.2. Participants should represent the target population. This includes gender, socio-economic status, age, and other salient characteristics. If not, it is possible the data may be skewed and may not adequately represent the target group, thus reducing the potential effectiveness of the program. In this program, formative research was conducted in two states in order to ensure the results were not relevant to teens in only one part of the country.3. Conduct enough focus groups or interviews to achieve theoretical saturation, or the point at which no new information emerges . In the 4. Although scripts are important for consistency in data collection, they should be semi-structured, allowing ample room for participants to share thoughts and opinions. Discrepant information  may be especially useful. Scripts should contain open-ended, neutral, and non-leading questions. Probes, prompts, and follow-up questions should be generously used to expand and more fully understand responses. The scripts used to guide discussions in the current project were semi-structured, and probes and prompts were used to explore responses and elicit additional information as needed.5. Member checks are important ,73. MembLimitations of this research include the use of qualitative research in only two locations, which limits generalizability of the findings. This research does not address desired frequency of user access, program components accessed, or time spent viewing or completing the various components. It also does not address the impact of the program on diet or physical activity behaviors. However, the outcome evaluation, which is currently underway, is collecting information to address these issues. Additional research is needed to understand triggers for participation , whether programs of this type meet their expectations, and long-term health effects in order to develop robust and effective online behavior change programs.Successful programs that encourage adolescents to adopt healthy diet and physical activity behaviors are needed to reduce obesity risk. Online behavior change programs designed in conjunction with youth may provide an important venue for achieving desired changes in these behaviors. The efficacy of this approach with adolescents is currently being tested and will provide valuable insights that can be used to guide future intervention research.