Disability-Adjusted Life Years (DALYs) and Quality-Adjusted Life Years (QALYs), which capture life expectancy and quality of the remaining life-years, are applied in a new method to measure socioeconomic impacts related to health. A 7-step methodology estimating the impact of health interventions based on DALYs, QALYs and functioning changes is presented. It relates the latter (1) to the EQ-5D-5L questionnaire (2) to automatically calculate the health status before and after the intervention (3). This change of status is represented as a change in quality of life when calculating QALYs gained due to the intervention (4). In order to make an economic assessment, QALYs gained are converted to DALYs averted (5). Then, by inferring the cost/DALY from the cost associated to the disability in terms of DALYs lost (6) and taking into account the cost of the action, cost savings due to the intervention are calculated (7) as an objective measure of socioeconomic impact. The methodology is implemented in Java. Cases within the framework of cardiac rehabilitation processes are analyzed and the calculations are based on 200 patients who underwent different cardiac-rehabilitation processes. Results show that these interventions result, on average, in a gain in QALYs of 0.6 and a cost savings of 8,000 €. Medical Health Records (MHRs), the socioeconomic impact of rehabilitation in a specific geographical area, considering, as a case study, the impact in Spain of cardiac rehabilitation. Cardiac rehabilitation attempts to reduce the levels of morbidity and impairment mainly caused by ischemic heart disease, the decrease in blood supply of the heart muscle usually due to coronary artery disease, which may result in angina pectoris, Acute Myocardial Infarction (AMI) or heart failure. Its role in improving the health of patients with ischemic heart disease has been recognized by the American Heart Association [The research described in this paper aims to provide a method to automatically assess, from existing ociation , the Eurociation  and the ociation , among oThis article is also aimed to validate the cost-effectiveness of rehabilitation, illustrating the gains obtained in terms of quality of life and, ultimately, of cost savings related to health recovery. Indicators of health outcomes combining mortality and morbidity can provide qualitative and quantitative measurements of these gains related to health interventions, and can be used to provide decision support to policymakers in order to enhance healthcare management. In the case of cardiac rehabilitation, validation of cost-effectiveness is crucial to encourage its usage; especially in patients that have suffered from AMI, since in most European countries less than 50% of them undergo rehabilitation .Instituto Nacional de Estadística or INE in Spanish,), ischemic cardiomyopathy is the main cause of mortality and one of the most common causes of morbidity and loss of quality of life. Patients may experience aggravating disability with increasing burden on their careers. They are also heavy users of healthcare and social-service resources, with an estimation of €2 billion per year being spent in Spain because of this disease [Ischemic cardiomyopathy is the leading cause of death in the World, accounting for 12% of total deaths, and the fourth leading cause of burden of disease, according to the World Health Organization (WHO) . In Spai disease .Disability-adjusted life years (DALYs) and Quality-adjusted life years (QALYs), which capture life expectancy and quality of the remaining life-years, can be used in methods to measure socioeconomic impacts related to health, while impairment is increasingly being measured according to the international classification of functioning, disability and health (ICF) (see below for more details on these concepts). Several studies  have  have Diss  and (2) years lived with disability (YLD). The second component is calculated as the discounted current value of years lived in a condition of impairment multiplied by a disability or severity weight for that condition, assigned in a scale of 0  to 1 (representing death). These weights are based on the outcomes of the Global Burden of Disease Study [DALYs are defined as the sum of the current value of future years of lifetime lost through premature mortality, and the current value of years of future lifetime adjusted for the average severity (frequency and intensity) of any mental or physical disability caused by a disease or injury . They arse Study , in whicse Study . Accordise Study ,13, an iBody Functions is the domain most closely related to a medical model as it refers to the physiological and psychological functions of body systems. Body Structures are defined by the ICF as anatomic parts of the body such as organs, limbs and their components. Activities refer to a wide range of deliberate actions performed by an individual. They are actions undertaken in order to accomplish a task, such as walking or climbing stairs. Participation refers to activities that are integral to economic and social life, such as attending school or holding a job. Environmental factors make up the physical, social and attitudinal environment in which people live. A given level of impairment in body function will not necessarily translate into activity or participation limitations if the environment accommodates a person’s functional condition. And environment refers not only to the physical environment, but the cultural and political environment as well. Disability comes from participation restrictions that result from the interaction of many factors [The ICF belongs to a family of international classifications developed by the WHO. Its aim is to provide a unified and standard language and framework for the description of health and health-related status; it defines components of health and some health-related components of well-being. According to the WHO, disability derives from the interaction between functional limitations and an unaccommodating environment. People are not described as having a disability based upon a medical condition, but are rather described according to a detailed representation of their functioning, which uses these main classes: body functions, body structures, activities, participation and environmental factors.  factors . Each ma factors ,15,16). 5) different possibilities of well-being status. The responses to the items in the EQ-5D-5L can be translated into health status weights using a utility-weighted algorithm [The precursor of EQ-5D-5L was the EQ-5D questionnaire from the EuroQol Group, a standardized, non-disease-specific instrument for describing and valuing health . Applicalgorithm , which hQALYs provide a common currency to assess the extent of benefits gained from a variety of interventions in terms of health-related quality of life and survival for the patient . QALYs aIndicators used when making decisions in public health policies  compare years of life saved (YLS), therefore they do not reflect gains in terms of quality of life, an outcome extremely relevant when estimating the impact in patients with functional limitation. Other evaluations are based on the assessment of the cost-utility (cost/QALY) ratio. The methodology presented in this paper proposes to calculate the cost-effectiveness ratio from QALYs gained after a health intervention. Moreover, it estimates the impact of rehabilitation using data from questionnaires currently used in clinical practice, and enables the automation of the calculation of DALYs and QALYs.Patients perform cardiac rehabilitation in three stages, namely: (1) hospital stay; (2) rehabilitation in the hospital 2–5 times a week; and (3) whole life . In the Cases of patients who had a myocardial infarction differ in their diagnosis, rehabilitation procedure and duration, the age of the patient and, consequently, the outcome after rehabilitation . The following examples correspond to three real cases of patients who suffered an acute myocardial infarction and performed different types of cardiac rehabilitation processes .Albert is the (anonymized) name of a 50-year-old man from Spain who suffered from an acute myocardial infarction. This refers to the condition when blood supply to part of the heart is interrupted because of a blockage in coronary arteries. When the myocardium (heart muscles) does not get enough oxygen, some of its cells die or become permanently damaged (necrosis). Two weeks after his hospital discharge, Albert starts the second stage of his cardiac rehabilitation. His initial evaluation shows he has some risk factors, such as smoking and hypertension, and some problems with his functional capacity . His left ventricular ejection fraction (LVEF) is normal, and he does not have problems with anxiety, depression, dyslipidemia, sedentary lifestyle, diabetes, alcohol, abdominal perimeter or body mass index. His therapist plans a personalized treatment for him including motor rehabilitation and, after 115 days of therapy, she makes a final evaluation in order to assess his progress, measured in QALYs gained, cost/DALY and the overall cost savings due to the rehabilitation program performed.Juan is the (anonymized) name of a 59-year-old man from Spain who suffered from an acute myocardial infarction. Two weeks after his hospital discharge, he starts the second stage of his cardiac rehabilitation. His initial evaluation shows that although his LVEF is normal, he has some risk factors, such as hypertension and dyslipidemia, and some problems with his functional capacity, anxiety and depression. His therapist plans a personalized therapy for him including motor and psychological rehabilitation and, after 157 days of rehabilitation, she makes a final evaluation in order to assess his progress, based on QALYs gained, cost/DALY and the overall cost savings due to the motor and psychological rehabilitation completed.Pedro is the (anonymized) name of a 77-year-old man from Spain who suffered from a dilated cardiomyopathy. He also suffered from an ischemic cardiopathy with acute coronary syndrome without ST elevation and with incomplete surgical revascularization. Two weeks after his last hospital discharge, Pedro starts the second stage of his cardiac rehabilitation. His initial evaluation shows his LVEF is normal, but it also shows that he has some risk factors, such as dyslipidemia and problems with his functional capacity, erectile dysfunction, anxiety and depression. His therapist plans a personalized therapy for him which includes motor and sexual rehabilitation during 123 days. Finally, after the treatment, she would like to make a final evaluation in order to assess QALYs gained, cost/DALY and the overall cost savings due to the motor and sexual rehabilitation performed.®, vardenafil, sold under the name of Levitra®, and tadalafil, sold under the name of Cialis®).Motor rehabilitation refers to physical training consisting of walking three times a week on a treadmill placed at the hospital gym. Exercise intensity is based on the maintenance of the heart rate, increasing progressively the distance or endurance of the session so as to reach the goal of walking 6 km per day or walking for an hour, maintaining the targeted heart rhythm. Psychological rehabilitation begins with the evaluation of the patient through an interview in order to assess his/her anxiety, depression and personality. Based on the results obtained, the patient receives individualized treatment by psychologists and, if necessary, the psychiatrist of the unit. Sexual rehabilitation refers to the treatment of erectile dysfunction through drugs such as phosphodiesterase-5 inhibitors  following the methodology of Cieza et al. [Cardiac Functional Classification [et al. [The present study took into account a population of 200 patients. Data about the above three sample cases are summarized in a et al. . This apfication  dependin [et al. ) into all patients (200 cases);(b)patients who performed only motor rehabilitation (92 cases);(c)patients who performed, at least, motor and psychological rehabilitation (24 cases);(d)patients who performed only motor and psychological rehabilitation (14 cases);(e)patients who performed, at least, motor and sexual rehabilitation (94 cases);(f)patients who performed only motor and sexual rehabilitation (86 cases);(g)patients who performed motor, psychological and sexual rehabilitation (10 cases).In order to compare the outcomes of performing different types of rehabilitation, the study has been segmented in seven cohorts, in which A 7-step methodology is suggested to assess the impact of performing cardiac rehabilitation . Step (1) The ICF values before and after rehabilitation for the ICF categories of et al. [et al. [f et al. . Table 6 [et al. . The visStep (2) The EQ-5D-5L values related to the ICF values found in Step 1 are identified. The 0–4 range of ICF values is mapped into the 1–5 range of the EQ-5D-5L questionnaire to obtain the weighted health status of each patient before and after cardiac rehabilitation.Step (3) The values of the five top items in the EQ-5D-5L can be calculated using a utility-weighted algorithm [lgorithm , which hStep (4) These health-status weights are used in QALYs as quality-weighted years of life. When used in order to calculate the gains in terms of life expectancy, quality-adjusted after rehabilitation, the number of QALYs gained is obtained as in Equation (1).L is the life expectancy for the subject at age a; i is the condition of a person after the rehabilitation process; t represents the individual years within the range of life expectancy; r is the discounting rate along years (r = 0.03); Q is the weight of the quality of life related to a year of life, calculated from the EQ-5D-5L values. Therefore, iL and iQ are the life expectancy and the weight of the quality of life after the rehabilitation process. We assume iL = L, which means the same years of life expectancy before and after rehabilitation, standardized to 80 years in males and 82.5 in females [ females .Step (5) In order to make a global economic assessment, we convert QALYs gained to DALYs averted, as in Equation (2). QALYs and DALYs are related by a conversion factor Ca, defined in Equation (3), where C = 0.1658 and β = 0.04, which takes into account the age-weighting implicit in DALYs, giving more weight to ages around 25 years [25 years .(2)(3)Step (6) The cost/DALY is identified, in order to give a global estimate of cost savings after having calculated the DALYs averted. The cost/DALY can be directly obtained from official documents or inferred from the cost associated to a disability in terms of DALYs lost, as in Equation (4).Step (7) The overall cost savings due to a health intervention is calculated from the DALYs averted and the cost/DALY, taking into account the cost of the intervention, as in Equation (5).ICF indicators before and after intervention, age, cost of the disease, DALYs lost due to the disease and cost of the intervention. Its outputs are: QALYs gained and cost savings.All steps except for steps 1 and 2 are implemented in a Java library, because if inputs are ICF indicators, they can be easily reused in other domains. The source code of the Java library and Javadoc documentation are freely available at . The libThe assessment of the methodology is performed from patients’ data and the literature about DALYs and QALYs. Following steps 1–3 described in previous section, ICF values are mapped into the EQ-5D-5L questionnaire and, subsequently, into health status weights see . The QALBeyond this comparison among interventions, in order to make a global economic assessment of the cases studied, we converted QALYs gained to DALYs averted (Step 5):myocardial ischemia implies the loss of 227,000 DALYs each year, the 48.0% of which  are because of acute myocardial infarction. Its cost is estimated in Spain at 1,955 million € [The National Health System of Spain and the INE state that illion € , which mCardiac rehabilitation cost is analyzed in recent reports ,35,36. WAlbert undertook a motor rehabilitation program consisting of walking. After the therapy, his exercise test shows an improvement of 3 METs in his functional capacity, translated into a decrease of 1 point (from 1 to 0) in the ICF scale related to mobility. Thanks to the rehabilitation program followed, Albert has experienced a significant improvement in his mobility function which represents a betterment in his quality of life, measured with a gain of 1.8 QALYs, an avoidance of 2 DALYs and a saving for the society of 30,000 €.Juan not only had some problems with his functional capacity, but also suffered from depression and anxiety. After his rehabilitation program consisting of walking and psychological treatment, the BDI registers a great improvement of 15 points translated into a decrease of two points in the ICF scale related to emotional functions. The outcome of completely eliminating moderate pain/discomfort and anxiety/depression is measured in a betterment of quality of life of 3 QALYs gained, 3 DALYs averted and 50,000 € saved.Pedro is a 77-year-old man who had some problems with his functional capacity, as well as erectile dysfunction. After performing motor and sexual rehabilitation, he has experienced an improvement in his mobility and emotional functions, translated into a decrease of three and two points, respectively, in each class of the ICF. This represents a betterment in quality of life of 1.1 QALYs gained, 0.6 DALYs averted  and, thus, 10,000 € saved.psychological rehabilitation at the same time results in little improvement in quality of life, and implies a positive comprehensive socioeconomic cost. As far as the other groups are concerned, they do not significantly diverge from the results obtained taking into account all patients for whom, on average, cardiac rehabilitation results in a gain in QALYs of 0.6 and cost savings of 8,000 €.Based on the results obtained, performing motor, psychological and sexual rehabilitation at the same time results in the highest gain in quality of life and the most saving in costs. On the other end of the spectrum, performing motor and psychological rehabilitation only and at the same time, it is suggested to allocate special attention when deciding about the eligibility for this combination of cardiac-rehabilitation processes. In these cases, according to the results obtained, adding sexual rehabilitation should be considered, if it makes sense for the specific patient under treatment. Nonetheless, it has to be taken into account that these results are based on changes in values of categories of the ICF, a classification which describes the functioning status using only five values. A patient could experience a slight recovery, to which the ICF is not sensitive. This representation in five levels particularly affects results when accounting for psychological changes using as data source the BDI questionnaire, described in Due to changes of little magnitude observed, on average, in the quality of life of patients who undergo motor and These results show the importance of cardiac rehabilitation after acute myocardial infarction. Although other studies had already proved its value by calculating gains in quality of life and cost-utility ratios, those measures were only able to compare interventions that modify QALYs in the same domain. From cost-utility ratios, it can be concluded the most effective intervention based on the minimum cost needed to gain a QALY. Although this outcome gives an objective measure when comparing interventions, it must be applied to the same disease or dysfunction, since costs of treating different pathologies are in most cases not comparable.This study provides a measure to more globally assess the cost savings of performing cardiac rehabilitation so it can be compared to any other intervention in the overall framework of healthcare. Moreover, the methodology proposed is also applicable to measure any kind of recovery that can be expressed as changes in ICF values before and after an intervention.Indicators of health outcomes can provide information about the quality of life gained after an intervention such as cardiac rehabilitation. These indicators, together with information provided by governments about the cost of lost quality of life in population and the cost of intervention to revert this, can be used to assess the impact of cardiac rehabilitation in patients who have suffered, e.g., an acute myocardial infarction. We presented a novel methodology to perform this assessment and validated the benefits of rehabilitation in terms of enhancing quality of life and giving decision support to healthcare policymakers. This methodology, which can be used with data from existing medical health records, takes into account changes in people’s functional status, represented by ICF values, resulting from interventions that produce quality-of-life gains but do not change an underlying medical diagnosis.In order to obtain an economic evaluation, QALYs were converted into DALYs and the cost/DALY indicator was calculated from the cost associated to a disability and the DALYs lost due to that disability. The proposed socioeconomic assessment, which was validated with data of 200 patients taking part in cardiac rehabilitation, showed that this health intervention offers, on average, a gain in QALYs of 0.6 and cost savings of 8,000 €. However, since the methodology presented is based on changes in the ICF, which describes functioning status using a scale of five levels, a patient could experience a slight recovery to which the ICF is not sensitive.We are planning to extend the use of the proposed approach for: (1) automatic socioeconomic impact assessment of other sorts of rehabilitation processes, such as functional, cognitive or respiratory rehabilitation; (2) QALYs evolution in periodic, comprehensive and multidimensional evaluations of people with disabilities of neurological origin; and (3) QALYs evolution in telemonitoring systems of people with functional diversity in which brain-neural computer interfaces are used. Furthermore, the generalization of this methodology to other diseases and rehabilitation processes can be a valuable tool to provide information on and evaluate health policies.


Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.−/−) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.Wild-type  and AR deficient . Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy. Diabetic retinopathy is a common complication of diabetes, and is the principal cause of blindness in working-aged adults. Hyperglycemia clearly initiates the disease process, but which of the sequelae of hyperglycemia are causal in development of the retinopathy is not clear.Activation of aldose reductase (AR) by elevated glucose was one of the first biochemical mechanisms postulated to explain the pathogenesis of diabetic complications Efforts to inhibit this pathway to inhibit diabetic retinopathy in patients have relied heavily on pharmacologic inhibitors of the enzyme −/− animals allow the opportunity to investigate the molecular pathways by which AR acts in hyperglycemia. Thus, the availability of AR−/− animals makes it worthwhile to revisit this topic. In the present study, we investigated the effect of AR deficiency on diabetes-induced degeneration of retinal capillaries in early diabetic retinopathy, and on diabetes-induced pro-inflammatory and pro-oxidant changes in the retina.Generation of animals that are totally deficient in AR is one way to overcome the shortcomings associated with the use of pharmacologic inhibitors of AR. Moreover, AR−/− mice were prepared −/− mice were randomly assigned to become diabetic or remain as nondiabetic group. Diabetes was induced by 5 sequential daily intraperitoneal injections of a freshly prepared solution of streptozotocin in citrate buffer (pH 4.5) at 45 mg/kg of body weight. Insulin was given as needed to prevent weight loss without preventing hyperglycemia and glucosuria . Glycohemoglobin (GHb) was measured by Bio-Rad Total Glycated Hemoglobin Assay  every 2–3 mos and just before animals were sacrificed. Food consumption and body weight were measured weekly. Animals were studied for durations of 10 months or 2 months of diabetes in order to investigate effects of the therapy on retinal histopathology, or molecular and physiologic changes, respectively. These durations were chosen because 2 mos diabetes has been found to result in numerous metabolic alterations which precede (and likely contribute to) the later appearance of vascular histopathology, and 10 mos duration of diabetes has been shown to have developed robust vascular histopathology characteristic of the early stages of the retinopathy.ARTreatment of animals conformed to the ARVO Resolution on Treatment of Animals in Research, as well as to institutional guidelines (Case Western Reserve University IACUC # 2010–0156).After 10 months of diabetes, one retina was fixed in 10% buffered formalin, washed in running water overnight, and digested in elastase  buffer  in an agitating water bath at 37° for 1½ h 2. To each tube, 0.5 mM lucigenin  was added, and photon emission was detected over the following 10 min using a luminometer. Retinal protein was quantified per samples  and luminescence was expressed per milligram protein.At 2 months of diabetes, fresh retinas from animals were analyzed for superoxide production as previously described At 2 months of diabetes, retinal homogenates separated by SDS-PAGE, and transferred to nitrocellulose membrane . Membranes were blocked in Tris-buffered saline containing 0.02% Tween 20 and 5% nonfat milk., washed, and were incubated with anti-rat ICAM-1 , anti-rat iNOS , anti-rat nitrotyrosine , anti-rat p38 MAPK and p-p38 MAPK  for 2 hrs, and then stained with respective horseradish peroxidase coupled secondary antibody  at a dilution of 1∶3000 for 1 hr. After extensive washing, immunostaining detected by the antibodies was visualized by enhanced chemiluminescence . The protein levels were quantitated relative to ß-actin-loading control  in the same samples. Results are expressed as a percent of values detected in the nondiabetic controls. Protein was quantified with the Bio-Rad protein assay .At 2 months of diabetes, tissues of interest were homogenized in 0.1 M phosphate buffer (pH 8.0) containing 5 mM EDTA. After centrifugation, a quantity of the resulting supernatant was removed for storage at −80°C for subsequent protein determination by the bicinchoninic acid protein assay method . GSH determinations were carried out on the freshly prepared tissue extract following the fluorometric method described previously 2 and 95% air, and the media was changed every other day for 5 days. When mRECs were 80% confluent , leukocytes  from male C57Bl/6J mice and AR−/− mice groups were added to the mREC and incubated for 24 hrs. After incubation, the leukocytes were carefully removed by gentle washing, and viability of remaining retinal endothelial cells was measured by trypan blue extrusion. Briefly, an aliquot of the endothelial cell suspension was diluted 1∶1 (vol/vol) with 0.1% trypan blue, and the cells were counted with a hemocytometer. Cell death was reported as the percentage of blue-stained cells (dead cells) of the total number of cells. Approximately 200–400 cells were counted in each sample.A mouse retinal endothelial cell line  were all significantly greater than nondiabetic values (132–137 mg/dl). The degree of hyperglycemia, as denoted by glycated hemoglobin, did not vary among diabetic mice , and was significantly greater in diabetic groups as compared with the corresponding nondiabetic groups . Body weight at 10 months of diabetes averaged 28 g±1in the wildtype group and 27 g±2 in AR−/− group, and these values were about half of those in nondiabetic mice of each group (51–60 g). Data from the 2 month experiment was similar (not shown). Deletion of AR had no apparent effect on glycemia or health of any animals. Cross-sections of retina in nondiabetic animals (12 mos of age) indicated that deletion of AR had no significant effects on retinal thickness or retinal architecture, and no effect on vascular density in nondiabetic animals.Diabetic mice were hyperglycemic over the entire duration of the 10 month experiment, and failed to gain weight at normal rate. Serum glucose levels of the diabetic groups , so we could not assess any effect of the AR deletion on neurodegeneration.Wild-type diabetics developed the expected increase in numbers of degenerate retinal capillaries by 10 mos of study (p<0.0001). In contrast, retinas from ARillaries . NondiabIn an effort to investigate the mechanism by which the AR deletion mediated the beneficial effect on degeneration of retinal capillaries in diabetes, we measured several parameters related especially to inflammation and nitrative (oxidative) stress, these being physiologic and molecular abnormalities that have been found in other studies to be associated with  the development of the early stages of diabetic retinopathy.−/− mice were totally protected from diabetes-induced increase in superoxide production (p<0.0001). In contrast, retinal GSH tended to be subnormal in wild-type diabetics, but deletion of AR had little effect on this diabetes-induced reduction , but deletion of AR inhibited the diabetes-induced enzyme induction only for iNOS . The expPrevious studies have demonstrated an important role of leukocytes in development of early stages of diabetic retinopathy AR and the polyol pathway were among the first molecular mechanisms considered to explain how hyperglycemia initiates diabetic retinopathy and other complications of diabetes. At that time, AR was regarded as the enzyme responsible for the reduction of glucose into sorbitol, a molecule that penetrates poorly through cell membranes, thus leading to cellular damage via the “osmotic hypothesis” Use of AR inhibitors revealed a variety of diabetes-induced alterations in retinal metabolism that could be manipulated. AR inhibitors were reported to inhibit diabetes-induced abnormalities in oxidative stress, inflammation, and apoptosis in the retina −/− mice studied by us κB, and both have been shown to be strongly related to the capillary degeneration occurring in retinas of diabetic animals To avoid potential uncertainties associated with pharmacologic inhibitors, mice genetically lacking AR were generated It has been assumed by many that the molecular abnormalities contributing to vascular pathology in diabetic retinopathy are present in vascular cells themselves. In vitro studies have indicated effects of AR within endothelial cells , however, led us to test the possibility that AR present in leukocytes might contribute to the capillary cell death. Our in vitro studies compared leukocyte-mediated killing of retinal endothelial cells by leukocytes having, and not having, AR. These results suggest that AR contributes only little to the hyperglycemia-induced killing of endothelial cells by leukocytes, suggesting that the beneficial effect of AR deletion might in fact be within cells of the retina itself.Our results utilizing AR mutant mice, together with results from other investigators using a similar approach


Common rehabilitation usually allows about 50% of patients with stroke to recover walking, leaving the others not independent in walking and other activities of daily living . For theDifferently from other fields of engineering, studies about the effectiveness of these technologies often occur after their development and their insertion in the rehabilitation settings. A number of studies showed the efficacy of these new technological approaches, whereas some others did not show any improvement in respect of conventional therapies. This uncertainty about efficacy, together with high purchase cost for some of these devices, some difficulties in their use by untrained staff, the absence of clear guidelines about better dosage and parameter values to select, and a somewhat diffuse scepticism by some members of the rehabilitation teams may limit the transfer of these new technologies from research laboratories to clinical settings, where patients are waiting to benefit from them. This special issue aimed to provide an overview on the use of new technologies for the rehabilitation of people with stroke. It contains two reviews and seven original researches. There is a generic review of M. Iosa and colleagues about seven promising technologies for stroke rehabilitation: robots, brain computer interfaces, noninvasive brain stimulators, neuroprostheses, virtual reality, wearable devices, and tablet-pcs. A more specific review is that of E. Q. van Delden and colleagues about twenty different devices for bilateral upper limb training. Upper limb rehabilitation was also the object of the study of G. Thielman and P. Bonsall, reporting the combined use of different technologies such as a unilateral robotic device used for interacting with a virtual task on a monitor, together with an auditory biofeedback for improving the trunk control. Also the study of P. Sale et al. was focused on the use of robotic rehabilitation of upper limb, in their case using a unilateral device designed for hand rehabilitation. Positive outcomes were found in all these studies, suggesting that robotic devices may provide a useful extension of currently available forms of therapy, as also stated in the paper by E. Q. van Delden and colleagues. But this special issue was not only focused on robotic devices. A. R. Bowers and colleagues investigated the on-road detection performance by drivers with hemianopia using oblique peripheral prisms. The study of G. Morone and colleagues showed the potentiality of using a neuroprosthesis not only for correcting the foot drop in chronic phase, but also for improving the sensorimotor relearning during rehabilitation in subacute phase of stroke, facilitating the gait recovery. P. J. Manns and R. G. Haennel investigated the use of a wearable device for assessing walking capacity in people after stroke in terms of energy expenditure and step measurement, concluding that this device is not enough accurate as step counter. So not all technologies can be used also in stroke population, and not in a nonspecific manner. The study of A. Fusco and colleagues, in fact, analysed three different montages of the electrodes of transcranial direct current stimulation, suggesting different effects among anodal montage acting on manual dexterity, cathodal montage improving manual force, and bilateral montage, the less effective. Also, G. Thielman and P. Bonsall suggested that the degree of changes varied per protocol and may be due to the appropriateness of the technique chosen, as well as based on patients impairments.These observations result in line with the proposal to change the research question from “is this specific technology effective?” into “how may I use this technology in an effective way?” as suggested by M. Iosa et al.  and “whiLife of Galileo.This special issue would contribute to provide clear results on the potential benefits of the use of new technologies for patients with stroke, avoiding overstatements of results, and, at the same time, reducing the scepticism of those who say “With machines they would perform miracles. What sort of miracles?” as asked by the Inquisitor in the drama of Bertolt Brecht Marco Iosa Marco Iosa Stefan HesseStefan HesseAntonio Oliviero Antonio Oliviero Stefano PaolucciStefano Paolucci


We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at aminoacid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 , electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies. ► The mitochondrial effects of idebenone in living cells are poorly characterized. ► Idebenone can be toxic or beneficial depending on its oxidation-reduction state. ► Idebenone is toxic through opening of the permeability transition pore. ► Idebenol stimulates respiration and ATP synthesis in cells lacking complex I. ► Short-chain quinones can be developed for the treatment of complex I defects. Here, we present a characterization of the mitochondrial effects of idebenone in human osteosarcoma-derived cybrids bearing wild-type mtDNA (HQB17) or the missense G3460A/MT-ND1 mutation of LHON (RJ206) 22.1p-trifluoromethoxyphenyl hydrazone (FCCP), decylubiquinone, dithiothreitol (DTT), ATP monitoring kit, P1,P5 - di(adenosine-5′) pentaphosphate pentasodium and protease inhibitors were from Sigma . Idebenone was from Apin Chemicals LTD . CsA was purchased from Calbiochem, whereas CsH was a generous gift of Dr. Urs Ruegg, Geneva. Calcium Green 5N was from Invitrogen . Tetramethylrhodamine methyl ester (TMRM) was purchased from Molecular Probes .Oligomycin, rotenone, antimycin A, malonate, malate, pyruvate, carbonylcyanide-2.2Mouse liver mitochondria were isolated in a buffer containing 250 mM sucrose, 10 mM Tris, 0.1 mM EGTA, pH 7.4, as described previously 2.32 + retention capacity (CRC) was determined by measuring external Ca2 + following addition of 10 μM Ca2 + pulses each minute to medium containing 0.5 mg/ml of mitochondria. External Ca2 + concentration was measured fluorimetrically in the presence of 1 μM Calcium Green 5 N, a membrane impermeant Ca2 + probe increasing its fluorescence upon Ca2 + binding . The CRC is the amount of Ca2 + taken up by mitochondria before the precipitous release that follows occurrence of the permeability transition. The CRC values observed at a given concentration of idebenone were normalized to the CRC obtained in the absence of the compound (CRC0).The mitochondrial Ca2.4− cells deprived of their own mtDNA 2.HQB17 and RJ206 cells cybrids were generated by fusion of enucleated fibroblasts derived from one control (HQB17) and one LHON patient  into osteosarcoma 143B.Tk2.5m) in isolated organelles was measured using a Perkin-Elmer LS50B spectrofluorometer and evaluated based on the fluorescence quenching of Rhodamine 123. Mitochondria (0.5 mg/ml) were added to 2 ml of 130 mM KCl, 10 mM MOPS-Tris, 1 mM Pi-Tris, 10 μM EGTA, 0.15 μM Rhodamine 123, pH 7.4. The fluorescence of Rhodamine 123 was monitored at excitation and emission wavelengths of 503 and 523 nm, respectively, with the slit width set at 2.5 nm. After a short incubation to reach stabilization of the signal, further additions were as indicated in the legend to Mitochondrial membrane potential . Cellular fluorescence images were acquired with an Olympus IX71/IX51 inverted microscope equipped with a xenon light source (150 W) for epifluorescence illumination and with a digital camera. For detection of fluorescence 568 ± 25 nm bandpass excitation and 585 nm longpass emission filter settings were used. Images were collected with an exposure time of 100 ms (6% illumination intensity) using a 40X, 1.3 NA oil immersion objective (Olympus). Data were acquired and analyzed using Cell R software (Olympus). Clusters of several mitochondria (10–30) were identified as regions of interest, and fields not containing cells were taken as the background. Sequential digital images were acquired every minute, and the average fluorescence intensity of all relevant regions was recorded and stored for subsequent analysis.For measurements of Δψ2.6Cells were seeded as described above and incubated in bicarbonate- and phenol red-free Hank's balanced salt solution supplemented with 10 mM Hepes and 1.6 μM CsH for 30 min. Alamethicin (50 μM) was added at the end of each experiment to allow complete release of pyridine nucleotides from mitochondria. For detection of fluorescence 340 nm bandpass excitation and 465 nm longpass emission filter settings were used. Images were collected with an exposure time of 800 ms (100% illumination intensity) using a 40X, 1.3 NA oil immersion objective (Olympus).2.74 cells/well in 200 μl of DMEM containing 4.5 g/l glucose (DMEM-high glucose) and incubated at 37 °C in 5% CO2 for 24 h. Assays were initiated by replacing the growth medium in each well with 670 μl of unbuffered DMEM-high glucose prewarmed at 37 °C. The cells were incubated at 37 °C for 30 min to allow temperature and pH equilibration. After an OCR baseline measurement, 70 μl of oligomycin, FCCP, rotenone and antimycin A were sequentially added to each well to reach final concentrations of 1 μg/ml for oligomycin, 0.2 μM (cybrids) or 0.1 μM (XTC.UC1 cells) for FCCP, and 1 μM for rotenone and antimycin A. Data are expressed as pmol of O2 per minute per 2 × 104 cells. At the end of each experiment the medium was removed and cells were incubated with 0.1 μM calcein-AM in 200 μl/well of Hank's balanced salt solution supplemented with 10 mM Hepes and 1.6 μM CsH for 30 min, at 37 °C. Calcein-labeled adherent cells were observed with the Olympus IX71/IX51 inverted microscope  using a 10X, 1.3 NA oil immersion objective (Olympus).Oxygen consumption rate (OCR) in adherent cells was measured with an XF24 Extracellular Flux Analyzer . HQB17 and RJ206 cybrids, and XTC.UC1 cells were seeded in XF24 cell culture microplates (Seahorse Bioscience) at 2 × 102.82, pH 7.4, in the presence of the inhibitor of adenylate kinase  pentaphosphate) and of complex I substrates  by using the luciferin/luciferase assay 4) increases the Mg2 + concentration to a level sufficient to allow activity of the enzyme, but still low enough to minimize the activity of oligomycin-insensitive ATPases. Thus, although the measured ATP synthesis rate was entirely of mitochondrial origin (as indicated by its full sensitivity to oligomycin) the actual figures may be underestimated because of the concomitant occurrence of ATP-hydrolyzing reactions. The reaction was started by addition of 0.1 mM ADP and chemiluminescence was determined as a function of time with a luminometer. The chemiluminescence signal was calibrated with an internal ATP standard after addition of 10 μM oligomycin. Data were normalized for citrate synthase activity The rate of mitochondrial ATP synthesis was measured in digitonin-permeabilized cells incubated in 150 mM KCl, 25 mM Tris–HCl, 2 mM EDTA, 0.1% BSA, 10 mM K-Pi, 0.1 mM MgCl2.9Unless otherwise stated in the figure legends, each experiment was repeated 3 times. Data are presented as average ± S.E. or, for clarity, as representative experiments (see figure legends for details).32 +, which is the expected response to Ca2 + accumulation (A). Subsequent addition of 50 μM idebenone triggered fast and complete mitochondrial depolarization (trace a) which was delayed and partially prevented by CsA (trace b), consistent with the PTP-inducing effects of idebenone Fig. 1B). The latter finding is novel and of some interest, because it suggests that idebenone may interact with a PTP regulatory site that is sensitive to oxidation-reduction events. This point was investigated further. Following accumulation of a permissive Ca2 + load isolated mitochondria treated with idebenone underwent the expected PTP-mediated swelling response , while pore opening was prevented by treatment with a low concentration of N-ethylmaleimide , which we have shown to block PTP-regulating sulfhydryl groups that are also sensitive to reduction with DTT Following energization with succinate, mitochondria underwent a small and transient depolarization upon the addition of Camulation A. Subseqm in intact cells based on accumulation of TMRM in HQB17 cybrids containing normal mtDNA. Addition of 50 μM idebenone was followed by a rapid drop of fluorescence of TMRM , which indicates fast mitochondrial depolarization. This fluorescence decrease was largely prevented by treatment with both CsA  and decylubiquinone , reagents that inhibit the PTP at different sites Fig. 2B, trace a) in a process that was prevented by CsA  or decylubiquinone  but still inducible with the pore-forming peptide alamethicin . Addition of rotenone after idebenone or alamethicin was not followed by NAD(P)+ reduction , indicating that PTP opening  causes the release of mitochondrial pyridine nucleotides rather than just their oxidation, at variance from treatment with FCCP alone (results not shown). The PTP-inducing effects of idebenone could be due to increased production of ROS, which has been shown to occur upon treatement of submitochondrial particles with this compound We next tested the effects of idebenone on Δψopen symbols). Addition of 50 μM idebenone was followed by a decreased OCR, which could no longer be stimulated by FCCP (gray symbols). The protective effects of CsA were negligible (closed symbols), in spite of preservation of the pyridine nucleotide pool.The bioenergetic consequences of treatment with idebenone were also assessed by measurements of oxygen consumption with the sensitive Seahorse technology. Respiration of HQB17 cells was substantially inhibited by oligomycin, indicating a good phosphorylation capacity; it was stimulated well above basal levels by uncoupler, indicating a large respiratory reserve; and it could be largely inhibited by rotenone with no further effects of antimycin A (A). Strikingly, however, the addition of DTT plus idebenone (i) prevented the depolarizing effects otherwise induced by the latter ; and (ii) allowed maintenance of the mitochondrial membrane potential after inhibition of complex I by rotenone, depolarization requiring the addition of antimycin A . These results are consistent with electron transfer to complex III. We assessed whether this effect could be also observed after treatment of cells with idebenol obtained by reduction of idebenone with sodium dithionite Fig. 5A), RJ206  or XTC.UC1 cells  with rotenone plus oligomycin caused a rapid depolarization  suggesting that oxidation of idebenol (i.e. generation of idebenone) causes sensitization of the PTP. Indeed, in the presence of CsA the Δψm was preserved and depolarization was only observed after the addition of antimycin A , indicating electron feeding at complex III.We then tested whether the PTP-inducing effects of idebenone observed in isolated mitochondria could be prevented by reduction with DTT in cells as well. DTT alone was unable to sustain the membrane potential after addition of rotenone to oligomycin-treated HQB17 cells A. StrikiA,A′), RJ206  and XTC.UC1 cells .The next questions we addressed were whether idebenol can improve respiratory performance in cells where activity of complex I is compromised, and whether inhibition of complex I occurs in cells with a normal respiratory function. To answer these questions we studied respiration of HQB17 A,A′, RJ2), RJ206 B,B′ and C1 cells C,C′.A; compare with dashed trace for the same experiment carried out in the absence of idebenol, data taken from Fig. 6A′). OCR could not be stimulated further by FCCP, but it became insensitive to rotenone while it could be inhibited by antimycin A, consistent with direct electron transfer to complex III . DTT alone inhibited maximal respiration, suggesting that the inhibitory effect on complex I may be caused by DTT rather than idebenol. In the absence of idebenol DTT was unable to sustain respiration after addition of rotenone .In HQB17 cells the addition of idebenol decreased basal respiration, which showed virtually no response to oligomycin, FCCP and rotenone . Like in HQB17 cybrids, treatment with idebenol made respiration totally insensitive to oligomycin, FCCP and rotenone (B). Also in this case, DTT plus idebenone allowed coupled (i.e. oligomycin-sensitive) respiration to occur in spite of a marked toxicity of DTT alone (B′).RJ206 cells displayed a lower OCR than wild-type cells and a normal response to oligomycin, but could not be stimulated beyond the initial rate by FCCP, indicating no reserve respiratory capacity B. Like irotenone B. Also iTT alone B′.C′).XTC.UC1 cells slightly improved their performance after the addition of idebenol, and displayed a very remarkable increase of the oligomycin-sensitive  OCR with DTT plus idebenone in spite of the inhibitory effect of DTT alone C′.2 + was added thus preventing PTP opening which is strictly Ca2 +-dependent. Remarkably, idebenol improved ATP synthesis in all cell types, and it allowed oxidative phosphorylation to proceed after inhibition of complex I with rotenone and of succinate dehydrogenase with malonate  that idebenone induces PTP opening in isolated mitochondria Reduced ATP synthesis and increased oxidative stress are believed to sensitize retinal ganglion cells to apoptosis, which leads to blindness Idebenone strongly activates glycerol phosphate oxidation in brown adipose tissue mitochondria, an effect that could be traced to release of the inhibition of glycerol phosphate dehydrogenase by endogenous free fatty acids A pathway for electron transfer from NADH to mitochondria has recently been identified with the demonstration that idebenone is a good substrate of cytosolic dicoumarol-sensitive NQO1 Besides anecdotal reports on the effects of idebenone therapy in various conditions The in vivo studies provided encouraging clinical evidence that idebenone is safe and well tolerated. Since idebenone is transformed into its metabolites QS-10, QS-6 and QS-4 within minutes of administration, and parent idebenone is not detectable in plasma after about 1 h


Sesamum indicum L.) is one of the most important oil crops; however, a lack of useful molecular markers hinders current genetic research. We performed transcriptome sequencing of samples from different sesame growth and developmental stages, and mining of genic-SSR markers to identify valuable markers for sesame molecular genetics research.Sesame  and 4,440 SSRs (SSRs ≥18 bp) were identified. On average, there was one genic-SSR per 6.55 kb (SSRs ≥15 bp) or 10.81 kb (SSRs ≥18 bp). Among perfect SSRs (≥18 bp), di-nucleotide motifs (48.01%) were the most abundant, followed by tri- (20.96%), hexa- (25.37%), penta- (2.97%), tetra- (2.12%), and mono-nucleotides (0.57%). The top four motif repeats were (AG/CT)n , (CA/TG)n [281 (7.65%)], (AT/AT)n [215 (5.85%)], and (GAA/TTC)n [131 (3.57%)]. A total of 2,164 SSR primer pairs were identified in the 4,440 SSR-containing sequences (≥18 bp), and 300 SSR primer pairs were randomly chosen for validation. These SSR markers were amplified and validated in 25 sesame accessions . 276 (92.0%) primer pairs yielded PCR amplification products in 24 cultivars. Thirty two primer pairs (11.59%) exhibited polymorphisms. Moreover, 203 primer pairs (67.67%) yielded PCR amplicons in the wild accession and 167 (60.51%) were polymorphic between species. A UPGMA dendrogram based on genetic similarity coefficients showed that the correlation between genotype and geographical source was low and that the genetic basis of sesame in China is narrow, as previously reported. The 32 polymorphic primer pairs were validated using an F2,164 genic-SSR markers have been developed in sesame using transcriptome sequencing. 276 of 300 validated primer pairs successfully yielded PCR amplicons in 24 cultivated sesame accessions. These markers increase current SSR marker resources and will greatly benefit genetic diversity, qualitative and quantitative trait mapping and marker-assisted selection studies in sesame. Sesamum indicum L., 2n = 26), belonging to the Pedaliaceae genus, is an ancient oilseed crop, considered important for its high quality seed oil [http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567). In China, one of the main long-term hindrances in sesame production is the lack of varieties with high disease resistance and water-logging tolerance. Genetic diversity among cultivars is relatively low since all varieties are derived from the one cultivated sesame species, Sesamum indicum L. The low level of polymorphism in sesame has been demonstrated using universal markers such as random amplified polymorphic DNA (RAPD) [Sesame (seed oil . Sesame A (RAPD) , inter-sA (RAPD) , amplifiA (RAPD) . InadequSince massive-scale cloning and sequencing of DNA or EST libraries has been relatively high-cost, low throughput and time-consuming, the development of SSR markers has been slow, making it more difficult to construct a detailed genetic linkage map that can be used in sesame genetics breeding programs. At present, including a recently published set of 40 sesame SSR markers derived from a transcriptome study -9, less 2 mapping population. Our set of SSR markers will provide a useful tool for sesame genetic research and comparative genome analysis.Recent advances in large-scale RNA-seq provide a fast, cost-effective, and reliable approach for the generation of large expression datasets in non-model species -13, and de novo assembled into 342,776 contigs with a length of over 100 bp, and then further assembled into 42,566 uni-scaffolds with a mean size of 1,127 bp using paired-end joining and TGI Clustering tools  and 4,440 (≥18 bp) SSRs, present in 17.21% and 10.43% of the uni-transcripts respectively, were identified in the data.We divided the SSRs into three groups according to the repeat motif classification criteria proposed by Weber , i.e., pIn the imperfect and compound SSR categories, only mono-, di- and tri-nucleotide SSR units were present. All repeat motifs in mono-nucleotide SSR units were of the A/T type. AG/CT, CA/TG and AT/AT repeat motif types were present in di-nucleotide SSR units, while only GAA/TTC repeat motifs were found in tri-nucleotide SSR units. Of the six types of SSR units, mono-mono, di-di-, tri-tri-, mono-di-, mono-tri- and di-tri-nucleotide types were found in both perfect and imperfect compound SSR categories. The di-di-nucleotide type was the most abundant, representing more than 80% of all SSRs.We noted that the proportion of six different SSR unit sizes was not evenly distributed among perfect SSR groups. Different repeat units occurred at frequencies of: 1.99% and 0.57% (mono-nucleotides) 39.97% and 48.01% (di-nucleotides), 28.45% and 20.96% (tri-nucleotides), 5.17% and 2.12% (tetra-nucleotides), 10.05% and 2.97% (penta-nucleotides), and 14.37% and 25.37% (hexa-nucleotides), for SSRs ≥15 bp and ≥18 bp, respectively  and 3,674 (SSRs ≥18 bp) perfect SSRs n di-nucleotides were the most abundant ; SSRs ≥18 bp, 1,268 (34.51%)), followed by (CA/TG)n di-nucleotides (469 (7.23%) and 281 (7.65%)), (AT/AT)n di-nucleotides (390 (6.01%) and 215 (5.85%)), and (GAA/TTC)n tri-nucleotides (279 (4.3%) and 131 (3.57%)).Further analysis indicated that the copy number of different repeat motifs in perfect SSRs sequences was distributed unevenly Table . The copUsing Primer3, 2,164 SSR primer pairs were detected in the 4,440 SSR-containing sequences (SSR ≥18 bp) and 300 SSR primer pairs were randomly selected and synthesized to validate their level of polymorphism  were polymorphic between the parents of our mapping population (M16 and M17). After screening the 96 FIn order to identify useful SSR markers and obtain transcriptomic information on disease resistance and developmental processes, we sequenced the transcriptomes of 24 sesame samples and identified 2,164 genic-SSR primer pairs (SSRs ≥18 bp).Genic-SSR markers are considered to have strong potential for genetic analysis and linkage map construction in crop species due to their specificity and high degree of conservation -21. AlthHere, to accurately analyze the frequency of SSRs in the transcribed regions of the sesame genome, we compared the numbers and types of SSR motif sequences of SSRs ≥15 bp and ≥18 bp. A total of 7,324 (17.21%) (SSRs ≥15 bp) and 4,440 SSRs (10.43%) (SSRs ≥18 bp) were identified in 42,566 uni-transcript sequences, with an average of one SSR per 6.55 kb and 10.81 kb, respectively. By the parameter of sequence length (Kb) per SSR marker, the distribution frequency of genic SSRs is both lower than that of previous EST-SSRs developed from EST sequences in sesame (8.68% (SSRs ≥18 bp), one EST-SSR per 4.99 kb) . FurtherRosaceae species, the most abundant repeat motif type was the di-nucleotide [Arabidopsis[Of the perfect repeat motifs types, tri-nucleotide repeats have generally been observed to have the highest frequency in many crops, including cotton, barley, wheat, maize, sorghum, rice and peanut -27. Howecleotide ,28. Hexacleotide ,30. As iabidopsis, soybeanabidopsis and peanabidopsis, but difabidopsis,32, the abidopsis,27, the To determine the level of polymorphism among our set of new genic-SSR markers, we validated 300 primer pairs using 25 sesame accessions. 276 (92.0%) successfully yielded PCR amplicons, in line with previously reported ratios of 60–92.2% amplification ,28,33-36Some reports indicated that the low polymorphism of SSR markers in sesame is likely due to its narrow genetic basis ,8. Dixitde novo RNA sequencing approach used here is well-suited for mining and developing large numbers of genic-SSRs in sesame, and can rapidly enrich the numbers of functional markers available to use in marker-assisted gene selection and QTL analysis.Our results indicate that large numbers of polymorphic SSR markers can be obtained when large volumes of transcript sequences or datasets are used, even though genetic diversity is restricted in sesame cultivars. Compared with other SSR detection methods, the Our dendrogram, based on genetic similarity results, did not divide our sesame accessions into clear groupings. The distribution of these sesame accessions was not based on their geographical sources, in agreement with some previous reports -5. The aWe anchored 14 of our newly developed genic-SSR markers in the sesame genetic map , one wild species  and their distant hybrid progeny. Samples were grown under normal conditions in a greenhouse at 25°C with 14 h light per day, or in an experimental field at Yuanyang Experimental station, HAAS. To evaluate biotic stress, seedlings were inoculated with a 106/mL conidiophore suspension of Fusarium oxysporum f. sp. sesami (No. HSFO 09030) for 0, 6, 24 or 48 h at 25°C in a greenhouse before harvesting. Control plants were inoculated with sterilized water. Plant parts, including the whole seedling, developing seeds (harvested at different days after flowering (DAF)), germinated seeds, and developing flowers (1-8 mm size), were harvested, immersed in liquid nitrogen and stored at -70°C before RNA extraction.The 24 samples analysed in RNA-seq experiments  according to the manufacturer’s instructions and total mRNA was then purified using oligo (dT) magnetic beads. cDNA libraries were prepared according to Illumina sequencing sample preparation protocols. In total, 24 paired-end cDNA libraries were constructed with an insert size ranging from 280 bp to 320 bp.de novo assembler Velvet and Oases [cDNA libraries were sequenced on an Illumina sequencing platform (GAII) using a 75 bp or 100 bp paired-end approach. Integrated high-quality paired-end Illumina reads (>Q20) were assembled using the nd Oases . After and Oases .To detect SSR markers, 42,566 uni-transcript sequences containing 2-6 repeat motifs were screened using SSRIT , and mon2, 0.1 mmol/L dNTPs, 1 μmol/L of each primer, 0.5 U Taq polymerase, and 80 ng template DNA. SSR-PCR amplification was performed on a PTC-225 machine  using the following profile: 1 cycle of 3 min at 94°C, 31 cycles of 1 min at 94°C, 50 s at 56–63°C, 1 min at 72°C and a final cycle of 6 min at 72°C. Amplicon electrophoresis was performed as described by Zhang et al. [To validate the SSR markers, genomic DNA was extracted from 25 accessions as described by Paterson et al. . DNA ampg et al. .PIC = 1-Pi2, where Pi is the frequency of the ith allele for a given SSR marker, and n is the total number of alleles detected for that SSR marker [2 segregation population, 14 of which were integrated into the first sesame genetic linkage map using JoinMap ver. 3.0 program [To estimate the allelic variation of SSRs in the 25 accessions, the polymorphism information content (PIC) of each SSR primer was calculated as following: R marker . CoefficR marker . A neighR marker ,47 of NT program .The authors declare that they have no competing interests.ZHY designed the study and finalized the manuscript. WLB carried out the SSR mining and validating experiment and drafted the manuscript. MHM coordinated the study, prepared the materials for transcriptome sequencing and performed the transcriptome information analysis. ZHT and WCY screened the SSR markers and mapping. Transcriptome sequencing and assembly was outsourced to Illumina, China. All authors read and approved the final manuscript.Characteristics of sesame genic-SSR primers used in this study. The SSR primer name, primer sequence, annealing temperature, repeat motif, product length, allele no., PIC value, E-value (nr) and annotation (nr) are given.Click here for file24 sesame samples used for RNA-seq.Click here for fileCharacteristics of the 25 sesame accessions used in the SSR validation. M1 ~ M8 are exotic sesame accessions from 8 countries; M9 ~ M16 are China released sesame cultivars, M17 ~ M24 are China local sesame accessions, M25 is a wild species (Sesamum radiatum). (DOC 51 kb)Click here for file


Genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-AR) have a pivotal role in the functions of the autonomic nervous system. Using heart rate variability (HRV) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-AR gene polymorphisms and heart rate dynamics.1-AR Ser49Gly, β2-AR Arg16Gly and β2-AR Gln27Glu. Each subject underwent two hours of electrocardiogram monitoring at rest. We applied an information-based similarity (IBS) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects.A total of 221 healthy Han Chinese adults  were recruited and genotyped for three common β-AR polymorphisms: β2-AR Arg16Gly genotype. Furthermore, the non-randomness index, a nonlinear HRV measure derived from the IBS method, was significantly lower in Arg16 homozygotes than in Gly16 carriers. The non-randomness index was negatively correlated with parasympathetic-related HRV variables and positively correlated with those HRV indices reflecting a sympathovagal shift toward sympathetic activity.With the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β2-AR polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control.We demonstrate a bottom-up categorization approach combining the IBS method and hierarchical cluster analysis to detect subgroups of subjects with HRV phenotypes associated with β-AR polymorphisms. Our results provide evidence that β Instantaneous heart rate in response to physiological perturbations often exhibits remarkable oscillations at multiple time scales. These oscillations, known as heart rate variability (HRV), are mainly mediated by the autonomic nervous system via parasympathetic and sympathetic innervations. Analysis of HRV has been suggested to reveal subtle patterns of heart rate dynamics that are relevant to the underlying physiological state and autonomic nervous system function AR) as an example.Despite increasing focus on investigating the genetic influence on autonomic functions, current approaches to genotype–HRV associations have largely been characterized by a top-down approach involving a direct comparison of continuous HRV variables among pre-defined groups of subjects , yet it is unclear how a particular genetic polymorphism may determine a similar pattern of autonomic heart rate control from one subject to another. Specifically, heart rate dynamics is a phenotypic “expression” of the autonomic nervous system, so comparing similar heart rate oscillation phenotypes among individuals may reveal a global profile of autonomic function relevant to genetic variants. With these considerations in mind, in the present study, we introduce a bottom-up genotype–phenotype analysis to investigate the association between genetic polymorphisms and autonomic control of heart rate dynamics, using three common polymorphisms in genes encoding β-adrenergic receptor  a non-randomness index AR genotype and to test the correlation of this index with standard HRV indices; and 2) using agglomerative hierarchical cluster analysis, we unsupervisedly categorized these subjects into clusters based on pairwise dissimilarity among heart rate dynamics, and then we investigated the association of these clustering patterns with β-AR gene polymorphisms. We show that this bottom-up, categorization approach combining the IBS method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with β-AR gene polymorphisms.We applied an information-based similarity index (IBS) Two hundred forty-seven healthy Han Chinese adult volunteers were recruited from two medical centers: Taipei Veterans General Hospital and Kaohsiung E-DA Hospital, Taiwan. Subjects were recruited by advertisement among medical employees, research laboratory staff working at both hospitals, and their relatives. All subjects gave informed consent before commencement of the study. The protocol was approved by the institutional review boards of the Taipei Veterans General Hospital  and E-DA Hospital . Each subject was given an interview using a standard questionnaire to carefully review the history of medical disease, psychiatric illness, and medication use. Subjects included in the study did not have a personal history of medical conditions , pregnancy, psychiatric illnesses or substance abuse/dependence. None of the subjects was taking any medication. The collected demographic data included age, sex, body mass index, and smoking. Of note, most volunteers were hospital colleagues, and the rate of smoking was low .BDNF gene Of these subjects, 228 were successfully contacted for ambulatory electrocardiogram (ECG) monitoring. Holter recordings  were used to obtain two hours of ECG signals. The E3-80 device continuously recorded three channels of ECG signals at a sampling rate of 250 Hz. All ECG monitoring took place in the daytime, and participants were asked to avoid smoking or drinking alcoholic beverages and to stay in a resting state while being monitored. Valid DNA samples were obtained in 221 subjects by drawing blood or by buccal swabs. The final study sample therefore consisted of 221 healthy adult subjects . Among the present study sample, 211 subjects have been included elsewhere in a previous report on the altered sympathovagal balance associated with Val66Met polymorphisms of the Each subject was genotyped for three polymorphisms , and genomic DNA was isolated using the PUREGENE DNA purification system . The genotypes of rs1042713 were determined using polymerase chain reaction and restriction fragment length polymorphism analysis. Briefly, primers and probes were designed with SpectroDESIGNER software . PCR was then performed, and unincorporated double-stranded nucleotide triphosphate bases (dNTPs) were dephosphorylated with shrimp alkaline phosphatase  followed by primer extension. The purified primer extension reaction product was spotted on to a 384-element silicon chip  and analyzed in a Bruker Biflex III MALDI-TOF SpectroREADER mass spectrometer (Sequenom). The resulting spectra were then processed with SpectroTYPER (Sequenom). All samples were genotyped for eight unrelated SNPs for DNA quality examination. The samples were diluted onto 96-well plates, and only the plates on which each of the eight unrelated SNPs had a successful genotyping rate greater than 95% were used for further study. All experiments were performed by investigators who were blind to phenotype. Failure in genotyping for rs1801252 polymorphism was noted in 6 cases.The ECG signals were automatically processed and analyzed by open-source HRV algorithms http://physionet.org), a research resource for complex physiologic signals In addition, we incorporated two nonlinear HRV indices: detrended fluctuation analysis (DFA) 1 (4 to 11 heartbeats) and the long-term scaling exponents α2 (>11 heartbeats) were also calculated In the DFA method, the root-mean-square fluctuation of integrated and detrended time series is measured at different observation windows and plotted against the size of the observation window on a log-log scale. The scaling exponent α is then derived from the slope of line fitting to the obtained log-log plot. The short-term exponent αMSE has been proposed as a biologically meaningful complexity measure by quantifying the entropy over multiple time scales inherent in physiologic signals. The procedure and calculation of the MSE is summerized as following three steps: 1) construction of coarse-grained time series, 2) quantification of the sample entropy of each coarse-grained time series, and 3) summation of the sample entropy values over a range of scales. In the present study, sample entropy was calculated using a pattern length (m) of 2 and a similarity factor (r) of 0.15. The sum of sample entropy over all scale factors from 1 to 20 was computed to represent the overall MSE measure. In addition, the sum of sample entropy over scale factors from 1–5 and 6–20 was calculated to represent short-term and long-term MSE measures, respectively Several methods of symbolic dynamic analysis of HRV have been proposed previously 0 and a deceleration of heart rate is designated as 1  is mapped to a symbolic sequence, according to a mapping rule that accelerated heart rate in consecutive heartbeats is designated as ted as 1 . This waTo compare the similarity between symbolic sequences, we plotted the rank number of each n-tuple sequence in the first sequence against that of the second sequence . If two n-tuples between two sequences, n is the number of different n-tuple sequences (or patterns). The absolute difference of ranks, H for http://www.physionet.org/physiotools/ibs/).We defined the distance AR genotypes.The applications of the IBS method in the present study were two-fold: 1) The pairwise distance of heart rate dynamics among individuals was calculated by an average of IBS distance using n-tuple symbolic sequences (n = 2–10) and was used in subsequent hierarchical cluster analysis. 2) To quantify the nonlinear aspect of heart rate time series, a non-randomness index was defined to measure the symbolic patterns of heart rate dynamics away from complete randomness  AR genotype. The spectral HRV indices were log-transformed to produce normalized distributions. To control for the effects of non-genetic factors, differences in HRV variables were compared for individual genotypes using a general linear model (GLM) followed by the Bonferroni post hoc test for corrections of multiple between-group comparisons., using age, gender, and body mass index as covariates. Effect sizes were calculated with Cohen d, defined as the difference between the means of two groups, divided by the pooled standard deviation of these groups. Partial correlation analysis was applied, controlling for age and BMI, to determine the associations between standard HRV indices and non-randomness index derived from the IBS method. A p value of less than 0.05 (two-tailed) was required for all statistical comparisons.We calculated allele and genotype frequencies and performed Hardy–Weinberg equilibrium tests for each β-http://gap.stat.sinica.edu.tw/). GAP is implemented here as an unsupervised clustering algorithm to visually categorize all subjects based on the dissimilarity matrix, which is derived from calculating the pairwise dissimilarity of heart rate dynamics among subjects using the IBS method. Cluster-specific association was analyzed by the chi-square test to assess the frequency of β-AR polymorphisms and by the GLM model to test the differences in standard HRV indices among resulting clusters. To validate cluster-specific associations, we tested the differences in HRV and β-AR genotypes in estimated clusters based on first- and second-half ECG data.A complete-linkage, hierarchical clustering tree was estimated using the generalized association plot (GAP) , β2-AR Arg16Gly genotype  or β2-AR Gln27Glu genotype .Demographic and clinical data for subjects with each β-AR genotypes with HRV indices determined by a GLM model using age, gender, and BMI as covariates. There was no significant effect of interaction on HRV indices detected between β-AR genotypes and demographic variables.1-AR Ser49Gly genotype was associated with mean heart rate and SDNN with borderline significance . The Gly16 and non-Gly16 carriers trended toward a significant difference in LF% (p = 0.069).There was no significant difference in HRV indices between heterozygotes Arg16/Gly16 and homozygous Gly16 carriers, indicating a dominant association of the Gly16 allele with HRV indices. Thus, when the sample was stratified according to the presence of the Gly16 allele, a significantly lower non-randomness index was detected in Arg16 homozygotes compared to subjects with one or two Gly16 alleles (p = 0.001). The effect size of the non-randomness index among Gly16 and non-Gly16 groups was 0.22  measure of heart rate dynamics. We first estimated the pairwise distance between interbeat interval time series of all subjects using the IBS method. No violation of the triangular inequality was observed. We then applied a hierarchical clustering algorithm to cluster these subjects based on the obtained distance matrix. Two major clusters were identified in the resulting dendrogram (AR genotypes and standard HRV characteristics according to two major clusters. A significant deviation in the distribution of homozygous β2-AR Arg16 carriers was detected between the two clusters (p = 0.011), whereas the other genotypes showed no deviation in genotype distribution.Next, we investigated the β-ndrogram . There w2-AR Arg16 carriers) showed significantly reduced LF power (p<0.001) and significantly increased HF power (p = 0.001). The clusters did not differ in other HRV indices. We verified the genotype distribution with clusters based on first- and second-half ECG data, and the results were consistent . First, our method enables us to cluster heart rate dynamics by an IBS method based on their acceleration/deceleration patterns of heartbeat sequence. The IBS method was not exclusively developed for the analysis of heartbeat sequence but also for other generic datasets consisting of repetitive elements 1-AR is the predominant β-AR subtype in the heart. Compared to activation of β2-AR, stimulation in β1-AR can result in more significantly increased frequency and contractility of heart. Although substantial efforts have been made to link β1-AR polymorphisms with cardiovascular function, studies have been mixed in this regard. For example, β1-AR Ser49Gly polymorphism was found to be associated with increased blood pressure and heart rate 1-AR, further research is needed to identify other possible β1-AR polymorphisms associated with HRV.β2-AR Arg16Gly genotype was associated with clusters based on acceleration/deceleration patterns of heart rate. Although β2-AR is expressed in the heart at lower concentrations than is the β1-AR subtype, it also expresses on the presynaptic sympathetic nerve terminals and expresses abundantly in vascular and bronchial smooth muscle. Therefore, changes in β2-AR function may not only alter sympathetic activity 2-AR Arg16Gly polymorphism and heart rate fluctuation patterns may simply reflect a functional genetic component nearby due to linkage disequilibrium and warrants future genetic research targeting on this region.This study found that β2-AR Arg16 carriers are in line with prior studies showing that the β2-AR Arg16 polymorphism is associated with lower sympathetic activity, manifested as lower blood pressure The non-randomness index was developed initially as a nonlinear HRV index AR genetic polymorphism with HRV seen in this study. Second, we were unable to evaluate the correlation of the non-randomness index with other commonly used sympathetic indicators, such as blood pressure or plasma catecholamine levels. Third, our findings are based on a healthy population and may not be generalizable to medically ill patients, such as those with cardiovascular diseases. The cross-sectional nature of our study also limited us examining the age modification of the effect of β-AR receptor polymorphism on autonomic function.Strength of this work include a larger sample size, matched gender distribution, and long recording of ECG signals using a Holter recorder, compared to prior genetic association study of HRV. Several limitations influence the interpretation of the findings presented in this study. First, we are unable to repeat findings of spectral HRV indices seen in a smaller male sample 2-AR gene polymorphisms. Further research should aim to identify the physiological mechanisms underlying these findings.In conclusion, this study shows that a bottom-up, categorization approach combining the IBS method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with βTable S1Demographic data according to β1-adrenergic receptor Ser49Gly genotype.(DOC)Click here for additional data file.Table S2Demographic data according to β2-adrenergic receptor Arg16Gly and Gln27Glu genotype.(DOC)Click here for additional data file.Table S3Partial correlations between non-randomness index and heart rate variability components.(DOC)Click here for additional data file.Table S4Genotype and standard heart rate variability characteristics according to two major clusters by a generalized association plot.(DOC)Click here for additional data file.


In primary visual cortex, spiking activity that evoked by stimulus confined in receptive field can be modulated by surround stimulus. This center-surround interaction is hypothesized to be the basis of visual feature integration and segregation. Spiking output has been extensively reported to be surround suppressive. However, less is known about the modulation properties of the local field potential (LFP), which generally reflects synaptic inputs. We simultaneously recorded spiking activity and LFP in the area 17 of anesthetized cats to examine and compare their modulation characteristics. When the stimulus went beyond the classical receptive field, LFP exhibited decreased power along the gamma band (30–100 Hz) in most of our recording sites. Further investigation revealed that suppression of the LFP gamma mean power (gLFP) depended on the angle between the center and surround orientations. The strongest suppression was induced when center and surround orientations were parallel. Moreover, the surround influence of the gLFP exhibited an asymmetric spatial organization. These results demonstrate that the gLFP has similar but not identical surround modulation properties, as compared to the spiking activity. The spatiotemporal integration of LFP implies that the oscillation and synchronization of local synaptic inputs may have important functions in surround modulation. The receptive field surround, which cannot elicit spikes when stimulated alone, can modulate the visual response of the classical receptive field (CRF) when they are stimulated together Previous studies on surround modulation have mostly focused on spiking activity, whereas another important neural signal – local field potential (LFP) is seldom explored in the center-surround interaction. LFP is the slow component (<250 Hz) of extracellular potentials. This potential is the result of the spatial and temporal summation of different electrical events on membranes of a local cell population. It was estimated that synaptic activities, intrinsic membrane oscillations, and spike after potentials, were the main contributors in the LFP of regular-structured brain tissues such as the neocortex Here, we examined the properties of LFP surround modulation and compared it to those of spiking. Our results showed that LFP modulation mainly occurred along the gamma band and the gamma mean power was suppressed by surround stimulation. Moreover, we found that the surround suppression of the gLFP was orientation-dependent, and the orientation selectivity was similar to that of spiking. Finally, we showed that the gLFP exhibited asymmetric surround suppression distributions. We interpreted the origin of our LFP signal and discussed its possible implications.Appropriate measures were observed to minimize pain and discomfort, in compliance with the US National Institutes of Health guidelines on the care and use of laboratory animals. All experimental procedures were approved by the Institutional Animal Care and Usage Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences (ID: SYXK(PTJ)2008-114).2 (3.5–4%), electrocardiogram (180–240 beats/min), and sometimes electroencephalogram, were continuously monitored. The pupils were dilated with homatropine, and the nictitating membranes were retracted with phenylephrine hydrochloride. The eyes were protected using contact lenses of the appropriate refractive power and covered with 4 mm artificial pupils. The location of the area centralis and optic disks were occasionally checked using a reversible ophthalmoscope. Craniotomy was performed at the Horsley-Clarke coordinates P0–P6 and L0.5–L4 to target the area of the primary visual cortex that represents the central visual field.A detailed description of the procedures used in this study has been previously reported Tungsten microelectrodes  were inserted perpendicular to the cortical surface. The reference electrodes were copper wires placed under the dura, at 3–5 mm away from the recording electrodes. The electrodes were advanced using a hydraulic microdrive . The extracellular potentials were pre-amplified and digitized (12 kHz) before they were sent to TDT System-3 RA16 processors  for filtering and recording. The spikes were extracted by band-pass filtering (Second-order Butterworth) raw signals between 300 Hz and 3000 Hz; after which, the waveform segments that exceeded 5 times the root mean square were cut. The local field potentials were extracted by band-pass filtering (Second-order Butterworth) the raw signals between 1 Hz and 200 Hz, and sampling at 3 kHz.http://stilib.codeplex.com) and were displayed on a cathode-ray-tube monitor  at 1024 × 768 resolution in full screen and 120 Hz refresh rate with vertical synchronization. The display was gamma corrected and placed 57 cm from the cat eyes. The ipsilateral eye was covered, and all stimuli were presented to the contralateral eye.Stimuli were generated by StiLib  was chosen as the center stimulus size, and the smallest size that substantially suppressed the center response was chosen as the surround stimulus size. Optimal center and surround gratings with different orientations were combined  to test The spatial structure of surround suppression was tested by the co-stimulation of the center and different surround positions at a square grid  centeredhttps://www.chronux.org) All data analyses were performed in the Matlab computing environment . Spectrum analysis was performed using Chronux .All spectra were estimated using the multi-taper method Spikes detected by the same electrode may contaminate the LFP even after the band-pass filtering. To determine the degree of this contamination, the LFP segments were cut around each spike to exclude the LFP components that originated from spike waveform, and the linear interpolations were used to replace the original ones s is the LFP power spectrum of the stimulus, and Pcontrol is the power spectrum of the uniform background. We also used the z-score rc are shown here.The visual response of LFP was defined as the change in the power spectrum relative to the control The power spectrum of the spikes was computed using the multi-taper method max is the maximum response and Smax is the corresponding stimulus size. Rmin_surround is the minimum response elicited by stimuli larger than Smax, and Smin is the corresponding stimulus size. When SI = 0, there is no suppression, and the response is either increasing or reaching a plateau. When SI = 1, the response is suppressed to the level of the control. When SI>1, the response is suppressed to a level lower than that of the control.The degree of surround suppression was represented by the suppression index (SI):e is the amplitude of the excitatory component, and σe is the width of the excitatory component. Ki is the amplitude of the inhibitory component, and σi is the width of the inhibitory component. K0 is the constant offset. The goodness of fit was assessed using Adjust-R2. We found the DoG model did not always coincide with the LFP responses (The size tuning curve was fitted using the difference of gaussian (DoG) function esponses . Therefok is the suppression amplitude of kth point in the stimulus grid . Second, we calculated the asymmetry suppression index (ASI) by normalizing the SVS, as follows:The asymmetrical distribution of the surround suppression was represented by the asymmetry vector (AV) lus grid , and θk We successfully acquired 75 recording sites. The majority of the recording sites  were premax = 2.5°, the most suppressive stimulus size Smin = 10°, and the suppression index SI = 0.59.The size-tuning curve is an effective indicator of the nature and spatial extent of surround modulation. max = 1°, Smin = 7.5°, and SI = 0.88, which was larger than that of the MFR. By contrast, the spike oscillation  monotonically increased with the surround size, with SI = 0°, Smax = Smin = 10°. In the gamma band  and high (60–100 Hz) sub bands. The results did not exhibit obvious differences from that of the whole gamma band . The mean SI and corresponding SD of gLFP and MFR were 0.89±0.33 and 0.71±0.26, respectively. However, the two Smax distributions were not significantly different , although they were significantly correlated . The mean Smax and corresponding SD of gLFP and MFR were 2.4° ±1.5° and 2.5° ±1.4°, respectively.The LFP gamma band was most sensitive to the stimulus. Thus, we performed a pairwise comparison of the size tuning properties of MFR and gLFP . The SI The surround suppression of the firing rate is dependent on orientation difference between the surround stimulus and the CRF stimulus To obtain the population responses, we normalized the surround orientation tuning curves under different center orientations. These normalized curves were then averaged across center orientations and across the recording sites. The additional surround stimulus suppressed both MFR and gLFP from the response level of the center stimulation alone . MoreoveThe spatial distribution of surround suppression was investigated by the co-stimulation of SSF and the different surround areas . The moda are presented in a was 0.14. The other ASI medians were all approximately 0.26, thereby indicating that almost half of the population had minimal asymmetrical suppression. Thus, we excluded the almost uniformly distributed surround, and the recording sites with ASI>0.2 were chosen for the analysis of the AD. The LSR of gLFP had the tendency to be located at the optimal orientation line, although, this trend was not statistically significant  that were aligned close to the optimal orientation and direction lines. Furthermore, we examined the relationship of the asymmetry suppression between MFR and gLFP. The distribution of AD difference between MFR and gLFP are illustrated in We used asymmetry vector to indicate the properties of surround suppression distribution. Some sites merely showed suppression asymmetry in our recordings, whereas others clearly showed asymmetrical surround suppression . The disnificant . By contIn the current study, we recorded local field potential and spiking activity simultaneously in anesthetized cats to investigate the surround modulation characteristics of LFP and to compare it to those of firing rate and spike oscillation. Our results can be summarized as follows: (1) The LFP power was suppressed by surrounding stimulus at the gamma band (30–100 Hz), and the size tuning properties of gLFP were similar to those of the firing rate. (2) The spike oscillation power was enhanced by surround stimulation at the beta band (20–30 Hz) and reached a plateau at the gamma band. (3) The surround suppression of gLFP was orientation-dependent, which is similar to that of the firing rate. Meanwhile, the surround facilitation of spike oscillation was not dependent on orientation. (4) The observed gLFP had both uniform and asymmetric surround suppression distributions.A receptors to prolong the decay time-constant of IPSC There have been a few studies, which investigated the LFP surround modulation By excluding possible effects on gamma oscillation caused by visual attention Bauer et al. Previous studies showed that hemodynamic signals have a strong correlation with high-frequency LFP oscillation Consistent with the previous studies Many studies have suggested that LFP mainly reflect synaptic activities and membrane potential oscillations. However, the contribution of different sources could substantially vary because of the network structure, temporal pattern, or functional regime min of gLFP and the firing rate were both approximately 8° in our results. Likewise, spike oscillation reached its maximum at approximately 6°–8°. These spatial ranges were roughly three times the mean diameter (2.4°) of SSF and covered all the adjacent non-overlapping RFs of similar size. This trend implies that the surround modulation in our results may mainly come from neighboring hypercolumns.Receptive field surround suppression has been understood to be primarily mediated by intra-cortical or feedback connections When the center column and its neighboring columns were both stimulated, the gLFP was decreased. As we discussed, this decrease may mainly come from excitatory inputs. Thus, the local excitatory synaptic activities and their synchronization were significantly reduced. This reduction may further decrease the total excitatory input conductance, which was observed in intracellular recordings Figure S1Population responses of LFP with segments around every spike replaced by linear interpolation. To determine whether spike waveforms contaminated LFP, we cut the LFP segments around each spike detected by the same electrode with  time window, and then used linear interpolation to replace the original LFP segments. The results of  (A) and  (B) were shown. The legends are the same as those in (TIF)Click here for additional data file.Figure S2Goodness of fit of the DoG model. Pairwise comparison of the Adjust-R2 of the fitted DoG curves. The mean and SD of Adjust-R2 for the firing rate and gamma power were 0.83±0.16 and 0.71±0.2, respectively. The legends are the same as those in (TIF)Click here for additional data file.Figure S3Distribution of recording depth. During each electrode penetration, we tried to record every isolatable unit. Therefore, the entire depth expansion may correspond to the entire depth of cortex. The depth ratio between layers is more or less constant. Thus, we estimated that majority of our recording sites were located in superficial layers. The black line indicates the median.(TIF)Click here for additional data file.Figure S4Surround suppression of low and high gamma power.  Population-averaged size-tuning curves. The legends are identical to those of (TIF)Click here for additional data file.