This study examined retention rates and associated predictors of methadone maintenance treatment (MMT) duration among 128 newly admitted patients in Taiwan. A semi-structured questionnaire was used to obtain demographic and drug use history. Daily records of methadone taken and test results for HIV, HCV, and morphine toxicology were taken from a computerized medical registry. Cox regression analyses were performed to examine factors associated with MMT duration. MMT retention rates were 80.5%, 68.8%, 53.9%, and 41.4% for 3, 6, 12, and 18 months, respectively. Excluding 38 patients incarcerated during the study period, retention rates were 81.1%, 73.3%, 61.1%, and 48.9% for 3 months, 6 months, 12 months, and 18 months, respectively. No participant seroconverted to HIV and 1 died during the 18-months follow-up. Results showed that being female, imprisonment, a longer distance from house to clinic, having a lower methadone dose after 30 days, being HCV positive, and in the New Taipei city program predicted early patient dropout. The findings suggest favorable MMT outcomes of HIV seroincidence and mortality. Results indicate that the need to minimize travel distance and to provide programs that meet women’s requirements justify expansion of MMT clinics in Taiwan. Treating heroin dependence and stemming the human immunodeficiency virus (HIV) infection amongst heroin users challenge public health professionals and health providers over the last three decades. The use of methadone maintenance treatment (MMT) to treat heroin dependence and reduce infectious diseases has gradually become prevalent. Countries in South East Asia and East Asia, including China, Indonesia, Malaysia, Thailand, and Vietnam, have been launching and expanding MMT programs as an essential response to reduce the harms of injection drug use. This trend of adopting harm reduction as an approach to reduce the ill effects of heroin dependency followed a series of important announcements and recommendations made by international organizations. In 2002, the United Nations Office on Drugs and Crime (UNODC) announced that based on systematically reviewed evidence from addiction studies, drug addiction should be considered a chronic and relapsing disease . Later oMethadone maintenance treatment (MMT) has been shown to reliably reduce drug use , overdosThis paper reports secondary analyses from a larger project entitled “A Prospective Study of Effectiveness of Methadone Maintenance Treatment,” an 18-month multi-site study that collected data on quality of life, mortality and HIV seroconversion as well as on the duration of MMT and associated factors . The resPatients had to meet the following criteria to be included in the study: at least 20 years of age, literate, self-reported usage of heroin within the past 6 months, and currently enrolled in an MMT program. In accordance with the Taiwan Civic Law, being 20 years old is considered as an adult. Of the 599 participants, we analyzed 128 newly admitted patients, who were approached with an explanation of the study’s purpose and procedures within 5 days of starting MMT. The participants were followed semi-annually for 18 months. All participants received 100 Taiwan dollars (approximately 3 US dollars) for each interview.A semi-structured questionnaire was used to obtain demographic and drug use history. The demographic items asked about the participant’s age, age at first heroin use, gender, education, living address, marital status, and employment. Education was categorized based on whether the patients had completed the compulsory 9 years of education in Taiwan. House-to-clinic distance was calculated from the patient’s home address to the MMT clinic using a Google map. Results of HIV and HCV tests, daily records of methadone taken, and morphine toxicology test results were taken from a computerized medical registry. Mortality data were obtained from the death records of the Taiwan Department of Health for the corresponding period. Arrest and incarceration records were provided by the Taiwan Ministry of Justice.Statistical analyses were performed using SPSS 19.0 for Windows. MMT duration was calculated from records of patients’ admission and dropout dates or the last day of follow-up . Cox multivariate regression analyses were performed twice, once on all 128 patients and once on non-incarcerated patients, to predict treatment dropout from age, age at first heroin use, gender, education, house-to-clinic distance, marital status, employment, incarceration, methadone dose after 30 days, and program site. Items with missing values were omitted from the analyses. The criterion for statistical significance was p < 0.05.As shown in The average number of days participants were in MMT was 344.65(203.68). Four patients withdrew from MMT within 30 days of treatment. Retention rates were 80.5% (n = 103), 68.8% (n = 88), 53.9% (n = 69), and 41.4% (n = 53) for individuals who consistently stayed in treatment for 3 months, 6 months, 12 months, and 18 months respectively. Of 128 participants, 38(29.7%) had to leave MMT during the study because they had been convicted of a crime and became incarcerated. For the remaining 90 patients, retention rates were 81.1% (n = 73), 73.3% (n = 66), 61.1% (n = 55), and 48.9% (n = 44) for those who stayed in treatment consistently for 3 months, 6 months, 12 months, and 18 months, respectively. None were seroconverted to HIV positive and one died because of accidents unrelated to overdose. The annual mortality rate was 0.5% per life year.The Cox regression analysis on the 128 patients revealed that patients who were women, imprisoned, HCV positive, lived a long distance from the clinic, low methadone dose after 30 days of admission, and treated in New Taipei City were associated with shorter duration of MMT . The CoxWe found that being male, married, living close to the clinic, living in Taipei City, and being prescribed a higher methadone dosage 30 days after admission, HCV positive and not imprisoned were positively associated with continuing (not dropping out of) MMT. No patient was tested HIV positive and one died because of accidents unrelated to overdose during the study period. These findings are consistent with prior studies showing that staying longer in MMT is associated with such positive patient outcomes as lower overdose mortality, fewer HIV/HCV infections, less drug use overall, fewer injections, and a better quality of life ,15.Studies from Western countries have shown that younger patients are the most likely to drop out of substance abuse treatment programs  and thatOne third of our participants became incarcerated during the 18months of the study, and incarceration was a significant predictor of early attrition. A previous study found involvement in the criminal justice system to be associated with less time in treatment . Legal pHigher methadone doses have also been associated with longer duration in MMT ,22. In aOur finding that distance from the clinic is associated with MMT retention is noteworthy but not surprising, because travel costs are important considerations for patients needing access to treatment. Because methadone is a 24-hour agonist that patients must take every day, they may have to take a half day off from work or spend more money on transportation to keep up with their medication. In such circumstances, reporting daily to the MMT clinic can be a heavy burden in terms of both time and cost for patients who live far from the clinic. Accessibility to clinics has been shown to be a crucial factor influencing MMT adherence and retention ,13. KellThe present study has a few limitations. As our participants were not recruited randomly from methadone clinics in northern Taiwan, the findings may not generalize to other Taiwanese methadone patients. Although our clinic-based sample was large, our participants still may have differed from the general population of heroin users in relevant respects. Examples of such possible differences are demographics, MMT modality, and patterns of drug use. Second, because ours was an observational study with no comparison group, confounding factors such as maturation, local policy change, and testing may have biased our results. The average number of days in treatment was fairly long in our study. Previous studies suggest that patients who remain in MMT programs more than three months are much more likely to stay in the program longer than other patients ,24. The An important strength of this study lies in its ability to use a prospective design. As a result, the conclusions of this study can provide evidence that MMT as a harm reduction program reduce the mortality and new HIV infections. Factors associated with duration of MMT are gender, methadone dose after 30 days, incarceration, program site, HCV and the geographic distance that clients travel to outpatient treatment clinics. Minimizing the travel distance might improve retention and duration of MMT.


The vast majority of deaths in the Kilifi study area are not recorded through official systems of vital registration. As a result, few data are available regarding causes of death in this population.To describe the causes of death (CODs) among residents of all ages within the Kilifi Health and Demographic Surveillance System (KHDSS) on the coast of Kenya.Verbal autopsies (VAs) were conducted using the 2007 World Health Organization (WHO) standard VA questionnaires, and VA data further transformed to align with the 2012 WHO VA instrument. CODs were then determined using the InterVA-4 computer-based probabilistic model.Five thousand one hundred and eighty seven deaths were recorded between January 2008 and December 2011. VA interviews were completed for 4,460 (86%) deaths. Neonatal pneumonia and birth asphyxia were the main CODs in neonates; pneumonia and malaria were the main CODs among infants and children aged 1–4, respectively, while HIV/AIDS was the main COD for adult women of reproductive age. Road traffic accidents were more commonly observed among men than women. Stroke and neoplasms were common CODs among the elderly over the age of 65.We have established the main CODs among people of all ages within the area served by the KHDSS on the coast of Kenya using the 2007 WHO VA questionnaire coded using InterVA-4. We hope that our data will allow local health planners to estimate the burden of various diseases and to allocate their limited resources more appropriately. In developed countries, cause-specific mortality data are readily available from well-established vital registration systems. In developing countries, however, where a majority of the world’s deaths occur, vital registration systems are poor , access Despite the fact that some studies have questioned the reliability of the Verbal Autopsy (VA) approach, where close relatives of the deceased are asked about the signs and symptoms that precede deaths , 3, it r2 and has a current resident population of ~265,000. The population register is updated three times each year and records between 1,200 and 1,500 deaths annually.The KHDSS study area has been described in detail previously . BrieflyVA, using the standard 2007 World Health Organization (WHO) VA tools , were fiCODs were assigned using the InterVA-4 computer-based probabilistic model . The oriAll VA forms were scrutinized by a clinician for anomalies before double entry into a computer database written in FileMaker Pro™ v11 . The VA data were further transformed to align with the WHO 2012 VA standard format and were then processed using the InterVA-4 model to assign COD. These VA standard formats and the specifications have been described in detail previously . Where aFinally, the variable ‘born_small’ requires either the birth weight or data to suggest that the child was small at birth as reported by the respondent. Some respondents indicated that the baby was born small while the birth weight recorded was more >2.5 kg or vice versa. Where responses were recorded for both indicators, therefore, we only considered the birth weight in mapping the ‘low birth weight’  or ‘big baby’ (born_big) indicators.http://www.r-project.org/).Deaths were aggregated for all individuals in the study population for the period January 2008 to December 2011. An analytical dataset was constructed from the model’s output in which each VA case had one or more records. Each record having one cause and a weight corresponding to the likelihood of that cause for the particular VA case. The model assigned multiple CODs if they reached half of the likelihood of the leading cause. Any residual margin of likelihood not accounted for by the likelihood of the first, second or third causes was then considered as indeterminate. The possible CODs determined by the InterVA-4 model were derived for all ages. Cause-specific mortality fractions (CSMF) were determined as the proportion of all deaths that were attributable to any specific COD. We stratified mortality analyses by sex and seven age groups . The age-group boundaries were chosen to reflect groups of public health importance. All statistical analyses were carried out using STATA V11  and the statistical software environment R  and by the KEMRI Scientific Steering Committee (SSC), both in Kilifi, and by the KEMRI/National Ethical Review Committee (ERC) in Nairobi.n=3,310), 10% were single, 55% were legally married, 28% were widowed, 4% were divorced, and 3% were separated. The InterVA-4 model assigned a single COD to 3,886 cases (87%), two CODs to 422 cases (9%), three CODs to 22 cases (1%), and 130 cases (3%) to ‘indeterminate’.A total of 5,187 deaths were recorded among the resident population of the KHDSS between January 2008 and December 2011. One thousand one hundred and eighty one cases (23%) were among children <5 years old, 242 cases (5%) were aged 5–14 years, and 3,764 cases (73%) were >15 years of age, of which 1,665 cases (32%) were >65 years old . Of the The top five CODs for the whole population overall were: HIV-/AIDS-related causes, acute respiratory infections including pneumonia, malaria, and pulmonary tuberculosis. Pneumonia was the most common COD among the infants while malaria and HIV were the commonest CODs among children 1–4 years and adults aged 15–49 years, respectively. Pulmonary tuberculosis was the leading COD among the elderly age groups.The majority (78%) of neonatal deaths occurred in the first 7 days of life: 31% occurred within the first 24 hours, and 47% between 2 and 6 days of age. The leading CODs in neonates were pneumonia (26%), birth asphyxia (24%), prematurity (6%), and sepsis (6%) . Five peThe top CODs in infants (excluding neonates) were pneumonia, HIV/AIDS, and malaria which accounted for 28, 21 and 12% of deaths, respectively. Five percent of deaths were attributed to sickle cell crisis while other COD categories totalled 14%. Twelve percent of deathswere indeterminate . No sex-Malaria, HIV/AIDS, and pneumonia were the predominant CODs in this age group, causing 23, 18, and 12% of the childhood deaths, respectively, while diarrheal diseases, severe malnutrition, sickle cell with crisis, and road traffic accidents explained 3, 2, 2, and 1% of deaths, respectively. All other CODs accounted for 7% while a further 9% of deaths were indeterminate . A slighRelatively fewer deaths were observed in this age group. The commonest CODs were HIV/AIDS related and malaria, which accounted for 23 and 13% of total deaths, respectively. Deaths due to pneumonia, road traffic accidents, meningitis/encephalitis, and sickle cell with crisis explained 8, 7, 5, and 4% of deaths, respectively. Eight percent of cases were indeterminate . Unlike A third of deaths in this age group were attributed to HIV/AIDS and pulmonary tuberculosis. The majority of those who died of HIV/AIDS were either divorced or separated (60%). Road traffic accidents, assault, and stroke accounted for 5, 3, and 3%, respectively. Overall, 7% of deaths were indeterminate . A higheAll neoplasms, pulmonary tuberculosis, HIV-/AIDS-related deaths and stroke were the predominant CODs among people of this age group, accounting for 16, 12, 12, and 10% of deaths, respectively. Other unspecified cardiac diseases explained 5% of deaths. Eight percent of the deaths were indeterminate . More maAll neoplasms, stroke, and pulmonary tuberculosis predominated, accounting respectively for 15, 14 and 10% of total deaths. Cardiac diseases accounted for 10% of deaths and 10% were indeterminate . A higheThe pattern of mortality as determined by the InterVA-4 model shows a high burden of infectious diseases, including HIV/AIDS, pneumonia, and pulmonary tuberculosis in the study population. These mortality patterns are consistent with existing knowledge on the burden of disease in many parts of sub-Saharan Africa.The majority of previous studies that have reported CODs among rural populations in the developing world have used the physician certified verbal autopsy (PCVA) approach to COD assignment–33. A VAIn general, our COD estimates appear credible and conform to expected patterns. As anticipated, we found a slight excess of deaths among males  in comparison to females . This observation was consistent across all age groups individually with the exception of the 15–49 years age group where, predictably, we found an excess of deaths among women, predominantly from pregnancy-related causes and HIV/AIDS. Of particular interest, external CODs such as road traffic accidents were more commonly observed among men than women. Since Kenya has recently experienced a surge in the use of motorcycles as a mean of transport, and a majority of riders lack proper training, this is, perhaps, not surprising, but is nevertheless a matter of considerable concern. Similarly, palm wine tapping, a common socioeconomic activity for men within the KHDSS, can sometimes result in fatal accidental falls, especially when men climb the trees having sampled the product. In the elderly, 65+ age group, malignancies and stroke were key concerns, the latter being consistent with data from our previous study in which we reported hypertension and diabetes as major risk factors .While in general the process of VA coding was straight forward, we did face some challenges with regard to the input and interpretation of our data. First, in comparison to that reported from previous studies , 37, a hA major advantage of computer-based coding methods such as InterVA-4 is their potential for providing standardized data across multiple sites and over long periods of time. For this to be viable, it will be important to agree on standardized methods for data input. For example, in our own data set some questions were missing from the WHO 2007 VA instrument but were present in the WHO 2012 instrument. However, these data were partially captured in the free-text sections of the VA form, for which we developed an automated search for the ‘keyword’ and mapped it onto the corresponding indicator. This was particularly important in capturing specific diagnoses such as SCD and specific forms of injury. While reviewing and modifying the questionnaires from time to time has certain advantages, it also leads to problems in the analysis and interpretation of longitudinal data. If the method is to be used successfully for the purpose of comparisons between multiple sites, collaborative discussion will be needed to agree on protocols for data input and interpretation.Our study suggests that both the WHO 2012 instrument and the InterVA-4 model are feasible tools to measure cause-specific mortality, which may potentially inform both health policy and program interventions in resource-limited settings. The model requires minimal time and labor resources, especially in comparison with the PCVA method. The current analysis, using InterVA-4, returned COD patterns that were generally credible at the population level and in subjects of all ages. While further work will be required to fine-tune and validate COD analysis using InterVA-4, particularly in children, we hope that our results will prove useful for informing health intervention policies both locally and internationally.


Appropriate dietary intake determines the disease progression and success of Anti-Retroviral Therapy (ART). Undernutrition unacceptably increases the risk of mortality among adults living with Human Immune Deficiency Virus (HIV). However in resource limited settings including Ethiopia, many of HIV positive clients lack access to sufficient quantities of nutritious food. There is limited evidences showing the magnitude of undernutrition in this segment of the community, particularly in the rural residents. Therefore, this study aimed to assess undernutrition and associated factors among HIV positive adults attending ART clinic in Dembia District.An institution based cross-sectional study was conducted in Dembia District from October 1 to 30, 2015. Systematic random sampling technique was used to recruit the study subjects. The anthropometric measurement, Body Mass Index, was computed to determine the nutritional status of the study participants. In order to identify factors associated with undernutrition a multivariable logistic regression analysis was employed. The Adjusted Odds Ratio (AOR) with 95 % Confidence Interval (CI) was calculated to show the strength of association. In multivariable analysis, variables with a P-value of <0.05 were considered as statistically significant.3 .Of the study participants, about 23.2 %  were undernourished in Dembia District. The result of adjusted analysis revealed that, the odds of undernutrition was higher among adults whose age ranged between 18-29 years , who had a Cluster of Differentiation (CD)4 count less than 200 cells/mm3 were positively associated with undernutrition. Therefore, efforts should be strengthened to mitigate the higher burden of undernutrition by considering the identified determinants.The prevalence of undernutrition among HIV positive adults was higher in the study area. Furthermore, being in the age range of 18-29 years, widowed, anemic, and having a CD4 count of less than 200 cells/mm Globally, 35.3 million people were living with HIV in 2012 .Proper nutrition directly affects immune status, moderates the drug efficacy and side effects, and enhances the quality of life . With thThe empirical evidences among HIV positive clients showed that, sex , residenIn Ethiopia since its detection, HIV/AIDS has badly costs the lives of millions. Unless the dispute of undernutrition is answered, this higher mortality rate could not be overwhelmed even in the era of ART.  Substant2 and had a total population of 271,053, of which 91.4 % are rural inhabitants  of HIV positive adults were undernourished. Only few, 0.4 %, HIV positive adults took nutritional support. Substantial proportion (88.7 %) of the respondents had high dietary diversity, and more than three quarter (81.6 %) were food secured , presence of anemia, and having CD4 counts of less than 200 cells/mmIn this study, the prevalence of undernutrition among HIV positive adults was 23.2 %. Based on the Nutrition Landscape Information System (NLIS) cut-off values, the burden of undernutrition in this study was higher . This fiHowever, the prevalence of undernutrition was lowest compared to the study reports from other developing countries, such as Botswana (28.5 %) , China (This study also tried to elicit the associated factors of undernutrition. Consequently, the odds of undernutrition was higher among clients whose age ranged between 18 to 29 years compared to those aged 45 years and above. The finding was unsupported by the study report from Brazil , in whicLikewise, the likelihood of undernutrition was higher among widowed study participants compared to married counter parts. This might be due to the emotion and grief encountered, and loss of spousal support. Obviously, the risk of mental illness was lower among married women than widowed . MaritalOne of the medical factors which showed a positive association with undernutrition was anemia. This finding was in line with a study conducted in Butajira . This as3. The finding of this study was comparable with a study done in Nepal [In addition, the odd of undernutrition was higher among clients with CD4 counts of less than 200cells/mmin Nepal . The CD4in Nepal . The proin Nepal . Weight in Nepal , 42.Obviously the previous limited study reports in Ethiopia showed the magnitude of undernutrition among the urban inhabitants. However, the current study tried show the magnitude of the problem by including the rural residents. Nevertheless, some of the limitation should be noted in interpreting the study results. Firstly, hence the study was institution based, the results could not be generalized to those HIV positive adults who are not enrolled for chronic HIV care. Secondly, measurement of some of the variables, such as food intake relies on memory, as a result the study is not free from the recall bias. But, we have made efforts to reduce recall bias, including pretest, regular supervision, and training on how to probe the clients to remember their food intake.3 were positively associated with undernutrition. Therefore, efforts should be strengthened to provide emotional support and psychological reassurance to younger and widowed HIV positive adults. Moreover, to curve the higher burden of undernutrition, emphasis should be given to the prevention and early management of anemia and low CD4 counts by strengthening the usual hemoglobin and CD4 count evaluation.This study revealed that, the prevalence of undernutrition among HIV positive adults was higher. Furthermore, being in the age range of 18-29 years, widowed, anemic, and having a CD4 count of less than 200 cells/mm4, cluster of differentiation4; CI, Confidence Interval; COR, Crude Odds Ratio; DDS, Dietary Diversity Score; FANTA, Food and Nutrition Technical Assistance Project; HIV, Human Immune Deficiency Virus; NLIS, Nutrition Landscape Information System; SPSS, Statistical Package for Social Science; WHO, World Health OrganizationAIDS, Acquired Immune Deficiency Syndrome; AOR, Adjusted Odds Ratio; ART, Anti-Retroviral Therapy; BMI, Body Mass Index; CD


Helicoverpa armigera, during the initiation, maintenance, and termination of pupal diapause. A total of 55 metabolites in the hemolymph and 52 metabolites in the brain were detected. Of these metabolites, 21 and 12 metabolite levels were altered in the diapause pupal hemolymph and brain, respectively. During diapause initiation and maintenance, the number of metabolites with increased levels in the hemolymph of the diapausing pupae is far greater than the number in the nondiapause pupae. These increased metabolites function as an energy source, metabolic intermediates, and cryoprotectants. The number of metabolites with decreased levels in the brain of diapausing pupae is far greater than the number in the nondiapause pupae. Low metabolite levels are likely to directly or indirectly repress the brain metabolic activity. During diapause termination, most of the metabolite levels in the hemolymph of the diapausing pupae rapidly decrease because they function as energy and metabolic sources that promote pupa-adult development. In conclusion, the metabolites with altered levels in the hemolymph and brain serve as energy and metabolic resources and help to maintain a low brain metabolic activity during diapause.A strategy known as diapause  has evolved in insects to increase their survival rate under harsh environmental conditions. Diapause causes a dramatic reduction in the metabolic rate and drastically extends lifespan. However, little is known about the mechanisms underlying the metabolic changes involved. Using gas chromatography-mass spectrometry, we compared the changes in the metabolite levels in the brain and hemolymph of nondiapause- and diapause-destined cotton bollworm,  Helicoverpa armigera that is an important agricultural noctuid moth in the world, its pupal diapause is induced by incubating larvae under short day-light and low-temperature conditions that down-regulate the expression of neuropeptide prothoracicotropic hormone (PTTH) in the pupal brain. The low PTTH expression level inhibits the prothoracic glands from synthesizing steroid hormone ecdysteroids (or ecdysone), which are needed to promote continuous development H. armigera pupal diapause is initiated by exposing 5th–6th instar larvae under short day (10 h light: 14 h dark) and 20 °C conditions, and the lifespan is over three times than in their nondiapause counterparts reared under long day (14 h light: 10 h dark) and 25 °C without diapause.Most insect species have evolved an adaptive strategy known as diapause  to survive an unfavorable season H. armigeraBombyx mori, glycogen is converted to sorbitol and glycerol during diapause Sarcophaga crassipalpisChilo suppressalis, larvae B. mori embryonic diapause The insect gradually reduce its metabolic activity during diapause initiation, and maintain the low metabolic activity during diapause maintenance for overwintering; when the environment is suitable, the insect physiological state will be reached in which direct development may activate S. crassipalpis, and many metabolite levels were observed to be altered in the diapause individuals using gas chromatography-mass spectrometry (GC-MS) Previous studies have primarily focused on a few metabolites, and little is known about the other metabolites that are involved in diapause. Metabolomics is an emerging field that it allows a more global picture of metabolic changes underpinning the phenotype of interest in biological systems and offers a broad and bias-free view of the biological processes involved H. armigera individuals during the initiation, maintenance, and termination of pupal diapause. A total of 55 metabolites in the hemolymph and 52 metabolites in the brain were detected, and these metabolites play various roles in insect diapause. The accumulation of proline and serine in hemolymph may serve as energy source during diapauses, the phosphoric acid levels affect phosphorylated protein levels in the brain, and a lot of metabolite levels decrease in hemolymph for resuming development during diapause termination.Insects have an open body cavity, and all of the organs and tissues are submerged in hemolymph, including the brain. Hemolymph transports substrates for development and diapause. Thus, the metabolic activity of the brain (brain activity) depends on the metabolites in the hemolymph. To provide clues about diapause mechanisms why there is low metabolic brain activity in diapausing individuals, we performed a GC-MS analysis to compare the metabolomic changes in the pupal hemolymph and brains of nondiapause and diapause H. armigera larvae were kindly provided by Dr. Jian-Ya Su, Nanjing Agricultural University . When the larvae were reared on an artificial diet and under long day conditions at 20 °C (L14: D10), all of the pupae developed without entering diapause. When the larvae were reared at 20 °C and under short day conditions (L10: D14), more than 90% of the individuals entered pupal diapause. The pupal samples collected on day 0, 1, 2, 3, 4, and 10 after pupation represent diapause initiation, and the samples collected on day 21 after pupation represent diapause maintenance. To investigate diapause termination, 1 µg 20-hydroxyecdysone (20E) was injected into diapausing pupae (day 21 after pupation), and the samples were collected at 24, 48, and 72 h after injection. Pupal brains were dissected in 0.75% NaCl medium and stored at −80°C. A sample consisted of 30 brains, and 4 replicates of these samples were prepared. The hemolymph (100 µL) was collected from 10 pupae and combined as a single sample, and 4 replicates of these samples were prepared. The samples were centrifuged at 12000 g for 5 min to remove the hemocytes, and the supernatant was transferred to a new 1.5 mL tube and stored at −80°C.For the hemolymph metabolomic analysis, 100 µL distilled water containing 0.1 mg/mL sucrose  and 800 µL methanol were added to each sample (100 µL hemolymph). Next, the sample was vortexed for 10 s, incubated on ice for 1 h, and centrifuged at 20000 g for 10 min. Then, 600 µL supernatant was transferred to a GC vial and dried using a vacuum for approximately 3 h. Next, 90 µL freshly prepared methoxylamine hydrochloride  was added to the GC vial, and the sample incubated for 16 h at room temperature. The sample was then added to 90 µL MSTFA  for trimethylsilylation for 1 h. Finally, the derivatization reaction was stopped by adding 120 µL hexane to the sample.et al. For the metabolomic analysis of the brain, the samples were derivatized following the method previously described by Zhang A total of 1 µL derivatization sample was auto-injected into the 7890A/5975C GC-MS (Agilent). The split ratio was 1∶50 for the hemolymph sample and 1∶10 for the brain sample. For the metabolomic analysis, a HP-5 ms column  was used. The injector temperature was maintained at 280 °C. The initial oven temperature was set at 50 °C. The oven temperature increased 5 °C/min to 300 °C and was then maintained at 300 °C for 5 min. Data were collected from the full scan mode across a mass range of 50 to 450 m/z.The individual peaks were identified by comparing the retention times with the metabolite standards. Other peaks were identified by matching the mass spectra against the National Institute of Standards and Technology (NIST). RSI value over 700 was considered a good match. The overlapping peaks were deconvoluted using traces for single ions. The individual integrated peak areas were converted to a relative response ratio relative to the internal standard (sucrose) peak area. One-way ANOVA analysis was used to analyze all of the metabolites, and Benjamini-Hochberg method was used to correct the p-value. A p-value<0.05 was considered a significant difference. For the principal component analysis (PCA), the relative response ratios for all of the metabolites were analyzed using MetaGeneAlyse 1.7  and separated by their principal components.H. armigera diapause- and nondiapause-destined pupae during diapause initiation, maintenance, and termination. A total of 55 metabolites were identified in the hemolymph, consisting of 16 amino acids, six sugar and polyols, six metabolic intermediates, three small molecules and 24 other metabolites , one polyol (myo-inositol), one small molecule (phosphoric acid), and one other metabolite (butanoic acid). Of these metabolites, the levels of four  were significantly lower in the diapause-destined pupae compared with the nondiapause-destined pupae. The tyrosine level in day 10 diapause-destined pupae is only half as much as its nondiapause-destined counterparts  and TCA cycle-related intermediates  in hemolymph and brains of diapause- and nondiapause-destined pupae during diapause initiation, maintenance and termination. At diapause initiation, we showed that 1) the levels of trehalose, succinate, and malate increase in the hemolymph, but glucose and pyruvate, decrease; 2) the levels of trehalose and glucose increase in the brain, but pyruvate, isocitrate, and succinate decrease terparts . Ten metterparts -2.Furthermore, we found 10 altered metabolite levels in the brains of the diapause-destined individuals, which included seven amino acids , one small molecule (phosphoric acid), and two other metabolites (D-gluconic acid and pantothenic acid). All of these metabolite levels were reduced in the diapause-destined pupal brain -1 exceptTo compare the changes in the metabolite levels between the nondiapause and diapausing individuals, day 3 nondiapause pupae (NP3) and day 21 diapausing pupae (DP21) were evaluated. First, all of the metabolites identified in the NP3 and DP21 pupae were subjected to PCA analysis. The hemolymph PCA score plot showed that the PC1 and PC2 accounted for 65.4% and 13.9% of total variability, respectively . The braβ-alanine, butanoic acid, glutamate, isoleucine, leucine, myo-inositol, pantothenic acid, phenylalanine, phosphoric acid, proline, serine, threonine, valine, and N-acetylglucosamine, were elevated. For example, the levels of isoleucine, proline, and serine in diapausing pupae are twice as much as it in nondiapause pupae . The hexadecanoic acid level in diapausing pupae is approximate one-fourth as much as it in nondiapause pupae -1. Only Finally, 20E was injected into diapausing pupae to investigate diapause termination, and changes in metabolite levels were evaluated at 24, 48, and 72 h after injection. The PC1 and PC2 accounted for 39.1% and 21.2% of total variability, respectively. The PCA score plots showed that there were clear changes in the metabolite levels 24 h after injection .β-alanine, proline, valine, cholesterol, octadecenoic acid, tyrosine, myo-inositol, pantothenic acid, linoleic acid, and phosphoric acid) was observed at 24 h after injection. Only the butanoic acid level demonstrated a slight increase. Of the 13 metabolites with reduced levels, the levels of 11 metabolites continued to decrease until 72 h after injection. At 72 h after injection, only the octadecenoic acid and tyrosine levels demonstrated significant increases , which have been reported in a previous study Insects cease to develop and their metabolic activity is reduced when they enter diapause At diapause initiation, trehalose, succinate, and malate increased in hemolymph of diapause-destined pupa, but glucose and pyruvate decreased, suggesting that low levels of glucose and pyruvate control brain metabolic activity to induce diapause entry β-alanine, isoleucine, leucine, proline, serine, threonine, and valine) were higher. These amino acids are closely correlated with carbohydrate metabolism, especially the TCA cycle.The amino acid profile was altered in the hemolymph of diapause pupae. The levels of four different amino acids  were lower, and the levels of seven different amino acids  pupae during overwintering H. armigera, which is consistent with a previous study H. armigera is not a glycerol-dependent diapause species and predominantly depends on trehalose, sorbitol and myo-inositol for cryoprotection. Furthermore, these cryoprotectants are synthesized during glycolysis, and an accumulation of these cryoprotectants implies that energy metabolism is reduced and affects the brain metabolic activity.An accumulation of cryoprotectants affects the supercooling point and enhances the resistance against low temperatures, which may influence an insect's survival rate. We observed higher levels of trehalose and sorbitol, which are consistent with previous reports Pantothenic acid (vitamin B5) is an essential nutrient and is used to synthesize co-enzyme A (CoA) in animals In a previous study, we found that the phosphorylated protein level in the brain is reduced during diapause initiation H. armigera individuals During diapause initiation, most of the metabolite levels increased in the hemolymph of diapause-destined pupae, whereas all of the metabolite levels, except for aspartate, decreased in the brain. An increase in metabolites in the hemolymph may serve as a resource for the long diapause phase and for pupal-adult development after diapause termination. The down-regulation of metabolites in the brain may serve to reduce brain activity, as previous studies have shown that there is low brain activity in diapause-destined Except for sugar and TCA-related metabolites, the levels of six metabolites decreased and the levels of 15 metabolites increased in the hemolymph of diapausing pupae. The levels of 12 metabolites decreased and only the aspartate level increased in the brains of the diapausing pupae.At diapause maintenance, glucose, pyruvate, fumarate, and malate decreased in diapause-destined pupal hemolymph, but trehalose and isocitrare increased β-alanine, proline, serine, threonine, valine, isoleucine, leucine, and phenylalanine) increased. As previously mentioned, the reduced levels of methionine and tyrosine in the hemolymph may be used as limiting factors to repress protein synthesis. The increased amino acid levels in the diapause pupal hemolymph may serve as a resource for pupal-adult development after diapause termination.In the diapausing pupal hemolymph, two amino acid levels (methionine and tyrosine) decreased, and nine amino acid levels  decreased, and only the aspartate level increased. The five amino acids with reduced levels are closely correlated with pyruvate, CoA, and succinate, which serve as TCA intermediates and suggest that low levels of these amino acids may be the limiting factor that reduces brain activity. Interestingly, a high level of aspartate was detected in the brain. Aspartate is the amino acid most closely associated with carbohydrate metabolism because aspartate can be converted to oxalate, which can be used to synthesize citric acid for the TCA cycle. In addition, other amino acids can be synthesized from aspartate, including four essential amino acids: methionine, threonine, lysine, and isoleucine. Therefore, aspartate can be considered an energy source and an amino acid resource for brain metabolism during a long diapause phase.The cryoprotectant myo-inositol level was higher in the brain during diapause, and as previously mentioned, myo-inositol may protect the brain from harsh environments. Fatty acids are the primary energy source, and we observed reduced levels of two fatty acids, linoleic acid and octadecenoic acid, in the diapause pupal hemolymph. Trehalose is the predominant sugar found in insect blood. The reduced fatty acid levels may be caused by trehalose synthesis, which can be easily used during the diapause phase. However, this method used does not allow detailed analysis of changes in fatty acids composition associated with diapause. Therefore, the function of these fatty acids with altered levels in diapause insects should be investigated in future studies.Both the pantothenic acid and phosphoric acid levels increased in the diapausing pupal hemolymph and decreased in the pupal brain. This finding suggests that pantothenic acid and phosphoric acid serve are source function and accumulate in the hemolymph and that the low pantothenic acid and phosphoric acid levels in the brain imply a reduced metabolic activity. It has also been shown that the pantothenic acid level increases in ground squirrel plasma during the hibernating period At diapause termination, trehalose and isocitrate decreased in hemolymph of diapause-destined pupa, but glucose, pyruvate, succinate, and malate increased β-alanine, proline, valine, cholesterol, octadecenoic acid, tyrosine, myo-inositol, pantothenic acid, linoleic acid, and phosphoric acid) were lower at 24 h after injection. Only the butanoic acid level was slightly increased. The glutamate, alanine, and proline levels decreased significantly, and these results suggest that there is an increase in the pyruvate level for TCA cycle activity, which would promote pupal-adult development When 20E was injected into the diapausing pupae to terminate diapause, 14 metabolite levels in the hemolymph were altered. The levels of 13 metabolites  At diapause initiation, the metabolite levels gradually increase in the pupal hemolymph and serve as a resource for pupa-adult development after diapause termination; the metabolite levels in the brain are reduced and correlated with low metabolic activity in the brain during diapause entry. 2) During diapause maintenance, the number of metabolites with increased levels in the pupal hemolymph is greater than the number of metabolites with decreased levels. These increased metabolites will serve as an energy source and as metabolic intermediates for maintaining the activity of the brain and other organs during a long diapause period and for pupal-adult development after diapause termination. The reduced metabolites in the diapause pupal hemolymph may contribute in the down-regulation of brain activity that helps to maintain the diapause state. In the diapause pupal brain, the number of reduced metabolites is far greater than the number of increased metabolites, which suggests that the low metabolite levels are crucial for low brain activity. 3) During diapause termination, most metabolite levels rapidly decrease in the hemolymph in response to the 20E signal and serve as energy and metabolic sources that promote pupa-adult development. Combing this study with previous reports, suggest that not only metabolites are energy resources and cryoprotectants, but also can serve as regulators The GC-MS analyses of diapause Figure S1Principal component analysis (PCA) score plots for metabolites during diapausing. The metabolites of hemolymph (A) and brain (B) during diapause maintenance. PC1 containing valine, leucine, threonine, tyrosine, linoleic acid, octadecenoic acid and phosphoric acid, etc. accounted for 65.4% of total variation, and PC2 containing glycine and D-gluconic acid accounted for 13.9% of total variation in hemolymph PCA plot (A). PC1 containing valine, alanine, leucine, isoleucine, and phosphoric acid accounted for 55.9% of total variation, and PC2 containing proline, phenylalanine, and inositol accounted for 16.8% of total variation in brain PCA plot (B). NP, nondiapause pupa; DP, diapausing pupa.(TIF)Click here for additional data file.


Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation. Lactococcus lactis belongs to the group of lactic acid bacteria and is encountered in a wide range of environments, such as animals and plant materials, and even in a great variety of traditional food products (as spontaneous growth)  status, is involved in milk acidification by the formation of lactate, proteolysis, and in the synthesis of aromatic compounds, polysaccharides, and bacteriocins, contributing therefore to the taste, texture, and safety of dairy products (– growth) , 2. Induproducts –7.L. lactis, which is by far the best characterized lactic acid bacterium (LAB) with respect to its physiology, metabolic pathways, and regulatory mechanisms, has become the model bacterium for most of the LAB research in biotechnology. The genomes of several L. lactis strains have thus been determined, including strains of L. lactis subsp. lactis, such as IL1403 .s IL1403 , KF147  using HiSeq paired-end sequencing. A total of 20,391,131 reads were used for assembly by using the Velvet and VelvetOptimiser softwares, leading to an average coverage of 730×. A total of 56 large contigs ranging from 1,013 to 431,322 bp in size were generated and annotated using AGMIAL, an integrated bacterial genome annotation platform  and inversely, the reduction of several amino acid biosynthetic capacities (auxotrophic for glutamate and aspartate) demonstrated this adaptation. However, the genome of L. lactis TOMSC161 still contains genes involved in the utilization of certain plant sugars  related to the original habitat of L. lactis. Furthermore, the analysis showed the presence of genes involved in exopolysaccharide and bacteriocin  production, which are interesting technological features.Analysis of the genome of L. lactis TOMSC161 will allow the powerful application of transcriptomics and proteomics to investigate the metabolic responses and thus the physiological processes involved in the adaptation to different environmental conditions.The availability of the genome sequence of L. lactis subsp. lactis TOMSC161 have been deposited in the EMBL database under accession numbers CBUJ010000001 to CBUJ010000062.The genome sequence and annotation of


Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism.The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed.It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively.Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization. Inflammatory mediators produced locally or systemically initiate and regulate cell migration and other processes during wound healing. Many types of inflammatory factors are produced after wounds occur, such as IL-1, IL-3, TNF-α and NO. NO is a small molecule that displays multiple functions in various physiological and pathological processes. Studies have demonstrated that treatment with an NO donor enhances wound healing, but the underlying mechanism is unclear . ResiduaMany studies have showed that NO can increase the expression and activation of Rho family members ,9. In tu2. The cells were passaged at a ratio of 1:5 using 0.25% trypsin  and cultured to 70 to 80% confluence for the following experiments.The HaCaT cell line was obtained from the American Type Culture Collection . The cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum  at 37°C in a humidified incubator containing 5% CO5 cells/2 ml per well and then incubated at 37°C until the cells reached 100% confluence. Mitomycin-C was added at a final concentration of 5 μg per ml, and the cells were incubated for an additional 4 hours to inhibit cell proliferation. A sterile plastic 10-μL pipette tip was used to scratch the confluent cell monolayer evenly in each well to generate a cell-free zone that was approximately 1 mm in width. The medium was aspirated and replaced with fresh RPMI-1640 medium in the absence or presence of different concentrations of sodium nitroprusside . Then, the cells were incubated for 48 hours. The migration of the cells in each well was evaluated under an inverted phase contrast microscope and quantified using ImageJ software  at designated time points, i.e., 0, 6, 12, 24, or 48 hours post-scratch. The migration rates were expressed as / × 100%. Each experiment was performed in triplicate. Also, other two NO donors i.e, spermine NONOate  and SNAP  were used to check the possible different effects on HaCat cell migration.An in vitro wound-healing model of cultured HaCaT cells was used according to previous studies ,17,18 wiActin cytoskeleton reorganization was assessed via filamentous actin (F-actin) staining, as reported previously . BrieflyAccording to the literature, the active proteins of CDC42, Rac1 and RhoA were quantified in pull-down assays ,23. AfteAt the designated time points, total RNA was extracted from HaCaT cells using TRIzol  according to the manufacturer’s instructions and then treated with DNase to decrease genomic DNA contamination. The purity and concentration of the RNA were assayed via electrophoresis and based on the ratio of the optical density at 260 nm to that at 280 nm, respectively.The Rho family PCR primers and reaction parameters employed in these assays followed Pan and were modified according to our own conditions . The PCREither a cGMP agonist  or a PKG agonist  was applied to stimulate the HaCaT cells. In some cases, the cells were first pretreated for 2 hours with a cGMP inhibitor , PKG inhibitor , Rho-specific inhibitor, , CDC42 inhibitor  or Rac1 inhibitor . Then, the cells were stimulated with SNP at a final concentration of 10 μM for another 24 hours. Subsequently, cell migration and the actin cytoskeleton were assayed as described in sections 2another respectively. Moreover, the mRNA expression and active proteins of CDC42, Rac1 and RhoA were quantified as described in section 4.1 and section 4.2, respectively.The data were expressed as the means ± SD of the indicated number of observations. Comparisons between two groups were performed using Student’s t-test. If necessary, one-way analysis of variance (ANOVA) was applied for comparisons among multiple groups. P values < 0.05 were considered statistically significant.As shown in Rhodamine-labelled phalloidin mainly stains polymeric F-actin. As shown in Based on pull-down assays, it was found that the expression of the active and total proteins of CDC42, Rac1 and RhoA in the 10 μM SNP treatment group was greater than in the control group . The levAs shown in The in vitro wound healing assay revealed that, similar to SNP treatment, treatment with either a cGMP or PKG agonist promoted HaCaT cell migration (P<0.05). There was no significant difference between these two groups. Migration was increased by 9.78% and 11.12% in these groups compared with the control group, respectively. However, the effect of SNP on cell migration was significantly decreased by pretreatment with a cGMP, PKG, RhoA, Rac1 or Cdc42 inhibitor (P<0.05) . Among tAs shown in RT-PCR analysis revealed that, similar to SNP-treated cells, the cells treated with a cGMP or PKG agonist displayed significantly increased mRNA expression of CDC42, Rac1 and RhoA compared with the control cells. Moreover, these effects of SNP were significantly inhibited by pretreatment with a cGMP or PKG inhibitor . CompareAs shown in Keratinocyte proliferation and migration are essential for re-epithelization, which is one of the key processes during wound healing, and it has been reported that many factors could affect these processes ,26. In tIn our experiments, SNP from Sigma was used as an NO donor, as in many other previous investigations ,28. It wThe guanylate cyclase/cGMP/PKG pathway is considered to be the key mechanism by which NO influences cellular functions . NO can Cytoskeletal reorganisation is considered to be a primary mechanism underlying cell migration . In our Like other members of the Rho small GTPase family, Rho, Rac, and Cdc42are small monomeric G proteins. They cycle between an inactive GDP-bound form and an active GTP-bound form, through which they regulate the actin cytoskeleton, cell migration, proliferation and other cellular processes ,42,43. IIn the present study, it was found that NO can upregulate RhoA, Rac1 and Cdc42 expression, not only at the mRNA level, but also at the active and total protein levels in HaCaT cells Figs.  and 4. TIn conclusion, the results of our primary experiment suggested that the stimulatory effects of NO on the cell migration of HaCaT cells may be mediated by the cGMP/PKG/Rho GTPase signalling pathway.


The regional management of trace elements in soils requires understanding the interaction between the natural system and human socio-economic activities. In this study, a social-ecological patterns of heavy metals (SEPHM) approach was proposed to identify the heavy metal concentration patterns and processes in different ecoregions of Beijing (China) based on a self-organizing map (SOM). Potential ecological risk index (RI) values of Cr, Ni, Zn, Hg, Cu, As, Cd and Pb were calculated for 1,018 surface soil samples. These data were averaged in accordance with 253 communities and/or towns, and compared with demographic, agriculture structure, geomorphology, climate, land use/cover, and soil-forming parent material to discover the SEPHM. Multivariate statistical techniques were further applied to interpret the control factors of each SEPHM. SOM application clustered the 253 towns into nine groups on the map size of 12 × 7 plane . The distribution characteristics and Spearman rank correlation coefficients of RIs were strongly associated with the population density, vegetation index, industrial and mining land percent and road density. The RIs were relatively high in which towns in a highly urbanized area with large human population density exist, while low RIs occurred in mountainous and high vegetation cover areas. The resulting dataset identifies the SEPHM of Beijing and links the apparent results of RIs to driving factors, thus serving as an excellent data source to inform policy makers for legislative and land management actions. Accumulation of heavy metals in agricultural soils may cause serious problems to human well-being by influencing soil quality, groundwater and food chains ,2,3,4,5.etc. [Identifying and quantifying the social-ecological patterns of heavy metals (SEPHM) is a challenging task to due to the variety and complexity of social-ecological data ,16,17,18etc. ,21,22 . etc. .etc. The SOM component planes can reveal very useful information to interpret results that remain hidden with the traditional approaches, such as the principal component analysis and hierarchical cluster analysis [SOM is a very interesting and promising classification approach employing an innovative and data-driven classification method based on unsupervised artificial neural networks. Its capabilities of clustering, classification, estimation, and prediction have been used in a widely spread range of disciplines, including engineering, agriculture, health, environment management, and remote sensing image classification, analysis ,25,26. et al. [et al. [et al. [et al. [The heavy metal contamination of Beijing soils has been widely reported. Huo et al.  assessedet al. ,28. Jian [et al.  and Wang [et al.  assessed [et al. ,33,34,35 [et al.  attempteTherefore, the objectives of this study were to explore the potential of the SOM approach to identify the SEPHM in Beijing, and to propose individualized approaches to the management of the soil heavy metal pollution.2, is located in the northwestern part of China’s north plain, generally between longitude 115°24'–117°30'E and latitude 39°38'–41°05'N. Its elevation slopes downward from 2,250 m in the northwest to 10 m in the southeast, and the mountainous area covers about 62% and plain 38% of the whole area . The annual average temperature difference is 30.4°, while the daily average temperature difference is 11.4°. Annual precipitation in this area is 470–660 mm, about 60% of which comes in July and August. Annual average evaporation is 1,800–2,000 mm. The area is the source of five big rivers, the Yongding, Chaobai, Beiyun, Jiyun and Daqing. Annual average runoff is about 1.8 × 10in China .3, and HClO4. Pb and Cd were analyzed by graphite furnace atomic absorption spectrophotometry, and the As concentration was determined by potassium borohydride silver nitrate spectrophotometry. In addition, the Hg concentration was determined by cold atomic absorption spectrophotometry after digestion with a mixture of H2SO4, HNO3, and KMnO4. Quality assurance and quality control procedures were conducted using the standard reference material Geochemical Standard Soil.In this study, 1,018 soil samples were collected in Beijing in 2006 using an irregular stratified sampling technique based on the agricultural land distribution and land use type maps . All theIn China, communities and/or towns are the smallest administrative units and usually act as the basic unit for planning and management purposes . EnvironNumerous factors need to be considered for ecoregional characteristics clusters. It is a fundamental point to understand the correlations between ecological factors and heavy metals concentration. According to the previous literature investigations, the major inputs of trace elements to agricultural soils including atmospheric deposition, livestock manures, fertilizers and agrochemicals, sewage irrigation, sewage sludge, and some other sources ,40. Tabl−2), livestock (unit·km−2), fertilizers and agrochemicals input (t·km−2), land use cover data (km·km−2 or percent distribution), elevation (m), precipitation (mm). Among them, land use cover data has six categories including Normalized Difference Vegetation Index (NDVI), industrial and mining land (percent distribution), river (km·km−2), road (km·km−2), single cropped and double cropped land (percent distribution). We gathered detailed information on bovine, ovine, porcine and avian livestock at the town-wise level for the year 2007. Livestock unit (LSU) was calculated in a standardized manner [Town-wise statistics metadata mainly consist of these parameters: human population density of each town  > Cd (30) > As (10) > Cu (5) = Pb (5) = Ni (5) > Cr (2) > Zn (1) [riE < 40, low potential ecological risk; 40 ≤ riE < 80, moderate potential ecological risk; 80 ≤ riE < 160, considerable potential ecological risk; 160 ≤ riE < 320, high potential ecological risk; and riE > 320, very high ecological risk. To facilitate clustering 253 towns by SOM, the biophysical dataset (such as CF and RI of each elements) was interpolated into a grid by Kriging, and the average of these indices were resampled at a town-wise level to match the socio-economic data for the further utilization of SOM.Land use cover data obtained from Beijing Municipal Bureau of Land and Resources, while the cropping patterns were classified by the Moderate Resolution Imaging Spectroradiometer (MODIS) image . All the applied . The Tri10) > Cu  = Pb (5)A SOM algorithm also known as Kohonen Map or Self-Organizing Feature Map, is an unsupervised neural network based on competitive learning ,51. It pi.e., unit towns) multiplied by 20 environmental parameters  in The distribution patterns of 20 input variables on the SOM plane are shown in When considering the differences in factors among the nine clusters, variance analysis was employed and the results are shown in A dozen regional patches were found in cluster 2 in the city’s peripheral suburbs. Cluster 3 and cluster 4 locate in the north and west quadrant of Beijing, respectively. Cluster 5 and cluster 6 were found in the vicinity of agricultural land in the east quadrant. Cluster 7, 8 and 9 were toward the city center area, relatively, where high eco-risk areas were observed.Correlation analysis was performed to investigate the relationships between environmental variables and soil heavy metals among the nine clusters. Most comparison cases in the Spearman correlation matrix showed a clear statistical significance when community characterizations were conducted . i.e., NDVI is strongly negatively related to the soil heavy metal risk in clusters 5, 6 and 8, while has no significant correlation coefficients in the others). Many more factors have significantly correlated coefficients (p < 0.01) in clusters 5, 6, and 8, indicating that the function of soil heavy metals risk is complicated in these clusters. In contrast, the structure and function are relatively simple in clusters 2, 7, and 9. Different relationships were detected among the nine clusters , biodiversity conservation classification and forest service classification. Social-ecological patterns represent the comprehensive interaction of substance and energy, which form the live organisms and the environment. Thus, difference-oriented policies should be made based on the local ecosystems, which share a number of basic structural and functional characteristics ,13. In tSOM have been proven to be a promising tool for describing the evolution of metal accumulations in terrestrial ecosystems. The SOM projection shifts the complicated structure from high dimensional arrays into the lower dimensional clusters based on the neighborhood relations, which is important to ecological classifications for environmental management. For instance, the spatial generalization of environmental data are measured at single sites as well as in dynamic global change modeling in land use or environmental planning ,60,61. LFrom the results of this study, it can be concluded that the heavy metals contamination patterns are strongly related to human populations, IML, and ROD distribution. Additionally, vegetation and geographical aspects of the land also affected heavy metals concentration. We could hypothesize the sequential pattern as the following: (1) high heavy metals risk areas locate in urbanized areas that exhibit high population and low elevation, which are suitable for increased road and IML density; (2) these areas, are more frequently invaded by vehicular traffic volumes and urbanization; (3) therefore, land use management on community units (at a town level) is needed in the more populated areas in order to reduce the heavy metals risk.Results from the ANOVA analysis of the element contents show differences among the clusters indicating that heavy element contents in clusters 7, 8 and 9 (the urban area) were higher than the other clusters. Cu, Zn, Cd, Pb and Hg in urban soils usually come from gasoline, car components, oil lubricants, and industrial and incinerator emissions ,62,63,64To meet the land-use management needs, besides the identification of metal accumulation spatial patterns, the consecutive spatial patterns are also an important component of zoning . IntegraZone I was located in the mostly mountainous areas and had relatively high vegetation cover in the west and northwest of Beijing A,B. ZoneIn China, the concept of major function oriented zone (MOFZ) was proposed to achieve coordinated regional development and environmental protection based on the territorial functions ,68. MoreEcoregional synthesis and management classification are the key issues of agricultural soil heavy metal environmental monitoring and management, linking the metals accumulation at individual soil sites with the social-ecological dataset of the area managed ,13,14. AThe main outcomes of the study can be drawn as follows: (i) SOM was verified to be a promising approach for pattern recognition and, in particular, for delineating social-ecological patterns of soil heavy metals; (ii) the main factors that influence the heavy metal concentration in Beijing were associated with the population density, vegetation index, industrial and mining land percent and road density—this is useful information for reference in future research; (iii) the social-ecological patterns of heavy metals in Beijing were detected in nine clusters and mainly categorized into three zones. The results are critical for improving the efficiency of the soil heavy metals management; (iv) classification of social-ecological patterns on soil heavy metals (SEPHM) provides a great deal of information enhancing the risk status source identification at the community scale, although the temporal spatial patterns were merely considered in this study. The spatialization of other factors such as the atmospheric deposition, sewage irrigation and sewage sludge may modify the patterns in a greater refinement. Designing a theoretical framework for combining these perspectives will be an exciting open problem for future analysis.


Maruca vitrata Fabricius (Lepidoptera: Crambidae), is a serious pantropical pest of grain legumes. A suitable artificial diet is desirable for producing uniform insects for commercial purposes or research. Three previously described artificial diets, 1 newly-developed artificial diet, and cowpea (Vigna unguiculata (L.) Walp. ), the natural hostplant of M. vitrata, were used for rearing M. vitrata, and the life parameters were examined. The results indicated that insects completed a full life cycle only when the larvae were fed cowpea or the diet reported by Onyango and Ochieng'-Odero (Glycine max (L.) Merr. , and wheat germ, Triticum aestivum L. . The egg production, egg hatching, larval developmental duration, and pupal survival of the M. vitrata reared on the new artificial diet were found to be significantly improved relative to the D-OO diet, but were not significantly better than on the host-plant cowpea. The optimum rearing density was 15–25 larvae per box. There were no significant changes in reproductive potential after 8 successive generations of rearing on the new diet. These results indicated that the newly developed diet could serve as a viable alternative to cowpea plant for continuous rearing of M. vitrata.The bean pod borer, g'-Odero , called  Maruca vitrata Fabricius (Lepidoptera: Crambidae), is an important pantropical pest of grain legumes, and has a wide distribution throughout Africa, Asia, South America, and the southern states of Australia  Walpers , and has caused significant yield losses yearly  in Wuhan City, Hubei Province, P. R. China. The population was maintained on the host-plant cowpea at 26 ± 1° C, with a photoperiod of 14:10 L:D and 60 ± 10% RH, except for adult mating and oviposition, when they were kept at at 85–90% RH. The eggs were collected daily, and the newly hatched larvae were used in the following experiments.Twenty newly hatched larvae were transferred to transparent plastic boxes (10 cm diameter) and reared with either artificial diets or on cowpea. The artificial diet was replaced every 5–6 days, and the cowpea was replaced every 2 days. A corrugated paper was put into the box as a pupation site when the larvae entered the prepupal stage, which was determined by a change in the body color from pink to light green. The pupae were sexed and kept indi vidually in plastic boxes. For mating, 15 pairs of newly emerged moths were transferred into a rearing cage (length × width × height = 30 × 40 × 50 cm) with a 10% honey solution. After 3 days, each pair was kept separately in an ovipositing container with a 10% honey solution. The container was composed of a transparent cylindrical plastic cup  with a Petri dish base on the bottom. Oviposition occurred on the inner surface of the cup. The cups were replaced daily. The cups with eggs were kept at 26 ± 1° C, with a photoperiod of 14:10 L:D and 60 ± 10% RH until hatching.Glycine max (L.) Merrill , and wheat germ, Triticum aestivum Triticum aestivum L. , were purchased from a local market. Wesson salt mix and vitamin mixture were prepared as the detailed list shown in the footnote of www.angelyeast.com). Other ingredients  were ordered from China National Medicine Corporation, Ltd. (www.cncm.com.cn).The ingredients of all the diets are shown in Three diets described in previous studies  were selThe results of Experiment 1 showed that enough adults for continuous rearing were obtained only when the larvae were fed on the D-OO diet. However, compared to the hostplant cowpea, the pupal survival, egg production, and egg hatching were lower and the developmental duration of the immature stage was longer on the D-OO diet. The diet was improved by changing the nutritional composition, shown in The new artificial diet was prepared in 5 steps as follows: (1) soybeans were baked in an oven at 100° C for 6 hr and ground to a fine powder in a 250 mL stainless steel blender; (2) agar was combined with 1/4 of the final volume of water; (3) the major nutritional and antimicrobial ingredients, soybean flour, wheat germ, sucrose, brewer's yeast, sorbic acid, and methyl-parahydroxybenzoate, were combined and added to the remaining volume of the water; (4) the first 2 parts (2 and 3) of the diet were blended and stirred for 3 min separately, then autoclaved at 121° C and 15 psi for 15 min together. After autoclaving, the 2 parts were blended for 5–6 min in the blender until cooled to approximately 60° C; then, the third part of the diet, including Wesson salt mix, ascorbic acid, choline chloride, a vitamin mixture, and approximately 50 mL of distilled water, was added to the mixture. Finally, (5) formaldehyde was added to the ingredients and blended for 2 min. After mixing all the ingredients and cooling to room temperature, the paste-like diet was dispensed into appropriate containers and stored at 4° C.The suitability of D-New for continuous rearing was estimated by comparing its effectiveness with those reared on cowpea. Life parameters were measured in terms of survival rates, developmental durations of the larvae and pupae, longevity, egg production, egg hatching, and egg developmental duration. Twenty larvae were reared in 1 box, as described by Liu and Hwang . Each diM. vitrata, 5 densities  were used to determine the optimum larval density for continuous rearing. Insect rearing methods were the same as described above, and 150 larvae were used in each density.To evaluate the effect of larval rearing density on the survival rate, developmental duration, and pupal weight of M. vitrata, all of the life parameters were compared for 8 successive generations on D-New. The methods of estimation were the same as described above, and the larval rearing density was 20 larvae per box. Ten boxes were reared for each generation.To evaluate the effect of D-New on the reproductive potential of www-01.ibm.com/software/analytics/spss). The data for larvae and pupae survival, egg hatching, developmental duration, body weight, longevity, and fecundity were analyzed by one-way analysis of variance (ANOVA), and Tukey's HSD test (p < 0.05) was used to compare the means. The data for all the life parameters measured on the artificial diet and the cowpea were analyzed using an independent-samples t test. Prior to the analysis, an arcsine squareroot transformation was performed on the data related to the egg hatching and survival of the larvae and pupae.Statistical analyses were performed using SPSS 11.5 . The developmental duration of M. vitrata reared on 3 diets was significantly longer than those on cowpea . For all the parameters measured in this experiment, the results of D-OO were superior to those D-JR and D-J, even though the differences were not statistically significant in some cases . Furthermore, enough adults for continuous rearing were obtained only for the larvae fed on D-OO or reared on cowpea. M. vitrata reared on the D-OO diet and on host-plant cowpea. Except for egg production, the measures of the three other parameters measured for the D-OO diet were not higher than the measures for the same parameters associated with rearing on the hostplant cowpea . The developmental durations of eggs and larvae on DNew were significantly shorter than those reared on D-OO but significantly longer than those reared on host-plant cowpea . The developmental duration of pupae on D-New was significantly longer than for those on the other two diets . Pupal body weight on host-plant cowpea was significantly greater than that on DOO and D-New . Male adult longevity for M. vitrata reared on these three diets changed slightly . Female adult longevity on D-New was longer than that on D-OO and shorter than that on host-plant cowpea, but the difference was significant only between D-OO and host-plant cowpea . The fecundity of females obtained on D-New was significantly greater than those on D-OO. Although females reared on D-New produced fewer eggs than those reared on host-plant cowpea, the difference was not statistically significant .The comparisons of all the life parameters of F = 1.893; df = 4, 43; p = 0.131; pupae: F = 0.325; df = 4, 43; p = 0.860). Pupal developmental times for the M. vitrata reared in medium density (15–25 larvae per box) were shorter than for those reared in high density (30 larvae per box) and low density (10 larvae per box) , whereas such differences were absent at the larval stage . There were no significant differences among the body weights of male pupae reared at different densities , whereas the body weights of the female pupae decreased with an increase in larval density from 10 to 30 larvae per box .The effects of larval rearing density on survival, the developmental duration of the larvae and pupae, and the pupal weight are shown in The biological performances of the 8 successive generations fed D-New were compared by measuring survival and hatching, developmental duration, body weight, longevity, and fecundity. Except for developmental duration and body weight, no decline in the survival rate and/or reproductive potential was found after 8 successive generations .M. vitrata performed excellently on D-New compared to other artificial diets, but not compared to hostplant cowpea. Some life parameters of insects reared on D-New, including immature survival, adult longevity, fecundity, and egg hatchability, were similar to those on hostplant cowpea, but the other parameters studied were significantly worse. These results imply that the new artificial diet based on soybean flour, wheat germ, brewers' yeast, and sucrose can be used as an alternative to natural hosts for rearing purposes.This study demonstrated that M. vitrata on artificial diet, e.g., proportions of different nutritional ingredients, physical conditions of the diet (including moisture levels and irregularities of the diet surface), microbial contamination, several specific compounds, and environmental conditions in culture rooms. The main protein sources in D-OO were soybean flour and brewer's yeast. Besides soybean flour  replacement because of the excellent preservatives, thus greatly reducing the frequency of diet changes and mechanical injury to the developing larvae.Maintaining high-quality and contaminationfree diet over a long period is an important feature of artificial diets for insects. It is a common practice to use antimicrobial compounds, separately or combined, to prevent microbial contamination of synthetic diets for insects. The use of different antimicrobials was a breakthrough in the diet reported by Onyango and Ochieng'-Odero . The addPrevious studies also showed that Wesson salt  and cholM. vitrata reared in a medium density condition  had a shorter developmental duration time from lar vae to pupae, while the survival rate did not differ significantly among the larvae raised at different densities. This study also revealed that one of the causes of larval mortality was cannibalism of both young and mature larvae, mostly during molting.Larval rearing density influenced the larval survival . UsuallyM. vitrata females was higher than that found in Onyango and Ochieng'-Odero's work  is influenced by many factors, such as sex ratio , delayedo's work , in whicM. vitrata to produce uniform insects of predictable performance without a loss of vigor or a decline in reproductive potential.In conclusion, this study demonstrated that the new artificial diet supported a high rate of survival comparable to those reared on the host-plant (cowpea). The new artificial diet was suitable for the continuous rearing of


The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not fully understood, and experimental models are an alternative to study this issue. We investigated the effects of a simple carbohydrate-rich diet on the development of obesity-related NAFLD and the impact of physical training on the metabolic abnormalities associated with this disorder. Sixty Wistar rats were randomly separated into experimental and control groups, which were fed with sucrose-enriched (18% simple carbohydrates) and standard diet, respectively. At the end of each experimental period , 6 animals from each group were sacrificed for blood tests and liver histology and immunohistochemistry. From weeks 25 to 30, 6 animals from each group underwent physical training. The experimental group animals developed obesity and NAFLD, characterized histopathologically by steatosis and hepatocellular ballooning, clinically by increased thoracic circumference and body mass index associated with hyperleptinemia, and metabolically by hyperglycemia, hyperinsulinemia, hypertriglyceridemia, increased levels of very low-density lipoprotein- (VLDL-) cholesterol, depletion of the antioxidants liver enzymes superoxide dismutase and catalase, and increased hepatic levels of malondialdehyde, an oxidative stress marker. Rats that underwent physical training showed increased high-density lipoprotein- (HDL-) cholesterol levels. In conclusion, a sucrose-rich diet induced obesity, insulin resistance, oxidative stress, and NAFLD in rats. Over the last decades, obesity has become a global epidemic and an important public health problem in many countries . This coIt has been considered that insulin resistance and hyperinsulinemia play a key role in the pathogenesis of NALFD (first causative step). Excessive deposition of fat in adipocytes and muscles determines insulin resistance with subsequent accumulation of fat in the liver , which, In spite of growing knowledge, several aspects of NAFLD pathogenesis are still unknown. Considering the difficulty in developing human studies to evaluate the influence of nutrition in the development of NAFLD and associated metabolic abnormalities, experimental models constitute a reliable alternative way. Different animal models of NAFLD/NASH have been developed, but few of them replicate the entire human phenotype , 14. TheSixty male Wistar rats, approximately 28 days old (after weaning), were housed individually and had free access to water and rat diet. The animals were randomly separated into the following groups: experimental group (EG), fed with highly palatable diet (see below) during 5 , 10 , 20 , and 30  weeks, and control group (CG), fed with standard rat chow during 5 , 10 , 20 , and 30  weeks. From week 25 to week 30, 12 animals belonging to the EG30 (6 rats) and CG30 (6 rats) were submitted to physical training (see below).At the end of each experimental period, after fasting for 10 hours, the animals were sacrificed. Blood samples were taken by cardiac puncture and stored at −20°C. The livers were immediately removed and fragments of about 1 mm thickness were fixed in 4% formaldehyde, dehydrated, immersed in xylene, and then embedded in paraffin for histology. Fresh tissue samples were collected to evaluate antioxidant enzymes activity.All experiments were approved by the Ethics Committee of the Universidade Federal de Minas Gerais for the Care and Use of Laboratory Animals (CETEA 53/2007) and were carried out in accordance with the regulations described in the Committee's Guiding Principles Manual. A rat belonging to the CG20 died and was excluded from all analyses.The standard rat chow  had the following nutrient composition: protein, 22%; fat, 4%; carbohydrate, 42%; minerals, 10%; phosphorus, 0.8%; vitamins, 1%; fiber, 8%; water, 12.5%. The chemical analysis revealed that 100 g of this diet contained 309 kcal, 24.8 g of protein, 3.4 g of fat, 44.8 g of carbohydrates, 8.2 g of fixed mineral residue, and 18.8 g of dietary fiber. The diet known as effective in inducing obesity in rats and described as highly palatable was composed of what follows: 33% of standard rat chow compacted to powder, 33% of condensed milk , 7% of sucrose , and 27% of water . The conThe diet was prepared daily, weighed, fractionated in portions, and stored in the feeder for 8–10 hours. The remaining food in the feeder was weighed to calculate the final amount of ingested food. The water content of the drinking bottles was renewed daily.On a weekly basis, the body weight, thoracic circumference (TC) (measured between the foreleg and hind leg), and nasoanal length were measured. Body mass index (BMI), that is, the ratio between body weight (g) and the square of body length (cm²), was calculated .−1 at 5% inclination for 5 minutes/day, during 5 consecutive days. After exercise familiarization, trained rats were submitted to the physical training protocol, which consisted of running sessions with gradual increase in intensity across 5 weeks, 5 days/week. The speed and duration of the exercise bouts were increased until the rats were able to run at 25 m·min−1, 5% inclination, during 60 minutes/day. The achievement of this exercise intensity ensures that a significant endurance training effect is produced [All animals were acclimatized to exercise on the motor-driven treadmill  by running at a speed of 10 m·minproduced . In ordeproduced .Measurement of glucose, total cholesterol, very low-density lipoprotein- (VLDL-) cholesterol, low-density lipoprotein (LDL-) cholesterol, high-density lipoprotein- (HDL-) cholesterol, and triglycerides was performed as recommended by the manufacturer  using an autoanalyzer .Serum concentrations of leptin and insulin were determined by radioimmunoassay  using a gamma-ray counter . The minimum detection value was 0.5 ng/mL.The determination of superoxide dismutase (SOD) activity was adapted from Dieterich et al. . Briefly2O2, 0.06 mL of liver homogenate, and 1.9 mL of potassium phosphate buffer  were mixed to give a final concentration of 6 mM of H2O2. It took 1 minute for the reaction to occur at room temperature. The decomposition of H2O2 by CAT was evaluated by the change in absorbance at 240 nm. The experiments were performed in duplicate. CAT activity was expressed as mmol of H2O2 decomposed per minute per milligram of protein. This procedure was adopted to avoid the possibility of interference in the activity of glutathione peroxidase, once the necessary cofactors were not present in the reaction medium.Catalase (CAT) activity was measured in the supernatant of liver homogenate as described by Nelson and Kiesow . BrieflyHistological sections were prepared from the material embedded in paraffin and stained with hematoxylin-eosin. The histological analysis was performed simultaneously by two examiners. The criteria established by Brunt et al. were used to describe the histological lesions. According to these criteria, macrovesicular steatosis is quantified based on the percentage of involved hepatocytes , and its zonal distribution and the presence of microvesicular steatosis are noted; hepatocellular ballooning is evaluated for zonal location, and the estimate of its severity  is based on the numbers of hepatocytes showing this abnormality .Hepatic expression of malondialdehyde (MDA), leptin, and the leptin receptor Ob-R was evaluated by immunohistochemistry in the animals sacrificed at weeks 20 and 30. From paraffin embedded tissues, sections on salinized slides (4 mm) were collected, deparaffinized, and hydrated. For immunohistochemistry, antigen reaction with ethylenediaminetetraacetic acid (EDTA) at pH 8.0, no steamer for 30 minutes at 98°C, was conducted, followed by Tris HCl pH 7.6 washing. The whole procedure was performed using Polymer Detection System kit . The primary antibodies used were anti-MDA monoclonal antibody (1F83)  diluted in 0.5 mL; anti-Ob (A-20) sc-84; and anti-Ob-R (H-300) sc-8325  at a dilution of 1 : 250 and 1 : 100, respectively.P value ≤0.25 at univariate analysis would be included initially. However, due to the high level of correlation between the explanatory covariates, we opted to adjust the final model with the following covariates: group, physical training, variation in BMI (ΔBMI), and variation in the amount of ingested calories . The adequacy of the models was assessed by analysis of the residues. For the categorical variables, logistic regression models were developed, with inclusion of the variables that showed on the univariate analysis a P value ≤0.25, and also clinical significance. The model fit was assessed by the Hosmer-Lemeshow test. Statistical analysis was performed using the R public domain software. Significance level was set at P value <0.05.Data are presented as frequencies and percentages, mean ± standard deviation (SD), and median and interquartile range (IQR). For each quantitative response's variables, we developed linear regression models in which all variables with The results of the anthropometric parameters, lipid and glucose profile, hormones levels, and antioxidant enzymes activity, as well the results of their comparative analyses between EG and CG along the time of follow-up, are described in Tables Liver histology was normal  in the CThe reaction for identifying MDA  was posiP = 0.007). No other clinical or metabolic variable was significantly different between the groups after the physical training.The comparison of the different variables between physical trained and untrained groups showed higher serum levels of HDL-cholesterol in the first group: medians 75 mg/dL and 52.2 mg/dL, respectively  and groups of rats, showed that the EG20 and EG30 had, respectively, lower insulin values of 83% and 89% compared to EG5. Furthermore, the animals of EG had an average insulin levels increased by 123% compared to the CG. The second model, including time (quantitative form), groups of rats, and Δkcal intake, showed that, for each increase of 1 unit in time, the average value of insulin decreased by 7% and, for each increase of 1 unit in Δkcal intake, the average value of insulin increased by 0.2%. The EG rats had an average insulin level increased by 100% compared to those of the CG.In EG20 and EG30, the leptin values were 33% and 40% higher, respectively, compared to the rats followed for 5 weeks. The EG had a mean value of leptin increased by 267% compared to the CG, and for every increase of 1 unit in ΔBMI the average value of leptin increased by 124.6%. The amount of SOD was 24% lower in the animals followed for 30 weeks in relation to those studied for 5 weeks. In the EG, the mean values of SOD were 11% lower compared to the CG; and, for each increase of 1 unit in ΔBMI, the mean SOD values decreased by 54%. The rats studied for 20 weeks presented an average of 2.4 less CAT units than those studied for 5 weeks, and in the EG an average of 1.8 less units of CAT relative to the CG was observed. Concerning the histological findings, it was found that, for each increase of 1 unit in the ΔTC, the chance of expressing ballooning and steatosis increased by 50%.This study demonstrates that a diet with high amount of simple carbohydrates, which resembles the current human dietary pattern, was able to induce obesity-related NAFLD, here characterized histologically by hepatic steatosis and hepatocyte ballooning, clinically by increased TC and BMI associated with hyperleptinemia, and metabolically by hyperglycemia, hyperinsulinemia (with subsequent insulin return to baseline levels), hypertriglyceridemia, increased serum levels of VLDL-cholesterol, depletion of antioxidants liver enzymes, and increased levels of MDA, an oxidative stress marker. Furthermore, rats that underwent physical training showed a significant increase in HDL-cholesterol in comparison to those that did not exercise.High-fat and methionine choline-deficient diets are widely used to produce hepatic steatosis and NASH in experimental animals , 21–26.  de novo hepatic lipogenesis, which is aggravated by diets with higher carbohydrate content than fat, plays an important role in glucose homeostasis and development of hypertriglyceridemia and hyperinsulinemia [ de novo hepatic lipogenesis increases by 10 times [In our study, free access to the sucrose-rich diet and high food consumption caused obesity/abdominal obesity in the EG rats from week 10. Obesity was associated with increased serum levels of glucose, triglycerides, VLDL-cholesterol, and insulin, which are manifestations of insulin resistance , 49. Theulinemia , 51. For10 times . Likewis10 times .A positive correlation between increase in serum levels of leptin and BMI was another finding of this study that corroborates human observations . The hyp in vivo enhances the activity of the fatty acid oxidative pathway in the liver, thus contributing to the reduction of triglycerides and VLDL-cholesterol in rats without leptin resistance [In an attempt to understand the action of leptin in the liver and its possible role in the pathogenesis of NAFLD/NASH, we evaluated the expression of leptin and Ob-R in the hepatic parenchyma and found intense leptin reaction in EG30, whereas Ob-R was observed in both groups, without difference between them. A possible role of leptin as an inducer of hepatic mitochondrial beta-oxidation has been postulated. Huang et al. demonstrated that leptinsistance . On the sistance . Cao et sistance . TherefoHepatic steatosis and hepatocellular ballooning—early stages of NAFLD—were present in all liver samples of the EG from week 10. At the final stage of the investigation, although more exuberant steatosis was expected, the pattern was similar to that observed at week 10. The duration of the study may not have been long enough to allow the development of more severe steatosis and the histological changes that characterize NASH. As the hepatic lesions that occur in NASH are associated with the expression of proinflammatory cytokines in the liver, it is possible that their investigation could have demonstrated NASH at an early stage. In addition, genetic factors could be acting. It is also possible to speculate that the high levels of leptin could be exerting a protective effect.MDA, a marker of lipid peroxidation, presented exuberant expression in the EG, whereas this reaction was negative in the CG. Oxidative stress induced by lipid peroxidation is a result of oxidant/antioxidant system imbalance . CellulaAlthough we observed hepatocellular ballooning denoting cell injury, one limitation of our study is the fact of not detecting NASH histologically. This was also a finding in several of the previous models in which NAFLD was induced by a simple carbohydrate-rich diet , 36–44. Exercise is considered an effective resource for controlling metabolic changes associated with obesity . The phyOur study demonstrated that a diet enriched with sucrose induced obesity, insulin resistance, diabetes, oxidative stress, and subsequent hepatic steatosis and hepatocellular ballooning. The lack of histologically evident inflammation and fibrosis in the liver parenchyma may have been due to the insufficient time of the experiment.