The aim of the study was to determine the prevalence and determinants of chronic non-communicable disease (NCD) risk factors in a rural community in the Limpopo Province of South Africa.This survey was conducted using the WHO "STEPwise approach to the surveillance of non-communicable diseases" (STEPS) methodology. Participants were residents of the Dikgale HDSS site and standardised international protocols were used to measure behavioural risk factors  and physical characteristics . Fasting blood glucose, triglyceride, cholesterol and HDL-C were determined in 732 participants. Data were analysed using STATA 12 for Windows.The prevalence of current smokers amongst the participants was 13.7%, of which 81.3% were daily smokers. Alcohol was consumed by 16.3% of the participants. The majority of participants (88.6%) had low daily intake of fruit and vegetables and low physical activity (66.5%). The prevalence of hypertension amongst the participants was 38.2%. Overweight, obesity and high waist circumference were prevalent in females. The cardio-metabolic risk profile was not significantly different between men and women. People who were older than 40 years, overweight or obese and those who consumed alcohol were more likely to be hypertensive. Smoking was associated significantly with older age, males, never married and divorced people. Alcohol consumption was associated with older age, males, low educational status and low income.High levels of risk factors for NCDs among adults in the Dikgale HDSS suggest an urgent need for health interventions to control these risk factors at the population level in order to reduce the prevalence of NCDs. Non-communicable diseases (NCDs) are amongst the leading causes of death in the world as they result in high mortality rates . Almost The available data in the scientific world reveal that nearly 80% of NCD-related deaths occur in low- and middle-income countries, despite the swift development of NCDs in high income countries. Most of the impacts resulting from mortality related to NCDs are preventable through innovative interventions which are cost-effective and achievable .Most NCDs share common risk factors, which are often categorised as behavioural or biological . TobaccoAn insight into the extent of the burden of risk factors for chronic non-communicable diseases in rural communities in the Limpopo Province of South Africa is crucial for effective advocacy and action. This could mainly contribute to the establishment of concerted and inclusive actions to improve chronic disease management irrespective of the cause by closely linking the development and global health agendas . SurveilThe Dikgale Health and Demographic Surveillance System (HDSS) centre  consistsThe participant’s demographic information, such as age, gender and educational achievement, were extracted from the Dikgale HDSS database. The WHO STEPwise approach to Surveillance (STEPS) for NCD risk factors  was usedCriteria used for the diagnosis of hypertension were those proposed by the World Health Organisation (WHO)/International Society of Hypertension, using the average systolic BP of 140mmHg or higher, or if the average diastolic BP was 90mmHg or higher if participants were on anti-hypertensive treatment –23. HeigSTATA statistical software  was utilised for all analyses and categorical variables were presented as percentages, whilst continuous variables were expressed as mean ± SD. The coding of data was done in line with WHO guidelines . CompariEthical approval of the study was obtained from the Medunsa Research and Ethics Committee (MREC) at University of Limpopo and Department of Health of the Limpopo Province. The Dikgale Tribal Authority gave permission for the study to be conducted and signed full informed consent forms were completed from each participant.The mean ages of men and women were 41.29±21.46 years and 45.74±20.39 years respectively. Sixty two percent of the participants were females and most of the participants were never married (55%), while 58% had low education. Literacy amongst men and women was 39.6% and 43.3% respectively, while the employment rate amongst men and women was 32.4% and 26.8% respectively. The definitions of variables used in the results are explained in p<0.001). Amongst current smokers, 81.3% were daily smokers ) (p<0.001)  was 13.7% and was significantly higher in males 29.2% than in females 4.5% (<0.001)) . The prep<0.001) .p<0.001) than in females. The prevalence of the use of smokeless tobacco products was significantly higher amongst older people of both genders (p<0.001). In females, use of smokeless tobacco products increased significantly from 1.1% in age group 15–25 years to 26.6% in age group 65 years and above (p<0.001)  was significantly higher in older males (from 13.0% in age group 15–25 years to 58.3% in age group 45–54 years) (p<0.001) .p<0.001) respectively (p<0.001) but a decline of 3.7% in age group 65 years and above was noticed (p<0.001)  was 16.3% of the total population and males had a significantly higher prevalence for alcohol consumption than females 28.9% vs 8.6% (ectively . The prep<0.001) .p = 0.91) (p<0.001). Among females there was no trend in the prevalence of low fruit and vegetable intake with age, p = 0.08  was 88.6% for both sexes and there was no significant difference between males and females ( = 0.91) . The prep = 0.08 .p<0.001 , was found in 65.5% of the total population. Females had a significantly higher prevalence for physical inactivity 70.8% as compared to 40.7% for males, p<0.001 . The prein males .p = 0.27. The prevalence of hypertension increased significantly with age in females . A similar trend was observed in males   .p<0.001) (p<0.001). In females, obesity was highest in the age group 45–64 years. The prevalence of obesity in females showed an increasing trend from 13.6% in age group 15–25 years to 41.9% in age group 55–64 years. (p<0.001)  . The prep<0.001) .p<0.001. There was no significant trend in the prevalence of high waist circumference with age  than did males (7.8%) with age .p = 0.017 and from 1.5% in age group 15–25 years to 19.6% in age group ≥63 years in males p = 0.003 . A similarly increasing trend was observed in males (11.1% in age group 15–25 years to 40.8% in age group 65 years and above) (p = 0.004)  .p<0.001). A similarly increasing trend was observed in males from 15.6% in age group 15–25 years to 48.2% in age group 65 years and above, p<0.001  levels was 32.6% of total population. Older females showed a higher prevalence of high total cholesterol (TC) levels when compared to younger female participants , 5.3 times more likely to use smokeless tobacco products (p<0.001), 2.4 times more likely to consume alcohol (p<0.005) and 1.1 times more likely to have low fruit and vegetable intake (p<0.005) (p<0.001), 0.1 less likely to use smokeless tobacco products (p<0.001), 4.9 times more likely to consume alcohol (p<0.001) and 0.6 times less likely to be physically inactive (p<0.001) (p<0.05) and 0.6 times less likely to consume alcohol (p<0.05) than were educated people. People who were never married were 2.5 times more likely to be smokers (p<0.001) than were married people, while divorced people were 5.8 times more likely to be smokers than were married people (p<0.001) and widowed people were 1.9 times more likely to be smokers. Marital status was not significantly associated with the use of smokeless tobacco products, alcohol consumption or intake of low fruit and vegetables. People in the low income category were 1.4 times more likely to consume alcohol than people in the high income category (p = 0<0.05) (Older people were 3.3 times more likely to be smokers (p<0.005) . Males wp<0.001)  than wer 0<0.05) .p<0.001), 1.8 times more likely to have high fasting blood glucose (p<0.05), 1.7 times more likely to have high cholesterol levels (p<0.005), 2.1 times more likely to have a high total cholesterol HDL-C ratio (p<0.05) and 2.2 times more likely to have raised triglycerides (p<0.001) than were young people (p = 0.189), 0.5 times less likely to have high cholesterol levels (p<0.05) and 0.3 times less likely to have high total cholesterol HDL-C ratio (p<0.05) than were non-smokers. People who consumed alcohol were 1.6 times more likely to be hypertensive (p<0.05), and 0.6 times less likely to have raised triglyceride (p<0.05) than people who did not consume alcohol. People who had low fruit and vegetable intake were found to be 1.8 times more likely to have high fasting blood glucose (p<0.05) than did people with normal fruit and vegetable intake  , but lowOur study demonstrated that risk factors for chronic non-communicable diseases are major public health problems, with a high percentage of men using tobacco and alcohol. The study findings reflect the cultural practices where smoking and alcohol consumption are more prevalent in men than in women. The prevalence of smoking and alcohol consumption in our study were found to be 13.7% and 16.3% of the population respectively, which is lower than the prevalence reported in a study comprising South African adults in all nine provinces aged 18 years and older (38% and 21%) , and werThe present study showed that smoking was associated with increasing age, male gender, people who were never married, those who were divorced and those who were widowed. This concurs with other studies conducted in rural HDSSs in Asia , 31. TheOur study findings also showed a strong association between alcohol consumption and increasing age, gender and low education, which concurs with the findings of other studies , 35. A hEngaging in physical activity is widely recognised as the primary means of prevention of chronic diseases, as well as for the treatment and rehabilitation of patients. Moreover, physical activity has beneficial effects on an individual’s health and well-being . Our stuThere may be under-reporting of physically active behaviour in rural communities when using self-report methods , 46. TheOur study showed a hypertension prevalence of 38.0% of the population, which is higher than the findings of the 2005 Alberts et al. study, where approximately 25% of participants had hypertension . Our stuContrary to the findings of a study conducted in Kerala, India , our stuObesity was found to be associated with hypertension in our study, which is consistent with findings observed elsewhere in South Africa and in India ,53. A hiThe prevalence of high fasting blood glucose generally increased with age in all groups assessed in this study, indicating diabetes, at a level of more than 7 mmol/l, in 12.5% of population. This is consistent with a study conducted by Bradshaw et al. in South Africa  and a stMales had a lower prevalence of high cholesterol levels than did females in our study, which is different from the results of a study conducted by Al-Nuaim in Saudi Arabia . The preIn the present study, the prevalence of total-to-HDL cholesterol ratio, a predictor of cardiovascular disease, showed no significant difference between males and females, which concurs with a study conducted in the same area in 2005 by Alberts et al. . In bothOur study findings need to be interpreted cautiously as the Dikgale HDSS is developed as a sub district level surveillance system in a rural setting in the Capricorn District of the Limpopo Province of South Africa. The survey was cross-sectional and was not conducted throughout the year. Some variables were self-reported, which may have resulted in self-report bias and we did not compute measures of agreement between self-reported conditions and those obtained from actual measurements. Again, some of the behaviours that vary seasonally  may not be representative; and cause and effect cannot be determined for associations between BMI and selected health conditions. Participants were requested not to eat anything after a certain time the previous evening before they came to participate in the study the following morning but it was difficult to measure adherence to this request, which is a limitation and a challenge to validity on the serum lipids and glucose results.It was not possible, therefore, to extrapolate our findings to a larger population at provincial or country level. However, the findings in our study are in line with most of the findings from other sub-national or national surveys from other parts of the world. Based on previous surveys, we expected a non-response rate of 10% but our study had a high proportion of non-respondents (32.6%), most of them were from the age group 25–54 years, which represents the working age group, contributing 55.4% of the non-respondents. Using the approach by firstly excluding the people who moved out of the community, deceased, therefore, the main reasons for the non-respondents were absent from home even after repeated visits (27.2%), refused to participate (35.1%), sick (1.3%), underage (2.1%) and prison (0.3%).The present study found that the prevalence of risk factors for non-communicable diseases, such as smoking, alcohol consumption, low fruit and vegetable consumption, physical inactivity, hypertension, overweight and high waist circumference is high. Due to the epidemiological transition which is occurring in this rural area, it is recommended that health interventions which aim to control risk factors at population level should be planned and implemented by the provincial government in order to slow the progress of the coming non-communicable diseases epidemic. In conclusion, our findings highlighted the importance of reaching out to poor rural communities with messages regarding the impact of diet, smoking and alcohol consumption on general health.This study in the Dikgale HDSS provides baseline data on risk factors for NCDs and these epidemiological data will be of value if used by the health policy makers in developing interventions for chronic disease risk factor prevention and control , 55 in t


Cerebral toxoplasmosis (Toxo) is rarely described in children. The Romanian pediatric cohort consists of children that have been infected parenterally in the late ’80s. We aimed to describe the prevalence, clinical findings and outcome of Toxo in children and adolescents from the Romanian Pediatric Cohort, that have been diagnosed in the “Dr. Victor Babeş” Hospital, one of the main reference centers for HIV from RomaniaCerebral toxoplasmosis was diagnosed based on CDC case definition (presumptive and definitive diagnosis). We reviewed retrospectively the charts of all 29 children diagnosed with Toxo starting 1996, recording the demographic, HIV markers, antiretroviral treatment, clinical and neuroimaging data, treatment and outcome of Toxo.T. gondii IgG antibodies in plasma and/or cerebrospinal fluid. Only 6 patients had treatment with cotrimoxazole, most of them being treated with pyrimethamine/sulphadoxine combination and alternatively with atovaquone. 69% of patients had any adverse reactions to Toxo treatment. Most adverse reactions were cutaneous in 10 patients (5 severe) and anemia in 7 patients. Nine patients had antiretroviral therapy (ART) before Toxo diagnosis, out of them 1 had mono, 1 dual therapy, 6 were failing cART and one patient had immune reconstitution syndrome. Ten patients (34.5%) died. The median survival time was 117 months. In univariate analysis survival was correlated with shorter time from onset to admission (p=0.04) while on multivariate analysis diagnosis in the post-cART period was the only factor associated with longer survival (p=0.03). 14 of 19 patients recovered with sequels, most of them motor deficits.The prevalence of Toxo was 4.8% from 604 patients followed in the Hospital. Out of 29 patients diagnosed with Toxo, 19 were girls and 28 had parenterally and 1 had vertically acquired HIV. The mean age at HIV diagnosis was 11.5±6.5 years, and 15.6±5.3 years at Toxo diagnosis. In 10 patients HIV was diagnosed concomitantly with Toxo. At onset 89.7% patients had focal neurological signs and 62.1% had headache. Median CD4 count was 60 (95% CI for median 27-95) lf/cmm. 28 patients had positive Cerebral toxoplasmosis in this particular pediatric cohort shared common features with that reported in adults pertaining to prevalence in pre-cART period and pathogenic mechanism. Survival was associated with a more rapid diagnosis of cerebral toxoplasmosis and with access to cART.


Schistosoma haematobium has been known to be endemic in several lowland areas of the country where it causes considerable public health problems, mainly among school-age children. However, information on recent magnitude and risk factors of the disease is lacking, particularly for Gambella area. Therefore, this study aimed to assess the prevalence of urinary schistosomiasis and associated risk factors among Abobo Primary School children in Gambella, southwestern Ethiopia.In Ethiopia, urinary schistosomiasis caused by A cross-sectional study involving 304 school children was conducted in Abobo Primary School, Gambella Regional State, southwestern Ethiopia, from February to June 2014. Ten ml of urine sample was collected from each study participant and processed for microscopic examination by the urine filtration method; egg load for positive individuals was determined per 10 ml of urine. Data on socio-demographic characteristics and risk factors were collected using an interview-based questionnaire. The data were entered into and analyzed with SPSS version 20. Logistic regression and odds ratio were used to measure association and strength between variables, respectively. P-value < 0.05 at 95% CI was considered as statistically significant.The prevalence of urinary schistosomiasis was 35.9% (109/ 304) with a mean egg intensity of 8.76 per 10 ml of urine. Being male [AOR (95%CI) = 2.15], having father as a farmer [AOR (95%CI) = 1.96] and children living apart from parents [AOR (95% CI): 3.09 ] were significantly associated with urinary schistosomiasis.The present study area in Gambella Regional State, southwestern Ethiopia, represents moderate-risk community for urinary schistosomiasis. Sex, father’s occupation and living apart from parents were found to be associated with infection. Treatment of all school-age children and fishermen is required once every 2 years until the prevalence of infection falls below the level of public health importance. It is also recommended to complement praziquantel treatment with supplementary measures such as provision of sanitation facilities and health education. Schistosoma. The main disease causing schistosome species are Schistosoma haematobium (S. haematobium), S. mansoni, S. japonicum, S. mekongi and S. intercalatum  and 9.36 per 10 ml of urine) than females (38.5% and 7.81per 10 ml). On the other hand, higher frequency of urinary schistosomiasis was identified among children in the 13 to 14 years age group (39.1%) than the 7 to 9 years age group (15.6%), and the 10 to 12 years age group (34.9%), . However, the mean egg count was higher among children in the 10 to 12 years age group (11.18 per 10 ml). Nevertheless, there was weak linear relationship (r = −0.007) between age of children and egg count of urinary schistosomiasis. Overall, there was no statistically significant association between mean egg count of urinary schistosomiasis and sex, age group, distance from the river, use of river for drinking and washing, and swimming habit (data not shown). Higher frequency of urinary schistosomiasis was identified among children with farmer as father’s occupation (56.0%)  and housewife as mother’s occupation (64.8%) . Children with a habit of swimming accounted for 91.7%  of the total urinary schistosomiasis prevalence.In this study, the prevalence of urinary schistosomiasis was 35.9% with a mean (SD) intensity of 8.76 (14.51) per 10 ml of urine. Among infected study participants, 106 (97.2%) presented with light intensity of urinary schistosomiasis and 3 (2.8%) with heavy intensity. The frequency and mean intensity of urinary schistosomiasis was higher among males , male [COR (95% CI): 1.94 and farmer as father’s occupation [COR (95% CI): 1.68] were significantly associated with urinary schistosomiasis. In the multivariate logistic regression analysis, only sex, father’s occupation and living with parent had statistically significant association with urinary schistosomiasis. Males were two times more likely to be infected with In the present study, the overall prevalence of urinary schistosomiasis among school children in Abobo area, Gambella, was 35.9% and can be categorized as moderate based on WHO categories of endemic communities . This prThis study showed lower mean intensity of urinary schistosomiasis (8.76 per 10 ml of urine) as compared to a study carried out in Afar Region, Ethiopia (14.8 eggs/10 ml of urine), Senegal (185eggs/10 ml of urine) and the Sudan (14.9 eggs/10 ml) ,25,26. TIn the current study, males were two times more likely to be infected with urinary schistosomiasis than females. This was comparable with studies conducted in the Sudan and Swaziland ,20,26. SS. haematobium eggs . This result agreed with studies from Sudan, Ghana and Nigeria ,26,29 whIn addition, children not living with their parents were three times more likely to be infected with urinary schistosomiasis [AOR (95% CI): 3.09 ] than those living with their parent. Similar result were reported from Nigeria . ChildreIt is interesting to observe high prevalence of urinary schistosomiasis among children with the habit of swimming without significant association. Similar result were reported from an earlier study in Nigeria . DespiteThe present study area in Gambella Regional State, southwestern Ethiopia, represents moderate-risk community for urinary schistosomiasis. Sex, father’s occupation and living apart from parents were the determinant factors for the infection. Treatment of all school-age children and fishermen is required once every 2 years until the prevalence of infection falls below the level of public health importance. It is also recommended to complement the praziquantel treatment with supplementary measures such as provision of sanitation facilities and health education.Swimming habit: child behaviour in recreational water contact.Swimming regularity: frequent recreational water contact of children.Living with parent: children living in the household of his or her biological parents for over three months.Distance from the river: the space between children’s home and the river.Heard about urinary schistosomiasis: information children have acquired about transmission, signs and symptoms, diagnosis and treatment of urinary schistosomiasis.Symptom of urinary schistosomiasis: information children have acquired about clinical symptoms of urinary schistosomiasis.Parasites penetrate skin: information children have acquired as to whether or not the schistosome parasites penetrates human skin during infection.Know the intermediate host: information children have acquired about schistosome intermediate hosts.Have prevention: information children have acquired about prevention and control measures of urinary schistosomiasis.Have treatment: information whether or not children knew the availability of treatment options against urinary schistosomiasis.


The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant (An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm Biosensors have wide applications ranging from the food industry to environmental monitoring and clinical analysis. The concept of a biosensor was first introduced by Clark and Lyons  in the f2O2) using molecular oxygen (O2) as the electron acceptor. The two-stage enzyme process, typical for the class of oxidases, consists of enzymatic oxidation of glucose by its cofactor FAD (flavin adenine dinucleotide) (redox centre) which is then reduced to FADH2  or the decrease in the cathodic current (O2 reduction) at the electrochemical cell at a fixed potential. Oxygen electrode-based glucose biosensors and hydrogen peroxide electrode-based glucose biosensors are two commonly studied amperometric glucose biosensors. The reactions on a cathodically polarized platinum electrode are shown below:2O2, where the current produced is proportional to its concentration. In an amperometric glucose biosensor, the working potential over which H2O2 is detected is typically between 500–750 mV vs. Ag/AgCl.In amperometry, the detection of glucose is usually based on measuring the increase in the anodic current -2-amino-2-deoxy-ß-D-glucopyranose], a cationic polysaccharide abundant in the shells of crustaceans. It is derived by partial deacetylation of chitin . The preThe sugar  content in ripened fruits is correlated with their glycaemic index. Diabetic patients often question and worry whether it is safe for them to eat fruit, which can contain large quantities of sugar. Glucose is a major monosaccharide found in almost all fruits and is easily absorbed through gastrointestinal tract to increase blood glucose levels. Thus, the glucose content attracts great attention as an indicator of the glycaemic index for fruits. Therefore, the development of simple, reliable and economical glucose biosensors is desirable for measuring the glucose content of fruits. Application of such biosensor for quality control in the fruit industry can have an economic impact as well as health benefits to end users who are diabetic patients. In this paper, we report the fabrication and characterization of an amperometric-based glucose biosensor for measuring glucose content in fruit homogenates.Aspergillus niger , glutaraldehyde  and glycerol were purchased from Sigma . Chitosan (Code #22742) was procured from Fluka (Switzerland). D(+)glucose monohydrate was supplied by System . Glacial acetic acid, citric acid monohydrate and potassium phosphate (KH2PO4) were obtained from R&M Marketing  while di-sodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) and sodium chloride were bought from Hamburg Chemical (Germany). Tri-sodium citrate was purchased from Grauwmeer . Hydrogen peroxide (>30% w/v) was bought from Fisher Scientific , while premounted dialysis membrane  was bought from Fisher Scientific . Aluminium oxide (highly pure for polishing) was purchased from BDH Laboratory Supplies (England). A PK-4 Polishing kit (MF-2060), platinum working electrode (MF-2013), and silver/silver chloride (Ag/AgCl) reference electrode (MF-2079) were purchased from Bioanalytical Systems Inc. . All chemicals were of analytical grade and were used without further purification. The molecular weight of chitosan was determined by dilute solution viscosity using an Ubbelohde viscometer U-tube  and the degree of deacetylation was determined using the first derivative UV-spectrophotometric method. The chitosan had viscosity-average molecular weight of 981.80 kDa and was 82.44% deacetylated.Glucose oxidase from 2.2.1 Electrochemical measurement. Amperometric detection of glucose was performed using a potentiostat , West Lafayette, Indiana, USA) poised at +0.6 V connected to an integrator-plotter  and a digital multimeter . The conventional three electrodes consisted of a silver/silver chloride (Ag/AgCl) reference electrode , a platinum wire  as counter electrode and a platinum working electrode  with the enzyme-chitosan layer and a protective dialysis membrane. Unless stated otherwise, all experiments were carried out in 10 ml of phosphate buffer  maintained at 25 ± 0.1°C using a digital temperature controller  with stirring to provide convective transport. Glucose stock solution (prepared in phosphate buffer) was allowed to mutarotate at 4°C for at least 24 h prior to use, since only β-D-glucose is a substrate for the enzymatic reaction. At stable background current, aliquots of the β-D-glucose stock solution were introduced into the stirred phosphate buffer and the steady anodic current produced by the enzymatically generated H2O2 was recorded.2.2.2 Preparation of chitosan membrane and characterization. One gram of chitosan was dissolved in 100 ml of 0.8% (w/v) acetic acid and stirred overnight to ensure complete dissolution. Varying volumes of chitosan solution were then pipetted into petri dishes at a pre-measured volume per surface area (ml/cm2) and then allowed to dry overnight in an oven at 60°C. The thickness of the membranes was measured using a micrometer  at five locations (the centre and four corners), and the mean thickness calculated. Mechanical properties such as tensile strength and elongation at break were measured with a texture analyser  equipped with a 5 kg load cell. The other prepared membranes were neutralized with 1% w/v sodium hydroxide (NaOH) for 30 minutes followed by rinsing with distilled water to remove excess NaOH. The neutralized membranes were cut into squares (1.5 x 1.5 cm2) for the diffusion study and for enzyme immobilization.2.3.3 Study on the diffusion of hydrogen peroxide through chitosan membranes. The diffusion properties of chitosan membranes cast in different thicknesses were determined by measuring the electrode response to H2O2 using amperometric detection. The anodic current generated by H2O2 which had diffused through the membrane was sensed by (i) a bare platinum electrode (bare PT), (ii) an electrode covered with dialysis membrane  and (iii) an electrode covered with blank chitosan membrane in addition to a dialysis membrane (CHIT/PT). A comparison of the effect of the chitosan membranes on the electrode response to H2O2 was then made.2.2.4 Enzyme immobilization. The method of Magalhães et al. [2) was coated with 20 μl of 1% (v/v) glutaraldehyde and allowed to dry at room temperature. Subsequently, 20 μl of 10 mg/ml (0.2 mg) GOD in phosphate buffer  containing 5% (v/v) glycerol was spread evenly onto the same surface of the membrane with the aid of an L-shaped rod. The immobilized membrane was then left to dry at room temperature. The small amount of glycerol in the enzyme solution acts as an emollient to facilitate even spreading of GOD on the membrane surface. The dried membrane was washed with distilled water and kept in phosphate buffer  at 4°C until further use.s et al.  was modi2.2.5 Construction of the glucose biosensor. The platinum electrode was first polished with 0.05 μm alumina on a polishing pad, washed with distilled water and finally sonicated for 2 minutes to remove the alumina particles. Then the GOD-chitosan membrane and a moist dialysis membrane as a lamination layer were fastened onto the surface of the platinum electrode with an O-ring.2.2.6 Optimization of experimental variables for the analysis of glucose using biosensor. The factors influencing enzymatic activity and ultimately biosensor performance were investigated. These included applied potential, membrane thickness, glutaraldehyde concentration, enzyme concentration, temperature, pH and buffer concentration.2O2 in phosphate buffer as well as by the biosensor (GOD-CHIT/PT) in 2 mM glucose in phosphate buffer was made.The effect of applied potential on the steady-state current response of the enzyme-electrode in the potential range from 0.30–0.80 V in 0.05 V increments was studied. The potential was set at the lowest voltage of 0.30 V and the background current allowed to decay to a steady-state value before increasing the applied potential stepwise to 0.80 V. A comparison of the response current at different applied potentials generated by the bare platinum electrode in phosphate buffer and in 0.05 mM H2. Glutaraldehyde concentration was investigated from 0.1% v/v to 1.0% v/v. Various amounts of GOD (0.05–0.8 mg) in phosphate buffer  were immobilised on the chitosan membrane. The effect of the temperature of analysis (from 15–50°C) on biosensor performance was studied by measuring the anodic current generated. The optimal pH for enzymatic activity was investigated by varying the pH value from 4.0–8.0. Buffers at different pH values were prepared with 0.1 M citrate buffer to obtain pH values from 4.0–5.5 and with 0.1 M phosphate buffer to attain pH values in the 6.0–8.0 range. The concentration of the working buffer at optimum pH was investigated from 0.01–0.20 M. The optimal value obtained for each parameter was used in subsequent experiments.The fabricated chitosan membranes were cast in thicknesses ranging from 0.21 to 0.42 ml/cm2.2.7 Calibration of the glucose biosensor. Six GOD-CHIT/PT electrodes were prepared according to the optimal conditions: 0.6 mg GOD was immobilized onto 0.21 ml/cm2 chitosan membrane and crosslinked with 0.2% v/v glutaraldehyde. The biosensors performed their electrochemical measurements at 35°C, in a supporting electrolyte of 0.1 M phosphate buffer, pH 6.0.Imax were determined by from a Eadie-Hofstee plot using the equation shown below:I is the steady state current after the addition of substrate, c is the bulk concentration of the substrate (glucose) and Imax is the maximum current measured under a saturated substrate condition. The Imax of the biosensor were then determined from the slope and intercept on the y-axis of the plot.Aliquots of β-D-glucose stock solution were successively added into 10 ml of stirred phosphate buffer in an electrochemical cell. Three different glucose stock solutions with concentrations of 0.01 M, 0.1 M and 1.0 M were prepared to obtain the hydrodynamic response for glucose from 0.01 to 130 mM. The mean value of the anodic current (μA) was plotted against the glucose concentration (in mM). The linear range of the biosensor was then determined from the saturation curve. The detection limit at which the signal to noise (s/n) > 3 was determined experimentally. The apparent Michaelis-Menten constant, 2.2.8 Repeatability and reproducibility. The repeatability generated by the glucose biosensor was studied by measuring the anodic current generated by 3.98 mM glucose in 10 ml phosphate buffer (pH 6.0) a total of 20 times in a single day. On the other hand, the reproducibility of the biosensors was studied by measuring the current generated by 3.98 mM glucose in 10 ml phosphate buffer (pH 6.0) by using six different glucose biosensors. Each biosensor was tested by replicate (n = 3) analysis. The total mean value was calculated and the relative standard deviation (RSD) provided the analytical precision. The RSD was calculated using the following equation:2.2.9 Storage stability study. Three good GOD-CHIT/PT biosensors were prepared. On the other hand, three free enzyme-electrodes (GOD/PT) were prepared with the same amount of GOD, where the enzyme was coated directly on the platinum electrode surface without immobilization, only protected with a layer of dialysis membrane and fastened with an O-ring. The storage stability of the two types of enzyme-electrodes was explored under optimal experimental conditions.The responses of the three GOD-CHIT/PTs and three GOD/PTs to 3.98 mM glucose were measured daily during the first two weeks. After 2 weeks, the biosensors were tested every 3–5 days over a period of 80 days. The mean of the relative current to the initial current sensed by these biosensors was plotted as a function of time. All enzyme electrodes were kept in 0.1 M phosphate buffer (pH 7.0) and stored at 4°C when not in use.2.2.10 Glucose determination in fruit. Fresh fruits  were obtained from the local market and homogenized with distilled water (10 or 1 g in 1 L). Further dilution using distilled water was made if necessary. The homogenate was centrifuged at 3000 rpm and 4°C and the supernatant was filtered through a nylon syringe filter (0.2 μm) prior to determining the glucose content using the glucose biosensor and a commercial glucose assay kit  that relies on spectrophotometric detection. The statistical analysis method (T-test) was used to compare the results obtained from the glucose biosensor and the commercial glucose assay kit at a level of significance P<0.05 using GraphPad v5.3.1.1 Mechanical properties. A concentration of acetic acid higher than 0.8% w/v was not suitable for use in enzyme immobilization [2. The mechanical strength of the membranes was described in terms of tensile strength whilst their brittleness was characterized by a decrease in the percentage of elongation at break. As shown in lization . Thus, 13.1.2 Studies on the diffusion of hydrogen peroxide through chitosan membranes. The effect of different membrane thicknesses on diffusion of the substrate was studied using H2O2 as an analyte at pH 7.0. The electrodes used were a bare platinum electrode (bare PT), a platinum electrode covered with dialysis membrane  and a third platinum electrode covered with both chitosan and dialysis membrane (CHIT/PT). The diffusion barrier was calculated as a permeation factor and expressed as a percentage as shown in the following equation:2 was too thin. It was not only brittle but also difficult to handle.3.2.1 Selection of applied potential.2O2 and GOD-CHIT/PT detecting 2 mM glucose. Phosphate buffer was used as the medium in both cases. The anodic current curve of the enzyme electrode in sensing glucose demonstrated a behaviour similar to that of the bare platinum electrode in sensing H2O2 over the same applied potential. The response current for both sensors increased from 0.3 to 0.6 V and then plateaued. The background current of the electrode in phosphate buffer increased only slightly with increasing applied potential from 0.3 to 0.8 V. An applied potential of 0.6 V was chosen as the working potential in subsequent experiments as it gave the highest response for the electrodes.3.2.2 Effect of membrane thickness on biosensor response. Chitosan membranes were cast in thicknesses ranging from 0.21 to 0.42 ml/cm2 and cut into 1.5 x 1.5 cm2 squares. The prepared membranes were immobilized with 10 mg/ml GOD and applied to the platinum electrode using the methods as stated in 2.2.4 and 2.2.5. The GOD-CHIT/PT electrodes were then utilized for electrochemical measurement. In general, the behaviour of an enzyme-electrode can be influenced by membrane thickness. The enzyme immobilized on a chitosan membrane with a measured volume of 0.21 ml/cm2 showed the highest response to glucose in amperometric detection. A decrease in current was observed with thicker membranes  of 0.9999 . Repeatability refers to the agreement between successive measurements of the same sample, whereas reproducibility describes the closeness of agreement between results  obtained using the same method under different conditions (using different glucose biosensors) .Repeated 20 measurements of 3.98 mM glucose by a biosensor had a precision (RSD) of 2.30%, with a mean current of 0.22 μA. The mean value of the current measured by six different biosensors was 0.23 μA and the precision (RSD) was 3.70%.Enzyme stability within the matrix is a vital consideration in developing a biosensor. As such, the storage stability of the biosensors was evaluated over a period of 80 days . The GODThe results implied that the immobilized enzyme on chitosan membrane could be used repeatedly for amperometric detection over a much longer period compared to free enzyme that was used only once. Crosslinking not only permits high enzyme loading but also improves the stability of the enzyme-chitosan membrane . This isHowever, there was still some loss of activity during the investigation period. We suggest that the gradual decrease in the current might be due to temperature changes in the enzyme electrodes occurring between storage and experimental temperatures . In addition, partial enzyme denaturation might have occurred over a period of time. The possibility of electrode fouling during storage could also affect the sensitivity of the biosensors.Both the commercial glucose assay and the biosensor were able to measure the glucose content of fruit homogenates accurately. Statistical analysis showed that there were no significant differences between these two methods of measurement . Thus, iChitosan is a suitable matrix for glucose oxidase immobilization via glutaraldehyde crosslinking. The resulting glucose biosensor shows good repeatability and reproducibility. The results of a storage stability trial suggest that the immobilization process permits the enzyme to be reused, resulting in operational stability over a period of time. The wide linear detection range provided good accuracy to the glucose content measurements. The small value of the Michaelis-Menten constant (


Gross target volumes (GTVs) of the primary tumor were contoured on the ten phases images of 4DCT to generate IGTV10. GTVPET were determined with eight different threshold using an auto-contouring function. The differences in the position, volume, concordance index (CI) and degree of inclusion (DI) of the targets between GTVPET and IGTV10 were compared.Twenty patients with non-small cell lung cancer (NSCLC) underwent enhanced three-dimensional CT (3DCT) scan followed by enhanced 4DCT scan of the thorax under normal free breathing with the administration of intravenous contrast agents. A total of 100 ml of ioversol was injected intravenously, 2 ml/s for 3DCT and 1 ml/s for 4DCT. Then PET (excluding GTVPET15%) and IGTV10 were observed only in z axes (P < 0.05). GTVPET15%, GTVPET25% and GTVPET2.0 were not statistically different from IGTV10 (P < 0.05). GTVPET15% approximated closely to IGTV10 with median percentage volume changes of 4.86%. The best CI was between IGTV10 and GTVPET15% (0.57). The best DI of IGTV10 in GTVPET was IGTV10 in GTVPET15% (0.80).The images from seventeen patients were suitable for further analysis. Significant differences between the centric coordinate positions of GTV10. At present 3D-PET/CT should not be used for IGTV generation.None of the PET-based contours had both close spatial and volumetric approximation to the 4DCT IGTV Worldwide, lung cancer is the most common cause of cancer-related mortality . About 810 is generated by combining all 10 individual gross tumor volumes (GTVs) contoured in each phase of the 4DCT dataset, which is thought to encompasses the motion information for the tumor in the whole respiratory cycle  was defined as the ratio of the intersection of A with B to the union of A and B . The maxThe definition of DI of volume A in volume B [DI (A in B)] is the percentage of the overlap between volume A and B in volume A . The forThe DI can represent the percentage of one volume included by another volume, and 1-DI can represent the percentage of one volume not included by another volume.PET and IGTV10, and in the CIs of GTVPET and IGTV10. The Wilcoxon test was performed to estimate the differences of centroid coordinate positions between GTVPET and IGTV10, and also used to estimate the variabilities of target volumes between GTVPET and IGTV10. We used the Spearman correlation test to analyze for associations between centroid shifts in the 3D directions and CIs. Values of P < 0.05 were regarded as significant for all the tests. Descriptive statistics were used as appropriate.Statistical analysis was performed using the SPSS software package (SPSS 17.0). The one-way ANOVA test was used to determine the variations in the DIs of GTVmax in the tumor was 6.09 and 6.07, we could not obtain GTVPET15% target volumes as the volume obtained from the SUV15% contours were indistinguishable from background lung activity.For lesion 15 and 16, the SUV10 volumes and the PET/CT volumes. The variations in the centroid coordinate positions in the CC direction of GTVPET20% and IGTV10, GTVPET25% and IGTV10, GTVPET30% and IGTV10, GTVPET35% and IGTV10, GTVPET40% and IGTV10, GTVPET2.0 and IGTV10, GTVPET2.5 and IGTV10 were significant , while in the LR and AP directions were not significant (P > 0.05). The variations in the LR, AP and CC directions of GTVPET15% and IGTV10 were not significant .Table 10, GTVPET15%, GTVPET20% and GTVPET2.0 showed no significant difference . Figure PET to IGTV10 standardized to the IGTV10 for each case. The SUV 15% contour approximated most closely to the IGTV10 with the lowest median percentage volume changes of 4.86%. The corresponding values with respect to the IGTV10 for the SUV20%, SUV25%, SUV30%, SUV35%, SUV40%, SUV2.0 and SUV2.5 contours were −8.87%, −24.51%, −37.51%, −43.57%, −52.24%, −7.03% and −21.93%, respectively.The volumes of primary tumors measured by PET/CT and 4DCT were shown in Table 10 volumes. The median CIs ranged from 0.30 to 0.57 . The best fit for CI was at SUV15% (0.57), followed by SUV 2.0 (0.56). The CIs of IGTV10 and GTVPET were inversely correlated with the centroid shifts in 3D directions .Figure PET in IGTV10, and IGTV10 in GTVPET. The median DIs of IGTV10 in GTVPET ranged from 0.31 to 0.80 . The best DI was IGTV10 in GTVPET15%. The median DIs of GTVPET in IGTV10 ranged from 0.60 to 0.85 . The best DIs were GTVPET35% and GTVPET40% in IGTV10.Figure 18 F-FDG PET/CT volumes of NSCLC [18 F-FDG PET/CT to IGTV definition is not currently well established. To the best of our knowledge, there is few study that has compared tumor sizes and CI values between GTVPET and IGTV10 in contouring NSCLC.Accurate definition of the target volume, ideally incorporating metabolic information, becomes paramount importance in the current trend in NSCLC treatment planning . A numbeof NSCLC –24. Howe10 from 10 phases of the 4DCT dataset and used them as the reference to find the optimal threshold that yield the best match between the GTVPET and IGTV10 in both the target size and the spatial conformity. Our study revealed that GTVPET using a threshold setting of SUV15% approximated most closely to the IGTV10 with the lowest median percentage volume changes. When using the threshold level of ≥ SUV25% and/or ≥ SUV2.5, the PET-based tumor sizes were estimated to be smaller than the IGTV10. Therefore, on the basis of the results of our study, the SUV threshold setting of ≥25% and/or ≥2.5 is not suitable for IGTV contouring in NSCLC.In this study, we determined the IGTVAnalogously, Hanna et al.  comparedPET15%, GTVPET20% and GTVPET2.0 showed no significant difference with IGTV10 in target volume. However, the CIs of them were significantly lower than 1.0. The best CI was between IGTV10 and GTVPET15%, which was only 0.57. It is not surprising as GTVPET15% is the biggest volume and hence has the greatest degree of potential overlap. Based on this consideration, this may not make it the most accurate. The poor CIs suggested great unconformity between what was indicated abnormal on PET image and on CT image. One of the reasons is that shape and/or positional alterations between IGTV10 and GTVPET had occurred. Our study showed that the minimum variation in the centroid coordinate position in the 3D direction of GTVPET and IGTV10 was 0.38 cm (median). Moreover, the CIs were inversely correlated with the centroid shifts in 3D directions. Although patients in our study were immobilized in the same position for both 4DCT and PET/CT, millimetric precision in set-up using immobilization devices may not be feasible. Furthermore, a rigid registration might not be sufficient for lung tumors. Hence, registration error may inevitable affect the spatial position between GTVPET and IGTV10. In addition, it is possible that some of this difference may be related to differences in the patient’s breathing pattern between acquiring the PET/CT and 4DCT. Different breathing pattern can influence tumor size, shape and distribution of activity on the free-breathing PET images [Similarity in absolute volume does not mean identity in the space location. Our results indicated that GTVT images .PET15% and IGTV10, there was 20% of (median) GTVPET15% not included in IGTV10 and 40% of (median) IGTV10 not included in GTVPET15%. It suggested that ITGV10 did not encompass GTVPET15% completely or vice versa. Therefore, we concurred with Gondi [Hanna et al.  used theth Gondi  who concOne limitation of our study is the small number of patients studied, so that subgroup analyses were limited. Therefore, a larger cohort of patients with many different tumor sizes and locations should be conducted to further investigate the relationship of using 3D-PET/CT and 4DCT for contouring IGTV of NSCLC. We are continuing our work to enroll more patients for further clinical investigations.10. At present 3D-PET/CT should not be used for IGTV generation.None of the PET based contours had both close spatial and volumetric approximation to the 4DCT IGTV


The aim of the study was to analyze the factors associated with hospital mortality in patients with severe community-acquired pneumonia (CAP) who required ICU admission.An observational, retrospective study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical, epidemiological and outcome variables. Quantitative variables were expressed as the mean and standard deviation. Qualitative variables are expressed as the percentage and absolute value. We applied the Mann-Whitney and Fisher's exact test, as needed, with an alpha error of 5%.P = 0.075). Patients chronically taking immunosuppressive therapy had a significantly higher mortality compared with the rest of the patients . The mortality rate was also higher in patients in whom NIV fail in the first 24 hours (42.9% vs. 17.6% with P = 0.09). Patients who required intubation and mechanical ventilation in the first 24 hours had a higher mortality rate . Regarding the etiology of pneumonia, in 11 patients the viral origin of infection was confirmed (10 patients had H1N1 pneumonia and one patient CMV pneumonia), with a mortality rate significantly lower than in patients with bacterial pneumonia . The use of the right antibiotic therapy at admission was associated with mortality (P = 0.0001).We analyzed 111 patients, 57.5 ± 17.7 years old, with 63.1% (70) males and APACHE II score on admission of 19.8 ± 17.7. ICU mortality was 29.7% (33) and in-hospital mortality was 32.4% (36). Ten percent of patients met criteria for medical care-associated pneumonia (HCAP); there were no significant differences in mortality between HCAP and CAP (Patients admitted to the ICU with severe CAP and immunosuppressive therapy have higher mortality, with no differences between HCAP and CAP. The delay in intubation as well as bacterial and inappropriate antibiotic treatment are factors that increase mortality.


Abelmoschus esculentus L. Moench (Lady’s Finger). A 6 weeks experiment was conducted under greenhouse conditions. Stress treatments were applied after 30 days of sowing. Plant height, leaf area, photosynthesis, transpiration rate (Tr), stomatal conductance and WUE were determined weekly under stressed [60% field capacity (F.C.)] and non-stressed (100% F.C.) conditions. Stomatal conductance and Tr decreased and reached near to zero in stressed plants. Stressed plants also showed resistance to water stress upto 5 weeks and gradually perished at sixth week. On the other hand, WUE improved in stressed plants containing biochar and gypsum as compared to untreated plants. Biochar alone is a better strategy to promote plant growth and WUE specifically of A. esculentus, compared to its application in combination with gypsum.Water being an essential component for plant growth and development, its scarcity poses serious threat to crops around the world. Climate changes and global warming are increasing the temperature of earth hence becoming an ultimate cause of water scarcity. It is need of the day to use potential soil amendments that could increase the plants’ resistance under such situations. Biochar and gypsum were used in the present study to improve the water use efficiency (WUE) and growth of  Drought is a long period of weather dryness due to lack of rainfall in any area that creates a serious imbalance of water and moisture deficiency for agriculture. Climate change and global warming are two basic environmental issues that are caused by anthropogenic activities making agricultural crisis even more severe. High temperature and severe weather conditions are the causes of flash floods, drought and glacier melting. Due to these climatic changes, earth is becoming warmer and drier and ultimately resulting in water scarcity.Water stress has become a worldwide problem, as it is a threat to the sustainability of agriculture . Asian c2 used during the process of photosynthesis . For the present study Lady’s finger was selected, as it requires short duration to complete its life cycle. In addition, it is a common crop which has an important nutritious value as containing carbohydrates, proteins, iron, Vitamin A, B, and C Soil was air-dried, ground and sieved through 2 mm sieve. It was analyzed for different parameters. Soil pH and electrical conductivity (EC) was measured by using modified glass electrode method . MoisturL. camara was dried and chopped into small pieces. Biochar was prepared by pyrolysis of stems of Lantana at 450°C using reactor according to procedure described by Plant biomass of A. esculentus L. Moench) before the application of the soil amendments. The pots of ∼5 kg capacity (17.7 cm high × 21.2 cm diameter) were used for the experiment. In each pot 4 kg soil with the mixture of soil  and sand in 3:1 ratio was taken (Table 1.The pot experiment was conducted by sowing the seeds of Lady’s finger (as taken . The expA. esculentus L. Moench) were sown in each pot planted in biochar and gypsum amended soil. The rate of biochar application was based on the previous study conducted by Five Seeds of lady’s finger  induction. Each time the total height of every plant was measured from the base to the apex of the stem using measuring tape (in centimeter).Leaf area was also measured at 15th and 30th DAS induction according to procedure described by Stomatal conductance, transpiration rate (Tr) and net photosynthesis were determined weekly using CIRAS 2 Portable Photosynthetic System (IRGA). A fresh and fully grown leaf was selected and enclosed in the cuvette of infrared gas analyzer (IRGA) to determine the values .At leaf level WUE is the ratio of photosynthesis and Tr. It was determined by the given formula:Where WUE is the WUE of plants, Pn is net photosynthesis rate and Tr is the transpiration rate .At the termination of experiment (week six), plant fresh weight was measured using digital balance. After harvesting all the plants were dried at 70°C for 72 h and again dry weights were measured. The difference in weight was calculated as the biomass of plants.Through MS Excel 2007 two way analysis of variance (ANOVA) was used to analyze the data statistically at 5% level of significance.Table 2.Before and after the application of treatments, the soil was analyzed for the physical and chemical parameters such as pH, EC, moisture content, soil texture and organic matter. These were determined for all the treatments including biochar and gypsum as well. Results of these parameters are presented in Before the start of experiment, pH of soil was 7.28 and categorized as neutral soil. The EC of soil was 3.03 μS/cm. The moisture content present in air-dried soil was 1.21 percent. The percentage of sand, silt and clay was 60, 15, 25 percent, respectively. Therefore, soil textural class was sandy loam. Organic matter was low in soil before the application of soil amendments. It was 0.47% in soil without any amendment.As the soil textural class was sandy loam, it has been observed that biochar and gypsum gave the positive response toward these soil parameters. Biochar pH, EC, moisture content and organic matter has been increased in all treated plants as compared to control soil.Table 3.Biochar was also analyzed for different parameters like pH, EC, moisture content, ash content and bulk density. Results of these parameters are presented in P ≤ 0.01) in biochar treated plants as compared to control . Plant height was higher in non-stressed (100% F.C.) plants as compared to the stressed (60% F.C.) ones. Among all these treatments  the plant height was higher in 1% biochar treated plants as compared to control and 3% biochar. It was 35 cm as compared to 0 and 3% where the heights were 29.6 and 23.8 cm respectively.Plant height measured at 15th and 30th DAS showed highly significant difference (P ≤ 0.01) in height. Plant height was privileged in non-stressed plants as compared to stressed plants. Therefore, 1% biochar application rate was found to be more effective than other rates.Similarly, at 60% F.C. of the same treatments, plant height was higher with 1% Biochar as compared to 0 and 3% Biochar. Moreover, non-stressed plants (100% F.C.) were also compared with stressed plants (60% F.C.) and also found significantly different  the plant height in all treatments increased throughout the experiment. But it was high in plants with 1% biochar + 0.1% gypsum (33.2 cm) as compared to other treatments. Similar trend was found in stressed plants (60% F.C.) and 1% biochar + gypsum was proved more beneficial soil amendment.Comparison of plant height between control and combination of biochar and gypsum treatments is shown in Figure 1C. Gypsum has also improved plant height as compared to control where no amendment was added as soil supplement. The highly significant results were obtained for plant height in all treatments (P ≤ 0.01). Among all these treatments , maximum plant height was observed in gypsum applied plants (32.4 cm) as compared to control (29.6 cm). On the other hand plant height was maximum in non-stressed plants as compared to stressed.Gypsum also has significant effect on the growth parameters of plants if treated alone. Comparison of plant height between control and gypsum is presented in Figure 2A. The highly significant results (P ≤ 0.01) were found for all treatments. An increasing trend was observed among all these treatments . But in T2 100, maximum leaf area was observed (120.2 cm2) as compared to T1 100 and T3 100 where it was just 86.24 and 47.36 cm2, respectively. In the same way, in stressed treatments  same trend was found and leaf area was also high with 1% B at 60% F.C. In contrast, leaf area was larger in non-stressed plants (100% F.C.) than stressed one (60% F.C.).Leaf area was also measured at 15th and 30th day of stress (DAS) for all stressed and non-stressed plants. Comparison of leaf area between control and biochar treatments is graphically presented in P ≤ 0.01) were also found when leaf area of control plants was compared with the combination of biochar and gypsum treated plants. Comparison of treated and untreated plants is graphically represented in Figure 2B. Among all treatments , leaf area was elevated in (1% B + 0.1% G) at 100% F.C. as compared to other treatments (0% B + 0% G and 3% B + 0.1% G). Therefore, at 100% F.C., 1% B + 0.1% G was considered to be more effective to increase the growth parameter as compared to other treatments. In addition, the same rates of biochar at 60% F.C. also showed the same trend.Effect of combination of biochar and gypsum was also observed. The highly significant results . The highly significant results (P ≤ 0.01) were found among all treatments  showing maximum leaf area (90.12 cm2) in gypsum treated plant as compared to control (86.24 cm2) with no gypsum application. At 60% F.C. similar trend was observed. Water has significant effect on the growth parameters of plants.Apart from biochar, gypsum alone is also effective for plants as obvious from the comparison of leaf area between control and gypsum  among all treatments. In all non-stressed plants  the stomatal conductance remained constant from week 1 to week 6 as these were maintained at 100% F.C. daily. The stomatal conductance was very high (364 mmolH2O m-2s-1) in 1% biochar as compared to 0 and 3% where it was 217 and 311 mmolH2O m-2s-1 respectively. On the other hand, in stressed plants  the conductance decreased very slowly up to the third week, until their F.C. was maintained at 60%. After 3 weeks when stress was increased, the sudden decline was observed in their stomatal conductance. Biochar showed stability at the intense stress and stomatal conductance was decreased upto 70 and 62 mmolH2O m-2s-1 in 1 and 3% B, respectively as compared to 0% B where it reached near to zero at the 6 week of experiment.At the start of experiment stomatal conductance was measured of all treated and untreated plants. Comparison of stomatal conductance between control and biochar treatments is shown in P ≤ 0.01). The stomatal conductance of control and soil amended plants is graphically presented in Figure 3B. Like biochar treatment, similar results were found for the combination of biochar and gypsum. At 100% F.C. of all treatments  stomatal conductance remained the same throughout the experimental period. On the other hand in stressed treatments i.e., at 60% F.C. for all treatments  it was decreased upto zero for 0% B + 0% G as compared to others where it didn’t reach to zero at the end of the experiment. Therefore, biochar and gypsum gave the resistance to plants under drought stressed conditions.Besides biochar alone, the combination of biochar with gypsum has also shown significant results , no change was observed in stomatal conductance of plants. In addition, stomatal conductance was higher in gypsum treated plants (236 mmolH2O m-2s-1) as compared to control (217mmol H2O m-2s-1). In contrast, at 60% F.C.  stomatal conductance was gradually decreased upto 3 weeks then it was decreased rapidly upto zero in 60% F.C. with no gypsum as compared to gypsum treated plants where it was 59 mmolH2O m-2s-1. Hence, gypsum and biochar have increased the soil moisture and ultimately their resistance and plants got maximum value of stomatal conductance.Gypsum alone also had positive effect to improve the stomatal conductance of plants. Stomatal conductance of control and gypsum applied plants is presented in Figure 4A. The results were highly significant (P ≤ 0.05) when Tr was compared between control and biochar treatments. Among all non-stressed treatment plants , the Tr was not significantly changed throughout the experiment as their F.C. was maintained at 100%, everyday. On the other hand, in stressed plants  the Tr was reduced slowly upto 3 weeks when their F.C. was maintained at 60%. After 3 weeks, plants were given stress and 60% of water loss was returned to all plants. At this stage, a rapid decline was observed in the rate of transpiration. Finally, it was reached near to zero at the end of the experiment in 0% biochar.Transpiration rate of control and biochar treated plants was also compared presented graphically in Figure 4B. Among all these treatments,  no change was observed throughout the experiment. On the other hand, at 60% F.C.  the Tr was gradually reduced upto 3 weeks and then suddenly decreased and reached near to zero at the end of experiment.The combination of biochar with gypsum has also significant impact on the rate of transpiration. The combination of biochar and gypsum has also significant effect on the Tr of plants. Comparison of Tr between control and biochar and gypsum amended soil is presented in Figure 4C. Like other treatments, same trend was observed with gypsum treatments at 100% F.C as well as 60% F.C. At 100% F.C. of all treatments (0% G and 0.1% G) Tr remained the same because plants were daily watered at 100% F.C. On the other hand, in case of 60% F.C. (0% G and 0.1% G) it was gradually decreased and reached near to zero at the end of the experiment.Besides the combination with biochar, gypsum alone has also significant effect on the Tr. Tr was compared between control and gypsum and graphically presented in At 100% F.C. the Tr was not altered because water loss was returned daily to the plants. Therefore it remained constant throughout the experiment. On the other hand at 60% F.C, only 60% of daily water loss was returned, so with the passage of time plants became sensitive and reached near to dry condition.P ≤ 0.01) were found for all treatments . At 100% F.C. of all treatments , a slight change was observed in the photosynthesis rate (Pn) of plants. Pn was higher in T2 100 (5.1 μmolCO2m-2s-1) as compared to T1 100 and T3 100 (2.7 and 4.8 μmolCO2m-2s-1), respectively. On the other hand an increasing trend was observed at 60% F.C. along with biochar (1 and 3% B) as compared to control (0% B) where it was reduced at the end of the experiment. Pn was higher in 1% B at 60% F.C. and increased from 4.6 to 6.9 μmol CO2m-2s-1 from week one to week six. Among all these treatments 1% B + 60% F.C. has shown significant impact on the Pn under drought stressed condition and proved effective treatment as compared to other treatments.Photosynthesis of all plants was also measured throughout the experiment. The highly significant results  among all the treatments. At 100% F.C. higher Pn was observed in combination of biochar and gypsum treatment as compared to control where no amendment was applied. Among all these treatments,  1% B was more effective at 100% F.C. than others. In contrast, Pn gradually increased in 60% F.C. in biochar treatments  than control (60% F.C. + 0% B). In these plants, Pn was elevated from 3.9 to 6.4 μmol CO2m-2s-1. Similarly, 1% biochar along gypsum has also shown positive impacts than other treatments.Comparison of photosynthesis between control and combination of biochar and gypsum treatments is presented in Figure 5C. At 100% F.C. (0% G and 0.1% G) like other treatments a slight change was observed in the Pn throughout the experiment. But gypsum treated plants gave high rate of photosynthesis (3.9 μmol CO2m-2s-1) as compared to control (2.7 μmol CO2m-2s-1). On the other hand, at 60% F.C. (0% G and 0.1% G) the photosynthesis was higher because gypsum prevents the water loss and ultimately increases the moisture and Pn. In gypsum applied plants, it was increased from 2.7 to 5.1 μmol CO2m-2s-1as compared to control where it was reduced from 2.3 to 0.8 μmolCO2m-2s-1.Photosynthesis rate of control and gypsum amended plants has also been compared . Like other treatments biochar has also improved the WUE of Lady’s finger as compared to control. At 100% F.C. of all treatments  a slight change was observed in WUE weekly throughout the experiment. An increased trend was observed in 1 and 3% B (0.8 and 0.83–1.14 and 1.16 mmolmol-1) as compared to control (0% B) where it was just 0.56 mmolmol-1. On the other hand at 60% F.C.  it was increased throughout the experiment. It has shown maximum value with 1% B (4.6 mmolmol-1) as compared to 0 and 3% B where it was (1.6 and 3.8 mmolmol-1). Therefore, 1% biochar at 60% F.C. was proved to be more effective in increasing the WUE of Lady’s finger as compared to other treatments.Comparison of WUE of control and biochar treated plants is presented in Figure 6B. At 100% F.C. same trend was observed for all the treatments  and maximum value was obtained at with 1% B + 0.1% G. In comparison, at 60% F.C.  among all treatments of biochar and gypsum, WUE was seen to increase throughout the experiment. Similarly, 1% biochar along with gypsum also proved to be more effective.Biochar along with any fertilizer has also improved the WUE. WUE of control and combination of biochar and gypsum is presented in Figure 6C. At 100% F.C. of all treatments (0% G and 0.1% G) very slight change was observed in all treatments. But in case of 60% F.C., (0% G and 0.1% G) significant increase was observed weekly throughout the experiment. In these treatments (60% F.C. + 0% G and 60% F.C. + 0.1% G), higher WUE was observed in gypsum treated plants (2.21 mmolmol-1) as compared to control where it only increased upto 0.91 mmolmol-1 at the end of the experiment.Comparison of WUE between control and gypsum is presented in Figure 7A. Dry matter was increased in all non-stressed plants . Among all these treatments it was higher (3.261 g) in 1% B treatment as compared to other stressed (60% F.C.) and non-stressed plants (100%).At the end of experiment, plants were harvested. Fresh and dry weights were calculated for each plant. Biomass of control and biochar treatments is graphically presented in Figure 7B. At 100% F.C.  maximum dry matter was found in 1% B + 0.1% G (2.093 g) than other treatments. On the other hand in stressed plants, i.e., at 60% F.C.  it was decreased due to water stress.Comparison of control and combination of biochar and gypsum treatment is presented in Figure 7C. In non-stressed plants an increased biomass was found as compared to stressed plant. At 100% F.C. (0% G and 0.1% G) higher biomass was found in gypsum treated plants (0.511 g) as compared to control (0.436 g).Comparison of control and gypsum treatments is shown in A. esculentus L. Moench (Lady’s Finger).A better understanding of different strategies is required to achieve drought resistance to cope with the water deficit. WUE is the ability of the crop to produce biomass per unit of water transpired. In this scenario of water stress, WUE can be regarded as an important adaptive trait to drought environment. Previous works have revealed the physiological basis of drought stress in different plants. The present study was made to check the potential of biochar along with gypsum in improving WUE under water stress conditions to study the effect of drought on growth parameters in Soil used for the experiment, represented typical water scares and stressed sandy loam, from dry sub-tropical conditions, with low organic matter, neutral pH, having EC 3.03 μS/cm. It has been observed that biochar and gypsum gave the positive response toward an overall improvement of the studied soil parameters. While pH, EC, moisture content and organic matter was increased in all treated plants as compared to the control soil. Our results followed similar trends established in different studies involving application of biochar in increasing soil pH and EC when applied on tomato  as well A. esculentus L. Moench. Several other studies report similar results, for other higher plants (It has been reported that leaf area of plants decreased by decreasing the amount of water. r plants .Cucmis melo) with no soil amendment. Moreover, same trend was also observed in chickpea  and clover (Trifolium subterranean) under drought stress condition as compared to control and fully watered plants (Robinia pseudoaccacia (Trifolium subterranean) with biochar treated plants under drought stressed conditions , rice (3.28 mmolmol-1), soybean (3.68 mmolmol-1) and maize (9.23 mmolmol-1). In the same study WUE was improved due to increased photosynthesis and decreased transpiration (In the present study, Tr was reduced and Pn was increased that caused an increase in WUE. In previous studies WUE was also determined for different species such as sweet potato (4.89 mmolmolpiration .In case of our findings for increase in biomass, a similar 20% increase in dry weight was obtained in biochar along with gypsum applied plants as compared to biochar and gypsum alone . For ourDifferent plants can withstand certain level of pH and lady’s finger can suitably grow under 6–8 pH. With the amendment of biochar, it overcomes the negative effects of pH at 1%. But at 3% concentration, the pH increased which resulted in reduction of positive effect of biochar . Our expWater being essential in growth and development of plants, a specific amount of water is needed for optimum growth. WUE in rain fed areas is the most important limiting factor for the plant growth. Increasing temperature of earth due to anthropogenic activities, Climate changes and severe weather conditions are reasons for flash floods, drought and glacial retreat, destabilized watersheds are heading toward water scarcity. Pakistan is facing increasing drought stress, being an agriculture based economy. In the current scenario soil amendments can prove to be the best methods to overcome drought stress.A. esculentus L. Moench seedlings were grown for 4 weeks and then stress treatments were imposed. The variations were noted and assessment of growth as well as physiological parameters was done in experimental conditions.In this study biochar and gypsum were used to improve the infiltration rate and water holding capacity of soil in which A. esculentus L. Moench, compared to the application of gypsum and combination of biochar and gypsum under both stressed and non-stressed conditions. Furthermore, at 60% F.C. Lady’s finger showed resistance as well as improved WUE. Therefore, it is considered beneficial in water stressed regions.Among all treatments, 1% biochar alone as well as along with gypsum gave significant results for all parameters as compared to control and other treatments. It is finally concluded that biochar alone is a better strategy to promote plant growth specifically of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bombus terrestris are essential pollinators in natural and managed ecosystems. In addition, this species is intensively used in agriculture for its pollination services, for instance in tomato and pepper greenhouses. Here we performed a quantitative trait loci (QTL) analysis on B. terrestris using 136 microsatellite DNA markers to identify genes linked with 20 traits including light sensitivity, body size and mass, and eye and hind leg measures. By composite interval mapping (IM), we found 83 and 34 suggestive QTLs for 19 of the 20 traits at the linkage group wide significance levels of p = 0.05 and 0.01, respectively. Furthermore, we also found five significant QTLs at the genome wide significant level of p = 0.05. Individual QTLs accounted for 7.5-53.3% of the phenotypic variation. For 15 traits, at least one QTL was confirmed with multiple QTL model mapping. Multivariate principal components analysis confirmed 11 univariate suggestive QTLs but revealed three suggestive QTLs not identified by the individual traits. We also identified several candidate genes linked with light sensitivity, in particular the Phosrestin-1-like gene is a primary candidate for its phototransduction function. In conclusion, we believe that the suggestive and significant QTLs, and markers identified here, can be of use in marker-assisted breeding to improve selection towards light sensitive bumblebees, and thus also the pollination service of bumblebees.Bumblebees such as  Bombus terrestris L., are used worldwide in greenhouses for the pollination of different crops , with α being the significance threshold value, m the number of performed tests and i the number of null hypotheses arranged by ascending P-values. Instead of the significance threshold of α = 0.05, we created with this formulae a ‘new threshold value’ for rejection of the null hypothesis, and this for the first i-value which has a lower calculated P-value than P(i). To achieve this, we searched for the first P-value which follows this formula. Here, with α = 0.05 and m = 190, we compared each P(i) with 0.05(i)/190, starting from P(190). As P(156) = 0.034 < (0.05*156)/190 our new significance threshold was 0.041. For datasets with many correlated traits, multivariate methods, like PCA, are often performed to reduce the dimensionality of the dataset without losing much of the original variation [Correlations between the different morphological characters were tested by the Spearman correlation test in SPSS (version 22.0.0.0). Instead of the more conservative sequential Bonferroni corrections for multiple significance tests , we calcariation . Therebyariation . Here, wariation . The PCsB. terrestris: 12 loci from Stolle et al. [B. lucorum [Apis mellifera [B. terrestris [Bumblebee DNA was extracted from one middle leg of each drone as described in Maebe et al. . Bumblebe et al. , 11 locie et al. , 106 loce et al.  by Stolle et al. , one newe et al.  and one e et al. –37 (S1 T lucorum  and one ellifera  (S1 Tablrrestris .5’-GAGTTTTCCCAGTCACGAC-3’) is coupled to a HEX, 6-FAM, VIC or NED fluorescent label to allow detection of the microsatellite alleles by capillary electrophoreses. Furthermore, for incorporation of this universal tail during PCR, the specific forward primers are prolonged at its 5’-end with the same (but unlabeled) sequence as the tail.For detection of the microsatellite alleles, we used a tailed-primer approach : a univeEach microsatellite locus was amplified in simplex by PCR. PCR reactions were carried out in 10 μl total volume. Each reaction contained 1.5 μl template DNA, 1 μl of 10x PCR buffer (Qiagen), 0.2 μl of 10 mM dNTP’s (Qiagen), 0.1 μl of 10 μM forward primer, 0.4 μl of 10 μM reverse primer, 0.4 μl of 10 μM labeled M13-primer and 0.05 μl of 2.5 units/reaction Hotstar Taq DNA Polymerase (Qiagen). Samples were initially denatured at 95°C for 15 min, followed by 30 cycles of denaturing at 94°C for 30 s, annealing at 48, 52 or 58°C for 30 s, and extension at 72°C for 30 s. The PCR protocol ended with a final extension step at 72°C for 10 min. After pooling the final PCR products, they were visualized on a ABI-3730xl sequencer (Applied Biosystems) using an internal size standard . The fragments were examined and scored manually using Peak Scanner Software v 1.0 (Applied Biosystems).-2md/L with m = number of markers, d = distance between markers (in cM), L = genome length and c = proportion of the genome within this distance d, as described in [First, a preliminary linkage mapping was established using 100 microsatellite loci with Kosambi’s mapping function. These loci were chosen based on their known distribution on the 18 linkage groups described in Stolle et al.  to obtairibed in  and usedribed in , showed B. terrestris colony as described by [Linkage analysis was performed with JoinMap software version 4.0 . Linkageribed by . Linkagep = 0.05 and p = 0.01. These permutation tests are less dependent on normal distributions to calculate significance thresholds. Third, we performed also a multiple QTL model mapping (MQM) within MapQTL 5.0. The selection of obtained suggestive QTLs in IM were used as cofactors during the MQM-mapping which allowed for the detecting of additional QTLs[First, we performed the Kruskal-Wallis (KW) test, a single marker non-parametric method imbedded in the software program MapQTL5.0  to deteconal QTLs. When thonal QTLs.t-test. Only the most significant interaction was reported when multiple significant epistatic interactions for several marker pairs between the same regions of two linkage groups were found.We scanned for epistatic interactions of QTLs using the software QTLMapper version 1.6 . Digenichttp://www.ncbi.nlm.nih.gov/genome/2739) and all genes on this sequence (± 500k bp) were selected as candidate genes. We searched in UniProt (http://www.uniprot.org/) for the known function of those candidate genes, and selected the candidate gene which function could be directly linked with vision or light perception as primary target gene.Candidate genes for light sensitivity were selected around the 95% confidence interval (= C.I.) of the QTL. The two SSR markers which determined the 95% C.I. of the QTL, were found in the bumblebee genome (P > 0.05). Most morphological parameters of the leg and the body size correlated significantly with body mass and the different eye morphology parameters ; and (ii) body mass did not correlate with tibia length and width , femur width , and both the trochanter length and width . Furthermore, we detected no correlation between birth order of the males and both bumblebee body size and body weight.In total, 96 drones were measured for 20 different traits . The disrameters . The onlP > 0.041). The CLS in blue and UV light conditions correlated only with the metatarsus length , the metatarsus width , and the tibia width . Furthermore, CLS in blue light sensitivity correlated also with the tarsus length , being the lowest light intensity at which a bumblebee is able to fly, of 4 days-old drones n = 96) in blue and UV light conditions was 3.58 ± 2.89 lux and 1.73 ± 0.47 lux, respectively . As ligh6 in blueqBLU3) explaining 10.6% of the genotypic variation, one significant and six suggestive QTLs for body mass, one significant and four suggestive QTLs for radial cell length, one significant and 47 suggestive QTLs for eye traits, and two significant and 26 suggestive QTLs for leg traits  and two suggestive QTLs (qDWE10 and qDWE15) while for the length of the radial cell we found one significant (qRAC15.2) and one suggestive QTL (qRAC1), cumulatively explaining 40.7% and 23.8% of the phenotypic variation, respectively. With MQM, we detected 1 to 3 suggestive QTLs for most of the eye traits: for the dorsal-ventral length , width  and total surface of the compound eye , the amount of ommatidia of a compound eye (qOMN3.2), the diameter of median ocellus , and the facet diameter (qFAC11) cumulatively explaining 40.1%, 57.2%, 33.9%, 21%, 23.8% and 9.7% of the phenotypic variation, respectively. For the amount of ommatidia of a compound eye we found also one significant QTL (qOMN9) explaining 16.7% of the phenotypic variation. For the different hind leg traits we found only suggestive QTLs for: (i) metatarsus length  explaining 34.2% of variation, (ii) tibia length and width  explaining 9.7% and 17.8% of variation, (iii) femur length and width  cumulatively explaining 28.8% and 9.3% of variation, (iv) trochanter width (qTRW1) explaining 7.8% of variation, and finally (v) three suggestive QTLs for tarsus length  explaining 72.9% of variation. Furthermore, we found one significant QTL with MQM for trochanter length (qTRL6) and metatarsus width (qMTW6) explaining 23.4% and 22.0% of variation, respectively.When considering the 19 traits for which we found a suggestive or significant QTL with IM, 15 traits had at least 1 QTL with multiple QTL model mapping (MQM). Indeed, with the MQM mapping we identified 5 QTLs significant at the genome wide significance level of 0.05%, and 27 and 20 QTLs significant at the LG specific significance level of 0.05% and 0.01%, respectively . These QqSPC1_6, qSPC1_15.1 and qSPC1_15.2) of PC1 are linked with body size in general as confirmed by the QTLs of the individual body size traits  were confirmed by the univariate suggestive QTLs for these three eye parameters, while QTL qEPC1_7 was only confirmed by ommatidia number  correlated with the univariate QTLs found for median occelus and ommatida number on LG6, LG7 and LG12. (The PCA on the different eye parameters showed 3 PCs which accounted for 74.1% (PC1), 10.9% (PC2) and 8.1% (PC3) of the total variance . Only PCa number . The thrnd LG12. .We identified epistatic interactions using the program QTLMapper version 1.6  Table 4Table 4. qBLU3. Therefore, we used SSR-marker BT08 which determine the QTL region, and the markers BT07 and 0291_60p14 as borders for the 95% C.I. of the QTL. The 64 genes around the markers BT07 and 0291_60p14 on the linkage group 3, were all identified as candidate genes  was comparable or smaller in comparison with the sample sizes of other QTL studies in bumblebees, such as in Wilfert et al. –18 wherePhosrestin-1-like gene as the primary candidate gene due to the known phototransduction function of Phosrestin-1 [Drosophila) Phosrestin-1, also known as Arrestin-B or Arrestin-2, is identified as interacting directly with light-activated rhodopsin thereby activating the phosphorylation of metarhodopsin [Arrestin-2 in the rhabdomeres will enhance the photoreceptor sensitivity in weak light conditions, and prevent hyperactivity of the photoreceptors in strong light conditions [In this project, we found a suggestive QTL for light sensitivity under blue light conditions in a region where there is no QTL linked with body size or any other related morphological parameter. We already showed before that although larger bumblebees are better equipped to capture light, other genetic parameters influence bumblebee light sensitivity . For thirestin-1 . Indeed,hodopsin . Furthernditions . FurtherqDWE2), LG3 (qDWE3), LG5 (qDWE5), and LG 10 (qDWE10), while radial cell and body size parameters had unique QTL regions at LG1, LG7 and LG15. These regions were confirmed by the PC-QTL. Indeed, PCs showed size related suggestive QTLs on LG6, LG10 and LG15. Only one suggestive QTL on PC4 overlapped with one of the unique univariate body mass suggestive QTLs on LG3 , and body size  at different places and on different linkage groups. Wilfert et al. [BS-8) was recovered in our study (qRAC15.1). This suggestive QTL, which accounts in our study only for 9.6% of the phenotypic variation, is a potential candidate for use as a genetic marker in MAS. Thus, in our study we were not only able to confirm a minor QTL for body size from Wilfert et al. [Although preliminary, these results support the idea of marker assisted breeding towards larger bumblebees, with the use of the identified markers at those unique QTLs. However, before these QTLs could be used they need to be validated in a broader genetic background, using multiple bumblebee populations. For QTL studies it is common that most of the QTLs found in one population will not withstand this validation, even if there are only very small differences in the experimental setup . Indeed,t et al.  found 10t et al. , but we Phosrestin-1-like gene is identified as the primary candidate gene. Finally, we also found several significant and suggestive QTLs for body weight, body size and the morphological parameters of the eye and hind leg. Further research needs to determine if the QTLs found here, resist validation in a broader genetic background and if some of the SSR markers linked with those QTLs could be used as genetic markers in marker assisted breeding, to improve the pollination service of bumblebees.In conclusion, our study identified one suggestive QTL for light sensitivity under blue light conditions explaining 10.6% of the phenotypic variation of the trait. Furthermore, we identified a list of 64 possible candidate genes for this trait of which the S1 Fig(PDF)Click here for additional data file.S1 TableTa), the observed size range of the PCR product, the location (LG) and the original reference.From each SSR marker we present the forward and reverse primer sequences, GenBank accession number, annealing temperature ((PDF)Click here for additional data file.S2 TableThe number of markers on each linkage group (n), the size of this linkage group (size LG), and the minimum (Min. d) and maximum (Max. d) distances between two markers on each linkage group.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableThe eigenvalues and eigenvectors of the PCA are given for the different body size traits and the eye parameters.(PDF)Click here for additional data file.S5 TableList of the place, accession number, name and annotation information of all genes, at suggestive QTL qBLU3 on LG 3, which can all be linked with the critical light sensitivity of bumblebee drones in blue light.(PDF)Click here for additional data file.


Malnutrition can be defined as a state of nutrition where the weight for age, height for age and weight for height indices are below -2 Z-score of the NCHS reference. It has posed a great economic burden to the developing world.The objective of this study is to assess the prevalence of malnutrition among pre-school children in abakiliki in Ebonyi state of Nigeria.This is a cross-sectional studies that assess the prevalence of malnutrition and associated factors among children aged 1-5 years attending nursery and primary schools. Nutritional assessment was done using anthropometry and clinical examination.A total of 616 children aged one to 5 years were enrolled into this study. Three hundred and sixty-seven (59.6%) were males while 249 (40.4%) were females. Sixty of the 616 children (9.7%) had acute malnutrition based on WHZ-score. Moderate acute malnutrition (MAM) was present in 33 children (5.3%) while 27 (4.4%) had severe acute malnutrition.The prevalence of global and severe acute malnutrition using z-score is 9.7% and 4.4% respectively while that of stunting is 9.9% with a male preponderance. Malnutrition is an unbearable burden not only on the health systems, but the entire socio-cultural and economic status of the society . MalnutrThe worldwide malnutrition estimation rates indicate that 35.8% of preschool children in developing countries are underweight, 42.7% are stunted, and 9.2% are wasted [None of these classifications address all the three indices of undernutrition - Stunting, Wasting and Underweight. Stunting (Low height-for-Age) is an indicator of chronic undernutrition due to prolonged food deprivation and/or illness; Wasting (Low weight for height) is an indicator of acute undernutrition, the result of more recent food deprivation and/or illness; Underweight (Low weight-for-age) is used as a composite measure to reflect both acute and chronic undernutrition .Currently, the WHO recommended the use Z-Score or SD system to grade undernutrition. This method measures all the three indices and expresses the results in terms of Z scores or standard deviation units. Children who are more than 2 SD below the reference median (i.e. a Z-Score of less than -2) are considered to be undernourished i.e. to be stunted, wasted or to be underweight. Children with measurements below 3 SD (a Z-Score of less than-3) are considered to be severely undernourished [Evaluation of prevalence of malnutrition among pre-school children using z-score is a very vital issue often under reported in pediatrics practice and in this part of the world, its importance therefore cannot be downplayed especially its impact on health which had been mentioned above.This study therefore is aimed at determining the prevalence and pattern of malnutrition among pre-school children using z-score. The results will help us to know the burden of this disease. This will enable us to establish a baseline data where other related issue will hinge on.We are not aware of any study of this nature from this environment. It is hoped that this will add to the body of knowledge available on these disorders and the findings of this study could form the template for intervention strategies in helping reduce this social malaise and managing such cases.This is a cross-sectional studies that assess the prevalence of malnutrition and associated factors among children aged 1-5 years.The study was conducted from selected two nursery and two primary schools in Abakiliki, Ebonyi state. Abakiliki is the capital of Ebonyi state, south-east Nigeria and has a population of 134102 according to the GeoNames geographical database [Source populations were all children aged 1-5 years attending nursery and primary schools in Abakiliki. Written Consent and approval were given by the school authorities where this study was carried out while verbal consent was obtained from parents of the pupils.Confidentiality of responses was also conveyed. Pupils were chosen by systemic sampling from nursery one to primary one. The study population are made up of people from mainly middle and lower class. Nutritional assessment was done using anthropometry. Children were weighed and measured as per the WHO guidelines on Anthropometry ,12.Stadiometer (Floor type model with sensitivity of 0.1 cm and 0.1 kg): was used to measure the height and weight of the children. It typically consists of a vertical ruler with a sliding horizontal rod or paddle which is adjusted to rest on top of the head. The mid arm circumference was measure with a measuring tape on two consecutive times. The sensitivity is 0.1 cm.Children aged 1-5 years who live and attend schools in nursery and primary schools in Abakiliki whose teachers gave consent were included in this study while children who were seriously ill and those whose parents did not give consents were excluded.The objective of this study is to assess the prevalence of malnutrition among children aged 1-5 years attending nursery and primary schools in Abakiliki in Ebonyi state of Nigeria.Moderate acute malnutrition was defined as weight for height of ≥ -3 and < -2 z-score and mid arm circumference (MAC) of 11 - <12.5 cm. Severe acute malnutrition was defined as weight for height z-score of < -3 and MAC of <11 cm. Stunting was defined as height for age z-score of < -2.Data analysis was with Statistical Package for Social Sciences (SPSS) version 19 (Chicago IL). Chi-square test was used to test for significant association of the proportion. A p-value of < 0.5 was regarded as significant. All reported p-values were 2-sided.A total of 616 children aged one to 5 years were enrolled into this study. Three hundred and sixty-seven (59.6%) were males while 249 (40.4%) were females. The distribution of the children according to age and gender is shown in Table Sixty of the 616 children (9.7%) had acute malnutrition based on WHZ-score. Moderate acute malnutrition (MAM) was present in 33 children (5.4%) while 27 (4.4%) had severe acute malnutrition. However, MAM and SAM based on MAC criteria was present in only 10 (1.6%) and 1 (0.2%) of the 616 children respectively. Thirty-seven of the 330 males (10.1%) compared to 23 of the 226 females (9.2%) had acute malnutrition (p = 0.78). Prevalence of acute malnutrition was highest among the one (12.4%) and two year olds (12.8%) and least among the four year old (4.7%) (p = 0.14). A higher proportion of the males (6%) than females (4.4%) had MAM. Conversely, a slightly higher proportion of the females (4.8%) than males (4.1%) had SAM. Nine of the 47 one year old females (19.1%) compared to 6 of 74 males (8.1%) had acute malnutrition. This difference showed a trend that failed to attain significance (p = 0.09). Conversely, 18 of 106 two- year old males (17.0%) compared to 5 of 74 females of same age had acute malnutrition. This difference also showed a trend that failed to attain statistical significance (p = 0.07). Detailed comparison of acute malnutrition between the genders according to their ages is shown in Table Sixty-one of the 616 children (9.9%) were stunted. Thirty nine of the 367 males (10.6%) were stunted compared to 22 of the 249 females (8.8%) although this difference was not statistically significant (p = 0.47). Across the age groups, a higher proportion of the two (19.4%) and three year olds (10.5%) compared to one (3.3%), four (2.8%) and five (2.7%) year olds were stunted (p < 0.001). However, 23 of 106 two-year old males compared to 12 of 74 females of same age were stunted (p = 0.45). Additionally, 13 of 112 males (11.6%) compared to 5 of 59 females aged 3 years were stunted (p = 0.61). Detailed comparison of stunting between the genders according to their ages is shown in Table The results of this study show acute malnutrition in this metropolis do exit, though there is lack of cultural and social recognition of this hidden malaise. The prevalence of stunting from our study is 9.9%. The result of this study revealed that, the prevalence of stunting is low when compared with a community cross-sectional study conducted in rural kebeles of Haramaya and NorthShewa district all in Ethiopia where a prevalence of 32.4% and 47.7% respectively were obtained ,14. The Possible reasons for the observed sex differences in these studies mainly centres on behavioural patterns . For insWe noted from this study that the prevalence of severe acute malnutrition and global malnutrition is 4.4% and 9.7% respectively. This is at variance with the global prevalence of 16.1 obtained by Casie et al.  who obtaThe prevalence of moderate acute malnutrition was 5.4% with a slight male preponderance. This is lower than the 16% reported by WHO with a female preponderance . A studyUsing MAC alone, a prevalence of severe malnutrition of 1.6% was obtained in this study. This is very low when compared with the 9% obtained by WHO . AlthougThe prevalence of global and severe acute malnutrition using z-score is 9.7% and 4.4% respectively while that of stunting is 9.9% with a male preponderance.Study of the prevalence of malnutrition in a larger community setting will make the impact and attendant problems to be appreciated better.