Volume 3, Issue 3

Advancements in Life Sciences, volume 3, issue 3

Published online: 25-May-2016
ISSN 2310-5380 


Short Communications:

Association patterns of volatile metabolites in urinary excretions among Type-2 Non-Insulin dependent diabetes patients
Muhammad Saqib Shahzad, Javed Iqbal Bajwa, Javed Iqbal Wattoo, Mushtaq A Saleem, pages 71-74
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 Background: Patterns of volatile metabolites in urine are important to detect abnormalities associated with diabetes. Present study was conducted to find out the excretion patterns of endogenously produced alcohols in urine for type 2 (Non-Insulin Dependent) diabetes mellitus. A cross sectional analytical study was conducted with duration extended from Jan to Mar 2015. Methods: The current study included 40 patients with chronic type 2 diabetes mellitus. In total, 10 sex and age matched subjects with no history of any disease were considered as controls. Blood sugar was estimated by autoanalyzer using standard kit of Merck following manufacturer`s instructions. Urine sugar was quantitatively detected by biuret reagent using titration technique. Urinary alcohol was identified and estimated by gas chromatography.  Urinary ketone bodies were estimated by urinary strip. Results: It was observed that level of fasting blood sugar was significantly increased (P<0.001) in patients as compared to their controls. The blood sugar and urinary alcohol in patients were 3.0% and 6.0% respectively. Urinary ketone bodies were found to be 2+. On the other hand urine sugar, alcohol and ketone bodies were not detected in the negative control subjects. Conclusions: It is concluded that urinary alcohol is endogenously produced in patients with type 2 diabetes due to uncontrolled hyperglycemia. However further work is needed to find out the ratio of urinary and blood alcohol which may confirm the present findings. 

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Genetic diversity and evolutionary analysis of Citrus Tristeza Virus p20 gene in Pakistan: insights into the spread and epidemiology
Noor-Ul-Huda Ghori, Muhammad Qasim Hayat, Salvatore Davino, pages 75-82
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 BackgroundCitrus tristeza virus (CTV) is a widespread disease and the most destruction causing agent of citrus. Pakistan is ranked amongst the top ten citrus producing countries around the globe and it contributes about 2% to its foreign exchange earnings. Based on this assumption it is very important to monitor and determine the evolutionary forces and the phylogeography of Pakistani CTV population. Methods: A total of 49 sequences of p20 gene from Pakistan were phylogenetically compared with CTV sequences worldwide. These sequences were analyzed for their genetic diversity and evolution using a Bayesian Probability approach and predicted secondary structure. Results: Phylogenetic analysis using Bayesian probability inference and predicted secondary structures diversity of CTV indicated that Pakistani isolates were not diverse from global isolates. Lineage analysis showed that CTV was introduced in Pakistan in three individual events from various parts of the world.  After that CTV dispersed in Pakistan via vector transmission or by use of infected propagating material by local farmers. Conclusions: Our study confirmed multiple introductions of CTV in Pakistan and also confirmed the dissemination of CTV within Pakistan. This study also shows that the mutations are present in the predicted secondary structure of the p20 protein, however, it is not known if it affects the pathogenicity of the virus. 

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Characterization of antibiotic resistant gene in Staphylococcus aureus isolated from surgical wounds
Aisha Nawaz, Abdul Razzaq, Sadia Ijaz, Aisha Nawaz, Arfan Ali, Afshan kaleem, pages 83-88

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 Background: In the field of surgery wound infections have been a problem. Staphylococcus aureusEscherichia coliProteus species, Klebsiella species, Streptococcus speciesEnterobacter speciesPseudomonas species and species of staphylococci are frequently isolated from wound. This study was carried out to determine the prevalence of different pathogens in surgical wounds and their antimicrobial susceptibility patterns. Methods: Pus swabs from each patient was collected aseptically, and inoculated on culture media. Isolates were characterized, identified, and their antibiotic susceptibility patterns were determined using the Kirby-Bauer diffusion method. Results: Out of 300 surgical wound specimens analyzed, 208 samples were positive culture among which Staphylococcus aureus was most frequent pathogen with 103 (49.51%). The other 33 (15.86%) isolates were Pseudomonas aeruginosa followed by 27 (12.98%) isolates ofKlebsiella 25 (12.01%) isolates of Proteus and 20 (9.61%). isolates of E.coli respectively. The antibiotic susceptibility of Staphylococcus aureus was checked. Conclusions: It was observed that 103 isolates of S. aureus were resistant to Augmentin 65.04% to Sparfloxacin 59.22% to Amikacin 57.28% to Cefuroxime 57.28% to cefotaxime 56.31% to Ceftazidime 53.39% to Fusidic acid 48.54% to Ciprofloxacin 39.80% to Methicillin 37.86% to Gentamicin 28.15% to Meropenem 13.59% to Imipenem 5.82%. PCR assay for the detection of clinically relevant antibiotic resistance gene of S. aureus was done. Fragments of mecA (encoding methicillin resistance) gene were amplified and commercially sequenced which showed insertion at three sites; 480-481: T, 484-485: T, 464-465: G. 

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Full Length Research Articles:

Analysis of reporter proteins GUS and DsRed driven under the control of CaMV35S promoter in syncytia induced by beet cyst nematode Heterodera schachtii in Arabidopsis roots
Muhammad Amjad Ali, Amjad Abbas, pages 89-96

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 Background: Cyst nematodes induce specialized feeding structures called syncytia in the plant roots. The expression of CaMV promoter in syncytia has remained topic of debate. The objective of this research was to study the activity of CaMV promoter by using reporter proteins like GUSand DsRed under the control of CaMV35S promoter in syncytia induced by H. schachtii in Arabidopsis roots. Methods: pMAA-Red and pPZP3425 plasmids were used to study expression of GUS and DsRedin syncytia.  The plants were grown in 2% Knop medium under sterile conditions in growth chambers at 25°C in long day conditions. GUS activity in syncytia was studied through staining of syncytia using X-gluc solution. Ds-Red fluorescence in syncytia was detected by using an inverse microscope equipped with UV filter. Results: The expression analysis of DsRed protein driven by CaMV promoter demonstrated that this promoter is active in syncytia at all the time points. All the syncytia showed DsRed expression at 5 dpi. At 7 dpi, 10 dpi and 15 dpi over 90%, 80% and 50% of the syncytia showed DsRedfluorescence respectively. There was very high fluorescence in the syncytia as compared to the uninfected root segments due to high expression. CaMV::GUS lines showed GUS expression in 80% of 5dpi syncytia. However, unlike expression of DsRed, the number of GUS stained syncytia decreased quickly to around 50% at 7 dpi and to about 5% in the 15 dpi syncytia. Conclusions: The results conclude that CaMV promoter is more active in younger syncytia as compared to older syncytia but can be used for expression in syncytia. Moreover, DsRed protein could be used as better reporter for evaluation of gene expression in syncytia as compared to GUS. 

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Biosafety assessment of transgenic Bt cotton on model animals
Ahmad Ali Shahid, Sadia Bano, Sana Khalid, Tahir Rehman Samiullah, Kamran Shahzad Bajwa, Muhammad Azam Ali, pages 97-108

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 Background: To know the effects of transgenic crops on soil microorganisms, animals and other expected hazards due to the introduction of GM crops into the environment is critical both scientifically and environmentally. The work was conducted to study the effect of insecticidal Bt protein on Rats and Earthworms. Methods: For this purpose, animals like rat and soil organisms like Earthworm were selected. Rats were selected on the basis of its 95% homology on genomic, cellular and enzymatic level with human while earthworm were preferred on the basis of their direct contact with soil to evaluate the impact of Bt (Cry1AC) crop field soil on earthworm, secreted by root exudates of Bt cotton. Several physical, molecular, biochemical and histological analyses were performed on both Rats/Earthworms fed on standard diet (control group) as well containing Bt protein (experimental group). Results: Molecular analyses such as immune Dot blot, SDS-PAGE, ELISA and PCR, confirmed the absence of Cry1Ac protein in blood and urine samples of rats, which were fed with Bt protein in their diet. Furthermore, histological studies showed that there was no difference in cellular architecture in liver, heart, kidney and intestine of Bt and non-Bt diet fed rats. To see the effect of Bt on earthworm two different groups were studied, one with transgenic plant field soil supplemented with grinded leaves of cotton and second group with non-Bt field soil. Conclusions: No lethal effects of transgenic Bt protein on the survival of earthworm and rats were observed. Bradford assay, Dipstick assay ELISA demonstrated the absence of Cry1Ac protein in the mid-gut epithelial tissue of earthworm. The results of present study will be helpful in successful deployment and commercial release of genetically modified crop in Pakistan. 

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