Expression, Purification, and Characterization of an Intrinsically Disordered Late Embryogenesis Abundant (LEA) Protein from Artemia franciscana Utilizing Escherichia coli and Nicotiana tabacum

Anhydrobiosis is an astounding strategy that allows certain species (both animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Despite the occurrence of several different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been most conclusive identified as a requirement for the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in different subcellular compartments including the cytosol and mitochondria. To understand the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins in the dry and/or hydrated states is still poorly defined and understood. We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) for land 7 days in the presence or absence of BSA or trehalose or other purified LEA protein. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via Intein Mediated Purification with an Affinity Chitin-binding Tag system; a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant. Tobacco plants with confirmed transgenic AfrLEA1.1 were subjected to water stress in the presence of polyethylene glycol (PEG; 10,000 MW) at increasing percentages to investigate the impact of osmotic stress on plant survival. PEG-stressed transgenic LEA plants showed significantly faster growth of roots compared to non-transgenic GUS control plants under the same conditions both if measured as an increase in fresh weight (P=0.033, P\u3c0.05) or dry weight (P=0.028, P\u3c0.05). This result clearly indicates a better capability to cope with water stress in presence of AfrLEA1.1 and points to a function of this protein not only during desiccation but also under less severe osmotic stress conditions. Transgenic LEA plants also showed a significantly increased level of total growth compared to controls, measured as an increase in total fresh weight (P=0.0461, P\u3c0.05) and total dry weight (P=0.0342, P\u3c0.05) under standard growth conditions. Along with the better growth of roots under osmotic stress condition and better overall somatic growth under control condition, they also showed a significantly higher amount of chlorophyll content after freezing condition compares to room temperature

Expression, Purification, and Characterization of an Intrinsically Disordered Late Embryogenesis Abundant (LEA) Protein from Artemia franciscana Utilizing Escherichia coli and Nicotiana tabacum

Anhydrobiosis is an astounding strategy that allows certain species (both animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Despite the occurrence of several different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been most conclusive identified as a requirement for the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in different subcellular compartments including the cytosol and mitochondria. To understand the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins in the dry and/or hydrated states is still poorly defined and understood. We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) for land 7 days in the presence or absence of BSA or trehalose or other purified LEA protein. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via Intein Mediated Purification with an Affinity Chitin-binding Tag system; a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant. Tobacco plants with confirmed transgenic AfrLEA1.1 were subjected to water stress in the presence of polyethylene glycol (PEG; 10,000 MW) at increasing percentages to investigate the impact of osmotic stress on plant survival. PEG-stressed transgenic LEA plants showed significantly faster growth of roots compared to non-transgenic GUS control plants under the same conditions both if measured as an increase in fresh weight (P=0.033, P\u3c0.05) or dry weight (P=0.028, P\u3c0.05). This result clearly indicates a better capability to cope with water stress in presence of AfrLEA1.1 and points to a function of this protein not only during desiccation but also under less severe osmotic stress conditions. Transgenic LEA plants also showed a significantly increased level of total growth compared to controls, measured as an increase in total fresh weight (P=0.0461, P\u3c0.05) and total dry weight (P=0.0342, P\u3c0.05) under standard growth conditions. Along with the better growth of roots under osmotic stress condition and better overall somatic growth under control condition, they also showed a significantly higher amount of chlorophyll content after freezing condition compares to room temperature

Molecular Characterization of Plasmid-Mediated Non-O157 Verotoxigenic Escherichia coli Isolated from Infants and Children with Diarrhea

أصبحت الزيادة الكبيرة في حدوث العدوى غير العدوى المسببة للسموم القولونية (VTEC) من المشاكل الصحية الخطيرة ، وهذا الوضع يزداد سوءًا بسبب انتشار الكائنات الحية الدقيقة المقاومة للبلازما متعددة الأدوية في جميع أنحاء العالم. تهدف هذه الدراسة إلى التحقق من وجود الجين verotoxin بوساطة البلازميد في غير E.157 E. القولونية. حددت التقنيات الميكروبيولوجية القياسية ما مجموعه 137 عزلة E. coli. تم الكشف عن البلازميد من خلال مجموعة إعداد Perfectprep Plasmid Mini. خضعت هذه العزلات لفحص انتشار القرص ومعالجة البلازميد بمعالجة بروميد إيثيديوم. تم إخضاع العزلات المحتوية على البلازميد إلى تفاعل سلسلة بوليميراز (PCR) للتحقيق في وجود جين ال verروتوكسين بوساطة البلازميد (VT1 و VT2) في غير E.157 E. coli. من بين عزلات E. coli 137 ، كانت 49 عزلة غير O157 E. coli بينما كانت عزلات 29 (59.1٪) من فيروتوكسين تنتج أنماط مصلية غير O157 و 26 عزلة غير O157 VTEC تمتلك بلازميدات. بعض العزلات تحتوي على بلازميد أحادي الحجم في حين أن البعض الآخر يحتوي على بلازميدات متعددة بأحجام مختلفة تتراوح من 1.8 كيلوبايت إلى 7.6 كيلوبايت. كان البلازميد الذي يحتوي على جميع العزلات (100٪) مقاوم للعديد من الأدوية. تغيرت ثماني عزلات في أنماط قابليتها للحساسية بينما وجد أن ثلاث عزلات تفقد البلازميد بعد المعالجة بالبلازميد وبقيت العزلات المتبقية (15) ثابتة. تتميز مجموعات PCR المختلفة بـ 3 سموم فيروتينية بوساطة البلازميد تنتج غير القولونية O157 E. coli. أوضحت الدراسة الحالية حدوث الجين verotoxin بوساطة البلازميد في غير O157 E. coli. وعلى حد علمنا ، هذا هو التقرير الأول في الأدبيات العالمية حول الجين verotoxin بوساطة البلازميد في غير E.157 E. coli. يجب أن يعطي التشخيص والمراقبة في الوقت المناسب للعدوى VTEC الأولوية لوقف أو إبطاء الجين للنشر عن طريق انتقال الجين بوساطة البلازميد بين نفس البكتيريا أو الأنواع الأخرى.A significant increase in the incidence of non-O157 verotoxigenic Escherichia coli (VTEC) infections have become a serious health issues, and this situation is worsening due to the dissemination of plasmid mediated multidrug-resistant microorganisms worldwide. This study aims to investigate the presence of plasmid-mediated verotoxin gene in non-O157 E. coli. Standard microbiological techniques identified a total of 137 E. coli isolates. The plasmid was detected by Perfectprep Plasmid Mini preparation kit. These isolates were subjected to disk diffusion assay, and plasmid curing with ethidium bromide treatment. The plasmid containing isolates were subjected to a polymerase chain reaction (PCR) for investigating the presence of plasmid mediated verotoxin gene (VT1 and VT2) in non-O157 E. coli. Among the 137 E. coli isolates, 49 isolates were non-O157 E. coli while 29 (59.1%) isolates were verotoxin producing non-O157 serotypes and 26 non-O157 VTEC isolates possessed plasmids. Certain isolates harboured single sized plasmid while others had multiple plasmids with different size varied from 1.8kb to 7.6kb. A plasmid containing all (100%) the isolates was multidrug-resistant. Eight isolates changed their susceptibility patterns while three isolates were found to lose plasmid after post plasmid curing treatment and the rest of the isolates (15) remained constant. Different PCR sets characterized 3 plasmid-mediated verotoxins producing non-O157 E. coli. This current study demonstrated the occurrence of plasmid mediated verotoxin gene in non-O157 E. coli. To the best of our knowledge, this is the first report in the global literature on plasmid-mediated verotoxin gene in non-O157 E. coli. Timely diagnosis and surveillance of VTEC infections should prioritize to stop or slow down the virulence gene for dissemination by plasmid-mediated gene transfer amongst the same bacteria or other species

Molecular studies of antibiotic resistance among the clinical isolates of pseudomonas aeruginosa isolated in Selayang Hospital, Malaysia / Md. Fazlul Karim Khan

This study was carried out to determine the molecular analysis of antibiotic resistance among the clinical isolates of Pseudomonas aeruginosa. For this study, 54 P. aeruginosa isolates were obtained from blood, skin, pus, respiratory, eyes, urine and sputum of both paediatric and adult patients. Antibiotic sensitivity analysis was carried out using showed Quinolone (Ciprofloxacin) to be the most active antimicrobial agents with 83.34 % susceptibility followed by imipenem (81.49%), aminoglycosides (amikacin,74.08% and gentamicin, 72.23%) and the beta- lactams (cefepime 62.97%, ceftazidime (15%) and amikacine (14%). Among all the P. aeruginosa isolates, 29% of the strains were resistant to one antibiotic, 20% strains were resistant to two antibiotics and 51% were multidrug resistance. All the 54 strains of P. aeruginosa were subjected to MIC determination for ceftazidime and piperacillin by E-test. Piperacillin and ceftazidime showed the maximum number of resistance against P. aeruginosa 50% and 29.63% respectively. Among all the P. aeruginosa isolates, 12.97% were ESBL positive in phenotypic test whereas the 11.12% were found positive using E-test. But none of the isolates were found to produce MBLs. All these isolates were examined for the production of plasmid and were further analysed. Supercoil DNA marker was used to determine the plasmid sizes. It was used in electrophoresis gel each time along with the plasmids as molecular weight marker. Plasmid profiling analysis shown that, DNA marker was used to estimate the plasmid size and was used as molecular weight markers in each gel running. Plasmids were detected in 10 isolates with the POR of (18.51 %) by using Close and Rodriguez with modification (1982) and QIAprep Spin Kit. The overall sizes of the plasmid DNA range from the lowest 1.8 kb to the highest 14 kb. These 10 isolates appear to harbor 1 or more plasmids with the maximum of 4 plasmids. Various plasmid profiles were observed in the isolates. The plasmid analyses revealed that detectable plasmids in 10 (29.42%) out of the 34 selected multi-drug resistant P. aeruginosa isolates. Twenty four of the isolates possessed no plasmids, 3 isolates possessed single sized plasmids (3400bp- 4600bp) while 3 isolates had four plasmids with sizes from (1800bp- 5800bp). Four isolates which had 2 plasmids size of (220bp-14000bp) respectively. PCR were used to detect the ESBL genes. In this study, we examined the molecular type of blaSHV, SHV, 7EMand CTX-M derived ESBL variants produced by clinical P. aeruginosa isolates. We identified the strains carrying blaSHV and SHV genes by PCR test and confirmed the results by nucleotide sequencing. Polymerase chain reaction used in this study proved to be rapid reproducible and specific. We have found that 6 P. aeruginosa isolates carried blaSHV gene whereas 2 P. aeruginosa isolates carried SHV gene. Polymerase chain reaction produces a fragment of 200bp of blaSHV gene and 475bp of SHV gene respectively. ESBL positive isolates were found to harbour 1 or more plasmids. But TEM and CTX-M genes were not found in all the ESBL positive strains. The SHV & blaSHV genes were amplified by PCR. Approximately, 10-12 PCR reaction was carried out to have adequate amount of gene for sequencing and combined together to pool and purified by PCR purification kit. The purified product for sequencing service was sent to Medigene Sdn Bhd Selangor, Malaysia. The sequencing result was analysed online using NCBI and BLAST software

Role of magnesium in essential hypertension

Beneficial role of magnasium supplementation in prevention of complications in coronary artery disease (Iseri,1984) in ischaemic heart disease (Altura,1988) and in the treatment of patients with AMI (Abraham,1990) has been well documented. One study showed that magnesium supplementation reduced arterial blood pressure in patients with hypertension (Dykner& Waster,1983).Renin profiling, nevertheless,has revealed higher,and lower magnesium levels in low renin and high renin hypertensives respectively (Resnick LM et a1 1983).The significance of this seems to be that the influence of magnesium intake on blood pressure may differ according to the underlying state of magnesium metabolism in hypertensive patients. These observations remain largely unconfirmed. Recently it has been reported that a disordered metabolism of magnesium and calcium ions is present in women with pregnancy induced hypertension (Singh, HJ 1993).So the objective of this study was to investigate the magnesium status in serum, erythrocyte and in urine of untreated hypertensive patients. Work was further extended to investigate the relationship between serum and urinary magnesium levels,plasma renin activity (PRA) and plasma renin concentration (PRC) in normotensive and age and sex-matched untreated hypertensives

Modelling the removal of an earth bund to maximise seawater ingress into a coastal wetland

Weed infestation is a critical management issue for maintaining the natural values of coastal wetlands across the world. Widespread use of herbicides to control weeds has resulted in environmental issues in the past and has led to the search for more natural control methods such as using saline water. This study investigates management options to restore the natural flow regimes between freshwater wetlands and seawater by removing a bund which was built to grow ponded pasture. The study was carried out in the Mungalla wetland in Queensland, which is an important Nywaigi aboriginal community asset for recreation, cultural and economic activities. The study used modelling and monitoring methods to (a) assess how effective the earth bund was in excluding seawater and (b) the extent to which seawater could enter the wetland without the bund. Eleven scenarios were investigated to estimate seawater ingress under different tide, onshore wind, low atmospheric pressure and sea level rise (SLR) conditions. Results show that removal of the bund reinstated periodic tidal ingress into the wetland. Seawater intrusion was also shown to increase when there were onshore winds and/or low atmospheric pressure associated with cyclones. The greatest impact was under future SLR, where large increases in the inundation frequency and extent are likely to cause a shift in the wetland vegetation towards native salt tolerant species. Findings of this study are useful for examining the potential impact of various management interventions that are being considered for wetland system repair. For example, the removal or height adjustment of tidal barriers, dredging of silted streams, removal of weeds from choked streams and reintroduction of tidal flows to control weed infestation, improve water quality and restore natural values to the wetlands

Regional flood frequency analysis using an artificial neural network model

This paper presents the results from a study on the application of an artificial neural network (ANN) model for regional flood frequency analysis (RFFA). The study was conducted using stream flow data from 88 gauging stations across New South Wales (NSW) in Australia. Five different models consisting of three to eight predictor variables (i.e., annual rainfall, drainage area, fraction forested area, potential evapotranspiration, rainfall intensity, river slope, shape factor and stream density) were tested. The results show that an ANN model with a higher number of predictor variables does not always improve the performance of RFFA models. For example, the model with three predictor variables performs considerably better than the models using a higher number of predictor variables, except for the one which contains all the eight predictor variables. The model with three predictor variables exhibits smaller median relative error values for 2- and 20-year return periods compared to the model containing eight predictor variables. However, for 5-, 10-, 50- and 100-year return periods, the model with eight predictor variables shows smaller median relative error values. The proposed ANN modelling framework can be adapted to other regions in Australia and abroad

Evaluating annual maximum and partial duration series for estimating frequency of small magnitude floods

Understanding the nature of frequent floods is important for characterising channel morphology, riparian and aquatic habitat, and informing river restoration efforts. This paper presents results from an analysis on frequency estimates of low magnitude floods using the annual maximum and partial series data compared to actual flood series. Five frequency distribution models were fitted to data from 24 gauging stations in the Great Barrier Reef (GBR) lagoon catchments in north-eastern Australia. Based on the goodness of fit test, Generalised Extreme Value, Generalised Pareto and Log Pearson Type 3 models were used to estimate flood frequencies across the study region. Results suggest frequency estimates based on a partial series are better, compared to an annual series, for small to medium floods, while both methods produce similar results for large floods. Although both methods converge at a higher recurrence interval, the convergence recurrence interval varies between catchments. Results also suggest frequency estimates vary slightly between two or morepartial series, depending on flood threshold, and the differences are large for the catchments that experience less frequent floods. While a partial series produces better frequency estimates, it can underestimate or overestimate the frequency if the flood threshold differs largely compared to bankfull discharge. These results have significant implications in calculating the dependency of floodplain ecosystems on the frequency of flooding and their subsequent management

Bund removal to re-establish tidal flow, remove aquatic weeds and restore coastal wetland services—North Queensland, Australia

The shallow tidal and freshwater coastal wetlands adjacent to the Great Barrier Reef lagoon provide a vital nursery and feeding complex that supports the life cycles of marine and freshwater fish, important native vegetation and vital bird habitat. Urban and agricultural development threaten these wetlands, with many of the coastal wetlands becoming lost or changed due to the construction of artificial barriers (e.g. bunds, roads, culverts and floodgates). Infestation by weeds has become a major issue within many of the wetlands modified (bunded) for ponded pasture growth last century. A range of expensive chemical and mechanical control methods have been used in an attempt to restore some of these coastal wetlands, with limited success. This study describes an alternative approach to those methods, investigating the impact of tidal reinstatement after bund removal on weed infestation, associated changes in water quality, and fish biodiversity, in the Boolgooroo lagoon region of the Mungalla wetlands, East of Ingham in North Queensland. High resolution remote sensing, electrofishing and in-water logging was used to track changes over time– 1 year before and 4 years after removal of an earth bund. With tides only penetrating the wetland a few times yearly, gross changes towards a more natural system occurred within a relatively short timeframe, leading to a major reduction in infestation of olive hymenachne, water hyacinth and salvina, reappearance of native vegetation, improvements in water quality, and a tripling of fish diversity. Weed abundance and water quality does appear to oscillate however, dependent on summer rainfall, as changes in hydraulic pressure stops or allows tidal ingress (fresh/saline cycling). With an estimated 30% of coastal wetlands bunded in the Great Barrier Reef region, a passive remediation method such as reintroduction of tidal flow by removal of an earth bund or levee could provide a more cost effective and sustainable means of controlling freshwater weeds and improving coastal water quality into the future

The role of NGOs in improving sanitation status in the rural areas of Bangladesh: challenges and expectations

NGOs are playing a significant role to ameliorate the sanitation situation in Bangladesh. Multi-stage 30 cluster sampling was adopted to collect quantitative data and 4,200 households were visited from 10 purposively selected sub-districts with and without NGO-led WASH programme. In every sub-district a focus group discussion was conducted to collect relevant information supplementing the findings from quantitative study. The overall sanitation coverage in areas with NGO intervention was significantly (p<0.001) higher than the areas without any such intervention. Logistic regression analyses showed that the existence of NGO-led programme, the level of education, poverty, land ownership and access to media had significant (p<0.001) influence on sanitation practice. Financial crisis was reported to be the predominant reason for households not having their own sanitary latrine, where NGO assistance was sought for. People acknowledged the role of NGOs in raising awareness, increasing sanitary latrine use and reported NGO assistance necessary for 100 per cent sanitation