Characterization and treatment of tunneling wastewater using natural and chemical coagulants.

Particles are a concern regarding tunneling wastewater, but gaps remain in understanding metal(loid)s content and coagulation efficiency. In this research, characterization of the wastewaters before and after treatment was investigated by chemical analysis and various techniques for particle characterization. Then, laboratory work was conducted to evaluate effectiveness of sedimentation and use of coagulants to remove particles and particle-associated contaminants. Both natural (chitosan) and chemical coagulants (ferric chloride sulfates and polyaluminium chloride solution), were applied in a jar test system. The results indicated that short-time sedimentation alone substantially reduced the particle content and particle-associated pollutants, including metal(loid)s, while subsequent chemical coagulation was required to comply with discharge limits. The optimum dosages of chitosan, PIX, and PAX for water 1 after pre-treatment (15 min sedimentation) were 1 mg/L, 3 mg Fe/L, and 3 mg Al/L, respectively, while a dosage of 2 mg Al/L gave the best results in water 3. Furthermore, chemical coagulation significantly decreased the volume density of particles in the diameter range of 1-100 µm, showing that coagulants are efficient for the removal of smaller particles not removed by conventional sedimentation.

Management of mung bean leaf spot disease caused by Phoma herbarum through Penicillium janczewskii metabolites mediated by MAPK signaling cascade.

Vigna radiata L., an imperative legume crop of Pakistan, faces hordes of damage due to fungi; infecting host tissues by the appressorium. The use of natural compounds is an innovative concern to manage mung-bean fungal diseases. The bioactive secondary metabolites of Penicillium species are well documented for their strong fungi-static ability against many pathogens. Presently, one-month-old aqueous culture filtrates of Penicillium janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum were evaluated to check the antagonistic effect of different dilutions (0, 10, 20, … and 60%). There was a significant reduction of around 7-38%, 46-57%, 46-58%, 27-68%, and 21-51% in Phoma herbarum dry biomass production due to P. janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum, respectively. Inhibition constants determined by a regression equation demonstrated the most significant inhibition by P. janczewskii. Finally, using real-time reverse transcription PCR (qPCR) the effect of P. Janczewskii metabolites was determined on the transcript level of StSTE12 gene involved in the development and penetration of appressorium. The expression pattern of the StSTE12 gene was determined by percent Knockdown (%KD) expression that was found to be decreased i.e. 51.47, 43.22, 40.67, 38.01, 35.97, and 33.41% for P. herbarum with an increase in metabolites concentrations viz., 10, 20, 30, 40, 50 and 60% metabolites, respectively. In silico studies were conducted to analyze the role of Ste12 a transcriptional factor in the MAPK signaling pathway. The present study concludes a strong fungicidal potential of Penicillium species against P. herbarum. Further studies to isolate the effective fungicidal constituents of Penicillium species through GCMS analysis and determination of their role in signaling pathways are requisite.

TcpA from the Clostridium perfringens plasmid pCW3 is more closely related to the DNA translocase FtsK than to coupling proteins.

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens, the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C. perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus.

Training Deep Neural Networks with Novel Metaheuristic Algorithms for Fatigue Crack Growth Prediction in Aluminum Aircraft Alloys.

Fatigue cracks are a major defect in metal alloys, and specifically, their study poses defect evaluation challenges in aluminum aircraft alloys. Existing inline inspection tools exhibit measurement uncertainties. The physical-based methods for crack growth prediction utilize stress analysis models and the crack growth model governed by Paris' law. These models, when utilized for long-term crack growth prediction, yield sub-optimum solutions and pose several technical limitations to the prediction problems. The metaheuristic optimization algorithms in this study have been conducted in accordance with neural networks to accurately forecast the crack growth rates in aluminum alloys. Through experimental data, the performance of the hybrid metaheuristic optimization-neural networks has been tested. A dynamic Levy flight function has been incorporated with a chimp optimization algorithm to accurately train the deep neural network. The performance of the proposed predictive model has been tested using 7055 T7511 and 6013 T651 alloys against four competing techniques. Results show the proposed predictive model achieves lower correlation error, least relative error, mean absolute error, and root mean square error values while shortening the run time by 11.28%. It is evident through experimental study and statistical analysis that the crack length and growth rates are predicted with high fidelity and very high resolution.

Neocosmospora rubicola, a stem rot disease in potato: Characterization, distribution and management.

Potato (Solanum tuberosum L.) is one of the most important crops in maintaining global food security. Plant stand and yield are affected by production technology, climate, soil type, and biotic factors such as insects and diseases. Numerous fungal diseases including Neocosmospora rubicola, causing stem rot, are known to have negative effects on potato growth and yield quality. The pathogen is known to stunt growth and cause leaf yellowing with grayish-black stems. The infectivity of N. rubicola across a number of crops indicates the need to search for appropriate management approaches. Synthetic pesticides application is a major method to mitigate almost all potato diseases at this time. However, these pesticides significantly contribute to environmental damage and continuous use leads to pesticide resistance by pathogens. Consumers interest in organic products have influenced agronomists to shift toward the use of biologicals in controlling most pathogens, including N. rubicola. This review is an initial effort to carefully examine current and alternative approaches to control N. rubicola that are both environmentally safe and ecologically sound. Therefore, this review aims to draw attention to the N. rubicola distribution and symptomatology, and sustainable management strategies for potato stem rot disease. Applications of plant growth promoting bacteria (PGPB) as bioformulations with synthetic fertilizers have the potential to increase the tuber yield in both healthy and N. rubicola infested soils. Phosphorus and nitrogen applications along with the PGPB can improve plants uptake efficiency and reduce infestation of pathogen leading to increased yield. Therefore, to control N. rubicola infestation, with maximum tuber yield benefits, a pre-application of the biofertilizer is shown as a better option, based on the most recent studies. With the current limited information on the disease, precise screening of the available resistant potato cultivars, developing molecular markers for resistance genes against N. rubicola will assist to reduce spread and virulence of the pathogen.

Outcome Of Short And Long Duration Steroid Therapy In Childhood Nephrotic Syndrome In Terms Of Frequency Of Relapse Rate.

BACKGROUND: Nephrotic syndrome is a clinical syndrome defined by massive proteinuria (greater than 40 mg/m2/hour) responsible for hypoalbuminemia (less than 30 g/L) resulting in oedema and hyperlipidaemia. Objective of the study was to compare the frequency of relapse rate with short and long duration steroid therapy in Nephrotic syndrome. It was a Quasiexperimental control group design, conducted at the Department of Paediatric Nephrology, The Children's Hospital and Institute of Child Health, Lahore. Duration of study: One year. METHODS: The data of 150 patients with steroid sensitive nephrotic syndrome was included with clinical presentation and diagnostic investigations. The children were randomly divided into long and short duration steroid treatment groups. Outcome was determined in terms of relapse rate after achieving remission with both treatment strategies. Independent sample t test was applied to compare the outcome in both groups with p≤0.05 considered as significant. Data was stratified for all the effect modifiers like age and gender and poststratification chi square test was applied to see the effect on the outcome, taking p≤0.05 as significant. RESULTS: The relapse rate of the disease was 0.8±0.72 per year in short-duration group and 1.28±0.61 per year in subjects receiving long-duration steroids, and difference between the two groups was found to be statistically significant (p<0.001). The relapse rate was less in the short duration therapy group as compared to the long duration therapy 62.7% (n=47) patients in group A had one or more relapses of the disease within one year of follow up in contrast to 94.7% (n=71) children in group B (p<0.001). CONCLUSIONS: Patients receiving short duration steroid therapy showed a lower relapse rate as compared to those who were administered long term steroids.

GC-MS analysis & antifungal activity of Datura metel L. against Rhizoctonia solani Kuhn.

The current study was designed to evaluate the antifungal properties of Datura metel L. against Rizoctonia solani Kuhn. To achieve this objective, six concentrations of leaves & stem methanol extract of D. metel viz. 1%, 1.5%, 2%, 2.5%, 3% & 3.5% were tested against R. solani in vitro. Leaf extract of D. metel was found more effective as its 3.5% concentration caused 75% retardation in test fungal growth as compared to the stem extract. D. metel methanolic leaf extract was fractioned between n-butanol, n-hexane, chloroform & ethyl acetate & bioactivities of isolated fractions were tested against R. solani. The chloroform fraction was found highly effective, as its concentrations 0.1% & 0.01% caused 27% & 21% growth inhibition respectively. So, this particular chloroform fraction was further analyzed to identify various chemical constituents through GC-MS (Gas chromatography mass spectroscopic) analysis. Twelve phyto-constituents viz. eugenol, 2-pentadecanone 6,10,14 trimethyl, pentadecanoic acid, pentadecanoic acid, 1 4-methyl- methyl ester, phytol, 9,12,15-octadecatrienoic acid, heptacosane, n-hexadecanoic, 6-octadecanoic acid, 9, 12 octadecanoic acid, dodecanoic & tetradecanoic acids were identified. So, the present study concluded that the presence of these bioactive constituents make D. metel as an effective antifungal agent against R. solani.

Characterization and Evaluation of Hydrothermal Liquefaction Char from Alkali Lignin in Subcritical Temperatures.

An evaluation of hydrothermal liquefaction (HTL) char is investigated in this work. Morphological studies, N2 adsorption behavior, FTIR analysis, thermal behavior, and elemental composition are studied. The HTL char yield showed an increase with higher operating temperatures. It increased from 11.02% to 33% when the temperature increased from 573 K to 623 K. At lower temperatures, the residence time showed an impact on the yield, while close to the critical point, residence time became less impactful. Elemental analysis showed that both higher operating temperatures and longer residence times increased the nitrogen content of the chars from 0.32% to 0.51%. FTIR analysis suggested the char became more aromatic with the higher temperatures. The aliphatic groups present diminished drastically with the increasing temperature. Residence time did not show a significant impact as much as the temperature when considering the functional group elimination. An increase in operating temperatures and residence times produced thermally stable chars. HTL char produced at the lowest operating temperature and showed both the highest surface area and pore volume. When temperature and residence time increase, more polyaromatic char is produced due to carbonization.

9-Acridinemethanamine and Acridine-9-Carboxaldehyde as Potential Fluorescence Lifetime pH Indicators.

A significant challenge concerning the development of fluorescence lifetime (FL) based pH sensors is the paucity of fluorophores with sufficiently large FL variation with pH. Acridine is amongst the indicators with highest fluoresce lifetime responses to pH, with a change in lifetime of about 13 ns within a pH range of 5-8. Here we examine the two acridine derivatives, 9-acridinemethanamine (9-AMA) and acridine-9-carbaldehyde (9-ACA) in terms of their FL pH sensitivity and pH sensing range. Both indicators are characterized when dissolved in buffer solutions, as well as when immobilized in support materials. 9-AMA has a change in FL of 11 ns between pH 2-5, both when dissolved in solution and when immobilized in surfactant-filled mesoporous silica. The FL of 9-ACA is not sensitive to pH when dissolved in buffer solutions; however, when covalently bound to amine-modified silica, its FL changes 15 ns between pH 3-6. 9-AMA and 9-ACA represent promising FL in the pH range of pH 2-6, and could potentially form the basis of new FL pH sensors. Graphical Abstract.

Molecular Diagnosis and Identification of Genetic Variants Underlying Distal Renal Tubular Acidosis in Pakistani Patients Using Whole Exome Sequencing.

Introduction: Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder characterized by an impaired urinary acidification process in distal nephrons that results in the production of alkaline urine. Loss of function variants in any of the three genes, ATP6V0A4, ATP6V1B1, or SLC4A1, which all play a role in normal acidification of urine by kidneys, may lead to dRTA. Objective: This study was designed to identify genetic variants underlying dRTA in Pakistani patients using whole exome sequencing, followed by confirmatory Sanger sequencing. Materials and Methods: Patients were identified following presentation with characteristic clinical features of dRTA including vomiting, dehydration, and highly alkaline urine with metabolic acidosis during the first few days of life. Whole exome sequencing and Sanger sequencing were employed for genetic analyses of the patients. In silico analyses of the identified variants were performed using web-based bioinfomatics programs. Results: Through whole exome sequencing, we identified two splice site variants (c.2257 + 1G>A and c.722 + 5G>A) in the ATP6V0A4 gene that likely underly the disease phenotype in the two families. Multiple in silico tools predicted these variants to affect the respective splice sites supporting their likely role in pathogenesis. Conclusion: The study extends the spectrum of ATP6V0A4 variants associated with dRTA and should benefit the genetic counseling and prenatal diagnosis of the affected families.

A Broad-Range Fluorescence Lifetime pH Sensing Material Based on a Single Organic Fluorophore.

A general drawback for optical based pH sensors is that their response is typically limited to within 2-3 pH units centered around the pKa of the indicator. Fluorescence lifetime (FL) is a particularly compelling basis for highly stable pH sensors since this is an intrinsic property of the indicator molecule. Here we demonstrate that it is possible to broaden the sensing range of FL based sensors significantly by placing the indicator in a support material where the indicator's chemical environment itself changes with pH. For acridine immobilized in amine-modified porous silica, a total FL change of 20 ns in the pH range 2-12 is achieved. A linear pH vs FL relationship is observed with three break points occurring at pH 4, 6 and 9 that are related to the pKa values of the indicator and the silica material. This proves the concept that tuning the fluorophore's chemical environment can broaden the FL pH sensing range, where currently available fluorophores do not cover the full pH range. Graphical Abstract.

Analysis of Antagonistic Potential of Secondary Metabolites and Organic Fractions of Trichoderma Species against Alternaria Alternata.

Role of Trichoderma species is well documented as antagonists as well as plant growth enhancers. Presently, the fungicidal potential of three Trichoderma species namely, T. koningii (FCBP769) , T. viride (FCBP904) , and T. harzianum (FCBP1277) was assessed against Alternaria alternata that causes leaf necrotic spots of Syzygium cumini and broad range of other plants using 0, 15, 30, 45, 60 and 75% dilutions of filtrates. There was a significant reduction of around 40-95%, 22-86% and 52-91% in fungal biomass by T. koningii, T. viride and T. harzianum, respectively. In fractionation bioassays, Trichoderma metabolites were partitioned using organic solvents viz., n-butanol, n-hexane, chloroform and ethyl acetate. Antifungal activity at different concentrations (10, 20, 30, 40 and 50 ppm) was assessed against the pathogen. Ethyl acetate fraction of T. koningii extract displayed the most promising activity resulting in 10-90% suppression in biomass. In case of T. viride butanol fraction proved the most effective in retarding the growth of pathogen from 20 to 80%. While T. harzianum extract revealed 55-85% arrest in fungal biomass due to n-hexane fraction. Present study concludes that test Trichoderma species demonstrated a strong fungicidal activity against A. alternata. Current research offers the possibility of developing strategies for controlling pathogens with bioactive metabolites of Trichoderma.

Pillars or Pancakes? Self-Cleaning Surfaces without Coating.

Surfaces that stay clean when immersed in water are important for an enormous range of applications from ships and buildings to marine, medical, and other equipment. Up until now the main strategy for designing self-cleaning surfaces has been to combine hydrophilic/hydrophobic coatings with a high aspect ratio structuring (typically micron scale pillars) to trap a (semi)static water/air layer for drag and adhesion reduction. However, such coating and structuring can distort optical properties and get damaged in harsh environments, and contamination, i.e., particles, oil droplets, and biofouling, can get trapped and aggregate in the structure. Here we present a radically different strategy for self-cleaning surface design: We show that a surface can be made self-cleaning by structuring with a pattern of very low aspect ratio pillars ("pancakes"). Now the water is not trapped. It can flow freely around the pancakes thus creating a dynamic water layer. We have applied the new pancake design to sapphire windows and made the first surfaces that are self-cleaning through structuring alone without the application of any coating. An offshore installation has now been running continuously with structured windows for more than one year. The previous uptime for unstructured windows was 7 days.

Molecular Study of Nephronophthisis in 7 Unrelated Pakistani Families.

Nephronophthisis is an autosomal recessive cystic kidney disease characterized by tubular interstitial infiltration, periglomerular fibrosis, and cysts, and is the most frequent genetic cause of end-stage renal disease in children. Nephronophthisis is pleiotropic as almost all the causative genes are involved in primary cilium and centrosome function which are found in almost all human cells. Genetic heterogeneity in nephronophthisis makes the molecular and genetic diagnosis somewhat difficult. Homozygous deletions in the nephronophthisis 1 (NPHP1) gene are the major contributor of nephronophthisis cases, while other genes accounts for less than 3% each. Nephronophthisis-related ciliopathy is a term used for extrarenal symptoms in addition to nephronophthisis. Herein, we are reporting the molecular study of 7 children from independent families fulfilling the criteria of nephronophthisis. A deletion analysis of the NPHP1 gene was performed in each case, and NPHP5 mutation screening was performed in the absence of such deletion in patients with Senior Loken syndrome.

COMPLICATIONS AND SAFETY OF POST PERCUTANEOUS RENAL BIOPSY IN PAEDIATRIC PATIENTS.

BACKGROUND: Renal biopsy is gold standard in diagnosis of various renal diseases. Though it is safe yet it requires adequate patient evaluation, preparation and monitoring after the procedure. METHODS: This descriptive case series was conducted in the Paediatric Nephrology Department, The Children's Hospital Lahore over a period of one year. Children aged less than 16 years were included who were suffering from renal diseases requiring renal biopsy for diagnosis. After renal biopsy patients were monitored for complications and were discharged after short stay of eight hours who got no complication during the observation period. RESULTS: Out of total 145 patients, 97 (66.1%) were males and 48 (33.9%) were females. The age range was from 0.7 years to 16 years. The duration of stay after renal biopsy was up to eight hours in 131 (90.34%) patients, while 14 (9.66%) stayed longer. Post-biopsy stay of 24 hours was observed in 8 patients and 48 hours in 5 patients. Only 1 patient stayed for 96 hours. Patients with gross haernaturia were 6 (4.1%) who were checked for perinephric hematoma formation by ultrasonography. Only one patient required blood transfusion along with fresh frozen plasma. The most common histopathological diagnosis was Mesangioproliferative with 67 (46.2%) cases, followed by Glomerulonephritis in 35 (24.1%) patients having Membranoproliferative Glomerulonephritis (MPGN) while Systemic Lupus Erythematous (SLE) nephritis was reported in 15 (10.35%) patients. CONCLUSION: Patients can be discharged after short stay post renal biopsy procedure provided done after proper screening of patients. As it not only reduces patient stay but also is cost effective.

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity.

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.

HISTOPATHOLOGICAL PATTERNS IN PAEDIATRIC IDIOPATHIC STEROID RESISTANT NEPHROTIC SYNDROME.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a common problem but difficult to treat for pediatric nephrologists. Due to paucity of studies done in few centres in southern Pakistan regarding the histopathological aspects in paediatric patients with SRNS, this study was conducted to determine the histopathological spectrum in children with SRNS at our centre. METHODS: This descriptive study has been conducted at the Nephrology department, The Children's Hospital Lahore from February 2014 to January 2015. Based upon history, physical examination and laboratory results, all patients diagnosed as idiopathic SRNS were included in the study and renal biopsy was done to determine the underlying pathology. Histopathology reports were retrieved and data analysis done using SPSS-20.0. RESULTS: There were a total of 96 patients, 64 (66.7%) males and 32 (33.3%) females. The age range was from 0.80 to 15 years with mean age of presentation being 6.34+3.75 years. The most common histo-pathological pattern was mesangio-proliferative Glomerulonephritis found in 79 (82.3%) cases followed by Focal segmental glomerulosclerosis (FSGS) in 9 (9.4%) patients while Minimal change disease (MCD) was seen in 5 (5.2%) subjects. CONCLUSION: Mesangioproliferative glomerulonephritis is the most common histological pattern seen in children presenting with idiopathic SRNS at our centre followed by FSGS and MCD.

TACROLIMUS DRUG LEVEL AND RESPONSE TO TREATMENT IN IDIOPATHIC CHILDHOOD STEROID RESISTANT NEPHROTIC SYNDROME.

BACKGROUND: The management of Steroid Resistant Nephrotic Syndrome (SRNS) is an uphill task for paediatric nephrologists as immunosuppressive agents are the mainstay of treatment in these patients. Tacrolimus is used along with steroids. This study is conducted to see the relationship between the tacrolimus dose, drug level and response in the management of SRNS. METHODS: This quasi experimental study was conducted at The Children's Hospital Lahore over a period of one year. Patients with SRNS of either sex and 1-10 years of age were included and those with secondary nephrotic syndrome were excluded. Tacrolimus was given at a dose of 0.05-0.1 mg/kg/day in 2 divided doses along with steroids. The follow-up was done for six months with proteinuria monitoring and tacrolimus drug levels done two weeks after initiation of treatment. RESULTS: Out of 42 patients, 27 (64.3%) were males and 15 (35.7%) were females. The most common histological diagnosis observed was mesangio-proliferative glomerulonephritis in 30 (71.4%) patients. The tacrolimus trough level range was 0.5-15.20 ng/ml with a mean value of 4.68 ng/ml ± 2.85. Forty-one (97.6%) children showed complete response to treatment while one patient showed partial response. CONCLUSION: This study suggests that tacrolimus is an effective drug for treatment of SRNS in paediatric patients and there is no linear relationship between the drug dose, response and drug level.

Self-assembly of ferromagnetic organic-inorganic perovskite-like films.

Perovskite-based organic-inorganic hybrids hold great potential as active layers in electronics or optoelectronics or as components of biosensors. However, many of these applications require thin films grown with good control over structure and thickness--a major challenge that needs to be addressed. The work presented here is an effort towards this goal and concerns the layer-by-layer deposition at ambient conditions of ferromagnetic organic-inorganic hybrids consisting of alternating CuCl4-octahedra and organic layers. The Langmuir-Blodgett technique used to assemble these structures provides intrinsic control over the molecular organization and film thickness down to the molecular level. Magnetic characterization reveals that the coercive field for these thin films is larger than that for solution-grown layered bulk crystals. The strategy presented here suggests a promising cost effective route to facilitate the excellently controlled growth of sophisticated materials on a wide variety of substrates that have properties relevant for the high density storage media and spintronic devices.

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs.

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Arg(87)respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368)and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Arg(368)and Asn(459)/Arg(87)pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.