Publications by authors named "Krishnan Sankaran"

43 Publications

Airway closing index in school-age children during exercise bronchoprovocation.

J Asthma 2020 Dec 2:1-9. Epub 2020 Dec 2.

New York Medical College, Valhalla, NY, USA.

Objective: Airway Closing Index (ACI), the ratio of % change in FVC to % change in FEV with bronchoprovocation, may represent changes in airflow due to airway closure, as opposed to airway narrowing. The objective of this study was to evaluate ACI during exercise bronchoprovocation (EB) in children.

Methods: Children, 6 to 18 years of age, who underwent EB using a stationary bicycle ergometer over a 6-year period were reviewed. Pulmonary function, including ACI, in patients with a positive exercise challenge, defined as ≥10% decrease in FEV following exercise, were compared to patients with a negative challenge.

Results: A total of 1030 children with a median age of 13 (IQR 11-15) underwent EB, of which 376 (37%) had a positive exercise challenge. There was wide variability in ACI, with a median of 0.75 (0.28-1.21). Median ACI in those with a positive test was 0.68 (IQR 0.41-0.93) compared to 0.84 (IQR 0.09-1.06) for those with a negative test,  = 0.017. Median ACI was higher in older children ( < 0.001) and females ( < 0.0001). Median percent change in FEV following bronchodilator for children in the highest quintile for ACI was 4.5 (IQR 1.3-8.1) compared to 5.5 (IQR 2-9.2) for children in the lowest quintile,  = 0.04.

Conclusions: There is wide variability in the ACI in children undergoing EB. ACI was lower in children with a positive challenge, the significance is unknown. Children with higher ACI may have increased airway closure with bronchoprovocation, and less response to bronchodilators.
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http://dx.doi.org/10.1080/02770903.2020.1850765DOI Listing
December 2020

Transmitted Home Oximetry and Duration of Home Oxygen in Premature Infants.

Pediatrics 2020 08 14;146(2). Epub 2020 Jul 14.

Division of Neonatology, Department of Pediatrics, College of Medicine, University of Kentucky, Lexington, Kentucky.

Objectives: To determine if a home oxygen therapy (HOT) management strategy that includes analysis of recorded home oximetry (RHO) data, compared with standard monthly clinic visit assessments, reduces duration of HOT without harm in premature infants.

Methods: The RHO trial was an unmasked randomized clinical trial conducted in 9 US medical centers from November 2013 to December 2017, with follow-up to February 2019. Preterm infants with birth gestation ≤37 + 0/7 weeks, discharged on HOT, and attending their first pulmonary visit were enrolled. The intervention was an analysis of transmitted RHO between clinic visits ( = 97); the standard-care group received monthly clinic visits with in-clinic weaning attempts ( = 99). The primary outcomes were the duration of HOT and parent-reported quality of life. There were 2 prespecified secondary safety outcomes: change in weight and adverse events within 6 months of HOT discontinuation.

Results: Among 196 randomly assigned infants (mean birth gestational age: 26.9 weeks; SD: 2.6 weeks; 37.8% female), 166 (84.7%) completed the trial. In the RHO group, the mean time to discontinue HOT was 78.1 days (SE: 6.4), compared with 100.1 days (SE: 8.0) in the standard-care group ( = .03). The quality-of-life scores improved from baseline to 3 months after discontinuation of HOT in both groups ( = .002), but the degree of improvement did not differ significantly between groups ( = .75).

Conclusions: RHO was effective in reducing the duration of HOT in premature infants. Parent quality of life improved after discontinuation. RHO allows physicians to determine which infants can be weaned and which need prolonged oxygen therapy between monthly visits.
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http://dx.doi.org/10.1542/peds.2020-0079DOI Listing
August 2020

SARS-CoV-2 Infection in Patients with Down Syndrome, Congenital Heart Disease, and Pulmonary Hypertension: Is Down Syndrome a Risk Factor?

J Pediatr 2020 Oct 28;225:246-248. Epub 2020 Jun 28.

Department of Pediatrics (Cardiology), Columbia University Irving Medical Center, New York, NY.

With increasing information available about the epidemiology, pathophysiology, and management of patients affected with severe acute respiratory syndrome corona virus-2 infection, patients with Down syndrome, congenital heart disease, airway obstruction, and pulmonary hypertension present a unique challenge. This case series describes 3 patients with Down syndrome and respiratory failure secondary to coronavirus infection.
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http://dx.doi.org/10.1016/j.jpeds.2020.06.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321054PMC
October 2020

Reply.

J Pediatr 2019 10 9;213:251. Epub 2019 Jul 9.

Pediatric Heart Lung Center, Section of Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, Aurora, Colorado.

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http://dx.doi.org/10.1016/j.jpeds.2019.06.020DOI Listing
October 2019

Evidence to Suggest Bacterial Lipoprotein Diacylglyceryl Transferase (Lgt) is a Weakly Associated Inner Membrane Protein.

J Membr Biol 2019 12 29;252(6):563-575. Epub 2019 Jun 29.

Centre for Biotechnology, Anna University, Chennai, 600025, India.

The unique and ubiquitous bacterial lipoprotein biosynthesis pathway is an attractive new antibiotic target. Crystal structures of its three biosynthetic enzymes have been solved recently. The first enzyme, Phosphatidylglycerol:proLipoprotein diacylglyceryl Transferase (Lgt), which initiates the post-translational modification at the metabolic interface of protein biosynthesis, phospholipid biosynthesis, protein secretion and lipid modification was reported to be a seven-transmembrane helical structure with a catalytic periplasmic head. Its complete solubilization in water or mild detergent in a fully active state, its chromatographic behaviour as an active monomer in the absence of detergent and recovery of active whole-length protein after proteolytic treatment of spheroplasts cast serious doubts about its proposed membrane association and orientation. Rather, it could be a seven-helical bundle partially embedded in the inner membrane's inner leaflet aided by hydrophobic interaction. In fact, there are examples where originally reported seven-transmembrane proteins were later shown to be seven-helical peripheral membrane proteins based on solubilization criterion and re-analysis. Validated computational tool, Membrane Optimal Docking Area (MODA), also predicted a weaker association of Lgt's helices with the membrane compared to typical transmembrane proteins. This insight is crucial to Lgt-based antibiotic design.
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http://dx.doi.org/10.1007/s00232-019-00076-3DOI Listing
December 2019

Active sulfite oxidase domain of Salmonella enterica pathogenic protein small intestine invasive factor E (SiiE): a potential diagnostic target.

Appl Microbiol Biotechnol 2019 Jul 18;103(14):5679-5688. Epub 2019 May 18.

Centre for Biotechnology, Anna University, Chennai, 600020, India.

Serovars of Salmonella enterica are common food-borne bacterial pathogens. Salmonella typhi, which causes typhoid, is the most dangerous of them. Though detailed molecular pathogenesis studies reveal many virulence factors, inability to identify their biochemical functions hampers the development of diagnostic methods and therapeutic leads. Lack of quicker diagnosis is an impediment in starting early antibiotic treatment to reduce the severe morbidity and mortality in typhoid. In this study, employing bioinformatic prediction, biochemical analysis, and recombinantly cloning the active region, we show that extracellularly secreted virulence-associated protein, small intestinal invasion factor E (SiiE), possesses a sulfite oxidase (SO) domain that catalyzes the conversion of sodium sulfite to sodium sulfate using tungsten as the cofactor. This activity common to Salmonella enterica serovars seems to be specific to them from bioinformatic analysis of available bacterial genomes. Along with the ability of this large non-fimbrial adhesin of 600 kDa binding to sialic acid on the host cells, this activity could aid in subverting the host defense mechanism by destroying sulfites released by the immune cells and colonize the host gastrointestinal epithelium. Being an extracellular enzyme, it could be an ideal candidate for developing diagnostics of S. enterica, particularly S. typhi.
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http://dx.doi.org/10.1007/s00253-019-09894-wDOI Listing
July 2019

Bronchodilators, Antibiotics, and Oral Corticosteroids Use in Primary Care for Children With Cough.

Glob Pediatr Health 2019 25;6:2333794X19831296. Epub 2019 Feb 25.

New York Medical College, Valhalla, NY, USA.

Real-world management decisions for acute cough in children in primary care practice are not well understood. This study is an analysis of 560 encounters for children with cough, 19 days to 18 years of age, seen in a predominantly suburban academic pediatric practice, over 1 year. Past history, cough duration, and cough characteristics significantly affected treatment decisions. Children with cough frequently had a history of preterm birth, allergies, asthma, and neurological conditions. Most common therapies were bronchodilators, antibiotics, and oral corticosteroids. Children prescribed antibiotics were older, more likely to have a wet or productive cough, history of sinusitis, pneumonia or dysphagia, and longer cough duration. Children prescribed oral corticosteroids were younger, less likely to be wet or productive and more likely to have history of asthma or dysphagia. Children prescribed bronchodilators were more likely to have fever, nasal congestion, and wheezing and history of previous asthma, pneumonia, or dysphagia.
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http://dx.doi.org/10.1177/2333794X19831296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390215PMC
February 2019

Optimizing a Metatranscriptomic Next-Generation Sequencing Protocol for Bronchoalveolar Lavage Diagnostics.

J Mol Diagn 2019 03 31;21(2):251-261. Epub 2018 Oct 31.

Department of Pathology, New York Medical College, Valhalla, New York; Department of Pathology and Clinical Laboratories, Westchester Medical Center, Valhalla, New York.

Compared with conventional serologic, culture-based, and molecular-based diagnostic tests, next-generation sequencing (NGS) provides sequence-evidenced detection of various microbes, without prior knowledge, and thus is becoming a novel diagnostic approach. Herein we describe an RNA-based metatranscriptomic NGS (mtNGS) protocol for culture-independent detection of potential infectious pathogens, using clinical bronchoalveolar lavage specimens as an example. We present both an optimized workflow for experimental sequence data collection and a simplified pipeline for bioinformatics sequence data processing. As shown, the whole protocol takes approximately 24 to 36 hours to detect a wide range of Gram-positive and -negative bacteria and possibly other viral and/or fungal pathogens. In particular, we introduce a spike-in RNA mix as an internal control, which plays a critical role in mitigating false-positive and false-negative results of clinical diagnostic tests. Moreover, our mtNGS method can detect antibiotic resistance genes and virulence factors; although it may not be comprehensive, such information is imperative and helpful for the clinician to make better treatment decisions. Results from our preliminary testing suggest that the mtNGS approach is a useful alterative in diagnostic detection of emerging infectious pathogens in clinical laboratories. However, further improvements are needed to achieve better sensitivity and accuracy.
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http://dx.doi.org/10.1016/j.jmoldx.2018.09.004DOI Listing
March 2019

The optimization of home oxygen weaning in premature infants trial: Design, rationale, methods, and lessons learned.

Contemp Clin Trials 2018 12 11;75:72-77. Epub 2018 Aug 11.

University of Massachusetts Memorial Medical Center, Division of Neonatology, Worcester, MA, United States; University of Massachusetts Memorial Medical Center, Division of Pediatric Pulmonology, Worcester, MA, United States. Electronic address:

Improved survival among preterm infants has led to an increase in diagnosis of chronic lung disease and infants discharged home from the NICU on supplemental oxygen. Despite this increased prevalence, no clearly defined guidelines for the management of home oxygen therapy (HOT) exist. This lack of consensus leads to significant variability in the duration of home oxygen therapy and a general paucity of evidence-based practice. Our team has identified recorded home oxygen therapy (RHO) as a potential new resource to guide clinical decision making in the outpatient pulmonology clinic. By recording extended O2 saturation data during the weaning process, RHO has the potential to save cost and improve the processes of HOT management. Our team is currently supporting a prospective, multi-center, randomized, controlled trial of RHO guided HOT weaning with the aims of determining effect upon duration of HOT, perceived parent quality of life and effect upon growth and respiratory outcomes. We plan to randomize 196 infants into one of two study arms evaluating standard HOT management versus RHO guided oxygen weaning. Our primary outcomes are total HOT duration and parental quality of life. This trial represents an unprecedented opportunity to test a novel home monitoring intervention for weaning within a vulnerable yet quickly growing population. If effective, the use of RHO may provide clinicians a tool for safe weaning.
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http://dx.doi.org/10.1016/j.cct.2018.08.001DOI Listing
December 2018

Asthma, Environment and Pollution: Where the Rubber Hits the Road.

Indian J Pediatr 2018 Oct 30;85(10):893-898. Epub 2018 May 30.

New York Medical College and Maria Fareri Children's Hospital at Westchester Medical Center, 40 Sunshine Cottage Road - Skyline 1N-D29, Valhalla, NY, 10595, USA.

The detrimental effects of environmental pollution on one's health are undeniable and have been demonstrated time and time again. Breathing in pollutants in ambient air often has consequences throughout the body, including cardiovascular disease, effects on the reproductive system, and oncologic implications. In the respiratory system, chronic exposure yields a number of outcomes, including chronic obstructive pulmonary disease (COPD) and asthma exacerbations, increased rates of hospitalizations, and increased severity of acute illnesses. On a macro-level, this morbidity and mortality then leads to vast and far-reaching public health consequences the world over, including the loss of billions of dollars' worth of labor. This is especially applicable in developing countries, which often undergo rapid growth, industrialization and urbanization with a resultant increase in vehicular traffic, coal combustion, and fuel emissions as a whole. For this reason, environmental pollutants have been studied extensively, and countries around the globe have established laws that regulate ambient air levels of so-called criteria pollutants. This article will explore several of these criteria pollutants, including particulate matter, nitrogen dioxide, sulfur dioxide, and ozone, and their individual relationships to asthma pathophysiology. However, it is also emphasized that though each one of these toxins yields its own effects, the group of them often works together to have cumulative consequences. For these reasons and many more, it is important to remain aware and educated about these omnipresent environmental pollutants.
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http://dx.doi.org/10.1007/s12098-018-2691-3DOI Listing
October 2018

Clinical characterization of children with resistant airflow obstruction, a multicenter study.

J Asthma 2019 06 14;56(6):611-617. Epub 2018 Jun 14.

j Pediatric Pulmonology , University of Virginia , Charlottesville , VA.

Objective: To characterize a cohort of children with airflow limitation resistant to bronchodilator (BD) therapy.

Methods: Pulmonary function tests performed in children 6-17 years of age at 15 centers in a clinical research consortium were screened for resistant airflow limitation, defined as a post-BD FEV and/or an FEV/FVC less than the lower limits of normal. Demographic and clinical data were analyzed for associations with pulmonary function.

Results: 582 children were identified. Median age was 13 years (IQR: 11, 16), 60% were males; 62% were Caucasian, 28% were African-American; 19% were obese; 32% were born prematurely and 21% exposed to second hand smoke. Pulmonary diagnoses included asthma (93%), prior significant pneumonia (28%), and bronchiectasis (5%). 65% reported allergic rhinitis, and 11% chronic sinusitis. Subjects without a history of asthma had significantly lower post-BD FEV% predicted (p = 0.008). Subjects without allergic rhinitis had lower post-BD FEV% predicted (p = 0.003). Children with allergic rhinitis, male sex, obesity and Black race had better pulmonary function post-BD. There was lower pulmonary function in children after age 11 years without a history of allergic rhinitis, as compared to those with a history of allergic rhinitis.

Conclusions: The most prevalent diagnosis in children with BD-resistant airflow limitation is asthma. Allergic rhinitis and premature birth are common co-morbidities. Children without a history of asthma, as well as those with asthma but no allergic rhinitis, had lower pulmonary function. Children with BD-resistant airflow limitation may represent a sub-group of children with persistent obstruction and high risk for life-long airway disease.
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http://dx.doi.org/10.1080/02770903.2018.1477956DOI Listing
June 2019

Bacterial Lipid Modification of ICP11 and a New ELISA System Applicable for WSSV Infection Detection.

Mar Biotechnol (NY) 2018 Jun 14;20(3):375-384. Epub 2018 Apr 14.

Centre for Biotechnology, Anna University, Guindy, Chennai, Tamil Nadu, 600025, India.

In ELISA, a popular analytical diagnostic tool, the stable non-covalent immobilization (coating) of hydrophilic proteins/peptides on to hydrophobic polystyrene surface has remained a major common challenge. Recombinant bacterial lipid modification of proteins in Escherichia coli system has been shown in this study to solve this problem owing to the hydrophobic anchorage provided by three fatty acyl groups in N-acyl-S-diacylglyceryl Cys at the N-terminus. Exploiting this first post-translational protein engineering, the most abundantly expressed white spot syndrome viral protein ICP11 was lipid-modified and tested as a new target in a new ELISA method useful to shrimp farming. The lipid served as a potent adjuvant to enhance the titer (16 times) of higher affinity antibodies where amino terminal lipoamino acid N-acyl-S-diacylglyceryl cysteine of bacterial lipoproteins induce inflammatory responses through TLR and stimulate humoral immune responses without additional adjuvant and also aided in the immobilization of even a few nanograms of ICP11. Competition between the immobilized and the free antigen from the sample provided a sensitive measure of antigen in the infected shrimp tissues. The detection limit for ICP11 protein using competitive ELISA was 250 pg and the linear range of the assay was 15-240 ng.
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http://dx.doi.org/10.1007/s10126-018-9815-7DOI Listing
June 2018

Stringency of bacterial prolipoprotein signal peptidase (LspA) in recognition of signal peptides - Structure-function correlation.

Biochem Biophys Res Commun 2017 06 12;488(2):413-417. Epub 2017 May 12.

Centre for Biotechnology, Anna University, Chennai, India.

Bacterial lipid modification of proteins is an essential post-translational event committed by Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) by catalysing diacyglyceryl transfer from Phosphatidylglycerol to cysteine present in the characteristic 'lipobox' ([LVI] [ASTVI] [GAS] C ) of prolipoprotein signal peptides. This is then followed by the cleavage of the signal peptide by lipoprotein-specific signal peptidase (LspA). It had been known for long that threonine at the -1 position allows diacylglyceryl modification by Lgt, but not signal peptide cleavage by LspA. We have addressed this unexplained stringency by computational analysis of the recently published 3D structure of LspA with its competitive inhibitor as well as transition state analogue, globomycin using PyMoL viewing tool and VADAR (Volume, Area, Dihedral Angle Reporter) web server. The propensity to form hydrogen bond (2.9a) between the hydroxyl group of threonine (not possible with serine) and the NH of the lipid-modified cysteine, possible only in the transition state, will prevent the protonation of NH of the leaving peptide and therefore its cleavage. This knowledge could be useful for designing inhibitors of this essential pathway in bacteria or for engineering LspA.
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http://dx.doi.org/10.1016/j.bbrc.2017.05.069DOI Listing
June 2017

An electrochemical immunosensor for efficient detection of uropathogenic E. coli based on thionine dye immobilized chitosan/functionalized-MWCNT modified electrode.

Biosens Bioelectron 2016 Aug 24;82:71-7. Epub 2016 Mar 24.

Environmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology University, Vellore 632014, Tamil Nadu, India; Nano and Bioelectrochemistry Research Laboratory, School of Advanced Sciences, Vellore Institute of Technology University, Vellore 632014, Tamil Nadu, India. Electronic address:

Uropathogenic Escherichia coli (UPEC) is the major cause of 150 million Urinary Tract Infections (UTI) reported annually world-wide. High prevalence of multi-drug-resistance makes it dangerous and difficult to cure. Therefore simple, quick and early diagnostic tools are essential for effective treatment and control. We report an electrochemical immunosensor based on thionine dye (Th) immobilized on functionalized-multiwalled carbon nanotube+chitosan composite coated on glassy carbon electrode (GCE/[email protected]) for quick and sensitive detection of UPEC in aqueous solution. This immunosensor was constructed by sequential immobilization of UPEC, bovine serum albumin, primary antibody and Horse Radish Peroxidase (HRP) tagged secondary antibody on the surface of GCE/[email protected] When analyzed using 2.5mM of hydrogen peroxide reduction reaction using cyclic voltammetry in phosphate buffer, pH 7.0, the immunosensor showed excellent linearity in a range of 10(2)-10(9)cfu of UPEC mL(-1) with a current sensitivity of 7.162μA {log(cfumL(-1))}(-1). The specificity of this immunosensor was tested using other UTI and non-UTI bacteria, Staphylococcus, Klebsiella, Proteus and Shigella. The clinical applicability of the immunosensor was also successfully tested directly in UPEC spiked urine samples (simulated sample).
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http://dx.doi.org/10.1016/j.bios.2016.03.062DOI Listing
August 2016

Crystal structure of E. coli lipoprotein diacylglyceryl transferase.

Nat Commun 2016 Jan 5;7:10198. Epub 2016 Jan 5.

National Laboratory of Macromolecules, National Center of Protein Science - Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.

Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer.
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http://dx.doi.org/10.1038/ncomms10198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728403PMC
January 2016

Simple and specific colorimetric detection of Staphylococcus using its volatile 2-[3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl] propanoic acid in the liquid phase and head space of cultures.

Appl Microbiol Biotechnol 2015 May 22;99(10):4423-33. Epub 2015 Apr 22.

Centre for Biotechnology, Anna University, Sardar Patel road, Guindy, Chennai, 600 025, India.

Spread of drug-resistant Staphylococcus spp. into communities pose danger demanding effective non-invasive and non-destructive tools for its early detection and surveillance. Characteristic volatile organic compounds (VOCs) produced by bacteria offer new diagnostic targets and novel approaches not exploited so far in infectious disease diagnostics. Our search for such characteristic VOC for Staphylococcus spp. led to the depiction of 2-[3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl] propanoic acid (ATMAP), a moderately volatile compound detected both in the culture and headspace when the organism was grown in tryptone soya broth (TSB) medium. A simple and inexpensive colorimetric method (colour change from yellow to orange) using methyl red as the pH indicator provided an absolutely specific way for identifying Staphylococcus spp., The assay performed in liquid cultures (7-h growth in TSB) as well as in the headspace of plate cultures (grown for 10 h on TSA) was optimised in a 96-well plate and 12-well plate formats, respectively, employing a set of positive and negative strains. Only Staphylococcus spp. showed the distinct colour change from yellow to orange due to the production of the above VOC while in the case of other organisms, the reagent remained yellow. The method validated using known clinical and environmental strains (56 including Staphylococcus, Proteus, Pseudomonas, Klebsiella, Bacillus, Shigella and Escherichia coli) was found to be highly efficient showing 100% specificity and sensitivity. Such simple methods of bacterial pathogen identification are expected to form the next generation tools for the control of infectious diseases through early detection and surveillance of causative agents.
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http://dx.doi.org/10.1007/s00253-015-6573-6DOI Listing
May 2015

A new fluorimetric method for the detection and quantification of siderophores using Calcein Blue, with potential as a bacterial detection tool.

Appl Microbiol Biotechnol 2015 Mar 30;99(5):2339-49. Epub 2015 Jan 30.

Centre for Biotechnology, Anna University, Sardar Patel road, Guindy, Chennai, 600 025, India.

The presence of microorganisms in biological fluids like urine and blood is an indication of vulnerability to infections. Iron is one of the important micronutrients required for bacterial growth. In an iron-deficit environment, bacteria release high-affinity iron-chelating compounds called siderophores which can be used as non-invasive target molecules for the detection of such pathogens. However, only limited reagents and procedures are available to detect the presence of these organic molecules. The present study aims at detecting the presence of siderophores in the iron-depleted media, exploiting the reversible quenching of Calcein Blue and iron(III) complex. The fluorescence of Calcein Blue is known to be quenched in the presence of iron(III); if a stronger chelator removes this ion from the fluorophore, the fluorescence of the fluorophore is regained. This behaviour of the fluorophore was exploited to detect and quantify siderophores down to 50 and 800 nM equivalent of standard siderophore, deferroxamine mesylate (desferal) in Dulbecco's PBS and siderophore quantification (SPQ) medium, respectively. The siderophores released by pathogens, equivalent to standard desferal, were in the range of 1.29 to 5.00 μM and those for non-pathogens were below 1.19 μM. The simple, sensitive and cost-effective method performed in a 96-well plate was able to detect and quantify iron chelators within 7-8 h of incubation.
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http://dx.doi.org/10.1007/s00253-015-6411-xDOI Listing
March 2015

Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor.

J Med Microbiol 2014 Dec 17;63(Pt 12):1595-1607. Epub 2014 Sep 17.

Department of Genetics, University of Leicester, Leicester, UK.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco's modified Eagle's medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.
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http://dx.doi.org/10.1099/jmm.0.076323-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250835PMC
December 2014

Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.

Mol Membr Biol 2014 Sep 26;31(6):183-94. Epub 2014 Aug 26.

Centre for Biotechnology, Anna University , Chennai , India.

Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.
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http://dx.doi.org/10.3109/09687688.2014.943819DOI Listing
September 2014

Differences in perspective on prognosis and treatment of children with trisomy 18.

Am J Med Genet A 2014 Oct 6;164A(10):2551-6. Epub 2014 Aug 6.

Department of Pediatrics, Hasbro Children's Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island.

Differences in perspective between physicians caring for children with trisomy 18 may be confusing and stressful for parents. The hypothesis of this study was that neonatologists and pediatric pulmonologists differ in their opinions regarding long-term prognosis and recommended interventions. Neonatologists and pediatric pulmonologists in New York State were surveyed. Respondents were asked to report their personal experience caring for affected children, opinions on prognosis, major influences on their opinions, and their likelihood of recommending specific medical or surgical interventions for two clinical vignettes. A total of 393 surveys were mailed, 327 to neonatologists and 66 to pediatric pulmonologists. Sixty-six (20%) neonatologists and 21 (32%) pediatric pulmonologists completed the survey. Neonatologists had cared for more patients with trisomy 18. Twenty-nine percent of pediatric pulmonologists had never cared for a patient with trisomy 18 compared to 2% of neonatologists, P < 0.001. Pediatric pulmonologists were more likely to recommend almost all interventions including antibiotics for pneumonia, mechanical ventilation, cardiac and orthopedic surgery, and "full code resuscitation." Neonatologists were more likely to recommend comfort care only or palliative care. Fifty-four percent of neonatologists and 5% of pediatric pulmonologists thought patients with trisomy 18 without significant congenital heart disease would die before age one despite aggressive medical care, P < 0.001. The major influences impacting these recommendations also varied. Pediatric pulmonologists are more optimistic about the prognosis for children than neonatologists and more likely to recommend medical and surgical interventions. Experience with the condition and perception of survivability may contribute to these differences in approach.
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http://dx.doi.org/10.1002/ajmg.a.36687DOI Listing
October 2014

Low-level environmental tobacco smoke exposure and inflammatory biomarkers in children with asthma.

J Asthma 2014 May 7;51(4):355-9. Epub 2014 Mar 7.

Division of Pulmonary & Critical Care and Sleep Medicine, Department of Medicine, Wayne State University , Detroit, MI , USA and.

Objective: The effects of low-level environmental tobacco smoke (ETS) exposure, on asthma control, lung function and inflammatory biomarkers in children with asthma have not been well studied. The objective of the study was to assess ETS exposure in school-age children with asthma whose parents either deny smoking or only smoke outside the home, and to assess the impact of low-level ETS exposure on asthma control, spirometry and inflammatory biomarkers.

Methods: Forty patients age 8-18 years with well-controlled, mild-to-moderate persistent asthma treated with either inhaled corticosteroids (ICS) or montelukast were enrolled. Subjects completed an age-appropriate Asthma Control Test and a smoke exposure questionnaire, and exhaled nitric oxide (FeNO), spirometry, urinary cotinine and leukotriene E(4) (LTE(4)) were measured. ETS-exposed and unexposed groups were compared.

Results: Only one parent reported smoking in the home, yet 28 (70%) subjects had urinary cotinine levels ≥1 ng/ml, suggesting ETS exposure. Seven subjects (18%) had FeNO levels >25parts per billion, six of whom were in the ETS-exposed group. In the ICS-treated subjects, but not in the montelukast-treated subjects, ETS exposure was associated with higher urinary LTE(4), p = 0.04, but had no effect on asthma control, forced expiratory volume in 1 s or FeNO.

Conclusions: A majority of school-age children with persistent asthma may be exposed to ETS, as measured by urinary cotinine, even if their parents insist they don't smoke in the home. Urinary LTE(4) was higher in the ETS-exposed children treated with ICS, but not in children treated with montelukast.
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http://dx.doi.org/10.3109/02770903.2013.823446DOI Listing
May 2014

The effects of low-level environmental tobacco smoke exposure on pulmonary function tests in preschool children with asthma.

J Asthma 2014 Sep 28;51(7):685-90. Epub 2014 Feb 28.

Division of Pulmonology, Winthrop University Hospital , Mineola, NY , USA and.

Objectives: Though parents of children with asthma smoke, they often avoid smoking in their homes or near their children, thus limiting exposure. It is not known if such low-level environmental tobacco smoke (ETS) results in measurable exposure or affects lung function. The objectives of this study were to measure urinary cotinine in preschool children with asthma, and to examine the relationship between low-level ETS exposure and pulmonary function tests (PFTs).

Methods: Preschool children with asthma were enrolled. Parents completed questionnaires on ETS exposure and asthma control, urinary cotinine concentrations were measured and PFTs were compared between subjects with and without recent ETS exposure.

Results: Forty one subjects were enrolled. All parents denied smoking in their home within the last 2 weeks, but 14 (34%) parents admitted to smoking outside their homes or away from their children. Fifteen (37%; 95%CI: 23-53) of the children had urinary cotinine levels ≥1 ng/ml, of which seven (17%; 95%CI: 8-32) had levels ≥5 ng/ml. FEV1 and FEV0.5 were lower in subjects with a urinary cotinine level ≥5 ng/ml as compared to those with levels <1 ng/ml or between 1 and 5 ng/ml; both at baseline and after inhalation of albuterol. Five of seven subjects with urinary cotinine levels ≥5 ng/ml had FEV0.5 less than 65% of predicted values. There were no significant differences in IOS measures.

Conclusions: Despite parental denial of smoking near their children, preschool children may be exposed to ETS. Such low-level ETS exposure may affect lung function, possibly in a dose-dependent manner.
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http://dx.doi.org/10.3109/02770903.2014.894054DOI Listing
September 2014

Biologic mechanisms of environmental tobacco smoke in children with poorly controlled asthma: results from a multicenter clinical trial.

J Allergy Clin Immunol Pract 2013 Mar 12;1(2):172-80. Epub 2013 Jan 12.

Center for Pharmacogenomics & Translational Research, Nemours Children's Clinic, Jacksonville, Fla.

Background: Environmental tobacco smoke (ETS) negatively affects children with asthma. The prevalence of ETS exposure among children with poor asthma control may be changing. Importantly, the mechanisms by which ETS worsens asthma control are poorly understood.

Objective: We describe how ETS affects gastroesophageal reflux (GER), respiratory infections, and leukotriene production among children with poor asthma control.

Methods: We analyzed data from 306 children between 6 and 17 years of age with poorly controlled asthma enrolled in a 6-month clinical trial. We evaluated prevalence and determinants of ETS exposure by interview, questionnaire, and urinary cotinine and the association of ETS exposure on leukotriene production, respiratory infections, GER, lung function, and asthma control. We used multivariable linear, logistic, and Poisson regressions to assess outcomes.

Results: ETS prevalence estimates ranged from 6% to 30%. Children with domestic indoor exposure had worse asthma control (c-Asthma Control Test, 17.8 vs 21.5; P = .04), worse FEV1 % predicted (84.1 vs 90.7; P = .02), and a trend for increased mean urinary leukotriene E4. ETS from any setting was associated with increased symptomatic respiratory infections (adjusted incidence rate ratio: 1.30; P = .02). However, children exposed to ETS did not have symptoms or pH probe results, suggestive of heightened GER.

Conclusions: Domestic smoking exposure was associated with both higher rates of symptomatic respiratory infection and poorer asthma control despite generally intensive controller therapy. ETS exposure is common among asthmatic children with poor control and may worsen asthma control by promoting respiratory infections. Further investigation is required to elucidate ETS mechanisms in poor asthma control.
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http://dx.doi.org/10.1016/j.jaip.2012.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5606191PMC
March 2013

2-methylbutanal, a volatile biomarker, for non-invasive surveillance of Proteus.

Appl Microbiol Biotechnol 2014 Jan 27;98(1):445-54. Epub 2013 Nov 27.

Centre for Biotechnology, Anna University, Sardar Patel Road, Guindy, Chennai, 600 025, India.

Pathogen detection needs a paradigm shift from time-consuming conventional microbiological and biochemical tests to much simpler identification methods with higher sensitivity and specificity. In this regard, a simple detection method for frequently isolated nosocomial uropathogen, Proteus spp., was developed using the characteristic volatile 2-methylbutanal released in Luria Bertani broth. The instant reaction of the compound with 5-dimethylaminonaphthalene-1-sulfonylhydrazine (DNSH) has been adapted to develop a sensitive fluorescence assay named "ProteAl" (Prote, "Proteus" & Al, "Aldehyde"). The assay was performed by direct addition of the fluorescence reagent to the culture after 7 h of growth. The distinct green fluorescence by Proteus (other organisms show orange fluorescence) served as the simplest and quicker identification test available for Proteus. In the laboratory, it exhibited 100% specificity and 100% sensitivity during testing of 95 strains including standard and known clinical isolates representing frequently encountered uropathogens.
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http://dx.doi.org/10.1007/s00253-013-5393-9DOI Listing
January 2014

Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).

Vaccine 2013 Nov 9;31(48):5672-9. Epub 2013 Oct 9.

Department of Food Safety & Infection Biology, Norwegian School of Veterinary Science, Postboks 8146 Dep, N-0033 Oslo, Norway.

The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.
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http://dx.doi.org/10.1016/j.vaccine.2013.09.058DOI Listing
November 2013

First ever isolation of bacterial prolipoprotein diacylglyceryl transferase in single step from Lactococcus lactis.

Protein Expr Purif 2013 Feb 17;87(2):120-8. Epub 2012 Nov 17.

Centre for Biotechnology, Anna University, Chennai 600 025, India.

The unique bacterial enzyme phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) is the least studied enzyme of the ubiquitous bacterial lipoprotein synthetic pathway, mostly due to the low abundance of the enzyme. So far, Lgt has been studied to a limited extent in gram-negative bacteria, mainly in Escherichia coli. We, for the first time, report the isolation of an adequate amount of Lgt from the gram-positive lactic acid bacteria, Lactococcus lactis and compare this wild-type bacterial enzyme with the E. coli enzyme. The L. lactis Lgt, when purified by cationic-exchange chromatography, showed a 20-fold increase in the specific activity compared to that of the load, and 75% of the total Lgt activity loaded was recovered. Kinetically, L. lactis Lgt was found to be similar to the E. coli enzyme with matching K(m) and V(max), whereas the specific activity of the L. lactis enzyme was about 20 times less than that of the E. coli enzyme. Comparative bioinformatic analysis of L. lactis, E. coli and Staphylococcus aureus Lgt revealed that the conserved and catalytically important His-103 residue in E. coli Lgt, was altered to Tyr in L. lactis. Investigations showed that other bacteria where this alteration is visible, form a diversion within the gram-positive bacteria in evolution. Further analysis revealed Mycobacterium smegmatis to be the species which evolved with the alteration of His to Tyr.
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http://dx.doi.org/10.1016/j.pep.2012.11.001DOI Listing
February 2013

High-throughput fluorescence-based early antibiogram determination using clinical Escherichia coli isolates as case study.

Microb Drug Resist 2012 Dec 28;18(6):586-96. Epub 2012 Aug 28.

National Hub for Healthcare Instrumentation Development, Anna University, Chennai, India.

The objective of this study is to develop an antibiogram (AB) method superior to a disc diffusion method (DDM) with respect to rapidity, reliability, and accuracy especially in view of an increasing threat from multidrug resistance (MDR) of infectious bacteria. A high-throughput liquid-phase fluorescent antibiogram method capable of providing results within 6-8 hours has been developed. The AB method has been optimally designed for ease of operation, growth, and dye stability. This new method was more reliable than DDM in differentiating AB sensitivity as susceptible, intermediate, and resistant within 6-8 hours and providing evidence for efflux mechanism in the MDR phenotype. The superiority of this method even over the standard liquid turbidity method was evidenced by more accurate determination of intermediary resistance in a set of 23 clinical Escherichia coli strains against five common antibiotics. In view of the demand for the right choice of an antibiotic in short time, the newly developed AB method is clinically applicable and useful in the rational use of antibiotic and minimizing of MDR emergence.
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http://dx.doi.org/10.1089/mdr.2011.0238DOI Listing
December 2012

Relationship between exhaled nitric oxide and exposure to low-level environmental tobacco smoke in children with asthma on inhaled corticosteroids.

J Asthma 2012 Sep 17;49(7):673-8. Epub 2012 Jul 17.

Division of Pediatric Pulmonology, Department of Pediatrics, Maria Fareri Children's Hospital at Westchester Medical Center and New York Medical College, Valhalla, NY 10595, USA.

Objectives: The relationship between exhaled nitric oxide (FeNO) and asthma severity or control is inconsistent. Active smoking lowers FeNO, but the relationship between passive smoking and FeNO is less clear. Children may be exposed to low-level environmental tobacco smoke (ETS) or thirdhand smoke, even if parents avoid smoking in the presence of their children. Our hypothesis was that FeNO is lower in children with asthma exposed to low-level ETS when compared with those who are not exposed.

Methods: Children with stable asthma, 8-18 years of age, on low- or medium-dose inhaled corticosteroids (ICS) were enrolled. Spirometry, Asthma Control Questionnaire (ACQ), FeNO, exhaled breath condensate pH (EBC pH), and EBC ammonia were compared between children with and without ETS exposure as determined by urinary cotinine.

Results: Thirty-three subjects were enrolled, of which 10 (30%) had urinary cotinine levels ≥1 ng/ml. There were no significant differences between the two groups in age, sex, BMI percentile, atopy status, FEV(1), EBC pH, or EBC ammonia. Median ACQ was 0.29 (IQR: 0.22-0.57) for those with cotinine levels <1 ng/ml and 0.64 (IQR: 0.57-1.1) for those with cotinine levels of ≥1 ng/ml, p = .02. Median FeNO (ppb) was 23.9 (IQR: 15.2-34.5) for unexposed subjects and 9.6 (IQR: 5.1-15.8) for exposed subjects, p = .008.

Conclusions: Children with asthma on low to medium doses of ICS and recent low-level ETS exposure have lower FeNO levels when compared with non-ETS-exposed subjects. Exposure to low-level ETS or thirdhand smoke may be an important variable to consider when interpreting FeNO as a biomarker for airway inflammation.
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http://dx.doi.org/10.3109/02770903.2012.701363DOI Listing
September 2012

Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

Biotechniques 2012 Mar;52(3):167-72

Centre for Biotechnology, Anna University, Chennai, India.

Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.
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http://dx.doi.org/10.2144/000113819DOI Listing
March 2012

The first nonradioactive fluorescence assay for phosphatidylglycerol:prolipoprotein diacylglyceryl transferase that initiates bacterial lipoprotein biosynthesis.

Anal Biochem 2012 Apr 27;423(1):163-70. Epub 2012 Jan 27.

Centre for Biotechnology, Anna University, Chennai 600 025, India.

The unique and physiologically vital bacterial enzyme, prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the committed first step in the posttranslational transfer of diacylglyceryl group from phosphatidylglycerol to the prospective N-terminal cysteine of prolipoproteins, remains to be characterized for want of a simpler but equally sensitive nonradioactive assay. We, for the first time, report a coupled enzymatic fluorescence assay for Lgt using the de novo synthetic peptide substrate MKATKSAVGSTLAGCSSHHHHHH. The assay is based on the conversion of the by-product, glycerol-1-phosphate, to dihydroxyacetone using an alkaline phosphatase-glycerol dehydrogenase combination and estimating the fluorescence of the coupled reduction of resazurin to resorufin. The minimum amount of glycerol-1-phosphate, and hence the modified peptide, detected by this method is approximately 20pmol, thereby making this assay a promising alternative to the radioactive assays. The assay is rapid, more convenient, less laborious, and suitable for purification and characterization of Lgt.
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http://dx.doi.org/10.1016/j.ab.2012.01.018DOI Listing
April 2012
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