Improvised method for urinary p-cresol detection and measurement using high performance liquid chromatography/mass spectrometry.

Gut microbiota has been implicated in many disorders including Autism Spectrum Disorder (ASD). ASD is a neurodevelopmental brain disorder affecting individuals leading to restricted and repetitive pattern of behaviour and disruption of communication and social interactions. Altered microbiome and the presence or absence of key species capable of affecting specific responses in levels of their fermentation products are reflected in the urinary metabolite profile of patients. The aim of our study is to develop an improvised method for the detection and quantification of urinary p-cresol levels which could serve as an indicator for GI microbial dysbiosis. The p-cresol analysis was achieved using HPLC by a reverse phase C18 column with mobile phase composition of Acetonitrile/water/formic acid (10:90:0.05, v/v/v) in an isocratic mode of elution with a flow rate of 1.0 mL/min. The mass analysis of p-cresol was performed using LC-MS [Triple Quadrupole Liquid Chromatography Mass Spectrometer] in negative ESI mode with electron multiplier detector. p-cresol was eluted at a retention time of approximately 3.4 min. The standard calibration curves had a superior regression coefficient of greater than 0.99 (R2 > 0.99) and were linear over a range from 0.0005 mg/mL to 0.015 mg/mL. The method was validated by analysis of six replicates with 0.08% relative standard deviation and method detection and quantification limits were 20 ng/mL and 50 ng/mL respectively. Further validation of method on real urine samples from two groups of children (Control population:< 10 years of age; 5M: 3F and ASD individuals: <10 years of age; All males) showed that detection was effective over a wide range of metabolite at levels as high as 149.73 µg/mL to as low as 0.897 µg/mL. This study reports a rapid, validated and sensitive method for the detection of p-cresol in urine samples.

Confluence of montmorillonite and Rhizobium towards the adsorption of chromium(vi) from aqueous medium.

Chromium in its hexavalent oxidation state is carcinogenic and wastewater from the electroplating industry is one of the principal sources of pollution. To reduce this toxicity and pave way towards environmental safety, a combination of environmental microbiology and chemistry is quite efficient for developing biosorbents to sequester chromium from waste water. Immobilization of Rhizobium in sodium montmorillonite provides a conducive environment to capture hexavalent chromium. Various characterization techniques such as FTIR, XPS and SEM-EDAX were performed and batch parameters such as pH variation, adsorbent dosage, concentration of metal ion and temperature were optimized. Pseudo second order kinetics coupled with a higher regression coefficient for Freundlich isotherm and a Langmuir adsorption capacity of 22.22 mg g-1 was achieved for the adsorption process. The adsorption was enhanced by the charge interactions between the protonated clay-Rhizobium surface and Cr(vi) ions in acidic medium. The biosorbent was stable and easily regenerated using NaOH. Preliminary column studies were performed to test the efficiency of the developed biosorbent at higher volumes on a laboratory scale.

Identification and Characterization of the microbial communities found in electronic industrial effluent and their potential for bioremediation.

Microbial communities are dynamic systems that develop depending on the ecological niche in which they survive. Electronic industry effluent, rich in heavy metals and salts is one such ecosystem where diverse heavy metal resistant microbes exist. Taxonomic identification of this microbial community would be interesting as no information on the microbial diversity from electronic industry effluent is available till date. Our paper attempts to characterize the microbial inhabitants of this niche. Culture dependent microbiological methods were used to establish and identify various microbial species from the effluent. Culture independent methods of identification involving biochemical tests and molecular biology based methods like 16 S- r DNA sequencing and lipid analyses (FAME analysis) were also carried out to confirm the identity of isolated species. Our study, first of its kind revealed the presence of a diverse group of resistant aerobic microbes and disclosed a total of ten bacterial and two fungal isolates. All these isolates were found to survive in presence of heavy metals like cadmium, lead and zinc and were resistant to antibiotics like ampicillin, tetracycline, streptomycin, penicillin and chloramphenicol as indicated by their Minimum Inhibitory Concentrations (MIC). Such resistant isolates harbor possibilities of metal adaptive/selective pathways which render them as economically beneficial bio-sorbent alternatives in bioremediation of heavy metals.

Potential Application of Saccharomyces cerevisiae and Rhizobium Immobilized in Multi Walled Carbon Nanotubes to Adsorb Hexavalent Chromium.

The presence of harmful contaminants in the waste stream is an important concern worldwide. The convergence of biotechnology and nanoscience offers a sustainable alternative in treating contaminated waters. Hexavalent chromium, being carcinogenic deserves effective and sustainable methods for sequestration. Here in, we report the immobilization of a prokaryote (Rhizobium) and eukaryote (Saccharomyces cerevisiae) in multiwalled carbon nanotubes (MWCNTs) for the effective adsorption of hexavalent chromium. The carboxylic groups were introduced into the MWCNTs during oxidation using potassium permanganate and were subjected to EDC-HOBT coupling to bind with microbial cell surface. FTIR, TGA, BET, FESEM-EDAX, HRTEM, XPS and confocal microscopy were the investigative techniques used to characterize the developed biosorbents. Experimental variables such as pH, adsorbent dosage, kinetics, isotherms and thermodynamics were investigated and it was observed that the system follows pseudo second order kinetics with a best fit for Langmuir isotherm. Electrostatic interactions between the functional groups in the microbial cell wall and hydrochromate anion at pH 2.0 propel the adsorption mechanism. The lab scale column studies were performed with higher volumes of the Cr(VI) contaminated water. Sodium hydroxide was used as the desorbing agent for reuse of the biosorbents. The sustainable biosorbents show prospects to treat chromium contaminated water.

Impact of heavy metal lead stress on polyamine levels in Halomonas BVR 1 isolated from an industry effluent.

In living systems, environmental stress due to biotic and abiotic factors triggers the production of myriad metabolites as a potential mechanism for combating stress. Among these metabolites are the small polycationic aliphatic amine molecules - polyamines, which are ubiquitous in all living organisms. In this work, we demonstrate a correlation between cellular concentration of three major polyamines (putrescine, spermidine and spermine) with lead exposure on bacteria for a period of 6-24 h. We report that indigenously isolated Halomonas sp. strain BVR 1 exhibits lead induced fluctuations in their cellular polyamine concentration. This response to lead occurs within 6 h post metal treatment. During the same time interval there was a surge in the growth of bacteria along with an enhancement in the putrescine levels. We conclude that in Halomonas sp. strain BVR 1, an early response is seen with respect to modulation of polyamines as a result of lead treatment and hypothesize that endogenous polyamines contribute towards scavenging lead in these bacteria.

Analysis and functional characterization of sequence variations in ligand binding domain of thyroid hormone receptors in autism spectrum disorder (ASD) patients.

Autism spectrum disorder (ASD) is a neuro developmental disorder, reported to be on a rise in the past two decades. Thyroid hormone-T3 plays an important role in early embryonic and central nervous system development. T3 mediates its function by binding to thyroid hormone receptors, TRα and TRß. Alterations in T3 levels and thyroid receptor mutations have been earlier implicated in neuropsychiatric disorders and have been linked to environmental toxins. Limited reports from earlier studies have shown the effectiveness of T3 treatment with promising results in children with ASD and that the thyroid hormone levels in these children was also normal. This necessitates the need to explore the genetic variations in the components of the thyroid hormone pathway in ASD children. To achieve this objective, we performed genetic analysis of ligand binding domain of THRA and THRB receptor genes in 30 ASD subjects and in age matched controls from India. Our study for the first time reports novel single nucleotide polymorphisms in the THRA and THRB receptor genes of ASD individuals. Autism Res 2017, 10: 1919-1928. ©2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Thyroid hormone (T3) and thyroid receptors (TRα and TRß) are the major components of the thyroid hormone pathway. The link between thyroid pathway and neuronal development is proven in clinical medicine. Since the thyroid hormone levels in Autistic children are normal, variations in their receptors needs to be explored. To achieve this objective, changes in THRA and THRB receptor genes was studied in 30 ASD and normal children from India. The impact of some of these mutations on receptor function was also studied.

In silico approach to ascertain the calcium dependent role of Plasmodium falciparum SERA5.

The P. falciparum serine repeat antigen (PfSERA5) is the most abundantly expressed protein in the parasitophorous vacuole during the asexual blood stage and serves as both drug and vaccine target. The processed central fragment (56 KDa) of PfSERA5 is implicated to play an important role in parasite exit (egress) during schizont rupture from erythrocytes. Structural characterization of its enzymatic domain supports protease-like function for this central domain. The understanding of exact functional role of PfSERA5 in parasite egress remains unconfirmed as recent studies also indicate an indispensable non-catalytic role for PfSERA5 putative enzyme domain in the blood stage. No structural insight into PfSERA5 prodomain is available. Structure prediction of PfSERA5 prodomain using in silico approach in our study, showed it to have structural similarity with calcium-binding proteins. An earlier observation of steep rise in intracellular calcium concentration as an important factor in egress makes the prodomain calcium-binding role significant. The implication of calcium on structure and activity of PfSERA5 putative enzyme domain is also unknown, and such information would aid to substantiating any calcium-dependent effects on PfSERA5. To understand this, we performed molecular dynamic (MD) simulation both in the presence and absence of calcium. MD results show secondary structure conformational differences in local regions of protein structure. Our results support calcium to be an important parameter for stability and function of PfSERA5. This computational assessment suggest a need to design future experiments like calcium-dependent inhibition studies to reveal exact functional role of PfSERA5 in parasite egress.

Mass spectrometric evaluation of neurotransmitter levels in IMR 32 cell line in response to Ayurvedic medicines.

RATIONALE: Ayurvedic herbal medicines are administered as part of disease management for many neurodevelopmental disorders like Autism Spectrum Disorder (ASD) and linked comorbid challenges. The biochemistry of the behavioral abnormalities as observed in comorbid conditions is already reported to involve neurotransmitters like gamma-aminobutyric acid (GABA), serotonin (5-HT) and dopamine (DA). The aim of our study is to evaluate the effect of ayurvedic medicines on neurotransmitter levels in IMR 32. Such a study will give some insight into the molecular mechanism of the action of these medicines and help us to understand their contributions in neurotransmitter homeostasis. METHODS: Solutions of Brahmi, Brahmi vati, Brahmi ghrita and Saraswata ghrita, each at 50 µM, were added to differentiated IMR 32 cells and grown for 24 h. The cell secretion was analysed by ultra-fast liquid chromatography/mass spectrometry (UFLC/MS) in electrospray ionisation (ESI) mode for the neurotransmitters DA, 5-HT and GABA. The mobile phase selected was 0.1% formic acid with 15 µg/mL Na2 -EDTA (A) and 0.1% formic acid in acetonitrile (B) introduced in the ratio of 92:8. RESULTS: All neurotransmitters under study were eluted within 7 min with GABA eluting at 3.82 min, 5-HT at 4.48 min and DA at 5.47 min, respectively. Linearity was excellent with a correlation coefficient (R(2) ) of 0.999; repeatability and accuracy were also within acceptable range. All herbal drugs evaluated increased the neurotransmitter levels and Brahmi vati increased the neurotransmitter levels to a larger extent. CONCLUSIONS: Decreased levels of neurotransmitters were observed in behavioral abnormalities which were also observed in children with ASD. Herbal medicines given as part of ayurvedic medicine increased the neurotransmitter levels in IMR 32. Thus, these ayurvedic medicines when prescribed to children with ASD might alleviate the abnormal behavioral symptoms by maintaining neurotransmitter homeostasis. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of the genetic basis of heavy metal resistance in an isolate from electronic industry effluent.

Halomonas BVR 1 isolated from an electronic industry effluent had high level of resistance to heavy metals like cadmium, lead, zinc and to various antibiotics. Minimum Inhibitory Concentration (MIC) of the strain toward cadmium and lead was found to be 200 mg L-1 and 400 mg L-1 respectively, while it could tolerate zinc up to 250 mg L-1 and chromium up to 150 mg L-1. The present study proved the genetic contribution of heavy metal resistance in this strain to be plasmid mediated. Isolation of the plasmid from Halomonas BVR 1 and its subsequent linearization with Bam H1 confirmed the presence of a plasmid of size >10 kb. Plasmid curing experiments affirmed plasmid mediated heavy metal resistance. Additionally, genetic transformation of a non metal resistant lab strain Escherichia coli and the cured strain of Halomonas BVR 1 with the isolated plasmid increased their metal tolerance level by 50% confirming the genetic determinant to be present in the plasmid.

An indigenous Halomonas BVR1 strain immobilized in crosslinked chitosan for adsorption of lead and cadmium.

Biomacromolecules play an important role in the adsorption of metals. In this context, we have studied the potential of an indigenous Halomonas BVR1 strain (isolated from an electronic industry effluent) immobilized in a glutaraldehyde crosslinked chitosan matrix for the adsorption of lead and cadmium. Adequate physico-chemical characterizations and the study of thermodynamic and kinetic parameters authenticated the experimental observations and the interaction mechanism. The adsorption was facile in the pH range 5-7 and pseudo second order kinetic model was favourable for both the metals. The Langmuir adsorption capacities for lead and cadmium were found to be 24.15 mg g(-1) and 23.88 mg g(-1) respectively. The negative ΔG values confirmed the thermodynamic feasibility and this lucid approach highlights the utility of green methodology for heavy metal adsorption.

Plasmodium vivax: N-terminal diversity in the blood stage SERA genes from Indian isolates.

Worldwide malaria risk due to Plasmodium vivax makes development of vaccine against P. vivax, a high priority. Serine Repeat Antigen of P. vivax (PvSERA) is a multigene family of blood stage proteins with 12 homologues. Sequence diversity studies are important for understanding them as potential vaccine candidates. No information on N-terminal diversity of these genes is available in literature. In this paper, we evaluate the genetic polymorphism of N-terminal regions of the highly expressed member PvSERA4 and PvSERA5 genes from Indian field isolates. Our results show that PvSERA4 has deletions and insertions in Glutamine rich tetrameric repeat units contributing to its diversity. PvSERA5 also exhibits high genetic diversity with non-synonymous substitutions leading to identification of novel haplotypes from India. Our first report helps in elucidating the allelic variants of PvSERA genes in this region and contributes to evaluating their efficacy as vaccine candidates.

Consanguinity in India and its association with autism spectrum disorder.

Autism Spectrum Disorder (ASD) has both genetic and environmental factors in its etiology. The risk for many disorders is increased by consanguinity, but it is not known whether it increases the risk for ASD. Our study from large population in India concludes that consanguinity increases the risk for ASD with an odds ratio of 3.22.

Microwave assisted preparation of n-butylacrylate grafted chitosan and its application for Cr(VI) adsorption.

Biopolymers such as chitosan possess excellent properties suited for varied applications. In this work, we describe a novel microwave assisted method for the preparation of n-butylacrylate grafted chitosan adsorbent and its utility for the adsorption of chromium(VI). A 3 min irradiation time was enough to prepare the adsorbent, and techniques such as FT-IR, powder XRD, SEM and EDS were used for comprehensive characterization. The adsorption was effective at pH 3.5 with 25 mL of 20 ppm Cr(VI) solution. Langmuir, Freundlich, Dubinin-Radushkevich, Temkin, Elovich and Redlich isotherms were studied in detail. The ΔG, ΔH and ΔS parameters were evaluated to understand the adsorption thermodynamics. The adsorption involves the interaction of Cr(VI) with the hydroxyl and amino groups in chitosan.

Genetic and structural characterization of PvSERA4: potential implication as therapeutic target for Plasmodium vivax malaria.

Plasmodium vivax malaria is geographically the most widely distributed and prevalent form of human malaria. The development of drug resistance by the parasite to existing drugs necessitates higher focus to explore and identify new drug targets. Plasmodial proteases have key roles in parasite biology and are involved in nutritional uptake, egress from infected reticulocytes, and invasion of the new target erythrocytes. Serine repeat antigens (SERA) of Plasmodium are parasite proteases that remain attractive drug targets and are important vaccine candidates due to their high expression profiles in the blood stages. SERA proteins have a unique putative papain-like cysteine protease motif that has either serine or cysteine in its active site. In P. vivax, PvSERA4 is the highest transcribed member of this multigene family. In this study, we have investigated the genetic polymorphism of PvSERA4 central protease domain and deduced its 3D model by homology modeling and also performed MD simulations to acquire refined protein structure. Sequence analysis of protease domain of PvSERA4 from Indian field isolates reveals that the central domain is highly conserved. The high sequence conservation of the PvSERA4 enzyme domain coupled with its high expression raises the possibility of it having a critical role in parasite biology and hence, being a reliable target for new selective inhibitor-based antimalarial chemotherapeutics. The 3D model showed the presence of an unusual antiparallel Beta hairpin motif between catalytic residues similar to hemoglobin binding motif of Plasmodial hemoglobinases. Our PvSERA4 model will aid in designing structure-based inhibitors against this enzyme.

Maternal hormonal interventions as a risk factor for Autism Spectrum Disorder: an epidemiological assessment from India.

Globalization and women empowerment have led to stressful life among Indian women. This stress impairs women's hormonal makeup and menstrual cycle, leading to infertility. National Family Health Survey-3 (NFHS-3) reports a decline in fertility status in India, indicating a rise in various infertility treatments involving hormonal interventions. No studies are available from India on the risk association link between maternal hormonal treatments and ASD. Hence, this study explores the association of maternal hormonal interventions with risk for ASD. Parents of 942 children (471 ASD and 471 controls) across 9 cities in India participated in the questionnaire-based study. The questionnaire was pilot tested and validated for its content and reliability as a psychometric instrument. Data collection was done at 70 centres through direct interaction with parents and with the help of trained staff. Statistical analysis of data was carried out using SAS 9.1.3. Out of the 471 ASD cases analysed, 58 mothers had undergone hormonal interventions (12.3 percent) while there were only 22 mothers among controls who underwent hormonal interventions (4.6 percent). According to logistic regression analysis maternal hormonal intervention (OR=2.24) was a significant risk factor for ASD.

Prenatal, perinatal and neonatal risk factors of Autism Spectrum Disorder: a comprehensive epidemiological assessment from India.

Incidence of Autism Spectrum Disorder (ASD) is increasing across the globe and no data is available from India regarding the risk factors of ASD. In this regard a questionnaire based epidemiological assessment was carried out on prenatal, perinatal and neonatal risk factors of ASD across 8 cities in India. A retrospective cohort of 942 children was enrolled for the study. 471 children with ASD, under age of 10, were analyzed for pre-, peri-, and neonatal factors and were compared with the observations from equal number of controls. The quality control of the questionnaire and data collection was done thoroughly and the observations were computed statistically. A total of 25 factors were evaluated by unadjusted and adjusted analysis in this study. Among the prenatal factors considered, advanced maternal age, fetal distress and gestational respiratory infections were found to be associated with ASD and had an odds ratio of 1.8. Evaluation of perinatal and neonatal risk factors showed labor complications, pre-term birth, neonatal jaundice, delayed birth cry and birth asphyxia to be associated with ASD with an odds ratio greater than 1.5. This important study, first of its kind in Indian population gives a firsthand account of the relation of pre-, peri- and neonatal risk factors on ASD from an ethnically and socially diverse country like India, the impact of which was unknown earlier. This advocates additional focused investigations on physiological and genetic changes contributed by these risk factor inducing environments.

Plasmodium vivax: C-terminal diversity in the blood stage SERA genes from Indian field isolates.

The burden of Plasmodium vivax malaria is huge in India, affecting a large population annually. Recent reports of P. vivax contributing to severe illness and death, makes vaccine research on P. vivax malaria, a high priority. Extent of sequence variation in antigen coding genes is known to be a major hurdle in vaccine initiatives against malaria. Serine repeat antigens of Plasmodium are promising asexual blood stage vaccine candidates against malaria and have been implicated to have a key role in merozoite invasion and egress. Among the P. vivax SERA proteins, SERA4 and SERA5 are the major transcribed members in erythrocytic stages, making them encouraging candidates to be explored for their polymorphism and vaccine potential. Earlier reports suggest that diversity in these PvSERA antigens is localized to the C-terminal region of the proteins. Hence, genetic diversity study of this region seems prudent. Moreover, as there are no reports available from India, the present study aims to investigate the polymorphism in the C-terminal region of two highly transcribed members PvSERA4 and PvSERA5 in Indian field isolates. Our result of PvSERA5 demonstrates extensive genetic diversity, with major deletions, insertions and SNPs and signifies the gene to be under positive selection. On the other hand, high sequence conservation was seen in the PvSERA4 C-terminal region in Indian field isolates which was contrasting to earlier report from Thailand where they have shown diversity. Research data showcased in this study will greatly aid in gaining better understanding of antigenic variations, immune mediated selection mechanisms and the functional significance of these two vivax proteins. This study also makes a striking contribution towards understanding the antigenic repertoire of PvSERA genes in Indian isolates.

Effective adsorption of hexavalent chromium through a three center (3c) co-operative interaction with an ionic liquid and biopolymer.

Biopolymers as well as ionic liquids are known for their potential applications. In this work, we report the utility of chitosan as an excellent platform for impregnating the ionic liquid, tetraoctylammonium bromide by ultrasonication and its subsequent adsorption for chromium(VI). The effective mass transfer due to sonication coupled with the hydrogen bonding interaction between chitosan-ionic liquid and the electrostatic interaction involving the amino groups in chitosan and hexavalent chromium governs this three center (3c) co-operative mechanism. The adsorption followed a pseudo second order kinetics with a Langmuir adsorption capacity of 63.69 mg g(-1). Various isotherm models were used to correlate the experimental data and the adsorption process is exothermic with a decreased randomness at the solid-solution interface. The thermodynamics of the spontaneous adsorption process could be explained through a positive co-operative effect between the host (chitosan) and the guest (ionic liquid). The adsorbed chromium(VI) could be converted to ammonium chromate using ammonium hydroxide, thereby regenerating the adsorbent. The method could be translated into action in the form of practical application to a real sample containing chromium.

An efficient ultrasound assisted approach for the impregnation of room temperature ionic liquid onto Dowex 1×8 resin matrix and its application toward the enhanced adsorption of chromium (VI).

The work discussed in this paper is based on the utilization of ultrasound in conjunction with an ionic liquid (Aliquat 336) impregnated Dowex 1×8 resin for the effective adsorption of chromium. Ionic liquids are known for their selectivity toward metal extraction and ultrasonic medium offers efficient energy transfer for impregnating the ionic liquid in the resin matrix. The molecular interaction between the ionic liquid impregnated resin and chromium was studied through various physicochemical and spectroscopic techniques. The influence of various analytical parameters on the adsorption of Cr(VI) such as pH, adsorbent dosage, temperature and interference of foreign ions was studied in detail. Chromium (VI) was quantitatively adsorbed in the pH range of 3.5-4, with a high adsorption capacity of 230.9 mg g(-1) in conformity with the Langmuir isotherm model. The study of thermodynamic parameters showed that the adsorption process is exothermic and spontaneous. The adsorbent could be regenerated using 1 mol L(-1) HCl-0.28 mol L(-1) ascorbic acid mixture. Chromium could be effectively detoxified from an industrial effluent and finally the developed method was validated with the analysis of a certified reference material (BCR-715). The obtained results indicated that the ultrasonic assisted impregnation of the room temperature ionic liquid significantly enhances and improves the removal efficiency of Cr(VI).

Microwave assisted solvent free green preparation and physicochemical characterization of surfactant-anchored cellulose and its relevance toward the effective adsorption of chromium.

The molecular interaction between tetrabutylammonium iodide (TBAI) and cellulose and its potential application for the adsorption of chromium is discussed in this paper. The biosorbent was prepared under solvent free conditions using microwave irradiation. A subtle balance between electrostatic and the hydrophobic effects governs the interaction of biopolymer with the surfactant. The interaction between the biosorbent and chromium was comprehensively studied using spectroscopic, thermal and surface analysis techniques. The various analytical parameters that influence the adsorption were optimized, and the maximum adsorption capacity of Cr(VI) obtained from the Langmuir isotherm model was found to be 16.67 mg g(-1). The sorption thermodynamics indicated the spontaneity and exothermic nature of adsorption. The biosorbent could be effectively regenerated using NaOH, which imparts greener aspect to the overall process.