Faculty of Applied Sciences
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Item Theoretical principles and applications of high performance capillary electrophoresis(Nova Science Publishers, 2015) Bathinapatla, Ayyappa; Kanchi, Suvardhan; Sabela, Myalowenkosi I.; Bisetty, KrishnaThis book chapter is aimed at addressing the theoretical principles and applications of capillary electrophoresis (CE) for the separation of high intensity artificial sweeteners. Electrophoresis is a technique in which solutes are separated by their movement with different rates of migration in the presence of an electric field. Capillary electrophoresis emerged as a combination of the separation mechanism of electrophoresis and instrumental automation concepts in chromatography. Its separation mainly depends on the difference in the solutes migration in an electric field caused by the application of relatively high voltages, thus generating an electro-osmotic flow (EOF) within the narrow-bore capillaries filled with the background electrolyte. Currently capillary electrophoresis is a very powerful analytical technique with a major and outstanding importance in separations of compounds such as amino acids, chiral drugs, vitamins, pesticides etc., because of simpler method development, minimal sample volume requirements and lack of organic waste. The main advantage of capillary electrophoresis over conventional techniques is the availability of the number of modes with different operating and separation characteristics include free zone electrophoresis and molecular weight based separations (capillary zone electrophoresis), micellar based separations (micellar electrokinetic chromatography), chiral separations (electrokinetic chromatography), isotachophoresis and isoelectrofocusing makes it a more versatile technique being able to analyse a wide range of analytes. The ultimate goal of the analytical separations is to achieve low detection limits and CE is compatible with different external and internal detectors such as UV or photodiode array detector (DAD) similar to HPLC. CE also provides an indirect UV detection for analytes that do not absorb in the UV region. Besides the UV detection, CE provides five types of detection modes with special instrumental fittings such as Fluorescence, Laserinduced Fluorescence, Amperometry, Conductivity and Mass spectrometry. Infact, the lowest detection limits attained in the whole field of separations are for CE with laser induced fluorescence detection. Regarding the applications of CE, the separation and determination of high intensity sweeteners were discussed in this chapter. The materials which show sweetness are divided into two types (i) nutritive sweeteners and (ii) non-nutritive sweeteners. The main nutritive sweeteners include glucose, crystalline fructose, dextrose, corn sweeteners, honey, lactose, maltose, invert sugars, concentrated fruit juice, refined sugars, high fructose corn syrup and various syrups. Non-nutritive sweeteners are sub-divided into two groups of artificial sweeteners and reduced polyols. On the other hand, based on their generation; artificial sweeteners can further be divided into three types as (a) first generation artificial sweeteners which includes saccharin, cyclamate and glycyrrhizin (b) second generation artificial sweeteners are aspartame, acesulfame K, thaumatin and neohesperidinedihydrochalcone (c) neotame, sucralose, alitame and steviol glycosides falls under third generation artificial sweeteners. Artificial sweeteners are also classified into three types based on their synthesis and extraction: (i) synthetic (saccharin, cyclamate, aspartame, acesulfame K, neotame, sucralose, alitame) (ii) semi-synthetic (neohesperidinedihydrochalcone) and (iii) natural sweeteners (steviol glycosides, mogrosides and brazzein protein). Polyols are other groups of reduced-calorie sweeteners which provide bulk of the sweetness, but with fewer calories than sugars. The commonly used polyols are: erythritol, mannitol, isomalt, lactitol, maltitol, xylitol, sorbitol and hydrogenated starch hydrolysates (HSH). The studies revealed that capillary electrophoresis was successfully used for the separation of high intensity artificial sweeteners such as neotame, sucralose and steviol glycosides. Additionally, the available methods for the other artificial sweeteners using capillary electrophoresis were reviewed besides the above indicated sweeteners.Item Biosynthesis of ZnO nanoparticles using Jacaranda mimosifolia flowers extract: Synergistic antibacterial activity and molecular simulated facet specific adsorption studies(Elsevier, 2016) Sharma, Deepali; Sabela, Myalowenkosi Innocent; Kanchi, Suvardhan; Mdluli, Phumlani Selby; Singh, Gulshan; Stenström, Thor-Axel; Bisetty, KrishnaThe naturally occurring biomolecules present in the plant extracts have been identified to play an active role in the single step formation of nanoparticles with varied morphologies and sizes which is greener and environmen-tally benign. In the present work, spherical zinc oxide nanoparticles (ZnO NPs) of 2–4 nm size were synthesized using aqueous extract of fallen Jacaranda mimosifolia flowers (JMFs), treated as waste. The microwave assisted synthesis was completed successfully within 5 min. Thereafter, phase identification, morphology and optical band gap of the synthesized ZnO NPs were done using X-ray diffraction (XRD), high resolution transmission elec-tron microscopy (HRTEM) and UV–Visible spectroscopy techniques. The composition of JMFs extract was ana-lyzed by gas chromatography–mass spectrometry (GC–MS) and the ZnO NPs confirmation was further explored with fourier transform infrared spectroscopy (FTIR). The GC–MS results confirmed the presence of oleic acid which has high propensity of acting as a reducing and capping agent. The UV–Visible data suggested an optical band gap of 4.03 eV for ZnO NPs indicating their small size due to quantum confinement. Further, facet specific adsorption of oleic acid on the surface of ZnO NPs was studied computationally to find out the im-pact of biomolecules in defining the shape and size of NPs. The viability of gram negative Escherichia coli and gram positive Enterococcus faecium bacteria was found to be 48% and 43%, respectively at high concentration of NPs.Item Functional insight into Putative Conserved Proteins of Rickettsia rickettsii and their Virulence characterization(Bentham Science Publishers, 2015) Shahbaaz, Mohd; Bisetty, Krishna; Ahmad, FaizanAbstract: Rickettsia rickettsii is an aerobic, Gram-negative and non-motile coccobacillus known to cause Rocky Mountain spotted fever. The sequenced genome of its 'Sheila Smith' strain contains 1,343 protein-coding genes, 3 rRNA genes and 33 transfer RNA genes. There are 680 hypothetical proteins (HPs) present in the genome of R. rickettsii. Since functions of these proteins are not validated ex-perimentally, characterization of these HPs may play a significant role in understanding the patho-genic mechanisms of R. rickettsii. Hence, functions of these HPs were annotated by in silico methods based on sequence similarity, protein clustering and protein-protein interactions. We have successfully predicted functions of 214 proteins among 680 HPs present in R. rickettsii. These annotated proteins were further classified into 88 enzymes, 59 transport and membrane proteins, 35 binding proteins, 12 structural motifs and the rest of the protein families. Moreo-ver, we identified HPs involved in virulence among 214 functionally annotated proteins. 15 HPs were classified as viru-lence factors and two proteins with the highest scores were selected for further analyses. Additionally, molecular dynam-ics simulations were performed on these selected virulent HPs in order to observe their conformational behaviors. These analyses can further be utilized in the identification of new drug targets for development of better therapeutic agents against the infections caused by R. rickettsii.Item Designing new kinase inhibitor derivatives as therapeutics against common complex diseases : structural basis of microtubule affinity-regulating kinase 4 (MARK4) inhibition(Mary Ann Liebert, Inc., 2015) Naz, Farha; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md. ImtaiyazDrug development for common complex diseases is in need of new molecular entities and actionable drug targets. MAP/microtubule affinity-regulating kinase 4 (MARK4) is associated with numerous diseases such as neurodegenerative disorders, obesity, cancer, and type 2 diabetes. Understanding the structural basis of ligands’ (inhibitors) and substrates’ binding to MARK4 is crucial to design new kinase inhibitors for therapeutic pur-poses. This study reports new observations on docking three well-known kinase inhibitors in the kinase domain of MARK4 variants and the calculated binding affinity. These variants of MARK4 are named as MARK4-F1 (59 N-terminal residues along with kinase domain) and MARK4-F2 (kinase domain of MARK4). We addi-tionally performed molecular dynamics (MD) simulation and fluorescence binding studies to calculate the actual binding affinity of kinase inhibitors, BX-912, BX-795, and OTSSP167 (hydrochloride) for the MARK4. Docking analyses revealed that ligands bind in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Simulations suggested that OTSSP167 (hy-drochloride) is forming a stable complex, and hence the best inhibitor of MARK4. Intrinsic fluorescence of MARK4 was significantly quenched by addition of ligands, indicating their potential binding to MARK4. A lower KD value of MARK4 with OTSSP167 (hydrochloride) suggested that it is a better interacting partner than BX-912 and BX-795. These data form a basis for designing novel and potent OTSSP167 (hydrochloride) derivatives as therapeutic candidates against common complex diseases. The inhibitors designed as such might possibly suppress the growth of tumor-forming cells and be potentially applied for treatment of a wide range of human cancers as well.Item Studies on bacterial proteins corona interaction with saponin imprinted ZnO nano-honeycombs and their toxic responses(ACS Publications, 2015-10-07) Sharma, Deepali; Ashaduzzaman, Md.; Golabi, Mohsen; Shriwastav, Amritanshu; Bisetty, Krishna; Tiwari, AshutoshMolecular imprinting generates robust, efficient and highly mesoporous surfaces for bio-interactions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understanding the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesised saponin imprinted zinc oxide nano-honeycombs (SIZnO NHs), average size 80-125 nm, surface area 20.27 m2/g, average pore density 0.23 pore/nm and number average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential anti-fungal and anti-bacterial agents have been studied on Sclerotium rolfsii (S. rolfsii), Pythium debarynum (P. debarynum) and Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), respectively. SIZnO NHs exhibited the highest antibacterial (~50%) and antifungal (~40%) activity against gram-negative bacteria (E. coli) and fungus (P. debarynum) respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalised into the cell. The molecular docking studies have been carried out using lipopolysaccharide and lipocalin Blc as binding proteins. It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies for lipopolysaccharide were higher than those of the lipocalin Blc. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalisation in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good agreement with the experimental data. Exploration of mechanistic interactions, as well as the formation of bioconjugate of proteins and ZnO NHs would play a key role to interpret more complex biological systems in nature.Item Towards new drug targets? Function prediction of putative proteins of Neisseria meningitidis MC58 and their virulence characterization(Mary Ann Liebert, Inc., 2015) Shahbaaz, Mohd; Bisetty, Krishna; Ahmad, Faizan; Hassan, Md. ImtaiyazNeisseria meningitidis is a Gram-negative aerobic diplococcus, responsible for a variety of meningococcal dis-eases. The genome of N. meningitidis MC58 is comprised of 2114 genes that are translated into 1953 proteins. The 698 genes (*35%) encode hypothetical proteins (HPs), because no experimental evidence of their biological functions are available. Analyses of these proteins are important to understand their functions in the metabolic networks and may lead to the discovery of novel drug targets against the infections caused by N. meningitidis. This study aimed at the identification and categorization of each HP present in the genome of N. meningitidis MC58 using computational tools. Functions of 363 proteins were predicted with high accuracy among the annotated set of HPs investigated. The reliably predicted 363 HPs were further grouped into 41 different classes of proteins, based on their possible roles in cellular processes such as metabolism, transport, and replication. Our studies revealed that 22 HPs may be involved in the pathogenesis caused by this microorganism. The top two HPs with highest virulence scores were subjected to molecular dynamics (MD) simulations to better understand their conformational behavior in a water environment. We also compared the MD simulation results with other virulent proteins present in N. meningitidis. This study broadens our understanding of the mechanistic pathways of pathogenesis, drug resistance, tolerance, and adaptability for host immune responses to N. meningitidis.Item Seasonal variation and distribution of Anionic Surfactants in and around Tirupati : a famous pilgrim centre in South India(Asian Publication Corporation, 2015) Ramanjulu, C.; Naidu, N. Venkatasubba; Kanchi, Suvardhan; Bisetty, KrishnaThis report presents the anionic surfactants concentration in water system collected in and around Tirupati, South India. The concentration of anionic surfactants such as sodium lauryl sulfate, sodium dodecyl sulfate, sodium hexadecyl sulfonate and sodium dodecyl benzene sulfonate were showed a rather homogeneous distribution (except sodium dodecyl benzene sulfonate) within the Tirupati, with high values of 80.0, 90.75, 46.90, 15.10 μg L–1 in Tirupati and slightly less concentration values of 75.0, 60.55, 35.40, 10.0 μg L –1 were found in samples collected from Renigunta Industrial Estate during pre-monsoon and monsoon seasons respectively. High concentration of these surfactants in open wells and open municipal wastewaters in Tirupati may be due to the huge discharge of domestic wastes into the drain system. The concentration of surfactants found in Renigunta Industrial Estate may be due to the direct discharge of industrial wastewater into the environment.Item Novel Dithiocarbamates for electrochemical detection of Nickel (II) in environmental samples(Asian Publication Corporation, 2015-06-22) Niranjan, T.; Kanchi, Suvardhan; Bisetty, Krishna; Naidu, N. VenkatasubbaAmmonium 4-phenylpiperazine-1-dithiocarbamate (Amm 4-PP-DTC) and ammonium 4-benzylpiperidine-1-dithiocarbamate (Amm 4-BP-DTC) were synthesized for the determination of nickel(II) using catalytic hydrogen currents (CHC’s) technique with DC Polarography. The method was based on the chelation of nickel(II) with Amm 4-PP-DTC/ Amm 4-BP-DTC in presence of NH 4 OH at pH 6.8 to produce catalytic hydrogen current at -1.50V and -1.41 V vs. SCE respectively. Optimized polarographic conditions were established by studying effect of pH, supporting electrolyte (NH4Cl), ligands and metal ion concentration and effect of adverse ions on peak height to improve the sensitivity, selectivity and detection limits of the present method. This technique is successfully applied for the analysis of nickel(II) in different matrices with recoveries ranging from 96.0-99.0 % and the results obtained were comparable with the atomic absorption spectroscopy.Item Analytical evaluation of steviol glycosides by capillary electrophoresis supported with molecular docking studies(Iranian Chemical Society, 2014-05-05) Ayyappa, Bathinapatla; Kanchi, Suvardhan; Singh, Parvesh; Dovey, Martin; Sabela, Myalowenkosi Innocent; Bisetty, KrishnaThis paper reports on a newly developed elec-trokinetic chromatographic method for the simultaneous separation and determination of steviol glycosides in real stevia samples by capillary electrophoresis and supported by molecular docking studies. Our results obtained using 30-mM heptakis-(2,3,6-tri-o-methyl betacyclodextrin) as a separating agent, suggest that at optimum experimental conditions the detection limits of 2.017 9 10-5 and 7.386 9 10-5 M and relative standard deviations (n = 5) of 1.10 and 1.17 were obtained for rebaudioside-A and stevioside, respectively. In addition, the molecular docking studies explained to a certain extent why the separation was successful. The calculated binding free energy results for the rebaudioside-A and stevioside complexes formed with the separating agent showed that although both ligands penetrated deeply into the hydrophobic cavity of the sep-arating agent, the presence of additional hydrogen bonding in the case of stevioside is probably responsible for its stronger binding affinity than that of rebaudioside-A.Item A Box-Behnken Design and Response Surface Approach for the Simultaneous Determination of Chromium (III) and (VI) Using Catalytic Differential Pulse Polarography(ESG, 2004-09-29) Sabela, Myalowenkosi Innocent; Kanchi, Suvardhan; Ayyappa, Bathinapatla; Bisetty, KrishnaThe present paper describes an optimized Box-Behnken design using a catalytic-differential pulse polarograhic technique for the simultaneous determination of chromium (III) and (VI) in wastewater samples using ammonium piperidine dithiocarbamate as a complexing agent. The optimization strategy was carried out using a two level full factorial design. The results of the experimental design were based on an analysis of variance and demonstrated that only the pH, concentrations of the buffer and the complexing agent were statistically significant throughout the experiment. The optimal values for the three variables were 8.0, 0.2 mM and 5.0 mM for pH, concentrations of the buffer and the complexing agent respectively. Under optimum experimental conditions the detection limit of the proposed method was found to be 0.0841 µg L-1 while the linear range was 1.0-10.0 and 0.5-25.0 µg L- for chromium (III) and (VI) respectively. The present method was also applied for the simultaneous determination of chromium in the presence of some foreign ions with satisfactory analytical responses.