Showing papers by "Bharathiar University published in 2012"

Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities.

Abstract: Objective To develop a novel approach for the green synthesis of silver nanoparticles using aqueous leaves extracts of Catharanthus roseus (C. roseus) Linn. G. Don which has been proven active against malaria parasite Plasmodium falciparum (P. falciparum).

A novel water soluble ligand bridged cobalt(II) coordination polymer of 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (isonicotinic) hydrazone: evaluation of the DNA binding, protein interaction, radical scavenging and anticancer activity.

Abstract: A novel water soluble ligand-bridged cobalt(II) coordination polymer has been synthesized by reacting the new ligand, 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (isonicotinic) hydrazone (H2L) with Co(NO3)2·6H2O and characterized by spectral, analytical and structural methods. Single crystal X-ray diffraction studies revealed that the Co(II) complex, {[Co(H2L)(H2O)2](NO3)2·3H2O}n has a slightly distorted octahedral geometry around the central Co(II) ion; the ligand is coordinated through the ONO donor atoms to one Co(II) metal center and bridged through the pyridine nitrogen atom to another similar Co(II) center so as to form a one-dimensional polymeric unit. The interaction of the ligand and the complex with calf thymus DNA (CT-DNA) has been explored by absorption and emission titration methods, which revealed that the compounds could interact with CT-DNA through intercalation. The interactions of the compounds with bovine serum albumin (BSA) were also investigated using UV–visible, fluorescence and synchronous fluorescence spectroscopic methods. The results indicated that the complex exhibited a strong binding to BSA over the ligand. Investigation of the antioxidative properties showed that the polymeric Co(II) complex has a strong radical scavenging potency against hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl radicals, nitric oxide and superoxide anion radicals. Further, the cytotoxic effect of the compounds examined on cancerous cell lines, such as human cervical cancer cells (HeLa), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (Hep G2), human skin cancer cells (A431) and non-cancerous NIH 3T3 mouse embryonic fibroblasts cell lines showed that the complex exhibited substantial anticancer activity.

Copper(I) and nickel(II) complexes with 1 : 1 vs. 1 : 2 coordination of ferrocenyl hydrazone ligands: Do the geometry and composition of complexes affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic activities?

Abstract: A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were synthesised by reacting suitable precursor complex [MCl2(PPh3)2] with the ligands HL1 or HL2 (where M = Cu(II) or Ni(II); HL1 = [Cp2Fe(CHN–NH –CO– C6H5)] (1) and HL2 = [Cp2Fe(CHN–NH–CO–C5H4N)]) (2). The new complexes of the composition [Cu(L1)(PPh3)2], (3) [Cu(L2)(PPh3)2] (4), [Ni(L1)2] (5) and [Ni(L2)2] (6) were characterised by various spectral studies. Among them, complexes 3 and 5 characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former with 1 : 1 metal–ligand stoichiometry, but a distorted square planar geometry with 1 : 2 metal–ligand stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the synthesised compounds and the results obtained were explained on the basis of structure–activity relationships. The binding constant (Kb) values of the synthesised compounds are found to be in the order of magnitude 103–105 M−1 and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in the presence of H2O2 as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon binding with the above complexes were also studied. In addition, concentration dependent free radical scavenging potential of all the synthesised compounds (1–6) was also carried out under in vitro conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and NIH 3T3 normal cells were also carried out.

Biolarvicidal and pupicidal potential of silver nanoparticles synthesized using Euphorbia hirta against Anopheles stephensi Liston (Diptera: Culicidae).

Abstract: Vector control is a critical requirement in epidemic disease situations, as is an urgent need to develop new and improved mosquito control methods that are economical and effective yet safe for nontarget organisms and the environment. Mosquitoes transmit serious human diseases, causing millions of deaths every year. Use of synthetic insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in addition to high operational cost. Insecticides of synthesized natural products for vector control have been a priority in this area. In the present study, activity of silver nanoparticles (AgNPs) synthesized using Euphorbia hirta (E. hirta) plant leaf extract against malarial vector Anopheles stephensi (A. stephensi) was determined. Range of concentrations of synthesized AgNPs (3.125, 6.25, 12.5, 25, and 50 ppm) and methanol crude extract (50, 100, 150, 200, and 250 ppm) were tested against larvae of A. stephensi. The synthesized AgNPs from E. hirta were highly toxic than methanolic crude extract against malarial vector, A. stephensi. The synthesized AgNPs were characterized by UV-vis spectrum, scanning electron microscopy (SEM), and X-ray diffraction. SEM analyses of the synthesized showed that AgNPs, measuring 30-60 nm in size, were clearly distinguishable. The synthesized AgNPs showed larvicidal effects after 24 h of exposure; however, the highest larval mortality was found in the synthesized AgNPs against the first to fourth instar larvae and pupae of values LC(50) (10.14, 16.82, 21.51, and 27.89 ppm, respectively), LC(90) (31.98, 50.38, 60.09, and 69.94 ppm, respectively), and the LC(50) and LC(90) values of pupae of 34.52 and 79.76 ppm, respectively. Methanol extract exhibited the larval toxicity against the first to fourth instar larvae and pupae of values LC(50) (121.51, 145.40, 169.11, and 197.40 ppm, respectively), LC(90) (236.44, 293.75, 331.42, and 371.34 ppm, respectively), and the LC(50) and LC(90) values of pupae of 219.15 and 396.70 ppm, respectively. No mortality was observed in the control. These results suggest that synthesized silver nanoparticles are a rapid, eco-friendly, and single-step approach; the AgNPs formed can be potential mosquito larvicidal agents.

Redox additive aqueous polymer gel electrolyte for an electric double layer capacitor

Abstract: A hydroquinone mediated PVA–H2SO4 gel electrolyte (PHHQ) and activated carbon from bio-waste were prepared for supercapacitor fabrication. PHHQ delivered a higher capacitance (941 F g−1 at 1 mA cm−2) and energy density (20 Wh kg−1 at 0.33 W g−1) than the PVA–H2SO4 gel electrolyte (425 F g−1 at 1 mA cm−2, 9 Wh kg−1 at 0.33 W g−1).

DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium(II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity.

Abstract: The coordination propensities of 4(N,N′)-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones (H2L1–4) were investigated by reacting with an equimolar amount of [PdCl2(PPh3)2]. The new complexes were characterized by various spectroscopic techniques. The structure determination of the complexes [Pd(DeaSal-tsc)(PPh3)] (1), [Pd(DeaSal-mtsc)(PPh3)] (2) and [Pd(DeaSal-etsc)(PPh3)] (3) by X-ray crystallography showed that ligands are coordinated in a dibasic tridentate ONS donor fashion forming stable five and six membered chelate rings. The binding ability of complexes (1–4) to calf-thymus DNA (CT DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed. The protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using lysozyme as a model protein. As determined by MTT assays, complex 3 exhibited a higher cytotoxic effect towards human lung cancer cell line (A549) and liver cancer cells (HepG2). LDH, NO assay and cellular uptake of the complexes have been studied. Further, antibacterial activity studies of the complexes have been screened against the pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, MIC50 values of the complexes showed that the complexes exhibited significant activity against the pathogens and among the complexes, 3 exhibited higher activity.

Influence of terminal substitution on structural, DNA, Protein binding, anticancer and antibacterial activities of palladium(II) complexes containing 3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones

Abstract: The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones was observed in equimolar reactions with [PdCl2(PPh3)2]. The new complexes were characterized by various analytical, spectroscopic techniques (mass, 1H-NMR, absorption, IR). All the new complexes were structurally characterized by single crystal X-ray diffraction. Crystallographic results showed that the ligands H2L1and H2L4 are coordinated as binegative tridentate ONS donor ligands in the complexes 1 and 4 by forming six and five member rings. However, the ligands H2L2 and H2L3 bound to palladium in 2 and 3 as uninegative bidentate NS donors by forming a five member chelate ring. From this study, it was found that the substitution on terminal 4(N)- nitrogen may have an influence on the chelating ability of thiosemicarbazone. The presence of hydrogen bonding in 2 and 3 might be responsible for preventing the coordination of phenolic oxygen to the metal ion. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic binding mode of DNA has been proposed. The protein binding studies were monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using Lysozyme as model protein. Antibacterial activity studies of the complexes have been screened against pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa.MIC50 values of the complexes showed that they exhibited significant activity against the pathogens and among them, 3 exhibited higher activity. Further, anticancer activity of the complexes on the lung cancer cell line A549 has also been studied.

Green synthesis of silver nanoparticles for the control of mosquito vectors of malaria, filariasis, and dengue.

Abstract: A biological method was used to synthesize stable silver nanoparticles that were tested as mosquito larvicides against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Annona squamosa leaf broth (5%) reduced aqueous 1 mM AgNO₃ to stable silver nanoparticles with an average size of 450 nm. The structure and percentage of synthesized nanoparticles was characterized by using ultraviolet spectrophotometry, X-Ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy methods. The median lethal concentrations (LC₅₀) of silver nanoparticles that killed fourth instars of Ae. aegypti, Cx. quinquefasciatus, and An. stephensi were 0.30, 0.41, and 2.12 ppm, respectively. Adult longevity (days) in male and female mosquitoes exposed as larvae to 0.1 ppm silver nanoparticles was reduced by ~30% (p<0.05), whereas the number of eggs laid by females exposed as larvae to 0.1 ppm silver nanoparticles decreased by 36% (p<0.05).

Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis orange peel extract against Anopheles stephensi , Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)

Abstract: Mosquitoes are the carriers of severe and well-known illnesses such as malaria, arboviral encephalitis, dengue fever, chikunguniya fever, West Nile virus and yellow fever. These diseases produce significant morbidity and mortality in humans and livestock around the world. The present study explored the effects of orange peel ethanol extract of Citrus sinensis on larvicidal, pupicidal, repellent and adulticidal activity against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. The orange peel material was shade dried at room temperature and powdered coarsely. From orange peel, 300 g powdered was macerated with 1 L of ethanol sequentially for a period of 72 h each and filtered. The yields of the orange peel ethanol crude extract of C. sinensis 13.86 g, respectively. The extracts were concentrated at reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4 °C. The larvicidal, pupicidal and adult mortality was observed after 24 h of exposure; no mortality was observed in the control group. For C. sinensis, the median lethal concentration values (LC50) observed for the larvicidal and pupicidal activities against mosquito vector species A. stephensi first to fourth larval instars and pupae were 182.24, 227.93, 291.69, 398.00 and 490.84 ppm; A. aegypti values were 92.27, 106.60, 204.87, 264.26, 342.45, 436.93 and 497.41 ppm; and C. quinquefasciatus values were 244.70, 324.04, 385.32, 452.78 and 530.97 ppm, respectively. The results of maximum repellent activity were observed at 450 ppm in ethanol extracts of C. sinensis and the mean complete protection time ranged from 150 to 180 min was tested. The ethanol extract of C. sinensis showed 100 % repellency in 150 min and showed complete protection in 90 min at 350 ppm against A. stephensi, A. aegypti and C. quinquefasciatus, respectively. The adult mortality was found in ethanol extract of C. sinensis with the LC50 and LC90 values of 272.19 and 457.14 ppm, A. stephensi; 289.62 and 494.88 ppm, A. aegypti; and 320.38 and 524.57 ppm, respectively. These results suggest that the orange peel extracts of C. sinensis have the potential to be used as an ideal eco-friendly approach for the control of the vector control programmes.

Ruthenium(II)/(III) complexes of 4-hydroxy-pyridine-2,6-dicarboxylic acid with PPh3/AsPh3 as co-ligand: impact of oxidation state and co-ligands on anticancer activity in vitro

Abstract: With the aim to develop more efficient, less toxic, target specific metal drugs and evaluate their anticancer properties in terms of oxidation state and co-ligand sphere, a sequence of Ru(II), Ru(III) complexes bearing 4-hydroxy-pyridine-2,6-dicarboxylic acid and PPh(3)/AsPh(3) were synthesized and structurally characterized. Biological studies such as DNA binding, antioxidant assays and cytotoxic activity were carried out and their anticancer activities were evaluated. Interactions of the complexes with calf thymus DNA revealed that the triphenylphosphine complexes could bind more strongly than the triphenylarsine complexes. The free radical scavenging ability, assessed by a series of in vitro antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion radical, hydrogen peroxide and metal chelating assay, showed that the Ru(III) complexes possess excellent radical scavenging properties compared to those of Ru(II). Cytotoxicity studies using three cancer lines viz HeLa, HepG2, HEp-2 and a normal cell line NIH 3T3 showed that Ru(II) complexes exhibited substantial cytotoxic specificity towards cancer cells. Furthermore, the Ru(II) complexes were found to be superior to Ru(III) complexes in inhibiting the growth of cancer cells.

Controllability of nonlinear fractional dynamical systems

Abstract: In this paper we establish a set of sufficient conditions for the controllability of nonlinear fractional dynamical systems. The results are obtained by using the recently derived formula for solution representation of systems of fractional differential equations and the application of the Schauder fixed point theorem. Examples are provided to illustrate the results.

Electrochemical properties of microwave-assisted reflux-synthesized Mn3O4 nanoparticles in different electrolytes for supercapacitor applications

Abstract: The nanosized Mn3O4 particles were prepared by microwave-assisted reflux synthesis method The prepared sample was characterized using various techniques such as X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR), Raman analysis, and transmission electron microscopy (TEM) Electrochemical properties of Mn3O4 nanoparticles were investigated using cyclic voltammogram (CV), electrochemical impedance spectroscopy (EIS), and galvanostatic charge–discharge analysis in different electrolytes such as 1 M KCl, 1 M Na2SO4, 1 M NaNO3, and 6 M KOH electrolytes XRD pattern reveals the formation of single-phase Mn3O4 nanoparticles The FT-IR and Raman analysis also assert the formation of Mn3O4 nanoparticles The TEM image shows the spherical shape particles with less than 50 nm sizes Among all the electrolytes, the Mn3O4 nanoparticles possess maximum specific capacitance of 94 F g−1 in 6 M KOH electrolyte calculated from CV The order of capacitance obtained by various electrolytes is 6 M KOH > 1 M KCl > 1 M NaNO3 > 1 M Na2SO4 The EIS and galvanostatic charge–discharge results further substantiate with the CV results The cycling stability of Mn3O4 electrode reveals that the prepared Mn3O4 nanoparticles are a suitable electrode material for supercapacitor application

Synthesis of novel heterobimetallic copper(I) hydrazone Schiff base complexes: A comparative study on the effect of heterocyclic hydrazides towards interaction with DNA/protein, free radical scavenging and cytotoxicity

Abstract: Two new copper(I) hydrazone complexes have been synthesised from bivalent copper precursor [CuCl2(PPh3)2] and ferrocene containing bidentate hydrazone ligands HL1 (1) or HL2 (2). Based on the elemental analyses and spectroscopic data, the complexes are best formulated as [CuL1(PPh3)2] (3) and [CuL2(PPh3)2] (4) of the monovalent copper ion. Solid state structures of ligand 2 and its corresponding complex 4 were also determined. The DNA/albumin interactions of all the synthesised compounds were investigated using absorption, emission and synchronous fluorescence studies. Further, antioxidant properties of all the compounds have also been checked against ABTS, O2− and OH radicals. Additionally, the in vitro cytotoxic activity of compounds 1–4 was assessed using tumour (HeLa, A431) and non-tumour (NIH 3T3) cell lines.

Mixed ligand palladium(II) complexes of 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazones with triphenylphosphine co-ligand: synthesis, crystal structure and biological properties.

Abstract: A series of new 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazone ligands (H2L1–H2L5) and their corresponding palladium(II) complexes [Pd(L1)(PPh3)] (1), [Pd(L2)(PPh3)] (2), [Pd(HL3)(PPh3)]Cl (3), [Pd(L4)(PPh3)] (4) and [Pd(L5)(PPh3)] (5), have been synthesized in order to evaluate the effect of terminal N-substitution in thiosemicarbazone moiety on coordination behaviour and biological activity. The new ligands and their corresponding complexes were characterized by analytical and various spectral techniques. The molecular structure of the complexes 2–5 were characterized by single crystal X-ray diffraction studies which revealed that the ligands H2L2, H2L4 and H2L5 are coordinated to palladium(II) as binegative tridentate (ONS2−) by forming six and five member rings whereas, the ligand H2L3 coordinated to Pd(II) as uninegative tridentate (ONS−). The interactions of the new complexes with calf thymus DNA (CT-DNA) have been evaluated by absorption and ethidium bromide (EB) competitive studies which revealed that complexes 1–5 could interact with CT-DNA through intercalation. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic methods, which showed that the new complexes could bind strongly with BSA. Antioxidant studies showed that all the complexes have a strong antioxidant activity against 2-2′-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azino-3-ethylbenzthiazoline-6-sulfonic acid diammonium salt (ABTS) cation radical. In addition, in vitro cytotoxicity of the complexes against human lung cancer (A549) cell line was assayed which showed that 4 has higher cytotoxic activity than the rest of the complexes and cisplatin.

Assessment of source water contamination by estrogenic disrupting compounds in China.

Abstract: Detection of estrogenic disrupting compounds (EDCs) in drinking waters around China has led to rising concerns about health risks associated with these compounds. There is, however, a paucity of studies on the occurrence and identification of the main compounds responsible for this pollution in the source waters. To fill this void, we screened estrogenic activities of 23 source water samples from six main river systems in China, using a recombinant two-hybrid yeast assay. All sample extracts induced significant estrogenic activity, with E2 equivalents (EEQ) of raw water ranging from 0.08 to 2.40 ng/L. Additionally, 16 samples were selected for chemical analysis by gas chromatography-mass spectrometry. The EDCs of most concern, including estrone (E1), 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), estriol (E3), diethylstilbestrol (DES), estradiol valerate (EV), 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP) and bisphenol A (BPA), were determined at concentrations of up to 2.98, 1.07, 2.67, 4.37, 2.52, 1.96, 89.52, 280.19 and 710.65 ng/L, respectively. Causality analysis, involving comparison of EEQ values from yeast assay and chemical analysis identified E2, EE2 and 4-NP as the main responsible compounds, accounting for the whole estrogenic activities (39.74% to 96.68%). The proposed approach using both chemical analysis and yeast assay could be used for the identification and evaluation of EDCs in source waters of China.

Homology modeling, molecular dynamics, e-pharmacophore mapping and docking study of Chikungunya virus nsP2 protease

Abstract: To date, no suitable vaccine or specific antiviral drug is available to treat Chikungunya viral (CHIKV) fever. Hence, it is essential to identify drug candidates that could potentially impede CHIKV infection. Here, we present the development of a homology model of nsP2 protein based on the crystal structure of the nsP2 protein of Venezuelan equine encephalitis virus (VEEV). The protein modeled was optimized using molecular dynamics simulation; the junction peptides of a nonstructural protein complex were then docked in order to investigate the possible protein-protein interactions between nsP2 and the proteins cleaved by nsP2. The modeling studies conducted shed light on the binding modes, and the critical interactions with the peptides provide insight into the chemical features needed to inhibit the CHIK virus infection. Energy-optimized pharmacophore mapping was performed using the junction peptides. Based on the results, we propose the pharmacophore features that must be present in an inhibitor of nsP2 protease. The resulting pharmacophore model contained an aromatic ring, a hydrophobic and three hydrogen-bond donor sites. Using these pharmacophore features, we screened a large public library of compounds (Asinex, Maybridge, TOSLab, Binding Database) to find a potential ligand that could inhibit the nsP2 protein. The compounds that yielded a fitness score of more than 1.0 were further subjected to Glide HTVS and Glide XP. Here, we report the best four compounds based on their docking scores; these compounds have IDs of 27943, 21362, ASN 01107557 and ASN 01541696. We propose that these compounds could bind to the active site of nsP2 protease and inhibit this enzyme. Furthermore, the backbone structural scaffolds of these four lead compounds could serve as building blocks when designing drug-like molecules for the treatment of Chikungunya viral fever.

Studies on larvicidal and pupicidal activity of Leucas aspera Willd. (Lamiaceae) and bacterial insecticide, Bacillus sphaericus, against malarial vector, Anopheles stephensi Liston. (Diptera: Culicidae)

Abstract: The efficacy of whole plant extracts of Leucas aspera and Bacillus sphaericus has been proven against larvicidal and pupicidal activities of the malarial vector, Anopheles stephensi. The present study investigated the larvicidal and pupicidal activity against the first to fourth instar lavae and pupae of the laboratory-reared mosquitoes, A. stephensi. The medicinal plants were collected from the area around Maruthamalai hills, Coimbatore, Tamil Nadu, India. L. aspera whole plant was washed with tap water and shade dried at room temperature. The dried plant materials were powdered by an electric blender. From the powder, 100 g of the plant materials was extracted with 300 ml of organic solvents of ethanol for 8 h using a Soxhlet apparatus. The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The crude plant extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure. All larval instars and pupae have considerably moderate mortality; however, the highest larval mortality was the ethanolic extract of whole plant L. aspera against the first to fourth instar larvae and pupae values of LC50 = I instar was 9.695%, II instar was 10.272%, III instar was 10.823%, and IV instar was 11.303%, and pupae was 12.732%. B. spaericus against the first to fouth instar larvae and pupae had the following values: I instar was 0.051%, II instar was 0.057%, III instar was 0.062%, IV instar was 0.066%, and for the pupae was 0.073%. No mortality was observed in the control. The present results suggest that the ethanolic extracts of L. aspera and B. sphaericus provided an excellent potential for controlling of malarial vector, A. stephensi.