Publications by authors named "Roquyya Gul"

13 Publications

  • Page 1 of 1

Molecular Cloning and Characterization of an Acidic Polygalacturonase from Grapes and Its Potential in Industry.

Crit Rev Eukaryot Gene Expr 2020 ;30(5):411-425

Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan.

Pectinase enzymes from different plants have many possible biotechnological applications in various industries. Considering industrial significance of pectinolytic enzymes, the polygalacturonase (PG) gene from grapes was cloned into Escherichia coli DH5α using pTZ57R/T vector. Homologous sequences established a close link to Vitis vinifera. Conserve domain analysis predicted PLN02218 domain belongs to the cl31843 superfamily, showing its function as polygalacturonase. After confirmation by PCR and restriction analysis, the PG gene was expressed in E. coli BL21 and induced by IPTG. Expression level was assessed by 12% SDS-PAGE, which showed a 47 kDa protein. High expression level in the soluble fraction indicated that the protein is intracellular or transmembrane. Maximum expression was obtained with 1 mM IPTG and 6 hour induction time, and autoinduction with lactose increased production of the recombinant enzyme. Zymogram analysis revealed that the induced protein was an active enzyme. Expressed PG showed pectinolytic effect on the physiochemical properties of lemon juice. Natural biopolymers are greatly needed because synthetic fibers can negatively affect health. Pectin hydrolysis of banana pseudostem by the PG enzyme produced better quality fiber. Morphological studies by scanning electron microscopy revealed pectin degradation within the fiber cell architecture, showing the effectiveness of PG treatment on banana pseudostem.
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http://dx.doi.org/10.1615/CritRevEukaryotGeneExpr.2020034410DOI Listing
January 2020

A Putative Prophylactic Solution for COVID-19: Development of Novel Multiepitope Vaccine Candidate against SARS-COV-2 by Comprehensive Immunoinformatic and Molecular Modelling Approach.

Biology (Basel) 2020 Sep 18;9(9). Epub 2020 Sep 18.

Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore 54590, Punjab, Pakistan.

The outbreak of 2019-novel coronavirus (SARS-CoV-2) that causes severe respiratory infection (COVID-19) has spread in China, and the World Health Organization has declared it a pandemic. However, no approved drug or vaccines are available, and treatment is mainly supportive and through a few repurposed drugs. The urgency of the situation requires the development of SARS-CoV-2-based vaccines. Immunoinformatic and molecular modelling are time-efficient methods that are generally used to accelerate the discovery and design of the candidate peptides for vaccine development. In recent years, the use of multiepitope vaccines has proved to be a promising immunization strategy against viruses and pathogens, thus inducing more comprehensive protective immunity. The current study demonstrated a comprehensive in silico strategy to design stable multiepitope vaccine construct (MVC) from B-cell and T-cell epitopes of essential SARS-CoV-2 proteins with the help of adjuvants and linkers. The integrated molecular dynamics simulations analysis revealed the stability of MVC and its interaction with human Toll-like receptors (TLRs), which trigger an innate and adaptive immune response. Later, the in silico cloning in a known pET28a vector system also estimated the possibility of MVC expression in . Despite that this study lacks validation of this vaccine construct in terms of its efficacy, the current integrated strategy encompasses the initial multiple epitope vaccine design concepts. After validation, this MVC can be present as a better prophylactic solution against COVID-19.
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http://dx.doi.org/10.3390/biology9090296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563440PMC
September 2020

Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable -Glucosidase from in .

Biomed Res Int 2019 7;2019:9308593. Epub 2019 Nov 7.

Institute of Molecular Biology and Biotechnology, Centre for Research in Molecular Medicine, The University of Lahore, Bhobatian Chowk, 1-Km Defence Road, Lahore 54500, Pakistan.

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from () by cloning in an () expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a of 19.83 mM and a of 0.12 mol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from a potential candidate in industrial applications.
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http://dx.doi.org/10.1155/2019/9308593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885295PMC
April 2020

Deleterious Variants in , and Causing Isolated and Syndromic Tooth Agenesis: A Structural Perspective from Molecular Dynamics Simulations.

Int J Mol Sci 2019 Oct 24;20(21). Epub 2019 Oct 24.

Institute of Molecular Biology and Biotechnology (IMBB), Center for Research in Molecular Medicine (CRiMM), The University of Lahore, Lahore 54000, Pakistan.

The dental abnormalities are the typical features of many ectodermal dysplasias along with congenital malformations of nails, skin, hair, and sweat glands. However, several reports of non-syndromic/isolated tooth agenesis have also been found in the literature. The characteristic features of hypohidrotic ectodermal dysplasia (HED) comprise of hypodontia/oligodontia, along with hypohidrosis/anhidrosis, and hypotrichosis. Pathogenic variants in , , , and , cause the phenotypic expression of HED. Genetic alterations in and cause particularly non-syndromic/isolated oligodontia. In the current project, we recruited 57 patients of 17 genetic pedigrees (A-Q) from different geographic regions of the world, including Pakistan, Egypt, Saudi Arabia, and Syria. The molecular investigation of different syndromic and non-syndromic dental conditions, including hypodontia, oligodontia, generalized odontodysplasia, and dental crowding was carried out by using exome and Sanger sequencing. We have identified a novel missense variant (c.311G>A; p.Arg104His) in in three oligodontia patients of family A, two novel sequence variants (c.207delinsTT, p.Gly70Trpfs*25 and c.1300T>G; p.Try434Gly) in in three patients of family B and four patients of family C, respectively. To better understand the structural and functional consequences of missense variants in WNT10A and EDAR on the stability of the proteins, we have performed extensive molecular dynamic (MD) simulations. We have also identified three previously reported pathogenic variants (c.1076T>C; p.Met359Thr), (c.1133C>T; p.Thr378Met) and (c.594_595insC; Gly201Argfs*39) in in family D (four patients), E (two patients) and F (one patient), correspondingly. Presently, our data explain the genetic cause of 18 syndromic and non-syndromic tooth agenesis patients in six autosomal recessive and X-linked pedigrees (A-F), which expand the mutational spectrum of these unique clinical manifestations.
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http://dx.doi.org/10.3390/ijms20215282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862269PMC
October 2019

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in : Insights from Molecular Dynamics Simulations.

Biomolecules 2019 04 3;9(4). Epub 2019 Apr 3.

Institute of Biochemistry and Biotechnology, Quaid-e-Azam Campus, University of the Punjab, Opp. Sheikh Zaid Hospital, Canal Bank Road, Lahore 54590, Pakistan.

The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the "alanine" alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔG = -140.62 kcal mol), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔG = -124.24 kcal mol). Here we report the dynamic behavior of change of "alanine" in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.
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http://dx.doi.org/10.3390/biom9040133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523802PMC
April 2019

Comparative Analysis of Serum Proangiogenic Biomarkers between those with and without Diabetic Retinopathy.

J Coll Physicians Surg Pak 2018 Sep;28(9):686-689

Medical Student, Faisalabad Medical University, Faisalabad.

Objective: To compare the serum proangiogenic biomarkers in diabetic patients suffering from with and without diabetic retinopathy (DR).

Study Design: An observational study.

Place And Duration Of Study: Arif Memorial Teaching Hospital, Lahore, Pakistan and Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular Medicine, The University of Lahore, Lahore, Pakistan, from March to December 2017.

Methodology: Forty patients with DR were included in group A and 15 patients without retinopathy (controls) were included in group B. Twelve serum pro-angiogenic biomarkers [Angiopoietin 2, Human Growth Factor (HGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Placental Growth Factor (PLGF), Vascular Endothelial Growth Factor-A and C (VEGF-A and VEGF-C), Bone Morphogenetic Protein 9 (BMP9), Follistatin, Leptin, Interleukin-8 (IL8), Endothelin (ET)] were analysed by xMAP flow cytometry technique, results were compared between the two groups and statistical analysis was done using Mann-Whitney U test.

Results: Serum ET, Follistatin and EGF were significantly raised in group A as compared to group B having p-values of 0.001, <0.001, and 0.033, respectively. Serum BMP9, Leptin, HGF, FGF and VEGF-C had p <0.001, 0.023, 0.020, and 0.009, respectively and were higher in group B than group A.

Conclusion: Serum ET, Follistatin and EGF were significantly higher in DR patients as compared to those without DR and should be considered to be significant biomarkers of retinal complications in diabetes mellitus.
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http://dx.doi.org/10.29271/jcpsp.2018.09.686DOI Listing
September 2018

Effect of intravitreal bevacizumab on macular thickness: exploring serum and vitreous proangiogenic biomarkers in patients with diabetic macular edema

Turk J Med Sci 2018 Aug 16;48(4):833-839. Epub 2018 Aug 16.

Background/aim: This study evaluates diabetic macular-edema (DME) patients for the effect of intravitreal bevacizumab (IVB) injection on macular thickness and proangiogenic biomarkers in serum and vitreous.

Materials And Methods: Forty DME patients were analyzed for macular thickness (MT). Twelve proangiogenic biomarkers in serum and vitreous were analyzed before and after IVB.

Results: Significant decrease in MT with vitreal vascular endothelial growth factor-A (VEGF-A) was observed as expected after IVB, while serum VEGF-A did not follow a decreasing trend in contrast to VEGF-C, which decreased both in serum and vitreous. Other vitreal factors like bone morphogenetic protein-9 (BMP9) and fibroblast growth factor (FGF) were also significantly decreased, while endothelial growth factor (EGF) increased following IVB. Before IVB, significant negative correlations were vitreous BMP9 with serum FGF, vitreous human growth factor (HGF) and interleukin-8 (IL-8) with serum endothelin, and vitreous and serum FGF and serum placental growth factor (PLGF) with EGF. After IVB, negative correlations in serum vs. vitreous were found for both HGF and PLGF with BMP9, and angiopoietin with FGF. Cube average thickness was negatively correlated with serum FGF and positively correlated with vitreous PLGF and endothelin.

Conclusion: Vascular endothelial growth factors are not the only factors that cause macular edema in diabetic patients. The effect of IVB on different proangiogenic biomarkers indicated a complex interplay of other factors in DME.
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http://dx.doi.org/10.3906/sag-1802-212DOI Listing
August 2018

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli.

Mol Biotechnol 2018 Aug;60(8):585-594

Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular Medicine, The University of Lahore, 1-km Defense Road, Bhobatian Chowk, Lahore, Pakistan.

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.
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http://dx.doi.org/10.1007/s12033-018-0094-3DOI Listing
August 2018

Microwave-assisted synthesis and evaluation of type 1 collagen-apatite composites for dental tissue regeneration.

J Biomater Appl 2018 07 2;33(1):103-115. Epub 2018 May 2.

4 Imam Abdulrahman Bin Faisal University College of Dentistry, Dammam, Saudi Arabia.

The aim was to develop an economical and biocompatible collagen-based bioactive composite for tooth regeneration. Acid-soluble collagen was extracted and purified from fish scales. The design was innovated to molecularly tailor the surface charge sites of the nano-apatite providing chemical bonds with the collagen matrix via microwave irradiation technique. The obtained collagen was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The composites were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis/differential scanning calorimetry, and scanning electron microscopy. MC3T3-E1 cell lines were used to assess the biological effects of these materials by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra zolium bromide (MTT) assay. Indirect contact test was performed by extracting representative elutes in cell culture media and sulforhodamine B analysis was performed. Chorioallantoic membrane assay was conducted to define the new vessels formation behavior. The purity of collagen extracts was determined and showed two α-chains, i.e. the characteristic of type I collagen. Fourier transform infrared spectroscopy showed the characteristic peaks for amide I, I, III, and phosphate for collagen and composites. Scanning electron microscopy images showed three-dimensional mesh of collagen/apatite nano-fibers. Nontoxic behavior of composites was observed and there were graded and dose-related effects on experimental compounds. The angiogenesis and vessels formation behavior were observed in bioactive collagen composite. The obtained composites have potential to be used for tooth structure regeneration.
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http://dx.doi.org/10.1177/0885328218773220DOI Listing
July 2018

Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli.

Appl Biochem Biotechnol 2018 Sep 24;186(1):256-270. Epub 2018 Mar 24.

Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular Medicine, The University of Lahore, 1-km Defense Road, Bhobatian Chowk, Lahore, Pakistan.

Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.
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http://dx.doi.org/10.1007/s12010-018-2736-0DOI Listing
September 2018

Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in .

Biomed Res Int 2017 23;2017:9350537. Epub 2017 Nov 23.

Gulab Devi Educational Complex, Gulab Devi Chest Hospital, Ferozpur Road, Lahore, Pakistan.

Recombinant human Bone Morphogenetic Protein 2 (rhBMP2) has important applications in the spine fusion and ortho/maxillofacial surgeries. Here we first report the secretory expression of biological active dimerized rhBMP2 from system. The mature domain of BMP2 gene was amplified from pTz57R/BMP2 plasmid. By using pHT43 expression vector two constructs, pHT43-BMP2-M (single BMP2 gene) and pHT43-BMP2-D (two BMP2 genes coupled with a linker to produce a dimer), were designed. After primary cloning (DH5 strain) and sequence analysis, constructs were transformed into for secretory expression. Expression conditions like media (2xYT) and temperature (30°C) were optimized. Maximum 35% and 25% secretory expression of monomer (~13 kDa) and dimer (~25 kDa), respectively, were observed on SDS-PAGE in SCK6 strain. The expression and dimeric nature of rhBMP2 were confirmed by western blot and native PAGE analysis. For rhBMP2 purification, 200 ml culture supernatant was freeze dried to 10 ml and dialyzed (Tris-Cl, pH 8.5) and Fast Protein Liquid Chromatography (6 ml, Resource Q column) was performed. The rhBMP2 monomer and dimer were eluted at 0.9 M and 0.6 M NaCl, respectively. The alkaline phosphatase assay of rhBMP2 (0, 50, 100, 200, and 400 ng/ml) was analyzed on C2C12 cells and maximum 200 ng/ml activity was observed in dose dependent manner.
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http://dx.doi.org/10.1155/2017/9350537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733156PMC
August 2018

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

Biomed Res Int 2017 10;2017:5046451. Epub 2017 Aug 10.

Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore 54590, Pakistan.

A thaumatin-like protein gene from Basrai banana was cloned and expressed in . Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against ; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.
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http://dx.doi.org/10.1155/2017/5046451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569749PMC
July 2018

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.

Appl Microbiol Biotechnol 2015 Aug 28;99(16):6791-801. Epub 2015 Jun 28.

School of Biological Sciences, University of the Punjab, Lahore, Pakistan.

This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.
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http://dx.doi.org/10.1007/s00253-015-6751-6DOI Listing
August 2015