Surface modified of polyacrylonitrile nanofibers by TiO2/MWCNT for photodegradation of organic dyes and pharmaceutical drugs under visible light irradiation.

This work describes the fabrication of two composite nanofibers systems containing polyacrylonitrile polymer (PAN), Multiwall carbon nanotubes (MWCNT) and Titania (TiO2) nanoparticles. Photodegradation experiments were performed to study the effect of various parameters including pH, catalyst dose, pollutant concentration and reaction time for three model compounds, methylene blue (MB), indigo carmine (IC), and ibuprofen (IBU) under visible light. Morphology and structure of the modified composite nanofibers were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), Photoluminescence (PL) spectroscopy, Raman spectra, and X-ray Photoelectron Spectra (XPS) analyses. The photocatalytic performance was achieved in a rather short time visible light (<30 min) and under low power intensity (125 W) compared to earlier reports. Kinetics data fitted well using pseudo-first order model to describe the mechanism of photocatalytic degradation processes. The stability and flexibility of the fabricated composite nanofibers allow their application in a continuous flow system and their re-use after several cycles.

Composite nanofibers for highly efficient photocatalytic degradation of organic dyes from contaminated water.

In this study highly efficient photocatalyst based on composite nanofibers containing polyacrylonitrile (PAN), carbon nanotubes (CNT), and surface functionalized TiO2 nanoparticles was developed. The composite nanofibers were fabricated using electrospinning technique followed by chemical crosslinking. The surface modification and morphology changes of the fabricated composite nanofibers were examined through SEM, TEM, and FTIR analysis. The photocatalytic performance of the composite nanofibers for the degradation of model molecules, methylene blue and indigo carmine, under UV irradiation in aqueous solutions was investigated. The results demonstrated that high photodegradation efficiency was obtained in a short time and at low power intensity compared to other reported studies. The effective factors on the degradation of the dyes, such as the amount of catalyst, solution pH and irradiation time were investigated. The experimental kinetic data were fitted using pseudo-first order model. The effect of the composite nanofibers as individual components on the degradation efficiency of MB and IC was evaluated in order to understand the overall photodegradation mechanism. The results obtained showed that all the components possess significant effect on the photodegradation activity of the composite nanofibers. The stability studies demonstrated that the photodegradation efficiency can remain constant at the level of 99% after five consecutive cycles.

High performance of PAN/GO-ZnO composite nanofibers for photocatalytic degradation under visible irradiation.

In this work, Polyacrylonitrile nanofibers were enhanced by graphene oxide and zinc oxide, in terms of enhanced the photocatalytic activity and mechanical properties for the composite nanofibers. Photocatalytic degradation of two organic dyes methylene blue dye and indigo carmine dye from the simulated industrial wastewaters has been investigated under visible light irradiation. Composite nanofibers fabricated by using electrospinning technique followed by chemical cross-linking with zinc oxide. The surface sensitization and morphology changes of the fabricated composite nanofibers were studied using Fourier transform infrared spectroscopy analysis, Scanning Electron Microscopy and transmission electron microscope. Sensitized zinc oxide with graphene oxide showed higher efficiency in the degradation of MB with a 96% of degradation efficiency after 70 min and 98% for IC after merely 27 min. The optimum catalytic parameters such as initial organic dye concentration and pH were discussed. The efficacy of degradation of methylene blue dye and indigo carmine increased as initial concentration decreased. The optimal pH was around 5 where the reaction rate decreases above and under this pH value.

Removal of hexavalent chromium by electrospun PAN/GO decorated ZnO.

A novel composite nanofibers material have fabricated by using electrospinning technique followed by chemical cross-linking with zinc oxide (ZnO). The surface sensitization and morphology changes of the fabricated composite nanofibers were studied by using X-Ray Diffraction (XRD) analysis, Scanning Electron Microscopy (SEM) and transmission electron microscope (TEM). The effect of operating parameters includes the amount of ZnO, initial solution PH, and hexavalent chromium concentration on adsorption were investigated. The maximum adsorption capacity was found to be 690 mg/g at pH 6, which is much higher than most of the reported adsorbents. The adsorption equilibrium reached within 25 and 180 min as the initial solution concentration increased from 10 to 300 mg/L, and the data fitted well using nonlinear pseudo first order model with determination coefficient (R2) in between 0.97 and 0.99. Adsorption isotherms correlate the data on equilibrium adsorption with different mathematical models to describe the behaviour of an adsorption process and provide valuable information for optimizing the design of an adsorption system.

Surface functionalized composite nanofibers for efficient removal of arsenic from aqueous solutions.

A novel composites nanofiber was synthesized based on PAN-CNT/TiO2-NH2 nanofibers using electrospinning technique followed by chemical modification of TiO2 NPs. PAN-CNT/TiO2-NH2 nanofiber were characterized by XRD, FTIR, SEM, and TEM. The effects of various experimental parameters such as initial concentration, contact time, and solution pH on As removal were investigated. The maximum adsorption capacity at pH 2 for As(III) and As(V) is 251 mg/g and 249 mg/g, respectively, which is much higher than most of the reported adsorbents. The adsorption equilibrium reached within 20 and 60 min as the initial solution concentration increased from 10 to 100 mg/L, and the data fitted well using the linear and nonlinear pseudo first and second order model. Isotherm data fitted well to the linear and nonlinear Langmuir, Freundlich, and Redlich-Peterson isotherm adsorption model. Desorption results showed that the adsorption capacity can remain up to 70% after 5 times usage. This work provides a simple and an efficient method for removing arsenic from aqueous solution.

Removal of chromium (VI) from aqueous solutions using surface modified composite nanofibers.

A novel material composite nanofibers (PAN-CNT/TiO2-NH2) based on adsorption of Cr(VI) ions, was applied. Polyacrylonitrile (PAN) and carbon nanotube (CNTs)/titanium dioxide nanoparticles (TiO2) functionalized with amine groups (TiO2-NH2) composite nanofibers have been fabricated by electrospinning. The nanostructures and the formation process mechanism of the obtained PAN-CNT/TiO2-NH2 composite nanofibers are investigated using FTIR, XRD, XPS, SEM, and TEM. The composite nanofibers were used as a novel adsorbent for removing toxic chromium Cr(VI) in aqueous solution. The kinetic study, adsorption isotherm, pH effect, initial concentration, and thermodynamic study were investigated in batch experiments. The composite nanofibers had a positive effect on the absorption of Cr(VI) ions under neutral and acidic conditions, and the saturated adsorption reached the highest when pH was 2. The adsorption equilibrium reached within 30 and 180min with an initial solution concentration increasing from 10 to 300mg/L, and the process can be better described using nonlinear pseudo first than nonlinear pseudo second order model and Intra-particle diffusion. Isotherm data fitted well using linear and nonlinear Langmuir, Freundlich, Redlich-Peterson, and Temkin isotherm adsorption model. Thermodynamic study showed that the adsorption process is exothermic. The adsorption capacity can remain up to 80% after 5 times usage, which show good durability performance. The adsorption mechanism was also studied by UV-vis and XPS.

TGMV replication protein AL1 preferentially binds to single-stranded DNA from the common region.

The AL1 protein of tomato golden mosaic virus (TGMV) is encoded by the viral DNA and has been shown to be essential for viral DNA replication. We have over-expressed the AL1 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. Using various different techniques we have studied the interaction of the AL1 protein with DNA. The AL1 protein is able to bind to DNA containing the common region of the viral genome, which can be demonstrated by photochemical cross-linking. Binding is 4-fold stronger to single-stranded than to double-stranded DNA. Antibodies against the AL1 protein can be used to precipitate the protein-DNA complex. The binding to single- and double-stranded DNA is specifically to the common region since a DNA fragment unrelated to TGMV is not shifted in a gel retardation assay.

Successful triple immunoenzymatic method employing primary antibodies from same species and same immunoglobulin subclass.

Protocols for immunohistochemical (IHC) detection of multiple antigens in the same tissue sections have been developed using primary antibodies directly conjugated to different enzymes or fluorochromes, or ones that have been raised in different species, or from different immunoglobulin (Ig) classes or subclasses. For antibodies lacking such dissimilarities, very few proposals have been published with varying degrees of generalizability. In this report we present a successful triple IHC protocol engaging three unconjugated monoclonal primary antibodies raised in the same species and of the same Ig subclass. Compared to other methods, our results showed that denaturation of the preceding reaction complex by microwave heating, combined with additional suppression of enzyme activity, enabled the detection of all three reactions by using the same detection system, with no cross reaction observed. Moreover, expression patterns of each of the three antigens in the triple stained sections, was found to be similar to the pattern observed when single staining was performed. Unlike previous reports, no damage of targeted antigens or tissues did occur following this protocol. Furthermore, the contrast of the colors employed was investigated by computerized color deconvolution, and the three reactions products were successfully separated into three individual images that could be used for further objective quantification.

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus.

A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with 32P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg2+ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3' ends, but different 5' ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.

The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3.

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.

Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants.

The movement protein of red clover necrotic mosaic virus (RCNMV) was expressed in Escherichia coli as a fusion with a maltose-binding protein using the vector pMAL-cRI and used to produce an antiserum. The RCNMV movement protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the movement protein antiserum. The movement protein could be detected 6 h after inoculation and reached a maximum after 24 h. In contrast, the virus capsid protein, detected in a soluble fraction by immunoblotting using a capsid antiserum, continued to increase for 72 h after inoculation.

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.

Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus.

Mutant movement proteins of red clover necrotic mosaic dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were produced in Escherichia coli using expression vectors. The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retardation. The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain.

Role of the 3' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase In vitro.

A template-dependent RNA polymerase has been used to determine the sequence elements in the 3' untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3'-terminal 4 nucleotides of domain D1 indicated the importance of the 3'-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3' untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

Cooperative binding of the red clover necrotic mosaic virus movement protein to single-stranded nucleic acids.

The movement protein of red clover necrotic mosaic dianthovirus was produced in Escherichia coli using an expression vector. Gel retardation analysis and u.v. cross-linking studies showed that the movement protein bound cooperatively to ssRNA and ssDNA, but not to dsDNA. Binding competition experiments established that the movement protein bound to ssRNA and ssDNA with similar affinities and that the binding was not sequence-specific in the experimental conditions employed. A truncated movement protein lacking the C-terminal 88 amino acids was also shown to bind to ssRNA.

Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure.

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.

Nucleotide sequence of RNA 2 of a Czechoslovakian isolate of red clover necrotic mosaic virus.

The complete nucleotide sequence (1448 nucleotides) of RNA 2 of a Czechoslovakian isolate TpM-34 of red clover necrotic mosaic virus (RCNMV-TpM-34) has been determined. The sequence contained one major open reading frame (ORF) with the potential to encode a protein of 326 amino acids (Mr 35755), designated P2. The nucleotide sequence of RNA 2 of RCNMV-TpM-34 and the previously published sequence of RNA 2 of an Australian isolate of the virus (RCNMV-Aus) were 83% identical and there was 80% amino acid sequence identity between the P2 proteins of these isolates. However the N-terminal two-thirds of the P2 proteins shared a higher degree of similarity than the C-terminal regions which were predicted to have a more flexible structure. An ORF in the 3' portion of RNA 2 of RCNMV-Aus, which could encode a protein of Mr 5000, was not present in RNA 2 of RCNMV-TpM-34. RNAs 1 and 2 of RCNMV-TpM-34 and RCNMV-Aus are bilaterally compatible.

A spontaneous red clover necrotic mosaic virus mutant with a truncated movement protein.

A spontaneous red clover necrotic mosaic virus mutant, TpM-341, was isolated by multiple passage of Czechoslovakian isolate TpM-34 in beans, followed by three cycles of single lesion isolation in cowpea and in Chenopodium quinoa. The symptoms induced in cowpea by TpM-34 and TpM-341 differed. TpM-34 gave rise to chlorotic lesions which expanded with time, often becoming confluent with adjacent lesions, and developed necrotic margins; the plants became systemically infected. TpM-341 induced necrotic lesions which, once developed, did not expand further; plants did not become systemically infected. Analysis of pseudorecombinants formed between the RNAs of TpM-34 and TpM-341 showed that RNA 2 determined the difference in symptoms. Comparison of the nucleotide sequence of the open reading frames (ORFs) encoding the P2 movement proteins of the two isolates revealed only one difference, a deletion of an A residue in a sequence of four A residues (nucleotides 790 to 793). Construction of full-length TpM-34 and TpM-341 RNA 2 cDNA clones, from which infectious RNA 2 could be transcribed in vitro, and in vitro mutagenesis of a cDNA clone of TpM-341, confirmed that the difference in the symptoms induced by TpM-341 was caused by the loss of this A residue. This single base deletion was predicted to cause a translational frame-shift in the P2 ORF causing the loss of 88 amino acids at the C terminus which were replaced by a sequence of 34 different amino acids, producing a truncated P2 protein. In vitro translation of RNA 2 transcribed from cDNA clones showed that RNA with four or three A residues starting at nucleotide 790 produced proteins of Mr 36K and 30K respectively, in agreement with the predictions based on the nucleotide sequence.

Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates.

We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.