Size Exclusion Chromatography Analysis Research Articles

Fast and efficient preparation of three-arm star block copolymers via tetrazine ligation

The inverse Electron Demand Diels-Alder (IEDDA) reaction between tetrazine and trans-cyclooctenol (TCO) is a fast and effective method employed to connect two or more molecules. In this study, an efficient and fast methodology based on tetrazine/trans-cyclooctene IEDDA reaction was developed for preparation of star-shaped block copolymers. Three-arm star polylactide-b-poly(ethylene glycol) (PLA-b-PEG) was chosen as a model system to show efficiency of the approach. First, three-arm PLAs with two different molecular masses were synthesized in a typical ring opening polymerization and functionalized with tetrazine end functionalities. while TCO end functionalized PEGs in two different molecular masses were perpared. Exact stoichiometric IEDDA click reaction between tetrazine moieties on three ends of the star polymer (s-3PLA-Tz) in the core and trans-cyclooctene (TCO) moieties of the periphery polymer (PEG-TCO) was performed in high click efficiency (up to 95.8%) in a short period (after 50–75 min). Ligation of PEG was confirmed by UV–vis, NMR and FT-IR spectroscopies, and size exclusion chromatography (SEC) and differential scanning calorimetry (DSC) analyses. In this way, various copolymers of desired architectures can be prepared in a short time and in high efficiency.

Purification, thermodynamics and kinetic characterization of fungal endoinulinase for the production of fructooligosaccharides from inulin

Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher Km (0.90 mM), Vmax (19.60 mM/min·mg), Kcat (1.3 × 10−3/min) and Vmax/Km ratio (21.77/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0–7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50–70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl2 and MnSO4, while CuSO4, CoCl2, AgNO3, CdCl2, NiCl2, ZnSO4, BaCl2, HgCl2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin.

Molecular Dispersion of Starch as a Crucial Parameter during Size-Exclusion Chromatography.

Starch, α-polyglucan consisting of a large number of anhydroglucose units joined by α-1,4- and α-1,6-glycosidic bonds, seems to be characterized by a simple structure when compared to other natural polymers. Nevertheless, starches of various botanical origins have different physicochemical properties that are related to the differences in molecular and supramolecular structure of this polymer. In terms of the functional value of starch, the behavior of its macromolecules in solution is the most important result of its structural features. Extremely high molecular mass is the fundamental structural property of starch. Water, considered simply as a solvent for solubilization, does not provide molecular dispersion of starch without its degradation. The objectives of this study are to characterize the suitability of a new aqueous media (urea/NaOH) for enhancing the dispersion of native corn and potato starches and its effect on the consequent size-exclusion chromatography (SEC) analysis. The results were referred to other aqueous base solvents used for dispersing starch (NaOH and KOH). The samples were separated using SEC with triple detection and phosphate buffer (pH 8.0) with urea as the eluent. The characteristics of tested normal and waxy starches were compared. The results revealed that urea/NaOH did not degrade starch during the dispersion process. The recovery of starches, however, was not higher than 42%. These results prove that while the urea/NaOH solvent allows to obtain cold-water-soluble starch, the degree of disintegration of the intramolecular interactions of amylopectin chains is still insufficient.

Evaluation of adhesive performance of a mixture of soy, sorghum and canola proteins

Soy protein has been the most widely researched protein-based adhesive; however, soy protein remains an expensive product because of its competitive uses in both food and feed industries. To mitigate such competition, this project was aimed to investigate protein composites by mixing low value sorghum and canola proteins into soy proteins. In addition to cost reduction, a novel composite with optimal composition of three protein were identified showing superior adhesion properties. For example, compared to soy protein alone, the optimum protein mixture (sorghum:canola:soy = 3:2:5) had a 63 % increase in dry adhesion strength, 103 % increase in wet adhesion strength, and 61 % increase in soaked adhesion strength. The physiochemical properties of different protein samples were characterized using rheometry, Fourier transform infrared spectroscopy, size exclusion chromatography, SDS-PAGE and surface hydrophobicity analysis. Protein analysis showed that the improved adhesive performance was likely due to optimizing protein molecular weight distribution and hydrophobicity. The present study demonstrates a simple, straight forward approach to improve soy protein-based adhesion performance and also reduce the overall cost of the plant protein-based composites as adhesives.

The phytopathogen Xanthomonas campestris utilizes the divergently transcribed pobA/pobR locus for 4-hydroxybenzoic acid recognition and degradation to promote virulence.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the β-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.

Sequence-Controlled Synthesis of Advanced Clickable Synthetic Oligonucleotides.

Through thiol-ene photopolymerization of presynthesized oligomers, advanced clickable nucleic acids (CNA-2G) are synthesized with sequence-controlled repeating units. As examples, poly(thymine-adenine) (polyTA) CNA-2G and poly(thymine-thymine-cytosine) CNA-2G are synthesized by polymerizing thiol-ene heterofunctional dimers with pendant thymine-adenine nucleobases and trimer with pendant thymine-thymine-cytosine nucleobases. Based on size exclusion chromatography (SEC) analysis, polyTA and polyTTC have number average molecular weights of 2000 and 1800, respectively, which contain 7-8 pendant nucleobases. Based on the different behavior of the CNA-2G monomers and CNA-2G oligomers with two or more pendant nucleobases in photopolymerization, an unusual thiol-ene chain-growth propagation mechanism is observed for the former and a common thiol-ene step-growth propagation mechanism for the latter. The uncommon thiol-ene chain-growth propagation is hypothesized to rely on a six-membered ring mediated intramolecular hydrogen atom transfer process.

Influence of molecular structure on the rheological properties and foamability of long chain branched polypropylene by “one-pot” reactive extrusion

In this work, reactive twin screw extrusion was conducted to synthesize long chain branched polypropylenes (LCB-PPs) in a “one-pot” process in which dicumyl peroxide (DCP) initiated maleic anhydride (MAH) grafting onto the linear PP, and the concomitant coupling reaction between ethylene diamine (EDA) and MAH grafted polypropylene (PP-g-MAH) proceeded in series. Fourier transfer infrared spectroscopy (FTIR) on the prepared materials confirmed the occurrence of both reactions. A series of LCB-PPs were prepared using different amounts of EDA, MAH and DCP to study their effects and determine the optimal synthesis conditions. The prepared materials were characterized by size exclusion chromatography (SEC) and rheological analysis to ascertain the polymer microstructure. The foamability of the LCB-PPs by supercritical carbon dioxide (scCO2) foaming and foam morphology were investigated. The LCB-PPs were found to have vastly improved foamability and cellular morphology. Under optimal conditions, a foam expansion ratio of over 20 was achieved.

Development of aqueous size exclusion chromatography conditions to characterize polyzwitterion-block-N-isopropyl acrylamide copolymers.

Block copolymers that exhibit both an upper critical solution temperature and a lower critical solution temperature are difficult to characterize due to inherent solubility difference between the two blocks. For example, accurate determination of both the molar mass and molar mass distribution is challenging for polyzwitterion-block-N-isopropyl acrylamide (NIPAM) copolymers in aqueous solutions due to self-assembly. However, there are a few examples of using size exclusion chromatography (SEC) for characterization, in which hexafluoro isopropanol (HFIP) is used in all cases. Yet, researchers are hesitant to use this solvent due to how expensive and hazardous HFIP is. Therefore, alternatives to HFIP for SEC analysis would be desirable. Here, a systematic methodology featuring aqueous SEC is demonstrated using several solvent conditions to enable the elution of polyzwitterion-block-NIPAM copolymers on Agilent PolarGel† and Tosoh TSKgel† column sets. These SEC conditions include 0.2M KI in water on the PolarGel columns and 0.2M KI/ 30% DMF in water on the PolarGel and TSKgel columns. These aqueous systems can be utilized for the characterization of similar water-soluble block copolymers that are relevant for drug delivery and other biomedical applications.

ASXL3 bridges BRD4 to BAP1 complex and governs enhancer activity in small cell lung cancer

BackgroundSmall cell lung cancer (SCLC) is a more aggressive subtype of lung cancer that often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. Consequently, such phenotypical characteristics of SCLC set limitations on viable procedural options, making it difficult to develop both screenings and effective treatments. In this study, we examine a novel mechanistic insight in SCLC cells that could potentially provide a more sensitive therapeutic alternative for SCLC patients.MethodsBiochemistry studies, including size exclusion chromatography, mass spectrometry, and western blot analysis, were conducted to determine the protein-protein interaction between additional sex combs-like protein 3 (ASXL3) and bromodomain-containing protein 4 (BRD4). Genomic studies, including chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing, and genome-wide analysis, were performed in both human and mouse SCLC cells to determine the dynamic relationship between BRD4/ASXL3/BAP1 epigenetic axis in chromatin binding and its effects on transcriptional activity.ResultsWe report a critical link between BAP1 complex and BRD4, which is bridged by the physical interaction between ASXL3 and BRD4 in an SCLC subtype (SCLC-A), which expresses a high level of ASCL1. We further showed that ASXL3 functions as an adaptor protein, which directly interacts with BRD4’s extra-terminal (ET) domain via a novel BRD4 binding motif (BBM), and maintains chromatin occupancy of BRD4 to active enhancers. Genetic depletion of ASXL3 results in a genome-wide reduction of histone H3K27Ac levels and BRD4-dependent gene expression in SCLC. Pharmacologically induced inhibition with BET-specific chemical degrader (dBET6) selectively inhibits cell proliferation of a subtype of SCLC that is characterized with high expression of ASXL3.ConclusionsCollectively, this study provides a mechanistic insight into the oncogenic function of BRD4/ASXL3/BAP1 epigenetic axis at active chromatin enhancers in SCLC-A subtype, as well as a potential new therapeutic option that could become more effective in treating SCLC patients with a biomarker of ASXL3-highly expressed SCLC cells.

The interaction of the mitochondrial protein importer TOMM34 with HSP70 is regulated by TOMM34 phosphorylation and binding to 14-3-3 adaptors

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

Acidic Versus Alkaline Bacterial Degradation of Lignin Through Engineered Strain E. coli BL21(Lacc): Exploring the Differences in Chemical Structure, Morphology, and Degradation Products.

There is increasing interest in research on lignin biodegradation compounds as potential building blocks in applications related to renewable products. More attention is necessary to evaluate the effects of the initial pH conditions during the bacterial degradation of lignin. In this study we performed experiments on lignin biodegradation under acidic and mild alkaline conditions. For acidic biodegradation, lignin was chemically pretreated with hydrogen peroxide. Alkaline biodegradation was achieved by developing the bacterial growth on Luria and Bertani medium with alkali lignin as the sole carbon source. The mutant strain Escherichia coli BL21(Lacc) was used to carry out lignin biodegradation over 10 days of incubation. Results demonstrated that under acidic conditions there was a predominance of aliphatic compounds of the C3–C4 type. Alkaline biodegradation was produced in the context of oxidative stress, with a greater abundance of aryl compounds. The final pH values of acidic and alkaline biodegradation of lignin were 2.53 and 7.90, respectively. The results of the gas chromatography mass spectrometry analysis detected compounds such as crotonic acid, lactic acid and 3-hydroxybutanoic acid for acidic conditions, with potential applications for adhesives and polymer precursors. Under alkaline conditions, detected compounds included 2-phenylethanol and dehydroabietic acid, with potential applications for perfumery and anti tumor/anti-inflammatory medications. Size-exclusion chromatography analysis showed that the weight-average molecular weight of the alkaline biodegraded lignin increased by 6.75-fold compared to the acidic method, resulting in a repolymerization of its molecular structure. Lignin repolymerization coincided with an increase in the relative abundance of dehydroabietic acid and isovanillyl alcohol, from 2.70 and 3.96% on day zero to 13.43 and 10.26% on 10th day. The results of the Fourier-transformed Infrared spectroscopy detected the presence of C = O bond and OH functional group associated with carboxylic acids in the acidic method. In the alkaline method there was a greater preponderance of signals related to skeletal aromatic structures, the amine functional group and the C – O – bond. Lignin biodegradation products from E. coli BL21(Lacc), under different initial pH conditions, demonstrated a promising potential to enlarge the spectrum of renewable products for biorefinery activities.

Effect of the aging temperature on soybean biodiesel oxidation, quantifying the formed gums and sediments by thermogravimetry

Biodiesel has been widely used as an alternative fuel in diesel engines. However, although its physicochemical properties make it a substitute for traditional diesel fuel, biodiesel is more susceptible to oxidation and degradation during storage than diesel. The high content of unsaturated fatty acid methyl (or ethyl) esters in biodiesel causes its fast oxidative degradation when in contact with precursor agents as oxygen. These reaction products are highly unstable, resulting in a mixture of different species, some of them of high molecular weight, producing sediments and undesirable polymeric compounds. In this study, a sample of commercial soybean biodiesel (BS100) was aged at 50 °C, 110 °C, 150 °C and 200 °C in a Rancimat apparatus. This was performed bubbling air into the sample at 10 L h−1 flow rate for 5 h. The original biodiesel and the aged samples were analyzed by thermogravimetry (TG/DTG), in dynamic mode, under nitrogen atmosphere. Data were compared with those obtained from the analyses performed by size-exclusion chromatography (SEC). Fourier transform infrared spectroscopy (FTIR) analyses were also performed. The objective of this work was to evaluate the effect of the aging temperature on the oxidation of biodiesel and on the degraded product composition, as well as to quantify the formed gums and sediments. The thermogravimetric analysis shows that biodiesel samples aged at 150 °C and 200 °C have a higher content of heavier products (gum), 33.4% and 44.9%, respectively, than the non-aged biodiesel (2.8%). The SEC analysis indicates molecular weight value of 283, 1241 and 1380 g mol−1 for samples aged at 150 °C, 200 °C and for the non-aged sample, respectively, which confirms the results of the TG/DTG analysis. The widening of ester carbonyl stretching band (C=O, 1740 cm−1) in the FTIR spectra, as the aging temperature rises, confirms the degradation of biodiesel into acids, ketones and other compounds containing this functional group. The decrease in percentage transmittance in the region corresponding to the fingerprint (1500–500 cm−1) indicates that heavy products (gums and sediments) are produced. These results indicate that the degradation product content in the aged biodiesel is strongly dependent on the temperature of the aging process.

Selenosugars Are the Major Selenometabolites in Liver of Turkeys Fed High Selenium

Abstract Objectives Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. Because the nature of this accumulated Se is not well characterized, we studied selenometabolomics in Se-deficient, Se-adequate, and high-Se liver. Methods Turkey poults were fed 0, 0.4, and 5 μg Se/g diet as Na2SeO3 in a Se-deficient (0.005 μg Se/g) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. Results In liver from turkeys fed a true Se-deficient diet and supplemented with inorganic Se (selenite), no selenomethionine (SeMet) was detected showing that the turkey cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient turkey liver, as expected in animals with negligible selenoprotein levels. Sec content in liver of turkeys fed high Se only doubled compared with Se-adequate liver, indicating that the 6-fold incryscease in liver Se in these birds was not due to increases in selenoproteins. What increased dramatically in high Se liver were the low molecular weight (MW) selenometabolites, glutathione-, cysteine- and methyl- conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). In addition, size-exclusion chromatography and followup analysis demonstrated that a substantial amount of Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide links, in addition to Sec-containing selenoproteins. In high-Se liver, these “selenosugar-decorated” proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to the low MW selenometabolites. Conclusions In Se-adequate liver, far more Se is present as the selenosugar moiety, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols, leading to the formation of methyl-SeGalNac. This suggested pathway, underlying adaptation to high Se status in animals, needs further investigation including study of the potential of selenosugar compounds as biomarkers of high Se status. Funding Sources USDA, Hatch project 1,004,389, and by the Wisconsin Alumni Foundation, Selenium Nutrition Research Fund 12,046,295.

Pilot study on nanofiltration membrane in advanced treatment of drinking water

Abstract In this study, nanofiltration (NF) was used to further treat the water from the disinfection process of a drinking water treatment plant (DWTP). The results indicate that the NF process reduced the dissolved organic carbon (DOC) by 92.87%, UV absorbance at 254 nm (UVA254) by 88.68%, conductivity by 94% and total dissolved solids by 94.49%. The removal efficiencies of trihalomethanes (THMs), 2-methylisoborneol (2-MIB) and trans-1,10-dimethyl-trans-9-decalol (geosmin) were also evaluated for both the DWTP and NF treatment processes. High-performance size-exclusion chromatography (HPSEC) analysis, three-dimensional excitation–emission matrix (3D-EEM) fluorescence spectroscopy and parallel factor (PARAFAC) analysis were applied to characterize the changes in different organic compounds in each process. A correlation analysis was carried out for samples of the raw water, feed water and permeate water to determine the correlations between the maximum fluorescence intensities (Fmax) of the PARAFAC components and the DOC, UVA254 and high specific UV absorbance (SUVA) of the samples. The results showed that a significant correlation (p < 0.01) existed between UVA254 and the Fmax of the three PARAFAC components as well as between DOC and Fmax.

Amylose content and specific fine structures affect lamellar structure and digestibility of maize starches

The molecular and lamellar structures and in vitro digestibility of high amylose maize starch granules were investigated. Size-exclusion chromatography (SEC) analysis of hydrodynamic radius (Rh) and relative contents (RC) of different amylose and amylopectin pools revealed that increased amylose content resulted in the decrease of RC of short amylopectin chains (AP1, DP < 36), and the increase of Rh and RC of long amylopectin chains (AP2, DP > 36). Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis showed the average chain lengths of defined structures of fa, fb1, and fb3, and the content of fb3 were increased with the amylose content and at AC content of 43%, the chain lengths decreased with the amylose content. The relative contents of fa and fb1and the degree of branching had the converse trends. Hence, HA-2, having an AC content of 43%, was the tipping point for these structural changes. Small angle X-ray scattering (SAXS) and its normalized 1D correlation function and SAXS scattering curve parametric fitting showed that the crystalline lamellar thickness was negatively correlated with proportion of short amylose chains (AM1, 100 <DP ≤ 150). The Bragg lamellar repeat distance was positively correlated to Rh of the intermediate amylose chains (AM2, 150< DP ≤ 800). The amorphous lamellae thickness was positively correlated with the Rh of long amylose chains (AM3, 800 <DP ≤ 40,000). The positive correlation between Rh of AM2 on the Gaussian peak area and the scattering invariant suggests AM2 chains stabilize the lamellar structure. Both the digestion rate of the digestible starch as well as the resistant starch content were positively correlated with amylose content. Pearson correlation analysis demonstrated that longer amylopectin chains and lower content of AP1 chains were positively correlated with the digestion rate.

Isolation, characterisation and application of a new antifungal protein from broccoli seeds – New food preservative with great potential

The worldwide problem of food loss and recent outbreaks of food-related diseases caused by microbial contamination has highlighted the urgent need of efficient preservation methods. Plants of Brassicaceae familyare particularly rich in compounds with antiviral, antimicrobial and anticancer properties. Herein, a water-soluble extract of broccoli (Brassica oleracea var. italica) seeds showed strong antifungal activity against fungal food contaminants. A 15-kDa polypeptide was selectively extracted with ammonium sulphate precipitation, ion-exchange and size exclusion chromatography Circular dichroism spectroscopy and analysis of broccoli seed transcriptome revealed high similarity to the previously isolated rapessed storage protein napins. This paper is the first report of the isolation and characterization of napin-like proteins from broccoli seedsIn addition, apH-dependent approach was used to increase the yield of extraction with a minimal use of chemicals. The potential of the broccoli napin, termed BoNap, as a novel antifungal agent was shown in in vitro assays against the spores of several fungal contaminants. BoNap prevented the germination of spores of Fusarium culmorum and Penicillium expansum, with a MIC value of 2.31 μM. In addition, the napin-like protein presented an arsenal of mechanisms for the protection of the seeds, including permeabilization of mycelium membrane and trypsin-inhibitory properties. Essential characteristics of heat stability and non-cytotoxicity towards mammalian cells highlight the promises of this compound in the area of food safety and crop protection. As shown here with a successful reduction of fungal contamination of cereals, the discovery of a readily extractable plant protein could contribute to its application as a natural stable preservative and to limit the global issue of food waste.

Tetrazole functional copolymers: Facile access to well-defined Rhenium(I)-Polymeric luminescent materials

Well-defined random copolymers containing tetrazole functional groups were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) radical homopolymerisation of pentafluorophenyl acrylate (PFPA), end-group modification and post-polymerisation modification of the activated ester side-groups with primary amines. PolyPFPA with a size exclusion chromatography (SEC) measured Mn of 10,800 and dispersity, Ð, of 1.05 was prepared with 2-cyano-2-propyl benzodithioate as the RAFT chain transfer agent and 2,2′-azobisisobutyronitrile (AIBN) as the source of primary radicals in 1,4-dioxane. Prior to modification of the pentafluorophenyl (PFP) ester side-groups, the thiocarbonylthio end-group was removed via a radical-mediated desulfurisation employing two equivalents of AIBN. Complete removal of the end-group was successful as judged by UV–vis spectroscopy with no effect on the molecular weight distribution of the parent homopolymer based on SEC analysis. The pendent PFP ester groups were subsequently modified in a sequential manner with n-butylamine and 5-aminotetrazole yielding the corresponding random amide species quantitatively, as judged by 19F NMR and FTIR spectroscopies, and of varying molar composition as determined by 1H NMR spectroscopy. The reaction of the tetrazole functional copolymers with Re(CO)3(phen)Br (phen = 1,10-phenanthroline) yielded the corresponding rhenium-polymer hybrids via coordination through the pendent tetrazole functional group. Successful, and quantitative coordination was confirmed by FTIR spectroscopy and detailed photophysical studies. In the case of the latter, the absorption profiles of all hybrid materials possessed broad bands centred around ca. 370 nm associated with metal-to-ligand charge transfer (MLCT) transitions; excitation as this wavelength results in a broad emission centred at 606 nm for all hybrid species and is due to emission from the triplet MLCT excited states.

Expression, purification and oligomerization of the S-adenosylmethionine transporter

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.

The Structural Characterization of AtLaRP6a C‐Terminal Domain

The La related proteins (LaRPs) are a superfamily of RNA binding proteins characterized by a conserved sequence called the La Module. This domain is composed of two distinct regions, the La Motif (LAM) and an RNA recognition motif (RRM), which form a functional RNA binding unit. The LaRP6 subfamily is characterized by a putative protein‐protein interaction motif in the C‐terminus, called the LAM‐ and S1 associated (LSA) motif. Interestingly, plants express three paralogs of LaRP6 (A, B, and C). We have recombinantly expressed and purified the LaRP6A protein from the plant model organism Arabidopsis thaliana, to compare the plant LaRP6A to the LaRP6 proteins of vertebrate organisms for the purpose of probing differences in structure and function. Previous work in our lab suggested that the C‐terminal domain of AtLaRP6A is intrinsically disordered. In this study, we have recombinantly expressed and purified only the C‐terminal domain. Biochemical and biophysical analyses, including analytical size exclusion chromatography, limited proteolysis, and circular dichroism, all support the prediction that the CTD is an intrinsically disordered region. This work provides a foundation to further study the role of the CTD in regulatory protein‐protein interactions to better understand the biological role for LaRP6.

Synthesis, structure analysis, investigation of conductivity, thermal properties of polyphenol derivatives containing a rhodanine moiety and their Cu(II), VO(IV) complexes

In this study, 5-benzylidenerhodanine derivatives namely 5-(2-hydroxybenzylidene)-2-thioxothiazolidin-4-one (SAR) and 5-((2-hydroxynaphthalen-1-yl)methylene)-2-thioxothiazolidin-4-one (HNAR) used as monomer. The new polymers of SAR and HNAR (PSAR and PHNAR respectively) were synthesized by oxidative polycondensation reaction. The polymers were characterized by elemental, FT-IR, UV–Vis, 1H NMR, 13C NMR, TG-DTA analyses techniques. The molecular weight distribution values of the PSAR and PHNAR were determined by size exclusion chromatography (SEC) analysis. According to the SEC analyses, the number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) values of PSAR and PHNAR were found to be 5300, 7250 g mol−1 and 1.368; 5750, 7350 g mol−1 and 1.278, respectively. The Cu (II), VO (IV) complexes of PSAR and PHNAR (PSAR-Cu and PSAR-VO) were successfully prepared using Cu (AcO)2xH2O and VOSO4x5H2O hydrate salts. The polymer-metal-complexes were characterized by FT-IR, elemental and TG-DTA analysis techniques. The weight losses of PHNAR, PHNAR-Cu, PHNAR-VO, PSAR, PSAR-Cu and PSAR-VO were found to be 69.50, 58.50, 62.25, 89.05, 74.55 and 61.75% at 1000 °C, respectively. TG analyses were showed to be stable of synthesized polymers against thermal decomposition. Electrical properties of doped polymers, undoped polymers and their complexes were determined. Also, the optical band gaps (Eg) of these complexes were calculated from their absorption edges.