8M Urea Research Articles

Eco-Friendly Spectrophotometric Estimation of Gliclazide using Hydrotropic Solubilization Technique

In the projected analysis work, a successful; attempt was made to develop simple, accurate, novel, ecofriendly and precise technique for the estimation of poorly water soluble drug Gliclazide in single drug containing dosage form. In the present investigation, a mixed hydrotropic blend of 2M urea and 6M trisodium citrate was used for quantitative determination. Gliclazide showed λ-max at 225 nm and beer's law was obeyed with in the concentration range of 4μg/ml-12μg/ml with mean recovery ranging from 95.33% to 96.35%. The developed technique was validated as per ICH guidelines.

Expression and purification of four truncated 56 kDa antigens from Orientia tsutsugamushi in Escherichia coli

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to humans by the bite of the larva of trombiculid mites. Diagnosis of scrub typhus is normally based on the clinical presentations. However, it is difficult to differentiate scrub typhus from other acute febrile illnesses, such as dengue fever, malaria and leptospirosis due to similar symptoms. For differential diagnosis of scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. In order to produce an ELISA kit for detection of antibodies against O. tsutsugamushi in Vietnam, four truncated 56 kDa antigenic genes of O. tsutsugamushi including Karp (HT-09), Gilliam (HT-11), TA763 (HT-49), and Kato (YB-50) íolated from the most prevalent cases in Vietnam were cloned and expressed in E. coli Rosetta 1 cells. The recombinant proteins formed inclusion bodies when expressed in E. coli. The recombinant 56 kDa proteins in insoluble form were solubilized in 6M urea and were successfully purified by Ni2+affinity column. The purity of four recombinant proteins,HT-09, HT-11, HT-49 and YB-50,reached more than 95% and their concentrations are 12,57 mg/ml; 11,6 mg/ml; 8,98 mg/ml và 8,02 mg/ml, respectively.

Synergistic Dual Inhibition of BCR-ABL1 and the Unfolded Protein Response Causes p38 MAPK-Mediated Cell Death and Sensitizes BCR-ABL1+ Acute Lymphoblastic Leukemia to Dexamethasone

Abstract Introduction The IRE1-XBP1 branch of Unfolded Protein Response (UPR) was previously shown to be pivotal for the survival of BCR-ABL1+ Acute Lymphoblastic Leukemia (ALL) cells. In this study, we identified a link between the IRE1 and BCR-ABL1 pathways which we harnessed for the pre-clinical application of a novel IRE1 inhibitor, MKC-8866, in combination with Nilotinib. The underlying mechanism was investigated by analysis of the phosphoproteome of BCR-ABL1+ ALL cell lines SUP-B15 and TOM-1. Upon dual treatment, the main force driving the cells toward death was ascribed to the activation of p38 MAPK. Additionally, the set of proteins affected under dual therapy suggested a possible regulation of glucorticoid receptor (GR) signaling that could modify the response to dexamethasone. Methods and Results SUP-B15 and TOM-1 cell lines were treated with vehicle, MKC-8866 (MKC, 30 µM) and nilotinib (NL, 0.5 µM) as single agents or in combination. Viability assays were performed in order to verify the synergistic effect of the treatment administered, evaluated using the Bliss formula. The combination of NL with MKC showed a striking synergism in both SUP-B15 and TOM-1 (Bliss calculation. Assumed additive effects: SUP-B15 24.2±20.6%, TOM-1 40.9±18.4% versus experimental values: SUP-B15 68.7±12.7%, TOM-1 78.8±4.9%). To validate the effect on a genetic level, pre-B cell derived from conditional Xbp1+/fl mice were transduced with BCR-ABL1and with either inducible cre or empty vector. The ALL-like cells were then treated with NL 0.5 µM and/or 4-hydroxytamoxifen (4OHT) 1µM to induce deletion. In agreement with the synergistic effect observed with the human cell lines, NL was shown to be significantly more effective in the presence of heterozygous deletion of Xbp1 (viable cells after 24 hours. NL: 65.2±0.3%, 4OHT + NL: 6.87±1.2%). To unravel the basis of this synergism, we performed a phosphoproteomic analysis of the two human cell lines treated for 16 hours with vehicle, MKC, NL and dual treatment, respectively. For this analysis, SUP-B15 and TOM-1 were lysed in 8M urea and digested using LysC/trypsin. The obtained peptides were labeled using the dimethyl isotope labeling method. Phosphopeptides were enriched using TiO2 beads and analyzed by nanoLC-MS/MS (Orbitrap Elite). The raw data were analyzed using MaxQuant and the Andromeda search engine against the human Uniprot database. The resulting information was further interpreted using the Perseus software package as well as the String and PhosphoPath apps in Cytoscape. The analysis of the regulated phosphorylated proteins highlighted a p38 MAPK differential regulation, with MKC alone or in combination causing its activation, and, in contrast, NL causing inhibition. Western Blot analyses investigating the activation of p38 MAPK showed increased phosphorylation of its main downstream target, heat shock protein beta-1 (HSPB1), in the presence of MKC alone or together with NL. Pharmacological inhibition of p38 MAPK activity by adding p38 MAPK inhibitor BIRB-796 (BIRB, 10µM) was able to rescue the cells from the concerted action induced by MKC and NL. Furthermore, the phosphoproteome pattern observed in the cells upon dual treatment suggested a regulation of crucial GR interactors. Therefore, we tested whether the studied combination would render the cells more responsive to the GR agonist dexamethasone (DEXA, 10 mg/L), cornerstone in ALL therapy. The viability assay revealed a striking enhancement of its cytotoxic effect. However, most of the effect was conveyed by the action of DEXA together with NL, with MKC adding only a small and non-significant accessory effect (percentages of viable cells compared to DMSO values after 24 hours treatment. SUP-B15: DEXA 83.9±4.2%; MKC + DEXA 81.6±5.1%; NL + DEXA 66.7±7.1% and MKC + NL + DEXA 59.8±7.3%. TOM-1: DEXA 78.2±8.4%; MKC + DEXA 74.1±5.1%; NL + DEXA 45.3±10.1% and MKC + NL + DEXA 45.2±8.1%). Conclusion By studying the phosphoproteome in BCR-ABL1+ ALL, we identified a possible underlying mechanism explaining the success of the combined action of NL and MKC-8866. The p38 MAPK axis was found responsible for the observed cytotoxic effect, elucidating important aspects of BCR-ABL1+ ALL biology. Overall, the successful treatment regime with NL and MKC in vitro, and its positive interaction with DEXA, provide a promising basis for further pre-clinical evaluation. Disclosures Patterson: Fosun Orinove PharmaTech, Inc.: Employment. Samali:Cell Stress Discoveries Ltd.: Other: co-founder, director and share holder . Brümmendorf:Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Research Funding; Takeda: Consultancy. Appelmann:Novartis: Research Funding. Kharabi Masouleh:Janssen Research & Development: Employment.

Extremely stable indole-3-glycerol-phosphate synthase from hyperthermophilic archaeon Pyrococcus furiosus.

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100°C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent Vmax and Km values of 20 ± 0.5μmolmin-1 mg-1 and 140 ± 10µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5kJmol-1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8M urea or 5M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200min at 100°C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7M urea with 38% α-helix and 14% β-sheet in solution and a molecular weight of 40.5kD. Tv-PSP1 models were used to perform dynamic simulations over 100ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity.

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea+1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95kDa) but also few dozens of peptides with molecular masses from 2 to 9.5kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.

Abstract 5660: Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies

Abstract Proteomic analysis is an important tool in classifying molecular architecture of tissue samples for subtyping and precision oncology applications. Tissues are commonly either flash frozen (FF) or embedded in optimal cutting temperature (OCT) compound for long-term storage. OCT provides a convenient specimen matrix for cryostat sectioning but contains glycols and resins which are incompatible with mass spectrometric analysis of proteins. In contrast, flash frozen preservation of tissue is historically considered to be more suitable and compatible with mass spectrometry applications. As a pilot study, we evaluated different tissue preservation methods, sample processing methods, and protein extraction methods, and their impact on quantitation and coverage of the proteome. In this pilot study, 30 breast cancer cases with matching flash frozen and OCT-embedded samples were used for proteomic analysis. Tissues were procured from the Clinical Breast Care Project (CBCP) in partnership with the Chan Soon-Shiong Institute of Molecular Medicine at Windber, and the Walter Reed National Military Medical Center (WRNMMC). Breast cancer subtypes were determined by clinical and expanded immunohistochemical panels. These subtypes were Her2, Luminal A (LA), Luminal B (LB) and Triple Negative (TN). Flash frozen tissues were cryosectioned to isolate 60-100 mg total tissue per case, and proteins were extracted using an 8M urea lysis buffer. The OCT specimens were laser microdissected to isolate primarily tumor cells from the samples. Proteins were extracted using the illustra TriplePrep kit. Digested proteins were then multiplexed using Tandem-Mass-Tag (TMT) 10plex isobaric labeling reagents, and chromatographically resolved using a Waters 2D nanoAcquity liquid chromatographer (LC) coupled to a Thermo Q Exactive Plus mass spectrometer (MS). Data was then analyzed using Proteome Discoverer. In total, 6130 proteins were identified using a 1% peptide FDR confidence filter in the 30 samples analyzed. Unsupervised hierarchical clustering of proteins across the samples resulted in two primary clusters, OCT and FF. Within the primary clustering, there were sub-clusters of Luminal, Her2, and Triple Negatives. At 2-fold threshold, 246 proteins were differentially expressed between OCT and FF tissues. However, the differential proteins between ER+ (LA and LB) and ER- (Her2 and TN) tumors correlate irrespective of the cryopreservation and processing methods. In conclusion, proteome analysis of samples stored under two different conditions provide subtyping information; however, there is differential expression of proteins between the two types of sample handling techniques which can impact biological interpretation of breast cancer precision oncology studies. Citation Format: Kiki Panagopoulos, Punit Shah, Albert Kovatich, Michael A. Kiebish, Jeffrey Hooke, Leigh Campbell, Mary Lou Cutler, Audrey Kovatich, Hai Hu, Brenda Deyarmin, Lori Sturtz, Praveen Kumar Raj Kumar, Stella Somiari, Emily Y. Chen, Elder Granger, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Niven Narain, Craig D. Shriver. Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5660.

DNA binding protein inhibitor ID2 in 8M urea pH2.3

DNA binding protein inhibitor ID2 in 8M urea pH2.3

Non-Enzyme Urea Sensing with Ag Covered ZnO with Different Morphologies on Carbon Papers

Urea is the nitrogenous waste substance of metabolism in the human body, and abnormal urea levels are associated with a variety of diseases such as renal failure, dehydration, and hepatic issues. For example, urea production and renal processing of urea, along with the causes of increased and reduced plasma/serum urea concentration are a major concern in physiological topic [1]. Thus, urea from the human urine and measurement of its concentration have been investigated for clinical application in the analysis of organic function [1-2]. Moreover, urea has attracted attention as a hydrogen source for fuel cells because of its stability, non-toxicity, and non-flammable properties [3]. Recently, many studies on non-enzyme urea sensing have been conducted due to the stability problems of enzymes. Non-enzymatic materials, e.g. Ni-based catalysts with peculiar structure [4] and 2-D graphene [5], have shown promising properties in the field of electrochemical sensors because of their unique properties such as remarkable surface area, good conductivity, and wide electrochemical window. Among them, approaches to high active surface areas are important in addition to exploration of various catalysts such as metal oxides, noble metals, and nickel-based catalysts due to its relation to the electro-catalytic activity towards urea oxidations. High surface area of the catalyst is critical for an excellent sensitivity, a low detection limit and a fast response time [6]. Although previous attempts have been made to have a high surface area for the urea detection, there is limited range of pH condition and Ni nanostructures for oxidation. As a result, ZnO or semiconducting metal oxide with a large surface to volume ratio and good crystallinity at nanoscale can be a good candidate for urea sensing [7]. In this work, covered Ag catalysts deposited on synthesized ZnO nanorods and nanoflakes on carbon substrates were prepared using low-temperature process for systematic investigation of surface area effect on non-enzymatic catalytic urea oxidation. The morphologies and structural properties of ZnO nanorods and nanoflakes with covered Ag were analyzed by SEM and XRD, and the quantity of the silver catalyst was measured by EDS. Thereafter electrochemical measurements of two catalysts have been conducted on a three-electrode cell in 1M urea containing 0.33 M urea. Electrochemical impedance spectroscopy (EIS) was also investigated for comparing the electron-transfer resistance. The surface effects of Ag/ZnO nanorods and nanoflakes electrodes for urea sensing were studied in terms of sensitivity and selectivity. Detailed discussion on the comparison of Ag/ZnO nanorods and nanoflakes surface effect will be given. References Thudichum, J. L. W. "On the analysis of urea in urine for clinical purposes." British medical journal38 (1857): 788.Green, W. E. "The practical utility of estimating the amount of urea passed daily." British Medical Journal1301 (1885): 1055.Boggs, Bryan K., Rebecca L. King, and Gerardine G. Botte. "Urea electrolysis: direct hydrogen production from urine." Commun.32 (2009): 4859-4861.Mahshid, Sahar Sadat, et al. "Template-based electrodeposition of Pt/Ni nanowires and its catalytic activity towards glucose oxidation." acta58 (2011): 551-555.Wang, Dan, et al. "Electrochemically reduced graphene oxide–nickel nanocomposites for urea electrolysis." Electrochim. Acta 89 (2013): 732-736.Li, Mian, et al. "Electrodeposition of nickel oxide and platinum nanoparticles on electrochemically reduced graphene oxide film as a nonenzymatic glucose sensor." Sens. and Actuat. B: Chem. 192 (2014): 261-268. Liu, Zhimin, et al. "A Mediator‐Free Tyrosinase Biosensor Based on ZnO Sol‐Gel Matrix." 17.12 (2005): 1065-1070. Acknowledgement This work was supported by Agency for Defense Development (ADD) as global cooperative research for high performance and light weight bio-urine based fuel cell (UD160050BD), the Ocean University of China-Auburn University (OUC-AU) Grants program, and the International Collaborative Energy Technology R&D Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (20158520000210).

Sputtering of Nickel-Palladium Bimetallic Anode Catalysts for Direct Urea/Urine Fuel Cell (DUFC) Application

In recent years, there has been growing interest in utilization of urea in terms of disposal of industrial urea wastewater and renewable energy. Urea with high energy density of 16.9 MJ/L corresponding to 10.1 wt% of hydrogen is considered as an attractive material that can produce hydrogen as the sources for fuel cells [1]. Urea also has the advantages of stability, non-toxicity, and non-flammable properties [2]. Therefore, urea can be a good candidate for alternative fuel in DUFC systems where urea is electrochemically oxidized to nitrogen (N2) and carbon dioxide (CO2) at the anode [3]. It can be economically feasible to produce hydrogen because the cell voltage required for urea electrolysis (0.37 V) is much lower than the cell voltage required for water electrolysis(1.23V) [2]. Various types of catalysts such as Ti-Pt [5], Pt-Co [6], and Pd-Co [7] have been researched because the urea can be oxidized in neutral medium when noble metals are used. However, due to the low activity to electrochemical oxidation of urea and high cost of noble metals, nickel-based catalysts have been investigated extensively as anode catalyst in DUFC. Ni-Co bimetal results in reduced onset voltage and improved electrochemical properties, but Co has a disadvantage of indirect contribution to urea oxidation [8]. Therefore, sputtered bimetallic and inexpensive Ni-Pd, catalysts were studied as a cathode catalyst for DUFC application. Moreover, sputter process can be simple and reproducible depositing catalysts on substrate. In this study, sputtered Ni-Pd catalysts on carbon substrate were prepared as an anode catalyst for DUFC platform. The morphologies and structural properties of Ni-Pd bimetals were analyzed by SEM and XRD, and the contents of Ni and Pd were measured by EDS. Thereafter electrochemical measurements have been performed on a three-electrode cell in 1M urea containing 0.33 M urea, and fuel cell testing was conducted using 0.33M urea or urine which is fed into flow channel as fuels. Detailed discussion on performance of Ni-Pd bimetal as anode catalyst in DUFC will be given. References Zhang, H., Wang, Y., Wu, Z., & Leung, D. Y. (2017). A direct urea microfluidic fuel cell with flow-through Ni-supported-carbon-nanotube-coated sponge as porous electrode. of Power Sources, 363, 61-69.Boggs, B. K., King, R. L., & Botte, G. G. (2009). Urea electrolysis: direct hydrogen production from urine. Commun., (32), 4859-4861.Wright, J. C., Michaels, A. S., & Appleby, A. J. (1986). Electrooxidation of urea at the ruthenium titanium oxide electrode. AIChE Journal, 32(9), 1450-1458. Simka, Wojciech, et al. "Electrochemical treatment of aqueous solutions containing urea." of applied electrochemistry39.7 (2009): 1137-1143.Huang, H., Fan, Y., & Wang, X. (2012). Low-defect multi-walled carbon nanotubes supported PtCo alloy nanoparticles with remarkable performance for electrooxidation of methanol. Acta, 80, 118-125.Wang, Z., Du, Y., Zhang, F., Zheng, Z., Zhang, Y., & Wang, C. (2013). High electrocatalytic activity of non-noble Ni-Co/graphene catalyst for direct ethanol fuel cells. of Solid State Electrochem., 17(1), 99-107.Xu, W., Zhang, H., Li, G., & Wu, Z. (2014). Nickel-cobalt bimetallic anode catalysts for direct urea fuel cell. Scientific reports, 4, 5863. Acknowledgement This work was supported by Agency for Defense Development (ADD) as global cooperative research for high performance and light weight bio-urine based fuel cell (UD160050BD) and the Ocean University of China-Auburn University (OUC-AU) Grants program.

Antiacanthain A: New proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae)

Crude extract (CE) from pulp of Bromelia antiacantha Bertol. mature fruit, contains at least 3 cysteine proteases with proteolytic activity. By single step cation exchange chromatography (Hi-trap SP-HP) of partially purified CE, the protease with the lowest pI, Antiacanthain A (AntA), was isolated. It showed maximum activity at pH9, and 75% of remaining activity was maintained over a wide pH range (pH6–10). The AntA activity exhibits a constant increase up to 70°C. Maintains almost 100% of its activity at 45 at pH6 and 9. A 60% of AntA was active by titration with specific inhibitor, E64. Amidasic activity was studied with pyroglutamyl-phenyl-leucyl-paranitroaniline (PFLNA) substrate having higher AntA catalytic efficiency of (kcat/Km=470s−1M−1) relative to stem bromelain (kcat/Km=305s−1M−1). Esterase activity using p-nitrophenyl esters of N-α-CBZ-l-Lysine (z-L-LysONp) showed a 10-fold higher catalytic efficiency for AntA (kcat/Km=6376s−1M−1) relative to stem bromelain (kcat/Km=688s−1M−1). Incubation with 8M Urea did not affect AntA activity and remained unchanged for 18h, with 6M GndHCl resulted in a 41% decrease in activity after 30min incubation, maintained this activity 18h. AntA exhibits high sequence identity with proteases of the Bromeliaceae family.

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods

ABSTRACTSample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

Enhancement of gene expression in Escherichia coli and characterization of highly stable ATP-dependent glucokinase from Pyrobaculum calidifontis.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95°C and 8.5, respectively. Catalytic efficiency (k cat/K m) towards glucose was 437mM-1s-1. The recombinant enzyme was highly stable against temperature with a half-life of 25min at 100°C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.

Influence of urea on single and mixed micellar systems of Tetronics®

Here we report micellar and micellization characteristics of single and mixed Tetronics® altered by the presence of urea using high sensitivity differential scanning calorimetry (HSDSC) and small angle neutron scattering (SANS). Three Tetronics® with almost identical polypropylene oxide (PPO) blocks but variable polyethylene oxide (PEO) blocks viz. T1301, T1304 and T1307, were chosen for studying their solution behavior in aqueous urea (0-8M) solutions. In the case of single micellar systems, the three copolymers showed markedly different behavior.T1301 being most hydrophobic forms vesicular structures at ambient temperature, T1304 with moderate hydrophobicity forms micelles with low polydispersity while very hydrophilic T1307 exhibits high polydispersity and occur as unimers, loose micelles and clusters. The addition of urea has a disruptive effect on all the three copolymers; smaller vesicles in T1301, smaller micelles with enhanced polydispersity in T1304 and T1307 completely demicellizes to unimers and clusters as evidenced by SANS. A similar trend has also been noticed for mixed micelles. However, the phase separation temperature/critical micelle temperature (CMT) of the copolymers in binary mixtures are slightly lower as compared to the copolymer solutions of individual block copolymers in the presence of 8M urea. HSDSC measurements revealed decreased enthalpy of micellization (∆H) and increased CMT in the presence of urea suggesting enhanced solubility of the copolymer and consequently delayed micellization.

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide – a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8M urea solution in the hybridization buffer. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60min at 100°C. It retained more than 60% of its activity even after an incubation of 72h at room temperature in the presence of 6M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats

The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments.

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8M urea (pH8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.

Counteraction of the deleterious effects of urea on structure and stability of mammalian kidney proteins by osmolytes

Owing to the urine concentrating mechanism of kidney cells, urea concentration is very high (3.0–5.0M) in mammalian kidneys which may denature many kidney proteins. Methylamines are known to counteract the deleterious effects of urea on structure, stability and function of proteins at 2:1 molar ratio of urea to methylamines. It is known that mammalian kidney cells also contain stabilizing osmolytes, non-methylamines (myo-inositol and sorbitol). A question arises: Do these non-methylmine osmolytes have ability to counteract the deleterious effects of urea on kidney proteins? To answer this question, we took two kidney proteins, namely, sheep serum albumin and Human carbonic anhydrase II. We measured their thermodynamic stability (ΔG0N↔D, the Gibbs free energy change in absence of GdmCl (guanidinium chloride) associated with the equilibrium, native (N) state↔denatured (D) state) from the GdmCl-induced denaturation curves in the presence of different concentrations of urea and each kidney osmolyte individually and in combination. For both proteins, we observed that (i) glycine betaine and myo-inositol provide perfect counteraction at 2:1 molar ratio of urea to osmolyte, i.e., denaturing effect of 2M urea is 100% neutralized by 1M of glycine betaine (or myo-inositol), and (ii) sorbitol fails to refold urea denatured proteins.

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state.

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone 1H, 13C, and 15N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.