Glycerol Buffer Research Articles

Paintable, Fast Gelation, Highly Adhesive Hydrogels for High-fidelity Electrophysiological Monitoring Wirelessly.

High-fidelity wireless electrophysiological monitoring is essential for ambulatory healthcare applications. Soft solid-like hydrogels have received significant attention as epidermal electrodes because of their tissue-like mechanical properties and high biocompatibility. However, it is challenging to develop a hydrogel electrode that provides robust contact and high adhesiveness with glabrous skin and hairy scalp for high-fidelity, continuous electrophysiological signal detection. Here, a paintable, fast gelation, highly adhesive, and conductive hydrogel is engineered for high-fidelity wireless electrophysiological monitoring. The hydrogel, consisting of gelatin, gallic acid, sodium citrate, lithium chloride, glycerol, and Tris-HCl buffer solution exhibits a reversible thermal phase transition capability, which endows it with the attributes of on-skin applicability and fast in situ gelation with 15 s, thereby addressing the aforementioned limitations. The introduction of gallic acid enhances the adhesive properties of the hydrogel, facilitating secure electrode attachment to the skin or hairy scalp. To accentuate the potential applications in at-home electrophysiological health monitoring, the hydrogel electrodes are demonstrated for high-fidelity electrocardiogram recording for one hour during various daily activities, as well as in simultaneous electroencephalogram and electrocardiogram recording during a 30 min nap.

Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period.

Glycerol-Based Green RP-HPLC Method for Simultaneous Determination of Methionine and Paracetamol in Pharmaceutical Tablets

Methionine and paracetamol are combined in dosage forms due to the ability of methionine to compensate the deficiency in glutathione in hepatic patients. In this work, an innovative simple time-saving green RP-HPLC method using glycerol as a solely green mobile phase with water for the first time was developed for the simultaneous determination of methionine and paracetamol in their standard and in Hepamol® tablets. The chromatographic conditions were optimized using factorial design with the aid of Minitab 17® Software. The method was performed on a C18 column at 38 °C, and a mobile phase consisting of glycerol and phosphate buffer (pH 2.4) (40:60, v/v), using diode array detector at 210 nm. The mixture was separated in 5 min. The developed method was validated in accordance with ICH requirement over linearity ranges of 10–90 μg/mL for both drugs, and LODs were 3.33 μg/mL for both methionine and paracetamol. Glycerol has high safety, low UV cut-off point, low flammability, and its viscosity can be manipulated when diluted with water. The procedure was compared to the reported reversed phase liquid chromatography method in the terms of their greenness with the green analytical procedure index and the analytical eco-scale. This work breaks new ground for scientists to use glycerol in greener RP-HPLC applications.

Effect of Heat Curing of Film-forming Solution on the Properties of Soy Protein Films

This study aimed to investigate the impact of heat treatment on the properties of soy protein isolate film-forming solutions. The solutions, consisting of soy protein isolate, glycerol, and phosphate buffer solution (pH 7.4), were subjected to heat treatment at 65, 75, and 85°C for 2, 4, or 6 h. The resulting films were then analyzed for tensile strength, elongation at break, film solubility, color parameters, and transparency. The results indicated that increasing the heat-curing temperature increased tensile strength, film transparency, and yellowish color in the films, as well as decreased water solubility and elongation at break. Notably, the film cured at 85°C for 2 h exhibited the highest tensile strength, film solubility, and elongation at break compared to both the control and other heat-cured films. However, the film heated at 75°C showed a better elongation at break. Overall, the findings suggest that heat curing of film-forming solutions at temperatures between 75 and 85°C has the potential to improve the properties of soy protein isolate films.

Magnetic Nano-Sized SDF-1 Particles Show Promise for Application in Stem Cell-Based Repair of Damaged Tissues.

Stem cell-based therapy is a promising option for repair of injured tissue. Stem cells have homing characteristics and can be mobilized to the injury sites following activation, under the regulation of the SDF-1/CXCR4 axis. However, a sufficient level of stem cell aggregation and retention is essential for ensuring favorable repair outcomes. Problems related to stem cell delivery/recruitment efficiency and retention in the injury site are among the main challenges faced during in vivo studies on stem cell therapy. In this study, we designed an SDF-1(alpha) magnetic nanoparticle delivery system for stem cell recruitment. We expressed and purified a biotin-labeled SDF-1(alpha) protein and immobilized it on streptavidin-modified magnetic nanoparticles (MNP) through the streptavidin–biotin linkage, with an efficiency of approximately 14%. The physicochemical properties of the SDF-MNP in glycerol buffer were similar to those of the streptavidin-modified MNP. Further evidence suggested that SDF-MNP barely show cytotoxicity even at a concentration of 125 µg/ml MNP and have a promising chemotaxis effect on mesenchymal stem cells in vitro and in vivo. Our study provides a strategy for the assembly of magnetic nanoparticle carrier systems for protein factors, as well as preliminary evidence for the application of SDF-MNP in stem cell-based therapy for the regeneration of injured bone tissue.

Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics

ABSTRACT Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug–microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at −80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

Optical Properties of Vibronically Coupled Cy3 Dimers on DNA Scaffolds.

We examine the effect of electronic coupling on the optical properties of Cy3 dimers attached to DNA duplexes as a function of base pair (bp) separation using steady-state and time-resolved spectroscopy. For close Cy3-Cy3 separations, 0 and 1 bp between dyes, intermediate to strong electronic coupling is revealed by modulation of the absorption and fluorescence properties including spectral band shape, peak wavelength, and excited-state lifetime. Using a vibronic exciton model, we estimate coupling strengths of 150 and 266 cm-1 for the 1 and 0 bp separations, respectively, which are comparable to those found in natural light-harvesting complexes. For the strongest electronic coupling (0 bp separation), we observe that the absorption band shape is strongly affected by the base pairs that surround the dyes, where more strongly hydrogen-bonded G-C pairs produce a red-shifted absorption spectrum consistent with a J-type dimer. This effect is studied theoretically using molecular dynamics simulation, which predicts an in-line dye configuration that is consistent with the experimental J-type spectrum. When the Cy3 dimers are in a standard aqueous buffer, the presence of relatively strong electronic coupling is accompanied by decreased fluorescence lifetime, suggesting that it promotes nonradiative relaxation in cyanine dyes. However, we show that the use of a viscous solvent can suppress this nonradiative recombination and thereby restore the dimer fluorescent emission. Ultrafast transient absorption measurements of Cy3 dimers in both standard aqueous buffer and viscous glycerol buffer suggest that sufficiently strong electronic coupling increases the probability of excited-state relaxation through a dark state that is related to Cy3 torsional motion.

Refractive index matching to develop transparent polyaphrons: Characterization of immobilized proteins

Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer (“soapy shell”) of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation.

Abstract A186: Low dose paclitaxel treatment increase the stability of p27Kip1

Abstract The purpose of our study is to better understand how taxanes (paclitaxel, docetaxel) induce cell death. Taxanes play a critical role in combination chemotherapy for many types of solid tumors, especially in advanced disease states. These drugs bind to tubulin in interphase and mitotic microtubules altering their dynamics. This leads to G2/M arrest that is thought to contribute to taxane-induced apoptosis, although the exact mechanisms remain elusive. We found that after treating MDA-MB-231 breast cancer cells with low dose (10X IC50 = 40 nM) paclitaxel over 4-48 h, the cyclin-dependent kinase inhibitor p27Kip1 progressively increases more than 2-fold. p27Kip1 is an important regulator of cell cycle, found in both the nucleus and cytoplasm, and its accumulation is associated with G1 arrest in most cells. Its tumor suppressor activity is associated with its nuclear localization, where it forms complexes with Cdk2 and cyclin E (or other cyclins) preventing DNA replication and cell cycle advance. In the cytoplasm, p27Kip1 was shown to enhance cell motility. When p27Kip1 becomes phosphorylated on Thr 187 leading to its ubiquitination and degradation, the cell cycle can progress. We pretreated cells with 10 μM cycloheximide for 1 h to prevent translational peptide elongation and found when cells were subsequently treated with 40 nM paclitaxel for up to 4 hours, quantitative Western blotting showed that p27Kip1 levels steadily increased, indicating that paclitaxel treatment stabilized p27Kip1 protein. Quantitative real-time PCR demonstrated a parallel increase in p27Kip1 mRNA over 8 hours and then a gradual decline in mRNA over 24-48 h, while the protein remained stable over this time period. Since paclitaxel binds to microtubules, we wanted to know whether association of p27Kip1 with microtubules might play a role in its stabilization. We isolated the intact microtubule cytoskeleton from cells using a 2M glycerol buffer (100 mM Tris pH 6.8, 0.5% Nonidet P-40, 1 mM MgCl2 and 2 mM EGTA) at 37°C and found that 20% of the total intracellular p27Kip1 is associated with the microtubule pellet in control cells and 40 nM paclitaxel treatment for 48 h reduces this amount by about half, demonstrating that paclitaxel treatment leads to an increased amount of p27Kip1 in the non-microtubule fraction of the cell lysate. Because the tumor suppressor function of p27Kip1 is associated with its nuclear localization, we isolated nuclear and cytoplasmic fractions from MDA-MB-231 cell lysates and found a 2-fold change in p27Kip1 with 40 nM paclitaxel treatment for 48 h, compared to cells treated with DMSO. The 2-fold increase in p27kip1 was found entirely in the cytoplasmic fraction. Western blotting with an antibody that recognizes p27Kip1 phosphorylated at Thr 187 surprisingly showed a nearly 2-fold increase in phospho- p27Kip1 with paclitaxel treatment in the cytoplasmic fractions. In summary, paclitaxel treatment of MDA-MB-231 breast cancer cells leads to a 2-fold increase in total p27Kip1. This increase is primarily in the cytoplasmic fraction and is associated with an increase in phospho-The 187-p27Kip1. Our data together suggest that paclitaxel treatment may block ubiquitination of phospho -Thr 187-p27Kip1 or its proteosomal degradation. These novel observations are important for understanding the regulation of p27Kip1 and the mode of action for paclitaxel. Citation Format: Sharon Lobert, Mary E. Graichen. Low dose paclitaxel treatment increase the stability of p27Kip1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A186.

English

Plant growth promoting bacteria (PGPB) enhances the growth of their hosts and can protect them from biotic and abiotic stresses. Bacterial inoculants contain one or more of these beneficial strains in a carrier material, which must be able to maintain the viability of the cells during the time of storage, and also guarantee the biological activity of the strains once applied in the soil. These inoculants can be solid, liquid, gel or oil-based, depending on the characteristics of the strains and the shelf life expected by the producers. In this study, we used a method of accelerated degradation to select a polymer and a concentration to maintain cell stability of a liquid inoculant based on the strain C16 Azospirillum brasilense. A screening at 45°C was made to compare the protectant effect of five polymers on the viability of the strain (p/v): carrageenan (1.5%), sodium alginate (1%), trehalose (10 mM), polyvinylpyrrolidone (2%), glycerol (10 mM) and phosphate saline buffer as control. Carrageenan and sodium alginate showed significant differences in cell viability over the use of other polymers (P < 0.05). We evaluated cell viability with these two polymers at three concentrations and three different temperatures (4, 28 and 45°C) for 60 days and determined the bacterial degradation rates. Based on the Arrhenius thermodynamic model, we calculated the time required to reduce cell concentration in three log units, and observed that the protectant activity of each polymer and each concentration depends on the temperature of storage. Cell viability was best preserved in all treatments at 4°C. In general, alginate prolonged cell viability at 28°C, and carrageenan at 45°C. Alginate at 1% and carrageenan at 0.75% showed a stable behavior (superior to the control) in the three evaluated temperatures, so we conclude that they can be used for a formulation of a liquid inoculant based on the strain C16 of A. brasilense. Key words: Energy of activation, degradation, cell death, kinetics, formulation.

Growth promotion effect of Pichia guilliermondii in chilli and biocontrol potential of Hanseniaspora uvarum against Colletotrichum capsici causing fruit rot

In the present investigation, we have attempted to identify the potential two epiphytic yeast strains for growth promotion and management of chilli fruit rot. Seed treatment with Pichia guilliermondii showed increased seedling vigour index (55%), fresh weight (96%) and dry weight (45%) over untreated control. Furthermore, P. guilliermondii showed higher root colonisation ability, indole-3-acetic acid (IAA) production and phosphate solubilisation ability. On the other hand, seedling dip with Hanseniaspora uvarum induced higher levels of defence-related compounds in chilli seedlings challenge-inoculated with Colletotrichum capsici under glasshouse conditions. Among the different media tested, higher biomass of P. guilliermondii and H. uvarum was obtained in pine juice broth and sugarcane juice broth, respectively. Glycerol buffer formulation showed viability (>70%) of P. guilliermondii up to 4 months and H. uvarum up to 9 months when stored at ambient conditions. Seedling dip and foliar sprays with H. uvarum showed 37– 40% reduction in chilli fruit rot incidence under field conditions. It also showed higher (cumulative) accumulation of defence-related compounds in chilli leaves and ripe fruits under field conditions. The results of current investigation indicated a clear difference among the two epiphytic yeast strains. P. guilliermondii was identified as growth promoter of chilli and H. uvarum as antagonist of chilli fruit rot pathogen, C. capsici.

Response of primary vectors and related diseases to impoundment by the Three Gorges Dam

To investigate the impact of the Three Gorges Dam on the local ecological environment. We conducted a 3-year cross-sectional survey of natural focus infectious diseases in the area before and after the water level rose to 156 m to evaluate the dam's health impacts. Direct and indirect immunofluorescence methods were applied to detect rat antigen and antibody of haemorrhagic fever with renal syndrome (HFRS). Fresh rat kidneys were inoculated in Korth's culture medium to detect Leptospira. A group of captured crabs were ground to observe the metacercariae of Paragonimus. Serum samples were collected from healthy local individuals. ELISA kits were used to detect human antibody against HFRS and paragonimiasis. Human Leptospira infections were detected by a microscopic agglutination test. Upstream rodent density increased significantly with Rattus flavipectus and Apodemus agrarius as the major pathophoric genera. The infection rate of human HFRS and Leptospira in the upstream human population samples was significantly higher than in the downstream samples and correlated with the increase in rodent density. Paragonimus infection rates remained at a low level during the study. Culex pipiens fatigan and Armigeres obturbans were the dominant species of mosquito. The creation of the Three Gorges Dam changed the proliferation of intermediary agents of diseases, but not notably. However, the ecological effects on the environment may require a prolonged period of time to manifest themselves; thus, long-term and effective surveillance of vectors and related diseases needs to be established.

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

English

&nbsp; Bacillus&nbsp;sp. SW1-2 producing poly glutamic acid (PGA), locally isolated from Eastern province in Saudi Arabia, was characterized and identified based on 16S rRNA gene sequencing. Phylogenetic analysis revealed its closeness to&nbsp;Bacillus megaterium. The homopolymer consists mainly of glutamic as indicated in the analysis of amino acid. Preliminary optimization of PGA production, through a series of one-variable-at-a-time (OVAT) experiments, revealed maximum PGA production of 10.5 (g/L). Statistically based experiments were applied to optimize culture conditions for production of PGA. Effect of 15 variables were examined for their significance on PGA production using Plackett-Burman factorial design. Among those variables; Na-citrate, (NH4)2SO4, glutamic acid and CaCl2.2H2O were most significant variables that encouraged PGA production, while those negatively affected were; glycerol, glucose and phosphate buffer. Significant parameters were further investigated using Box-Behnken design to define the optimal medium composition. Based on statistical analysis, maximal PGA production on optimized medium was 36.5 (g/L) which was more than 4-folds the basal production medium. Verification experiment was carried out to examine model validation and revealed more than 95% validity. &nbsp; Key words:&nbsp;Poly glutamic acid,&nbsp;Bacillus megaterium,&nbsp;statistical experimental design, optimization, one-variable-at-a-time (OVAT).

Immunotherapy – 2066. A study on skin prick test patterns of atopic patients managed in a tertiary care setting of Sri Lanka

according to AREA classification 1 . Presence of coexisting Asthma and allergic conjunctivitis was documented. The severity of asthma was assessed using the GINA guidelines. Informed written consent was obtained by a member of the research team. SPT was performed using a panel of 19 allergens. Positive control was done using a standard solution of histamine and the negative was performed with glycerol buffer. The resultant induration was measured using a standard scale and recorded in millimeters. A wheel of 3 mm was considered as positive. The results were analyzed in percentages. Results We studied 25 males and 29 females. The age range of patients was from 09 to 68 yrs with a mean age of 33.5yrs. A positive reaction to at least one allergen was seen in 87.1% of patients. Intermittent Rhinitis was seen in 53.7% and persistent disease was seen in 46.3%. Moderate to severe disease was found in 72.2% and the rest had mild disease. Coexisting asthma was seen in 43.6% and conjunctivitis was seen in 29.6% of the study population. The results of SPT expressed in percentages showed, Dermatophagoides farina spp. 59.3%, Dermatophagoides pteronyssinus spp. 63%, Wheat Dust 33%, Cotton Gin 33%, Rice Dust 14%, Aspergillus flavus 18.5%, Aspergillus fumigates 7.4% Aspergillus niger 11.1%, Aspergillus glaucus/amstelodami 11%, Tricho. mentagrophytes 7.4%, Candida spp. 18.5%, Cladosporum herbarium 11%, Epidermo floccosum 22.2%, German Cockroach 51.9%, American Cockroach 40.7%, Bermuda Grass 11%, Red Mulberry 7.4%, White Mulberry 11.1% and Acacia 3.7% positivity.

Application of Carbon Quantum Dots in Fluorescent Imaging of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A new method of carbon quantum dots (CQDs) fluorescent imaging for human serum proteins detection after polyacrylamide gels electrophoresis (PAGE) is established. Polyacrylamide gel electrophoresis is one of the most general and powerful technique to separate complex biosamples, and it is widely used in molecular biology, biochemistry and medicine. With the evolution of clinical proteomics, the development of a novel method to detect serum proteins after PAGE with high resolution and high sensitivity is of great significant. As far as we know, the carbon quantum dots have not been applied in the detection of serum proteins after PAGE. The fluorescent carbon quantum dots were synthesized by a one-step microwave pyrolysis method: glycerol and phosphate buffer (7.1 mmolL -1 , pH 7.4) (φ=70%) was mixed evenly, and then put the solu- tion into the advanced microwave digestion system and heated for 14 min (750 W), the colorless solution turned to yellow after reaction. The yellow solution was diluted into the incubation solution with HOAc-NaOAc buffer (80 mmolL -1 , pH 2.7), and then used for staining serum proteins. The emission and excitation spectra of carbon quantum dots were measured, and the excitation wavelength allowed the use of an ultraviolet lamp at 365 nm for the fluorescent imaging. In order to inves- tigate the performance of CQDs fluorescent imaging, the dilution of human serum samples separated by electrophoresis was used for detection, and was compared with traditional method of CBB-R250 staining and silver staining. The results sug- gested that CQDs based on fluorescent imaging could effectively detect human serum proteins with high sensitivity and resolution. The sensitivity of CQDs imaging was higher than CBB staining and comparable to silver staining. Therefore, the CQDs fluorescent imaging could be an inexpensive, time-saving, pollution-free and convenient method with high sensitivity and resolution for the detection of human serum proteins. It demonstrates the CQDs fluorescent imaging technology has great significant prospects in the development of biotechnology and nanotechnology. Keywords carbon quantum dot; polyacrylamide gel electrophoresis; protein; fluorescent imaging

Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus

BackgroundDetection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. ObjectivesTo develop an isothermal ‘recombinase polymerase amplification (RPA)’ assay on an ESEquant tubescanner device. Study designRPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. ResultsThe one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log10 step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.

Formation of dAMP-glycerol and dAMP-Tris Derivatives by Thermococcus kodakaraensis DNA Primase

In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.