Search results for: fluorescent proteins

Authors: Sumayah Mohammed Asiri A., Aviva Levina B., Elizabeth New C., Peter Lay D.

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Pt compounds have been among the most successful anticancer drugs in the last 40 years, but the development of resistance to them is an increasing problem. Cellular homeostasis of an essential metal, Cu, is known to be involved in Pt resistance, but mechanisms of this process are poorly understood. We used a novel ratiometric Cu(I)-sensitive fluorescent probeInCCu1 dye to detect Cu(I) in the mitochondria. Total Cu and labile Cu pool measured using AAS and InCCu1 dye in A2780 cells and their corresponding resistant cells A2780-cis.R cells treated with Cu and cisplatin. The main difference between both cell lines in the presence and absence of Cu(II) is that resistant cells have lower total Cu content but higher labile Cu levels than cisplatin-sensitive cells. This means that resistant cells can metabolize and export excess Cu more efficiently. Furthermore, InCCu1 has emerged not only as an indicator of labile cellular Cu levels in the mitochondria but as a potentially versatile multi-organelle probe.

Keywords: AAS and ICPMS, A2780 and its resistant cells, ratiometric fluorescent sensors, inCCu1, and total and labile Cu

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Authors: Irene Alevizos, Jessica Lewis, Marina Harper, John Boyce

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Acinetobacter baumannii causes a range of severe nosocomial-acquired infections, and many strains are multi-drug resistant. A. baumannii possesses survival mechanisms allowing it to thrive in competitive polymicrobial environments, including a Type VI Secretion System (T6SS) that injects effector proteins into other bacteria to give a competitive advantage. The effects of T6SS firing are broad and depend entirely on the effector that is delivered. Effects can include toxicity against prokaryotic or eukaryotic cells and the acquisition of essential nutrients. The T6SS of some species can deliver ‘specialised effectors’ that are fused directly to T6SS components, such as PAAR proteins. PAAR proteins are predicted to form the piercing tip of the T6SS and are essential for T6SS function. Although no specialised effectors have been identified in A. baumannii, many strains encode multiple PAAR proteins. Analysis of PAAR proteins across the species identified 12 families of PAAR proteins with distinct C-terminal extensions. A. baumannii AB307-0294 encodes two PAAR proteins, one of which has a C-terminal extension. Mutation of one or both of the PAAR-encoding genes in this strain showed that expression of either PAAR protein was sufficient for T6SS function. We employed a heterologous expression approach and determined that PAAR proteins from different A. baumannii strains, as well as the closely related A. baylyi species, could complement the A. baumannii ∆paar mutant and restore T6SS function. Furthermore, we showed that PAAR fusions could be used to deliver artificially cloned protein fragments by generating Histidine- and Streptavidin- tagged PAAR specialised effectors, which restored T6SS activity. This provides evidence that the fusion of protein fragments onto PAAR proteins in A. baumannii is compatible with a functional T6SS. Successful delivery by this mechanism extends the scope of what the T6SS can deliver, including user designed proteins.

Keywords: A. baumannii, effectors, PAAR, T6SS

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Authors: Mohammed Hakim Jafferali, Balaje Vijayaraghavan, Ricardo A. Figueroa, Ellinor Crafoord, Veronica J. Larsson, Einar Hallberg, Santhosh Gudise

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The nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail. The results show that the Samp1 binds stronger to RanGTP than RanGDP. Samp1 is the first transmembrane protein known to bind Ran and it is tempting to speculate that Samp1 may provide local binding sites for RanGTP at membranes.

Keywords: MCLIP, nuclear envelope, ran, Samp1

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Authors: Mostafa Ghasemi, Andrew Urquhart

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In this study, fluorescent carbon dots (CDs) were synthesized in a green way using a one-step hydrothermal method. Carbon dots are carbon-based nanomaterials with a size of less than 10 nm, unique structure, and excellent properties such as low toxicity, good biocompatibility, tunable fluorescence, excellent photostability, and easy functionalization. These properties make them a good candidate to use in different fields such as biological sensing, photocatalysis, photodynamic, and drug delivery. Fourier transformed infrared (FTIR) spectra approved OH/NH groups on the surface of the as-synthesized CDs, and UV-vis spectra showed excellent fluorescence quenching effect of Fe (III) ion on the as-synthesized CDs with high selectivity detection compared with other metal ions. The probe showed a linear response concentration range (0–2.0 mM) to Fe (III) ion, and the limit of detection was calculated to be about 0.50 μM. In addition, CDs also showed good sensitivity to the pH value in the range from 2 to 14, indicating great potential as a pH sensor.

Keywords: carbon dots, fluorescence, pH sensing, metal ions sensor

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Authors: M. N. Hanina, M. Hairul Shahril, I. Ismatul Nurul Asyikin, A. K. Abdul Jalil, M. R. Salina, M. R. Maryam, M. Rosfarizan

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The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Keywords: Bacillus subtilis ATCC21332, streptomycin sulfate, extracellular proteins, antibiotics

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Authors: Misty Attwood, Helgi Schioth

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Transmembrane proteins are important components in many essential cell processes such as signal transduction, cell-cell signalling, transport of solutes, structural adhesion activities, and protein trafficking. Due to their involvement in diverse critical activities, transmembrane proteins are implicated in different disease pathways and hence are the focus of intense interest in understanding functional activities, their pathogenesis in disease, and their potential as pharmaceutical targets. Further, as the structure and function of proteins are correlated, investigating a group of proteins with the same tertiary structure, i.e., the same number of transmembrane regions, may give understanding about their functional roles and potential as therapeutic targets. In this in silico bioinformatics analysis, we identify and comprehensively characterize the previously unstudied group of proteins with five transmembrane-spanning regions (5TM). We classify nearly 60 5TM proteins in which 31 are members of ten families that contain two or more family members and all members are predicted to contain the 5TM architecture. Furthermore, nine singlet proteins that contain the 5TM architecture without paralogues detected in humans were also identifying, indicating the evolution of single unique proteins with the 5TM structure. Interestingly, more than half of these proteins function in localization activities through movement or tethering of cell components and more than one-third are involved in transport activities, particularly in the mitochondria. Surprisingly, no receptor activity was identified within this family in sharp contrast with other TM families. Three major 5TM families were identified and include the Tweety family, which are pore-forming subunits of the swelling-dependent volume regulated anion channel in astrocytes; the sidoreflexin family that acts as mitochondrial amino acid transporters; and the Yip1 domain family engaged in vesicle budding and intra-Golgi transport. About 30% of the proteins have enhanced expression in the brain, liver, or testis. Importantly, 60% of these proteins are identified as cancer prognostic markers, where they are associated with clinical outcomes of various tumour types, indicating further investigation into the function and expression of these proteins is important. This study provides the first comprehensive analysis of proteins with 5TM regions and provides details of the unique characteristics and application in pharmaceutical development.

Keywords: 5TM, cancer prognostic marker, drug targets, transmembrane protein

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Authors: I. Sani, A. Abdulhamid, F. Bello, Isah M. Fakai

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This research has focused on the application of green fluorescent protein (GFP) as a new technique for direct monitoring of fermentation processes involving cultured bacteria. To use GFP as a sensor for pH and oxygen, percentage ratio of red fluorescence to green (% R/G) was evaluated. Assessing the magnitude of the % R/G ratio in relation to low or high pH and oxygen concentration, the bacterial strains were cultivated under aerobic and anaerobic conditions. SCC1 strains of E. coli were grown in a 5 L laboratory fermenter, and during the fermentation, the pH and temperature were controlled at 7.0 and 370C respectively. Dissolved oxygen tension (DOT) was controlled between 15-100% by changing the agitation speed between 20-500 rpm respectively. Effect of reducing the DOT level from 100% to 15% was observed after 4.5 h fermentation. There was a growth arrest as indicated by the decrease in the OD650 at this time (4.5-5 h). The relative fluorescence (green) intensity was decreased from about 460 to 420 RFU. However, %R/G ratio was significantly increased from about 0.1% to about 0.25% when the DOT level was decreased to 15%. But when the DOT was changed to 100%, a little increase in the RF and decrease in the %R/G ratio were observed. Therefore, GFP can effectively detect and indicate any change in pH and oxygen level during fermentation processes.

Keywords: Escherichia coli SCC1, fermentation process, green fluorescent protein, red fluorescence

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Authors: Cristina Núñez, Rufina Bastida, Elena Labisbal, Alejandro Macías, María T. Pereira, José M. Vila

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A highly selective quinoline-based fluorescent sensor L was designed in order to functionalize gold nanoparticles (GNPs@L). The cytotoxicity of compound L and GNPs@L on the MCF-7 breast cancer cells was explored and it was observed that L and GNPs@L compounds induced apoptosis in MCF-7 cancer cells. The cellular uptake of the hybrid system GNPs@L was studied using confocal laser scanning microscopy (CLSM).

Keywords: cytotoxicity, fluorescent probes, nanoparticles, quinoline

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Authors: Yao Xing, Hong Ling Liu, Wei Dong Yu

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With the increase of global population, it is of importance for the safe water supply, while, the water-monitoring method with the capability of rapidness, low-cost, green and robustness remains unsolved. In this paper, gold nanoclusters-wool keratin is added into copper ions measured by fluorescent method in order to probe copper ions in aqueous solution. The fluorescent results show that gold nanoclusters-wool keratin exhibits high stability of pHs, while it is sensitive to temperature and time. Based on Gauss fitting method, the results exhibit that the slope of gold nanoclusters-wool keratin with pH resolution under acidic condition is higher compared to it under alkaline solutions. Besides, gold nanoclusters-wool keratin added into copper ions shows a fluorescence turn-off response transferring from red to blue under UV light, leading to the dramatically decreased fluorescent intensity of gold nanoclusters-wool keratin solution located at 690 nm. Moreover, the limited concentration of copper ions tested by gold nanoclusters-wool keratin system is around 1 µmol/L, which meets the need of detection standards. The fitting slope of Stern-Volmer plot at low concentration of copper ions is larger than it at high concentrations, which indicates that aggregated gold nanoclusters are from small amounts to large numbers with the increasing concentration of copper ions. It is expected to provide novel method and materials for copper ions testing with low cost, high efficiency, and easy operability.

Keywords: gold nanoclusters, copper ions, wool keratin, fluorescence

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Authors: Ergun Sakalar, Kubra Bilgic

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In the last decades, real-time PCR has become a reliable tool preferred to use in many laboratories for pathogen detection. This technique allows for monitoring target amplification via fluorescent molecules besides admit of quantitative analysis by enabling of convert outcomes of thermal cycling to digital data. Sensitivity and traceability of real-time PCR are based on measuring of fluorescence that appears only when fluorescent reporter dye bound to specific target DNA.The fluorescent reporter systems developed for this purpose are divided into two groups. The first group consists of intercalator fluorescence dyes such as SYBR Green, EvaGreen which binds to double-stranded DNA. On the other hand, the second group includes fluorophore-labeled oligonucleotide probes that are separated into three subgroups due to differences in mechanism of action; initial primer-probes such as Cyclicons, Angler®, Amplifluor®, LUX™, Scorpions, and the second one hydrolysis probes like TaqMan, Snake assay, finally hybridization probes, for instance, Molecular Beacons, Hybprobe/FRET, HyBeacon™, MGB-Eclipse, ResonSense®, Yin-Yang, MGB-Pleiades. In addition nucleic acid analogues, an increase of probe affinity to target site is also employed with fluorescence-labeled probes. Consequently, abundant real-time PCR detection chemistries are chosen by researcher according to the field of application, mechanism of action, advantages, and proper structures of primer/probes.

Keywords: fluorescent dye, food safety, molecular probes, nucleic acid analogues

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Authors: F. Nameni, H. Poursadra

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The present study investigated resistance and progressive training alters the expression of chaperone proteins. These proteins function to maintain homeostasis, facilitate repair from injury, and provide protection. Nineteen training female in 2 groups taking part in the intervention volunteered to give blood samples. Levels of chaperone proteins were measured in response to resistance and progressive training. Hsp 70 levels were increased immediately after 2 h progressive training but decreased after resistance training. The data showed that human skeletal muscle responds to the stress of a single period of progressive training by up-regulating and resistance training by down-regulating expression of HSP70. Physical exercise can elevate core temperature and muscle temperatures and the expression pattern of HSP70 due to training status may be attributed to adaptive mechanisms.

Keywords: resistance training, heat shock proteins, leukocytes, Hsp 70

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Authors: Raana Babadi Fathipour

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Food packaging plays a crucial role in protecting food from unwanted external factors. Antibacterial edible films are a promising option for food packaging due to their biodegradability, environmental friendliness, and safety. This paper reviews recent research progress on antimicrobial edible films, focusing on those made from polysaccharides, proteins, and lipids. Polysaccharides and proteins are the primary components of antimicrobial edible films, while lipids primarily serve as plasticizers and carriers for active substances in composite films. For instance, second-generation liposomes have shown great potential as carriers for antimicrobial substances and other bioactive compounds due to their exceptional stability. Furthermore, this paper analyzes recent advancements and future trends in antimicrobial edible films. One promising direction is the integration of antimicrobial edible film materials with delivery systems, such as nanoemulsion and microencapsulation technologies, to ensure stable loading of bioactive substances. Another emerging area of interest is the development of smart and active packaging that allows consumers to assess the freshness of food products without opening the package. pH-sensitive films and smart fluorescent "on-off" sensors for humidity are currently being explored as materials for smart and active packaging to monitor food product freshness, with further exploration anticipated in the future.

Keywords: antimicrobial edible film, biopolymer, antimicrobial agent, encapsulation, antimicrobial assay

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Authors: Wei Tian, Jie Liang, Hammad Naveed

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Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods can help to understand their biophysical principles. We have developed a novel computational method to predict the 3D structure of beta-barrel membrane proteins using evolutionary coupling (EC) constraints and a reduced state space. Combined with an empirical potential function, we can successfully predict strand register at > 80% accuracy for a set of 49 non-homologous proteins with known structures. This is a significant improvement from previous results using EC alone (44%) and using empirical potential function alone (73%). Our method is general and can be applied to genome-wide structural prediction.

Keywords: beta-barrel membrane proteins, structure prediction, evolutionary constraints, reduced state space

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Authors: Maria Toader, Gheorghe Valentin Roman, Alina Maria Ionescu

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Crops diversity and maintaining and enhancing the fertility of agricultural lands are basic principles of organic farming. With a wider range of crops in agroecosystem can improve the ability to control weeds, pests and diseases, and the performance of crops rotation and food safety. In this sense, the main objective of the research was to study the productivity and chemical composition of some alternative crops and their adaptability to soil and climatic conditions of the agricultural area in Southern Romania and to cultivation in the organic farming system. The alternative crops were: lentil (7 genotypes); five species of grain legumes (5 genotypes); four species of oil crops (5 genotypes). The seed production was, on average: 1343 kg/ha of lentil; 2500 kg/ha of field beans; 2400 kg/ha of chick peas and blackeyed peas; more than 2000 kg/ha of atzuki beans, over 1250 kg/ha of fenugreek; 2200 kg/ha of safflower; 570 kg/ha of oil pumpkin; 2150 kg/ha of oil flax; 1518 kg/ha of camelina. Regarding chemical composition, lentil seeds contained: 22.18% proteins, 3.03% lipids, 33.29% glucides, 4.00% minerals, and 259.97 kcal energy values. For field beans: 21.50% proteins, 4.40% lipids, 63.90% glucides, 5.85% minerals, 395.36 kcal energetic value. For chick peas: 21.23% proteins, 4.55% lipids, 53.00% glucides, 3.67% minerals, 348.22 kcal energetic value. For blackeyed peas: 23.30% proteins, 2.10% lipids, 68.10% glucides, 3.93% minerals, 350.14 kcal energetic value. For adzuki beans: 21.90% proteins, 2.60% lipids, 69.30% glucides, 4.10% minerals, 402.48 kcal energetic value. For fenugreek: 21.30% proteins, 4.65% lipids, 63.83% glucides, 5.69% minerals, 396.54 kcal energetic value. For safflower: 12.60% proteins, 28.37% lipids, 46.41% glucides, 3.60% minerals, 505.78 kcal energetic value. For camelina: 20.29% proteins, 31.68% lipids, 36.28% glucides, 4.29% minerals, 526.63 kcal energetic value. For oil pumpkin: 29.50% proteins, 36.92% lipids, 18.50% glucides, 5.41% minerals, 540.15 kcal energetic value. For oil flax: 22.56% proteins, 34.10% lipids, 27.73% glucides, 5.25% minerals, 558.45 kcal energetic value.

Keywords: adaptability, alternative crops, chemical composition, organic farming productivity

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Authors: Ertuğ Yıldırım, Dile Kara, Salih Zeki Yıldız

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Metal containing polymers (MCPs) are macro molecules usually containing metal-ligand coordination units and are a multidisciplinary research field mainly based at the interface between coordination chemistry and polymer science. The progress of this area has also been reinforced by the growth of several other closely related disciplines including macro molecular engineering, crystal engineering, organic synthesis, supra molecular chemistry and colloidal and material science. Schiff base ligands are very effective in constructing supra molecular architectures such as coordination polymers, double helical and triple helical complexes. In addition, Schiff base derivatives incorporating a fluorescent moiety are appealing tools for optical sensing of metal ions. MCPs are well-known systems in which the combinations of local parameters are possible by means of fluoro metric techniques. Generally, without incorporation of the fluorescent groups with polymers is unspecific, and it is not useful to analyze their fluorescent properties. Therefore, it is necessary to prepare a new type epoxy polymers with fluorescent groups in terms of metal sensing prop and the other photo chemical applications. In the present study metal containing polymers were prepared via poly functional monomeric Schiff base metal chelate complexes in the presence of dis functional monomers such as diglycidyl ether Bisphenol A (DGEBA). The synthesized complexes and polymers were characterized by FTIR, UV-VIS and mass spectroscopies. The preparations of epoxy polymers have been carried out at 185 °C. The prepared composites having sharp and narrow excitation/emission properties are expected to be applicable in various systems such as heat-resistant polymers and photo voltaic devices. The prepared composite is also ideal for various applications, easily prepared, safe, and maintain good fluorescence properties.

Keywords: Schiff base ligands, crystal engineering, fluorescence properties, Metal Containing Polymers (MCPs)

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Authors: Zonglin Yang, Tatsuya Akiyama, Kerry S. Williamson, Michael J. Franklin, Thiruvarangan Ramaraj

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Pseudomonas aeruginosa is an opportunistic pathogen that forms surface-associated microbial communities (biofilms) on artificial implant devices and on human tissue. Biofilm infections are difficult to treat with antibiotics, in part, because the bacteria in biofilms are physiologically heterogeneous. One measure of biological heterogeneity in a population of cells is to quantify the cellular concentrations of ribosomes, which can be probed with fluorescently labeled nucleic acids. The fluorescent signal intensity following fluorescence in situ hybridization (FISH) analysis correlates to the cellular level of ribosomes. The goals here are to provide computationally and statistically robust approaches to automatically quantify cellular heterogeneity in biofilms from a large library of epifluorescent microscopy FISH images. In this work, the initial steps were developed toward these goals by developing an automated biofilm detection approach for use with FISH images. The approach allows rapid identification of biofilm regions from FISH images that are counterstained with fluorescent dyes. This methodology provides advances over other computational methods, allowing subtraction of spurious signals and non-biological fluorescent substrata. This method will be a robust and user-friendly approach which will enable users to semi-automatically detect biofilm boundaries and extract intensity values from fluorescent images for quantitative analysis of biofilm heterogeneity.

Keywords: image informatics, Pseudomonas aeruginosa, biofilm, FISH, computer vision, data visualization

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Authors: Xue Ma, Yang Fu, Dangyuan Lei

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Passive daytime radiative cooling is an emerging technology which has attracted worldwide attention in recent years due to its huge potential in cooling buildings without the use of electricity. Various coating materials with different optical properties have been developed to improve the daytime radiative cooling performance. However, commercial cooling coatings comprising functional fillers with optical bandgaps within the solar spectral range suffers from severe intrinsic absorption, limiting their cooling performance. Fortunately, it has recently been demonstrated that introducing fluorescent materials into polymeric coatings can covert the absorbed sunlight to fluorescent emissions and hence increase the effective solar reflectance and cooling performance. In this paper, we experimentally investigate the key factors for fluorescence-assisted radiative cooling with TiO2-based white coatings. The surrounding TiO2 nanoparticles, which enable spatial and temporal light confinement through multiple Mie scattering, lead to Purcell enhancement of phosphors in the coating. Photoluminescence lifetimes of two phosphors (BaMgAl10O17:Eu2+ and (Sr, Ba)SiO4:Eu2+) exhibit significant reduction of ~61% and ~23%, indicating Purcell factors of 2.6 and 1.3, respectively. Moreover, smaller Stokes shifts of the phosphors are preferred to further diminish solar absorption. Field test of fluorescent cooling coatings demonstrate an improvement of ~4% solar reflectance for the BaMgAl10O17:Eu2+-based fluorescent cooling coating. However, to maximize solar reflectance, a white appearance is introduced based on multiple Mie scattering by the broad size distribution of fillers, which is visually pressurized and aesthetically bored. Besides, most colored pigments absorb visible light significantly and convert it to non-radiative thermal energy, offsetting the cooling effect. Therefore, current colored cooling coatings are facing the compromise between color saturation and cooling effect. To solve this problem, we introduced colored fluorescent materials into white coating based on SiO2 microspheres as a top layer, covering a white cooling coating based on TiO2. Compared with the colored pigments, fluorescent materials could re-emit the absorbed light, reducing the solar absorption introduced by coloration. Our work investigated the scattering properties of SiO2 dielectric spheres with different diameters and detailly discussed their impact on the PL properties of phosphors, paving the way for colored fluorescent-assisted cooling coting to application and industrialization.

Keywords: solar reflection, infrared emissivity, mie scattering, photoluminescent emission, radiative cooling

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Authors: Abdul Musaweer Habib, Habibul Hasan Mazumder, Saiful Islam, Sohel Sikder, Omar Faruk Sikder

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The plethora of genome sequence information of bacteria in recent times has ushered in many novel strategies for antibacterial drug discovery and facilitated medical science to take up the challenge of the increasing resistance of pathogenic bacteria to current antibiotics. In this study, we adopted subtractive genomics approach to analyze the whole genome sequence of the Fusobacterium nucleatum, a human oral pathogen having association with colorectal cancer. Our study divulged 1499 proteins of Fusobacterium nucleatum, which has no homolog in human genome. These proteins were subjected to screening further by using the Database of Essential Genes (DEG) that resulted in the identification of 32 vitally important proteins for the bacterium. Subsequent analysis of the identified pivotal proteins, using the KEGG Automated Annotation Server (KAAS) resulted in sorting 3 key enzymes of F. nucleatum that may be good candidates as potential drug targets, since they are unique for the bacterium and absent in humans. In addition, we have demonstrated the 3-D structure of these three proteins. Finally, determination of ligand binding sites of the key proteins as well as screening for functional inhibitors that best fitted with the ligands sites were conducted to discover effective novel therapeutic compounds against Fusobacterium nucleatum.

Keywords: colorectal cancer, drug target, Fusobacterium nucleatum, homology modeling, ligands

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Authors: Abdorrasoul Malekpour, Mohammad Ghorbani Mojtaba Mortazavi, Mohammadreza Fattahi, Mohammad Hassan Meshkibaf, Ali Fakhrzad, Saeid Salehi, Saeideh Zahedi, Amir Ahmadimoghaddam, Parviz Farzadnia Dr., Mohammadreza Hajyani Asl Bs

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Hepatitis B as an infectious disease has eight main genotypes (A–H). The aim of this study is to Bioinformatically identify Rare Codon Clusters (RCC) in proteins structure of HBV. For detection of protein family accession numbers (Pfam) of HBV proteins; used of uni-prot database and Pfam search tool were used. Obtained Pfam IDs were analyzed in Sherlocc program and RCCs in HBV proteins were detected. In further, the structures of TrEMBL entries proteins studied in PDB database and 3D structures of the HBV proteins and locations of RCCs were visualized and studied using Swiss PDB Viewer software. Pfam search tool have found nine significant hits and 0 insignificant hits in 3 frames. Results of Pfams studied in the Sherlocc program show this program not identified RCCs in the external core antigen (PF08290) and truncated HBeAg protein (PF08290). By contrast the RCCs become identified in Hepatitis core antigen (PF00906) Large envelope protein S (PF00695), X protein (PF00739), DNA polymerase (viral) N-terminal domain (PF00242) and Protein P (Pf00336). In HBV genome, seven RCC identified that found in hepatitis core antigen, large envelope protein S and DNA polymerase proteins and proteins structures of TrEMBL entries sequences that reported in Sherlocc program outputs are not complete. Based on situation of RCC in structure of HBV proteins, it suggested those RCCs are important in HBV life cycle. We hoped that this study provide a new and deep perspective in protein research and drug design for treatment of HBV.

Keywords: rare codon clusters, hepatitis B virus, bioinformatic study, infectious disease

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Authors: Elise M. Shauf

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Beryllium can be found naturally in some fruits and vegetables (carrots, garden peas, kidney beans, pears) at very low concentrations, but is typically not clinically significant due to the low-level exposure and limited absorption of beryllium by the stomach and intestines. However, acute or chronic beryllium exposure can result in harmful toxic and carcinogenic biological effects. Beryllium can be both a workplace hazard and an environmental pollutant, therefore determining the presence of beryllium at trace levels can be essential to protect workers as well as the environment. Analysis of gallein, C₂₀H₁₂O₇, to determine if it is usable as a fluorescent dye for beryllium detection. The primary detection method currently in use includes hydroxybenzoquinoline sulfonates (HBQS), for which alternative indicators are desired. Unfortunately, gallein does not have the desired aspects needed as a dye for beryllium detection due to the peak shift properties.

Keywords: beryllium detection, fluorescent, gallein dye, indicator, spectroscopy

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Authors: Waroton Paisuwan, Mongkol Sukwattanasinitt, Mamoru Tobisu, Anawat Ajavakom

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Novel dihydroquinoline derivatives (DHP and DHP-OH) were synthesized in one pot via a tandem trimerization-cyclization of methylpropiolate. DHP and DHP-OH possess strong blue fluorescence with high quantum efficiencies over 0.70 in aqueous media. DHP-OH displays a remarkable fluorescence quenching selectively to the presence of Au3+ through the oxidation of dihydropyridine to pyridinium ion as confirmed by NMR and HRMS. DHP-OH was used to demonstrate the quantitative analysis of Au3+ in water samples with the limit of detection of 33 ppb and excellent recovery (>95%). This fluorescent probe was also applied for the determination of Au3+ residue in the gold nanoparticle solution and a paper-based sensing strip for the on-site detection of Au3+.

Keywords: Gold(III) ion detection, Fluorescent sensor, Fluorescence quenching, Dihydropyridine, Gold nanoparticles (AuNPs)

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Authors: Abdul Matin, Muhammad Ajmal, Uzma Yunus, Noaman-ul Haq, Hafiz M. Shohaib, Ambreen G. Muazzam

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Carbon nanoparticles (CNPs) have unique properties that are useful for the diagnosis and treatment of cancer due to their precise properties like small size (ideal for delivery within the body) stability in solvent and tunable surface chemistry for targeted delivery. Here, highly fluorescent, monodispersed and water-soluble CNPs were synthesized directly from a suitable carbohydrate source (glucose and sucrose) by one-step acid assisted ultrasonic treatment at 35 KHz for 4 hours. This method is green, simple, rapid and economical and can be used for large scale production and applications. The average particle sizes of CNPs are less than 10nm and they emit bright and colorful green-blue fluorescence under the irradiation of UV-light at 365nm. The CNPs were characterized by scanning electron microscopy, fluorescent spectrophotometry, Fourier transform infrared spectrophotometry, ultraviolet-visible spectrophotometry and TGA analysis. Fluorescent CNPs were used as fluorescent probe and nano-carriers for anticancer drug. Functionalized CNPs (with ethylene diamine) were attached with anticancer drug-Methotrexate. In vitro bioactivity and biocompatibility of CNPs-drug conjugates was evaluated by LDH assay and Sulforhodamine B assay using human lung carcinoma cell lines (H157). Our results reveled that CNPs showed biocompatibility and CNPs-anticancer drug conjugates have shown potent cytotoxic effects and high antitumor activities in lung cancer cell lines. CNPs are proved to be excellent substitute for conventional drug delivery cargo systems and anticancer therapeutics in vitro. Our future studies will be more focused on using the same nanoparticles in vivo model system.

Keywords: carbon nanoparticles, carbon nanoparticles-methotrexate conjugates, human lung carcinoma cell lines, lactate dehydrogenase, methotrexate

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Authors: Olivier de Backer, Alexis Khelfi, Olivier Svensek, Axelle Nolmans, Dominique Desnoeck

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The SMC5-SMC6 complex helps replication and repair of DNA double-strand breaks. Nse1, Nse3 and Nse4 are non-SMC components of the complex in which Nse3 stimulates the E3 ubiquitin ligase activity of Nse1 and is required for recruiting the complex on DNA. In most eukaryotes, Nse3 is a single protein, but in eutherians (placental mammals), it belongs to a large family of proteins called MAGE (Melanoma antigen) that share a conserved domain of about 200 aa known as MHD (Mage homology domain). MAGE assembles specific RING and HECT ubiquitin ligases and determines new substrates for ubiquitination. The MHD is required for the interaction with the cognate E3 ligase. Some MAGEs (referred to as Type I) are exclusively expressed in germ cells of the testis but are often expressed ectopically in cancer cells as the result of epigenetic modifications. The 12 MAGE-A genes belong to this category. Serval MAGE-A proteins could promote tumorigenesis by targeting tumor suppressor proteins (including p53) for ubiquitination and degradation. We showed that depletion of MAGE-A proteins in melanoma cells results in impaired DNA damage response and increased double-strand breaks after exposure to camptothecin. Moreover, it was shown that other actors of the DNA Damage Response were impacted when cells were depleted of MAGEA proteins.

Keywords: DNA damage response, melanoma, camptothecin, new role, MAGEA

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Authors: J. J. Lee, S. R. Sung, E. J. Yum, S. C. Kim, B. H. Hyun, M. Her, H. S. Lee

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Canine brucellosis is a critical problem in dogs leading to reproductive diseases which are mainly caused by Brucella canis. There are, nonetheless, not clear symptoms so that it may go unnoticed in most of the cases. Serodiagnosis for canine brucellosis has not been confirmed. Moreover, it has substantial difficulties due to broad cross-reactivity between the rough cell wall antigens of B. canis and heterospecific antibodies present in normal, uninfected dogs. Thus, this study was conducted to characterize the immunogenic proteins in cytoplasmic antigen (CPAg) of B. canis, which defined the antigenic sensitivity of the humoral antibody responses to B. canis-infected dogs. In analysis of B. canis CPAg, first, we extracted and purified the cytoplasmic proteins from cultured B. canis by hot-saline inactivation, ultrafiltration, sonication, and ultracentrifugation step by step according to the sonicated antigen extract method. For characterization of this antigen, we checked the sort and range of each protein on SDS-PAGE and verified the immunogenic proteins leading to reaction with antisera of B. canis-infected dogs. Selected immunodominant proteins were identified using MALDI-MS/MS. As a result, in an immunoproteomic assay, several polypeptides in CPAg on one or two-dimensional electrophoresis (DE) were specifically reacted to antisera from B. canis-infected dogs but not from non-infected dogs. The polypeptides with approximate 150, 80, 60, 52, 33, 26, 17, 15, 13, 11 kDa on 1-DE were dominantly recognized by antisera from B. canis-infected dogs. In the immunoblot profiles on 2-DE, ten immunodominant proteins in CPAg were detected with antisera of infected dogs between pI 3.5-6.5 at approximate 35 to 10 KDa, without any nonspecific reaction with sera in non-infected dogs. Ten immunodominant proteins identified by MALDI-MS/MS were identified as superoxide dismutase, bacteroferritin, amino acid ABC transporter substrate-binding protein, extracellular solute-binding protein family3, transaldolase, 26kDa periplasmic immunogenic protein, Rhizopine-binding protein, enoyl-CoA hydratase, arginase and type1 glyceraldehyde-3-phosphate dehydrogenase. Most of these proteins were determined by their cytoplasmic or periplasmic localization with metabolism and transporter functions. Consequently, this study discovered and identified the prominent immunogenic proteins in B. canis CPAg, highlighting that those antigenic proteins may accomplish a specific serodiagnosis for canine brucellosis. Furthermore, we will evaluate those immunodominant proteins for applying to the advanced diagnostic methods with high specificity and accuracy.

Keywords: Brucella canis, Canine brucellosis, cytoplasmic antigen, immunogenic proteins

Procedia PDF Downloads 118

Authors: Jiang Xu, Daoping Wang, Yinghong Pan

Abstract:

The jointing-booting stage is a critical period of both vegetative growth and reproductive growth in rice. Therefore, the proteomic analysis of the mutant Osbri1, whose corresponding gene OsBRI1 encodes the putative BRs receptor OsBRI1, at jointing-booting stage is very important for understanding the effects of BRs on vegetative and reproductive growth. In this study, the proteomes of leaves from an allelic mutant of the DWARF 61 (D61, OsBRI1) gene, Fn189 (dwarf54, d54) and its wild-type variety T65 (Taichung 65) at jointing-booting stage were analysed by using a Q Exactive plus orbitrap mass spectrometer, and more than 3,100 proteins were identified in each sample. Ontology analysis showed that these proteins distribute in various space of the cells, such as the chloroplast, mitochondrion, and nucleus, they functioned as structural components and/or catalytic enzymes and involved in many physiological processes. Moreover, quantitative analysis displayed that 266 proteins were differentially expressed in two samples, among them, 77 proteins decreased and 189 increased more than two times in Fn189 compared with T65, the proteins whose content decreased in Fn189 including b5-like Heme/Steroid binding domain containing protein, putative retrotransposon protein, putative glutaminyl-tRNA synthetase, and higher content proteins such as mTERF, putative Oligopeptidase homologue, zinc knuckle protein, and so on. A former study founded that the transcription level of a mTERF was up-regulated in the leaves of maize seedling after EBR treatment. In our experiments, it was interesting that one mTERF protein increased, but another mTERF decreased in leaves of Fn189 at jointing-booting stage, which suggested that BRs may have differential regulation mechanisms on the expression of various mTERF proteins. The relationship between other differential proteins with BRs is still unclear, and the effects of BRs on rice protein contents and its regulation mechanisms still need further research.

Keywords: bri1 mutant, jointing-booting stage, proteomic analysis, rice

Procedia PDF Downloads 218

Authors: Jamaludin Sallim, Rozlina Mohamed, Roslina Abdul Hamid

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In this paper, we proposed an Ant Colony Optimization (ACO) algorithm together with Traveling Salesman Problem (TSP) approach to investigate the clustering problem in Protein Interaction Networks (PIN). We named this combination as ACOPIN. The purpose of this work is two-fold. First, to test the efficacy of ACO in clustering PIN and second, to propose the simple generalization of the ACO algorithm that might allow its application in clustering proteins in PIN. We split this paper to three main sections. First, we describe the PIN and clustering proteins in PIN. Second, we discuss the steps involved in each phase of ACO algorithm. Finally, we present some results of the investigation with the clustering patterns.

Keywords: ant colony optimization algorithm, searching algorithm, protein functional module, protein interaction network

Procedia PDF Downloads 571

Authors: Indu Barwal, Shloka Thakur, Subhash C. Yadav

Abstract:

The plant virus capsid protein based nanoparticles are extensively studied for their application in biomedical research for development of nanomedicines and drug delivery systems. We have developed a chimeric drug delivery system by controlled in vitro assembly of separately bioconjugated fluorescent dye (as reporting molecule), folic acid (as receptor binding biomolecule for targeted delivery) and doxorubicin (as anticancer drug) using modified EDC NHS chemistry on heterologously overexpressed (E. coli) capsid proteins of cowpea chlorotic mottle virus (CCMV). This chimeric vehicle was further encapsidated on gold nanoparticles (20nm) coated with 5≠ thiolated DNA probe to neutralize the positive charge of capsid proteins. This facilitates the in vitro assembly of modified capsid subunits on the gold nanoparticles to develop chimeric GNPs encapsidated targeted drug delivery system. The bioconjugation of functionalities, number of functionality on capsid subunits as well as virus like nanoparticles, structural stability and in vitro assembly were confirmed by SDS PAGE, relative absorbance, MALDI TOF, ESI-MS, Circular dichroism, intrinsic tryptophan fluorescence, zeta particle size analyzer and TEM imaging. This vehicle was stable at pH 4.0 to 8.0 suitable for many organelles targeting. This in vitro assembled chimeric plant virus like particles could be suitable for ideal drug delivery vehicles for subcutaneous cancer treatment and could be further modified for other type of cancer treatment by conjugating other functionalities (targeting, drug) on capsids.

Keywords: chimeric drug delivery vehicles, bioconjugated plant, virus, capsid

Procedia PDF Downloads 453

Authors: Hung-Chieh Chen, Kuo-Hui Wang, Tsu-Lin Yeh

Abstract:

Proteomics represent the performance of genomic complement proteins and the protein level on functional genomics. This study adopted proteomic strategies for comparing serum proteins among three groups: elite sprinter (sprint runner group, SR), long-distance runners (long-distance runner group, LDR), and the untrained control group (control group, CON). Purposes: This study aims to identify elite sprinters and long-distance runners’ serum protein and to provide a comparison of their serum proteome’ composition. Methods: Serum protein fractionations that separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by a quantitative nano-LC-MS/MS-based proteomic profiling. The one-way analysis of variance (ANOVA) and Scheffe post hoc comparison (α= 0.05) was used to determine whether there is any significant difference in each protein level among the three groups. Results: (1) After analyzing the 307 identified proteins, there were 26 unique proteins in the SR group, and 18 unique proteins in the LDR group. (2) For the LDR group, 7 coagulation function-associated proteins’ expression levels were investigated: vitronectin, serum paraoxonase/arylesterase 1, fibulin-1, complement C3, vitamin K-dependent protein, inter-alpha-trypsin inhibitor heavy chain H3 and von Willebrand factor, and the findings show the seven coagulation function-associated proteins were significantly lower than the group of SR. (3) Comparing to the group of SR, this study found that the LDR group’s expression levels of the 2 antioxidant proteins (afamin and glutathione peroxidase 3) were also significantly lower. (4) The LDR group’s expression levels of seven immune function-related proteins (Ig gamma-3 chain C region, Ig lambda-like polypeptide 5, clusterin, complement C1s subcomponent, complement factor B, complement C4-A, complement C1q subcomponent subunit A) were also significantly lower than the group of SR. Conclusion: This study identified the potential serum protein markers for elite sprinters and long-distance runners. The changes in the regulation of coagulation, antioxidant, or immune function-specific proteins may also provide further clinical applications for these two different track athletes.

Keywords: biomarkers, coagulation, immune response, oxidative stress

Procedia PDF Downloads 91

Authors: Shigenori Togashi, Kei Takenaka

Abstract:

We developed new processes which can collect and detect rapidly airborne pathogens such as the avian flu virus for the pandemic prevention. The fluorescence antibody technique is known as one of high-sensitive detection methods for viruses, but this needs up to a few hours to bind sufficient fluorescence dyes to viruses for detection. In this paper, we developed a mist-labeling can detect substitution viruses in a short time to improve the binding rate of fluorescent dyes and substitution viruses by the micro reaction process. Moreover, we developed the rapid detection system with the above 'mist labeling'. The detection system set with a sampling bag collecting patient’s breath and a cartridge can detect automatically pathogens within 10 minutes.

Keywords: viruses, sampler, mist, detection, fluorescent dyes, microreaction

Procedia PDF Downloads 435

Authors: Vineeta Kaushik, Manisha Goel

Abstract:

Chaperones are proteins that help other proteins fold correctly, and are found in all domains of life i.e., prokaryotes, eukaryotes and archaea. Various comparative genomic studies have suggested that the archaeal protein folding machinery appears to be highly similar to that found in eukaryotes. In case of protein folding; slow rotation of peptide prolyl-imide bond is often the rate limiting step. Formation of the prolyl-imide bond during the folding of a protein requires the assistance of other proteins, termed as peptide prolyl cis-trans isomerases (PPIases). Cyclophilins constitute the class of peptide prolyl isomerases with a wide range of biological function like protein folding, signaling and chaperoning. Most of the cyclophilins exhibit PPIase enzymatic activity and play active role in substrate protein folding which classifies them as a category of molecular chaperones. Till date, there is not very much data available in the literature on archaeal cyclophilins. We aim to compare the structural and biochemical features of the cyclophilin protein from within the three domains to elucidate the features affecting their stability and enzyme activity. In the present study, we carry out in-silico analysis of the cyclophilin proteins to predict their conserved residues, sites under positive selection and compare these proteins to their bacterial and eukaryotic counterparts to predict functional divergence. We also aim to clone and express these proteins in heterologous system and study their biophysical characteristics in detail using techniques like CD and fluorescence spectroscopy. Overall we aim to understand the features contributing to the folding, stability and dynamics of the archaeal cyclophilin proteins.

Keywords: biophysical characterization, x-ray crystallography, chaperone-like activity, cyclophilin, PPIase activity

Procedia PDF Downloads 181