ResearchPad - Applied Microbiology and Biotechnology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[PEGylated crushed gold shell-radiolabeled core nanoballs for in vivo tumor imaging with dual positron emission tomography and Cerenkov luminescent imaging]]> https://www.researchpad.co/product?articleinfo=5b58ec17463d7e5414116038

Background

Radioactive isotope-labeled gold nanomaterials have potential biomedical applications. Here, we report the synthesis and characterization of PEGylated crushed gold shell-radioactive iodide-124-labeled gold core nanoballs (PEG-124I-Au@AuCBs) for in vivo tumor imaging applications through combined positron emission tomography and Cerenkov luminescent imaging (PET/CLI).

Results

PEG-124I-Au@AuCBs showed high stability and sensitivity in various pH solutions, serum, and in vivo conditions and were not toxic to tested cells. Combined PET/CLI clearly revealed tumor lesions at 1 h after injection of particles, and both signals remained visible in tumor lesions at 24 h, consistent with the biodistribution results.

Conclusion

Taken together, the data provided strong evidence for the application of PEG-124I-Au@AuCBs as promising imaging agents in nuclear medicine imaging of various biological systems, particularly in cancer diagnosis.

Electronic supplementary material

The online version of this article (10.1186/s12951-018-0366-x) contains supplementary material, which is available to authorized users.

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<![CDATA[Response surface methodology (RSM) modeling to improve removal of ciprofloxacin from aqueous solutions in photocatalytic process using copper oxide nanoparticles (CuO/UV)]]> https://www.researchpad.co/product?articleinfo=5b4c8951463d7e0cf6817989

Ciprofloxacin (CIP) antibiotic is considered as an emerging and biological resistant pollutant. This study aimed to improve of the removal of CIP from synthetic aqueous solutions in photocatalytic process through copper oxide nanoparticles as catalyst (CuO/UV). The effect of CIP concentration (10–200 mg/l), catalyst dosage included CuO (0.01–0.1 g/l) and pH (3–11) as independent variables on the COD removal efficiency as response in photocatalytic process using UV-C lamps with three different powers of 8, 15 and 30-W were optimized through the central composite design in response surface method using design-expert software. A second order model was selected as the best model with R2 values and lack of fit as 0.85 and 0.06 for lamp 8-W, 0.89 and 0.11 for lamp 15-W, and 0.86 and 0.19 for lamp 30-W, respectively. Optimum conditions were obtained in CIP concentration of 11.2 (mg/l), CuO dosage of 0.08 (g/l), and pH value of 8.17. In this condition, predicted maximum COD removal was respectively found 83.79, 93.18, and 98.90% for lamps 8, 15 and 30-W. According to the results, photocatalytic process using copper oxide nanoparticles can effectively compose CIP in aqueous solutions.

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<![CDATA[Intensified Sampling in Response to a Salmonella Heidelberg Outbreak Associated with Multiple Establishments Within a Single Poultry Corporation]]> https://www.researchpad.co/product?articleinfo=5b4c5a3c463d7e09f8ec4a81

Abstract

On June 28, 2013, the Food Safety and Inspection Service (FSIS) was notified by the Centers for Disease Control and Prevention (CDC) of an investigation of a multistate cluster of illnesses of Salmonella enterica serovar Heidelberg. Since case-patients in the cluster reported consumption of a variety of chicken products, FSIS used a simple likelihood-based approach using traceback information to focus on intensified sampling efforts. This article describes the multiphased product sampling approach taken by FSIS when epidemiologic evidence implicated chicken products from multiple establishments operating under one corporation. The objectives of sampling were to (1) assess process control of chicken slaughter and further processing and (2) determine whether outbreak strains were present in products from these implicated establishments. As part of the sample collection process, data collected by FSIS personnel to characterize product included category (whole chicken and type of chicken parts), brand, organic or conventional product, injection with salt solutions or flavorings, and whether product was skinless or skin-on. From the period September 9, 2013, through October 31, 2014, 3164 samples were taken as part of this effort. Salmonella percent positive declined from 19.7% to 5.3% during this timeframe as a result of regulatory and company efforts. The results of intensified sampling for this outbreak investigation informed an FSIS regulatory response and corrective actions taken by the implicated establishments. The company noted that a multihurdle approach to reduce Salmonella in products was taken, including on-farm efforts such as environmental testing, depopulation of affected flocks, disinfection of affected houses, vaccination, and use of various interventions within the establishments over the course of several months.

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<![CDATA[Transcriptome analysis of a taxol-producing endophytic fungus Cladosporium cladosporioides MD2]]> https://www.researchpad.co/product?articleinfo=5b4c4250463d7e06c7b529de

The shortage of molecular information for taxol-producing fungi has greatly impeded the understanding of fungal taxol biosynthesis mechanism. In this study, the transcriptome of one taxol-producing endophytic fungus Cladosporium cladosporioides MD2 was sequenced for the first time. About 1.77 Gbp clean reads were generated and further assembled into 16,603 unigenes with an average length of 1110 bp. All of the unigenes were annotated against seven public databases to present the transcriptome characteristics of C. cladosporioides MD2. A total of 12,479 unigenes could be annotated with at least one database, and 1593 unigenes could be annotated in all queried databases. In total, 8425 and 3350 unigenes were categorized into 57 GO functional groups and 262 KEGG pathways, respectively, exhibiting the dominant GO terms and metabolic pathways in the C. cladosporioides MD2 transcriptome. One potential and partial taxol biosynthetic pathway was speculated including 9 unigenes related to terpenoid backbone biosynthesis and 40 unigenes involved in the biosynthetic steps from geranylgeranyl diphosphate to 10-deacetylbaccatin III. These results provided valuable information for the molecular mechanism research of taxol biosynthesis in C. cladosporioides MD2.

Electronic supplementary material

The online version of this article (10.1186/s13568-018-0567-6) contains supplementary material, which is available to authorized users.

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<![CDATA[Molecular Networks of Postia placenta Involved in Degradation of Lignocellulosic Biomass Revealed from Metadata Analysis of Open Access Gene Expression Data]]> https://www.researchpad.co/product?articleinfo=5b4c1729463d7e0329bcc442

To understand the common gene expression patterns employed by P. placenta during lignocellulose degradation, we have retrieved genome wide transcriptome datasets from NCBI GEO database and analyzed using customized analysis pipeline. We have retrieved the top differentially expressed genes and compared the common significant genes among two different growth conditions. Genes encoding for cellulolytic (GH1, GH3, GH5, GH12, GH16, GH45) and hemicellulolytic (GH10, GH27, GH31, GH35, GH47, GH51, GH55, GH78, GH95) glycoside hydrolase classes were commonly up regulated among all the datasets. Fenton's reaction enzymes (iron homeostasis, reduction, hydrogen peroxide generation) were significantly expressed among all the datasets under lignocellulolytic conditions. Due to the evolutionary loss of genes coding for various lignocellulolytic enzymes (including several cellulases), P. placenta employs hemicellulolytic glycoside hydrolases and Fenton's reactions for the rapid depolymerization of plant cell wall components. Different classes of enzymes involved in aromatic compound degradation, stress responsive and detoxification mechanisms (cytochrome P450 monoxygenases) were found highly expressed in complex plant biomass substrates. We have reported the genome wide expression patterns of genes coding for information, storage and processing (KOG), tentative and predicted molecular networks involved in cellulose, hemicellulose degradation and list of significant protein-ID's commonly expressed among different lignocellulolytic growth conditions.

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<![CDATA[Oxygen effects on rhamnolipids production by Pseudomonas aeruginosa]]> https://www.researchpad.co/product?articleinfo=5b4bd974463d7e7e0dcdb89d

Background

Rhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa. This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl-genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements.

Results

Quantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC–MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C8-C10, Rha–Rha-C10-C12:1 and Rha–Rha-C8-C10; anaerobically produced rhamnolipids mainly contained Rha-C10-C12 and Rha-C10-C10. Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air–water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI24 = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI24 = 62.3%. Both two rhamnolipids products retained surface activity (ST < 35.0 mN/m) and emulsifying activity (EI24 > 60.0%) under temperatures (4–121 °C), pH values (4–10) and NaCl concentrations less than 90 g/L.

Conclusions

Oxygen affected the rhl-genes expression in P. aeruginosa, thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

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<![CDATA[Ten hub genes associated with progression and prognosis of pancreatic carcinoma identified by co-expression analysis]]> https://www.researchpad.co/product?articleinfo=5b4b2557463d7e737c241d59

Since the five-year survival rate is less than 5%, pancreatic ductal adenocarcinoma (PDAC) remains the 4th cause of cancer-related death. Although PDAC has been repeatedly researched in recent years, it is still predicted to be the second leading cause of cancer death by year 2030. In our study, the differentially expressed genes in dataset GSE62452 were used to construct a co-expression network by WGCNA. The yellow module related to grade of PDAC was screened. Combined with co-expression network and PPI network, 36 candidates were screened. After survival and regression analysis by using GSE62452 and TCGA dataset, we identified 10 real hub genes (CCNA2, CCNB1, CENPF, DLGAP5, KIF14, KIF23, NEK2, RACGAP1, TPX2 and UBE2C) tightly related to progression of PDAC. According to Oncomine database and The Human Protein Atlas (HPA), we found that all real hub genes were overexpressed in pancreatic carcinoma compared with normal tissues on transcriptional and translational level. ROC curve was plotted and AUC was calculated to distinguish recurrent and non-recurrent PDAC and every AUC of the real hub gene was greater than 0.5. Finally, functional enrichment analysis and gene set enrichment (GSEA) was performed and both of them showed the cell cycle played a vital role in PDAC.

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<![CDATA[Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions]]> https://www.researchpad.co/product?articleinfo=5b47a4d9463d7e73d008efd1

Background

The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis.

Methods

Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents.

Results

Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification.

Conclusions

Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.

Electronic supplementary material

The online version of this article (10.1186/s12934-017-0856-9) contains supplementary material, which is available to authorized users.

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<![CDATA[Graphene oxide conjugated with polymers: a study of culture condition to determine whether a bacterial growth stimulant or an antimicrobial agent?]]> https://www.researchpad.co/product?articleinfo=5bf2b9bdd5eed0c48412c521

Background

The results showed that the deciding factor is the culture medium in which the bacteria and the graphene oxide (GO) are incubated at the initial manipulation step. These findings allow better use of GO and GO-based materials more and be able to clearly apply them in the field of biomedical nanotechnology.

Results

To study the use of GO sheets applied in the field of biomedical nanotechnology, this study determines whether GO-based materials [GO, GO-polyoxyalkyleneamine (POAA), and GO-chitosan] stimulate or inhibit bacterial growth in detail. It is found that it depends on whether the bacteria and GO-based materials are incubated with a nutrient at the initial step. This is a critical factor for the fortune of bacteria. GO stimulates bacterial growth and microbial proliferation for Gram-negative and Gram-positive bacteria and might also provide augmented surface attachment for both types of bacteria. When an external barrier that is composed of GO-based materials forms around the surface of the bacteria, it suppresses nutrients that are essential to microbial growth and simultaneously produces oxidative stress, which causes bacteria to die, regardless of whether they have an outer-membrane-Gram-negative-bacteria or lack an outer-membrane-Gram-positive-bacteria, even for high concentrations of biocompatible GO-POAA. The results also show that these GO-based materials are capable of inducing reactive oxygen species (ROS)-dependent oxidative stress on bacteria. Besides, GO-based materials may act as a biofilm, so it is hypothesized that they suppress the toxicity of low-dose chitosan.

Conclusion

Graphene oxide is not an antimicrobial material but it is a general growth enhancer that can act as a biofilm to enhance bacterial attachment and proliferation. However, GO-based materials are capable of inducing ROS-dependent oxidative stress on bacteria. The applications of GO-based materials can clearly be used in antimicrobial surface coatings, surface-attached stem cells for orthopedics, antifouling for biocides and microbial fuel cells and microbial electro-synthesis.

Electronic supplementary material

The online version of this article (10.1186/s12951-017-0328-8) contains supplementary material, which is available to authorized users.

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<![CDATA[Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc]]> https://www.researchpad.co/product?articleinfo=5b476a9f463d7e6ff2423d80

Background

Mushroom showed pellet, clump and/or filamentous mycelial morphologies during submerged fermentation. Addition of microparticles including Talc (magnesium silicate), aluminum oxide and titanium oxide could control mycelial morphologies to improve mycelia growth and secondary metabolites production. Here, effect of microparticle Talc (45 μm) addition on the mycelial morphology, fermentation performance, monosaccharide compositions of polysaccharides and enzymes activities associated with polysaccharide synthesis in G. frondosa was well investigated to find a clue of the relationship between polysaccharide biosynthesis and morphological changes.

Results

Addition of Talc decreased the diameter of the pellets and increased the percentage of S-fraction mycelia. Talc gave the maximum mycelial biomass of 19.25 g/L and exo-polysaccharide of 3.12 g/L at 6.0 g/L of Talc, and mycelial polysaccharide of 0.24 g/g at 3.0 g/L of Talc. Talc altered the monosaccharide compositions/percentages in G. frondosa mycelial polysaccharide with highest mannose percentage of 62.76 % and lowest glucose percentage of 15.22 % followed with the corresponding changes of polysaccharide-synthesis associated enzymes including lowest UDP-glucose pyrophosphorylase (UGP) activity of 91.18 mU/mg and highest UDP-glucose dehydrogenase (UGDG) and GDP-mannose pyrophosphorylase (GMPPB) activities of 81.45 mU/mg and 93.15 mU/mg.

Conclusion

Our findings revealed that the presence of Talc significantly changed the polysaccharide production and sugar compositions/percentages in mycelial and exo-polysaccharides by affecting mycelial morphology and polysaccharide-biosynthesis related enzymes activities of G. frondosa.

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<![CDATA[A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli]]> https://www.researchpad.co/product?articleinfo=5b4730ca463d7e6c0337a131

Background

The high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner.

Methods

Based on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags.

Results

The expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase.

Conclusions

Observations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins.

Electronic supplementary material

The online version of this article (10.1186/s12934-017-0845-z) contains supplementary material, which is available to authorized users.

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<![CDATA[Comparison of tolerance of four bacterial nanocellulose-producing strains to lignocellulose-derived inhibitors]]> https://www.researchpad.co/product?articleinfo=5bf1b216d5eed0c484d2ceab <![CDATA[Spherules and IBV]]> https://www.researchpad.co/product?articleinfo=5bc163a640307c11974ebee9

Infectious bronchitis virus (IBV) is an economically important virus infecting chickens, causing large losses to the poultry industry globally. While vaccines are available, there is a requirement for novel vaccine strategies due to high strain variation and poor cross-protection. This requires a more detailed understanding of virus-host cell interactions to identify candidates for targeted virus attenuation. One key area of research in the positive sense RNA virus field, due to its central role in virus replication, is the induction of cellular membrane rearrangements by this class of viruses for the assembly of virus replication complexes. In our recent work, we identified the structures induced by IBV during infection of cultured cells, as well as primary cells and ex vivo organ culture. We identified structures novel to the coronavirus family, which strongly resemble replication sites of other positive sense RNA viruses. We have begun to extend this work using recombinant IBVs, which are chimera of different virus strains to study the role of viral proteins in the induction of membrane rearrangements.

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<![CDATA[Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha]]> https://www.researchpad.co/product?articleinfo=5b7d0aef463d7e30de717cf2

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-γ and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L−1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFα1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.

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<![CDATA[Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae]]> https://www.researchpad.co/product?articleinfo=5b7d0af2463d7e30de717cf5

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies μm−2. For relative quantitation, we compared wild-type cells to gas1Δ cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.

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<![CDATA[Specific transcriptional responses induced by 8-methoxypsoralen and UVA in yeast]]> https://www.researchpad.co/product?articleinfo=5b7d0aec463d7e30de717cf0

Treatment of eukaryotic cells with 8-methoxypsoralen plus UVA irradiation (8-MOP/UVA) induces pyrimidine monoadducts and interstrand crosslinks and initiates a cascade of events leading to cytotoxic, mutagenic and carcinogenic responses. Transcriptional activation plays an important part in these responses. Our previous study in Saccharomyces cerevisiae showed that the repair of these lesions involves the transient formation of DNA double-strand breaks and the enhanced expression of landmark DNA damage response genes such as RAD51, RNR2 and DUN1, as well as the Mec1/Rad53 kinase signaling cascade. We have now used DNA microarrays to examine genome-wide transcriptional changes produced after induction of 8-MOP/UVA photolesions. We found that 128 genes were strongly induced and 29 genes strongly repressed. Modifications in gene expression concern numerous biological processes. Compared to other genotoxic treatments, c. 42% of the response genes were specific to 8-MOP/UVA treatment. In addition to common DNA damage response genes and genes induced by environmental stresses, a large fraction of 8-MOP/UVA response genes correspond to membrane-related functions.

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<![CDATA[Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish]]> https://www.researchpad.co/product?articleinfo=5b7af55e463d7e5e66b93aca

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.

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<![CDATA[Bio-Bites!]]> https://www.researchpad.co/product?articleinfo=5ae685e8463d7e502ebb38bb ]]> <![CDATA[A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation]]> https://www.researchpad.co/product?articleinfo=5add682e463d7e355c484539

Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable ‘omics’ studies of the subcellular compartments of S. cerevisiae and other yeasts.

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<![CDATA[Recent Advances in Drug Repositioning for the Discovery of New Anticancer Drugs]]> https://www.researchpad.co/product?articleinfo=5ad69394463d7e08f36bfecb

Drug repositioning (also referred to as drug repurposing), the process of finding new uses of existing drugs, has been gaining popularity in recent years. The availability of several established clinical drug libraries and rapid advances in disease biology, genomics and bioinformatics has accelerated the pace of both activity-based and in silico drug repositioning. Drug repositioning has attracted particular attention from the communities engaged in anticancer drug discovery due to the combination of great demand for new anticancer drugs and the availability of a wide variety of cell- and target-based screening assays. With the successful clinical introduction of a number of non-cancer drugs for cancer treatment, drug repositioning now became a powerful alternative strategy to discover and develop novel anticancer drug candidates from the existing drug space. In this review, recent successful examples of drug repositioning for anticancer drug discovery from non-cancer drugs will be discussed.

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