Generation of a tumor- and tissue-specific episomal non-viral vector system

Rudolf Haase1, Terese Magnusson1, Baowei Su, Florian Kopp, Ernst Wagner, Hans Lipps, Armin Baiker and Manfred Ogris

Abstract
Background:

A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the
generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR).

Results:

Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer.

Conclusions:

In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

Keywords: Tumor Targeting, pDNA, Episomal, AFP, SM22, Tissue-specific Replication

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Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli

Yi Liu1, Wenjing Cui1†, Yueqin Fang2, Yuechun Yu1, Youtian Cui1, Yuanyuan Xia1, Michihiko Kobayashi  and Zhemin Zhou1*

Abstract
Background:

Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was overexpressed, its low expression level and low stability affect the further analysis.

Results:

We successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K.

Conclusions:

Our results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.

Keywords: NHase, N-end rule, Pseudomonas putida, P14K, Stability

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Optimization and validation of mitochondria-based functional assay as a useful tool to identify BH3-like molecules selectively targeting anti-apoptotic Bcl-2 proteins

Jianting Long, Liu Liu, Zaneta Nikolovska-Coleska, Sanjeev Shangary, Han Yi, Shenming Wang and Shaomeng Wang

Abstract
Background:

Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins delicately regulate mitochondrial outer membrane integrity through protein-protein interactions, which makes the mitochondrion an ideal cell-free system for screening molecules targeting the Bcl-2 anti-apoptotic proteins. But assay conditions need to be optimized for more reliable results. In this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins. In this study, high ionic strength buffer was adopted during the initiation of MOMP. Mitochondria isolated from human breast cancer cell lines with distinct expression patterns of Bcl-2 anti-apoptotic proteins were permeabilized by different BH3 peptides alone or in combination, with or without the presence of recombinant anti-apoptotic Bcl-2 family proteins. Cytochrome C and Smac/Diablo were tested in both supernatants and mitochondrial pellets by Western blotting.

Results:

Sufficient ionic strength was required for optimal release of Cytochrome C. Bad and Noxa BH3 peptides exhibited their bona fide antagonistic effects against Bcl-2/Bcl-xL and Mcl-1 proteins, respectively, whereas Bim BH3 peptide antagonized all three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergized with each other in the induction of MOMP when mitochondria were dually protected by both Bcl-2/Bcl-xL and Mcl-1.

Conclusions:

This method based on MOMP is a useful screening tool for identifying BH3 mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major Bcl-2 anti-apoptotic proteins.

Keywords: Mitochondrion, B cell lymphoma 2 (Bcl-2), Bcl-2 homolog domain 3 (BH3), Mitochondrial outermembrane permeabilization (MOMP)

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Diffusion of small molecules into medaka embryos improved by electroporation

Gerlinde Jung, Markus Hug, Christian Halter, Andrea Friesenhengst, Johann Walzer and Thomas Czerny

Abstract

Background:

Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.

Results:

Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.

Conclusions:

The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

Keywords: Medaka, Small molecules, Diffusion, Toxicology, Electroporation, LiCl

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High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

Volker Jäger, Konrad Büssow, Andreas Wagner, Susanne Weber, Michael Hust, André Frenzel and Thomas Schirrmann

Abstract
Background:

The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and
purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.

Results:

In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.

Conclusion:

Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

Keywords: Recombinant Antibodies, Single Chain Fv, scFv-Fc, ImmunoRNase, Transient Mammalian Protein Production, Serum-free medium

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Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum

Hafiz Muhammad Nasir Iqbal and Muhammad Asgher

Abstract

Background:

An eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP)
produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents.

Results:

A large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity
parameters were below the permissible limits.

Conclusions:

In conclusion, G. lucidum MnP was immobilized by sol–gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective
capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.

Keywords: Bio-catalysis, G. lucidum, MnP, PAGE, Sol–gel, Immobilization, Textile effluents, Decolorization,Toxicity reduction

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Monoclonal antibody humanness score and its applications

Sean H Gao, Kexin Huang, Hua Tu and Adam S Adler

Abstract
Background:

Monoclonal antibody therapeutics are rapidly gaining in popularity for the treatment of a myriad of diseases, ranging from cancer to autoimmune diseases and neurological diseases. Multiple forms of antibody therapeutics are in use today that differ in the amount of human sequence present in both the constant and
variable regions, where antibodies that are more human-like usually have reduced immunogenicity in clinical trials.

Results:

Here we present a method to quantify the humanness of the variable region of monoclonal antibodies and show that this method is able to clearly distinguish human and non-human antibodies with excellent specificity. After creating and analyzing a database of human antibody sequences, we conducted an in-depth analysis of the humanness of therapeutic antibodies, and found that increased humanness score is correlated with decreased immunogenicity of antibodies. We further discovered a surprisingly similarity in the immunogenicity of fully human antibodies and humanized antibodies that are more human-like based on their humanness score.

Conclusions:

Our results reveal that in most cases humanizing an antibody and confirming the humanness of the final form may be sufficient to eliminate immunogenicity issues to the same extent as using fully human antibodies. We created a public website to calculate the humanness score of any input antibody sequence based
on our human antibody database. This tool will be of great value during the preclinical drug development process for new monoclonal antibody therapeutics.

Keywords: Therapeutic antibody, Humanization, Immunogenicity

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Prospecting endophytic fungal assemblage of Digitalis lanata Ehrh. (foxglove) as a novel source of digoxin: a cardiac glycoside

Sanjana Kaul •Maroof Ahmed •Khalid Zargar •Pooja Sharma •Manoj K. Dhar

Abstract :

Endophytes, the chemical synthesizers inside plants, are the microorganisms having mutualistic relationship with the host plant. They can be used by plants for defense in addition to the production of a wide variety of beneficial bioactive secondary metabolites. There are reports that microbial endophytes mimic the bioactive compounds as produced by the plant itself thus making them a promising source of novel compounds. During the present study, endophytes were isolated from the symptomless leaves and stem of the angiosperm, Digitalis lanata (foxglove). Digitalis lanata belongs to the family Plantaginaceae and is an important medicinal plant known for the production of an important glycoside, digoxin having valuable medicinal importance. Glycosides from Digitalis have been reported to be cardiotonic and are widely used in the treatment of various heart conditions namely atrial fibrillation, atrial flutter, heart failure, etc. Endophytic fungi were isolated from Digitalis to screen them for such glycosides as have been found in the plant itself. A total of 35 fungal endophytes were isolated and screened for the production of secondary metabolites. After preliminary analysis by thin layer chromatography for the presence of bioactive compounds, crude extracts of five fungal cultures were selected for HPLC. HPLC chromatograms revealed the production of glycoside digoxin from the five selected endophytic cultures, thus providing a novel, alternative and eco-friendly source for the production of such a pharmaceutically important and valuable drug.

Keywords : Endophytes [1] Secondary metabolites [1]Glycosides [1] Digitalis [1] Digoxin [1] HPLC

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Studies on bioflocculant production by a mixed culture of Methylobacterium sp. Obi and Actinobacterium sp. Mayor

Ntsaluba Luvuyo,Uchechukwu U Nwodo,Leonard V Mabinya,Anthony I Okoh

Abstract :
Background : 
Bioflocculants effect the aggregation of suspended solutes in solutions thus, a viable alternative to inorganic poly-ionic and synthetic organic flocculants which are associated with deleterious health problems. Consequently, a consortium of two bacteria species were evaluated for optimized bioflocculant yield following the inadequacies of axenic cultures.

Results :
16S rDNA nucleotide sequencing and BLAST analysis of nucleotide sequences were used to identify the bacterial species, carbon and nitrogen sources optimally supporting bioflocculant production were assessed and the purified bioflocculant characterized. Nucleotide sequences showed 97% and 96% similarity to Methylobacterium sp. AKB-2008-KU9 and Methylobacterium sp. strain 440. The second isolate, likewise, showed 98% similarity to Actinobacterium OR-221. The sequences were deposited in GenBank as Methylobacterium sp. Obi [accession number HQ537130] and Actinobacterium sp. Mayor [accession number JF799090]. Flocculating activity of 95% was obtained in the presence of Ca2+ and heat-stability was exhibited with retention of above 70% activity at 100°C in 30
min. In addition, bioflocculant yield was about 8.203 g/l. A dose of 1 mg/ml of purified bioflocculant was optimal for the clarification of Kaolin suspension (100 ml) following Jar test. FTIR spectrum revealed the presence of carboxyl and hydroxyl functional groups amongst others.

Conclusions :
The mixed culture produced bioflocculant with high flocculating activity and an improved yield. The efficiency observed with jar test may imply industrial applicability.

Keywords : Bioflocculant, Consortium, Flocculating activity, Thermostable, Functional groups

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Expression of arachidonic acid-metabolizing cytochrome P450s in human megakaryocytic Dami cells

Yazun Bashir Jarrar & Jae-Gook Shin & Su-Jun Lee

Abstract :
Cytochrome P450s (P450s) are involved in the metabolism of arachidonic acid (ARA), and ARA metabolites are associated with various cellular signaling pathways,such as blood hemostasis and inflammation. The present study demonstrates the expression of ARA-metabolizing P450s in the human megakaryocytic Dami cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunublotting analysis followed by activity assays using ARA as a substrate. In addition to the previously identified CYP5A1, both protein and mRNAs of CYP1A1, 2U1, and 2J2 bands were detected. Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P<0.001). CYP1A1 protein expression in Dami cells was induced by 3-methylenecholantheren. This increase in CYP1A1 protein level was correlated with enhanced EROD activity (fourfold increase vs. the control), as well as with increased metabolites, such as 20-hydroxyeicosatrienoic acid (20-HETE), 14, 15-EET (14-,15-epoxyeicosatrienoic acid), and 14, 15-dihydroxyeicosatrienoic acid (14,15-DHET). The expression of soluble epoxide hydrolase, an enzyme responsible for the synthesis of DHETs from EETs,was confirmed by RT-PCR. Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A.The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.

Keywords : Arachidonic acid .Dami cells .P450s .Megakaryocytes .Platelets

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Nutrient improvement for simultaneous production of exopolysaccharide and mycelial biomass by submerged cultivation of Schizophyllum commune AGMJ-1 using statistical optimization

Mayur Joshi • Harshad Patel • Shilpa Gupte • Akshaya Gupte

Abstract:

Exopolysaccharides (EPS) of fungal origin have attracted special attention from researchers due to their multifarious applications in the food and pharmaceutical industries. In the present study, optimization of the process parameters for the production of exopolysaccharide by Schizophyllum commune AGMJ-1 was studied using one factor at a time (OFAT) method, Plackett–Burman design (PBD) and response surface methodology (RSM). OFAT method revealed xylose and yeast extract to be the most effective carbon and nitrogen sources and pH 5.3 as an optimum for maximum EPS production. Xylose, yeast extract and KCl were screened as statistically significant variables for EPS production using PBD. RSM based on the central composite design estimated that maximum EPS (4.26 g L-1), mycelial biomass (14 g L-1) and specific yield (0.45 g g-1) were obtained when concentration of xylose, yeast extract and KCl were set at 2.5 g % (w/v), 0.83 g % (w/v) and 6.53 mg % (w/v), respectively, in the production medium.

Keywords: Exopolysaccharide Mycelial biomass, Schizophyllum commune, One factor at a time, Plackett– Burman design Response surface methodology

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Phytochemicals and antioxidative enzymes defence mechanism on occurrence of yellow vein mosaic disease of pumpkin (Cucurbita moschata)

Namrata Jaiswal • M. Singh • R. S. Dubey • V. Venkataramanappa • D. Datta

Abstract :

Pumpkin (Cucurbita moschata) samples showing yellow vein mosaic disease in Varanasi region were identified with begomovirus infection using PCR amplification. A sequencing analysis of the full genome revealed that it is a strain of Tomato leaf curl Palampur virus (GenBank ID. FJ931537). Phytochemical composition and antioxidative enzyme levels were compared in infected and healthy plants. The study revealed that the amount of total protein declined in the infected leaves but elevated up to 135 % in the fruits of infected plants, whereas vitamin C and antioxidants declined in infected leaves as well as fruits. There was substantial increase in total phenol content in leaves (72 %) and fruits (300 %) of infected plants. In infected samples, substantial increase in activities of superoxide dismutases (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT) was observed as compared to the uninfected control plants. The native PAGE showed alterations in the intensities of isozyme bands in the infected plants. The APX, GPX, CAT, SOD and glutamate dehydrogenase (GDH) bands were intense in the infected plants, whereas the GR isozyme showed reduced intensity in diseased plants.

Keywords: Catalase Glutathione reductase Begomovirus Peroxidase Superoxide dismutase

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