Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS. Methods:In the current investigation, a recombinant scFv mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results:Increased efficiency was achieved by EnBase® compared to LB broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC₅₀ for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion:Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv     

Evaluation of different types of compost tea to control rose powdery mildew (Sphaerotheca pannosa var. rosae)

Powdery mildew (Sphaerotheca pannosa var. rosae) is an important foliar disease which is found in all rose-producing greenhouses. An experiment was carried out in a commercial greenhouse in Hashtgerd, Iran, in 2014 to investigate whether compost teas can be used to control this disease. Taguchi's design of experiment (Taguchi's DOE) method was used to plan a minimum number of experiments and optimization to control rose powdery mildew. The test included three factors, namely the different types of compost tea (aerated and non-aerated compost tea, aerated and non-aerated vermicompost tea), the dosage (1:8 and 1:16, compost:water), and the number of spray applications (once and twice per week). The experiment was set up as an orthogonal array L₈ with three replicates. Based on the experimental results, the optimum treatment combination for controlling rose powdery mildew was found to be aerated compost tea with 1:8 dilution with an application of twice per week.     

Soil fertilization and pH effects on meadowfoam growth and flowering 1

Meadowfoam (Limnanthes alba Hartweg ex. Bentham) is a new oilseed crop. Published information on nutritional requirements and optimum soil pH for growing meadowfoam in controlled environments is currently lacking. This study evaluated the effects of application rates and timing of a modified Hoagland's solution and a range in initial potting medium pH on meadowfoam (L. alba subsp. alba, cv Mermaid) growth and development under controlled environment conditions. Weekly application of 50 mL of nutrient solution at a nitrogen (N) concentration of 840 mg/L (four times higher than the concentration in modified Hoagland's solution) resulted in good Mermaid growth and flower production. Optimum fertilizer application timing is apparently from one week after transplanting (38 days after seeding) until the flowering stems begin to elongate above the rosette leaves. Optimum soil pH seemed to be between 5 and 6, although Mermaid grew relatively well under initial soil pH ranging from 3.8 to 7.3.     

Goal programming approach for sustainable forest management （case study in Iranian Caspian forests）

We used a goal programming technique to determine the optimal harvest volume for the Iranian Caspian forest. We collected data including volume, growth, wood price at forest roadside, and variable harvesting costs. The allometric method was used to quantify seques- trated carbon. Regression analysis was used to derive growth models. Expected mean price was estimated using wood price and variable harvesting costs. Questionnaire was used to determine the constraints and the equation coefficients of the goal programming model. The optimal volume was determined using the goal programming method according to multipurpose forest management. LINGO software was used for analysis. Results indicated that the optimum volumes of species were 250.25 m3.ha-1 for beech, 59 m3.ha-1 for hornbeam, 73 m3.ha-1 for oak, 41 m3.ha-1 for alder, and 32 m3.ha-1 for other species. The total optimum volume is 455.25 m3.ha-1.     

Weed Control Efficacy: Response of Chickpea to Pre- and Post-Emergence Herbicides

Lack of control options for cool-season broadleaf weeds is a major deterrent to autumn-sown chickpea. Weed control and chickpea tolerance to PRE (pre-emergence) and POST (post-emergence) application of isoxaflutole and oxyflurofen, PRE metribuzin, POST pyridate, and flumetsulam were investigated at three locations, including Kermanshah, Kurdistan, and Hamedan provinces during 2017–2018. Untreated and weed-free checks were added for comparison. Pyridate and PRE oxyflurofen 125?g ai ha^?1 caused the minor visual crop injury according to EWRS score (1–1.8), while the highest crop injury occurred with metribuzin (EWRS score 3.5–8.5) in whole locations. The most effective herbicides for weed reduction were pyridate (70–75%), PRE oxyfluorfen (69–76%), and POST oxyfluorfen (65–73%) at Kermanshah, PRE oxyfluorfen at 125 and 175?g ai ha^?1 (70–78%), POST oxyfluorfen (70–76%) and pyridate (70–78%) at Kurdistan, PRE oxyfluorfen at 125 and 175?g ai ha^?1 (88–96%), metribuzin (91–100%) and Pyridate (80–97%) at Hamedan. Pyridate and PRE oxyfluorfen at 125?g ai ha^?1 resulted in the highest chickpea grain yield at the three locations. In general, PRE oxyfluorfen (125?g ai ha^?1) was similar to pyridate in terms of efficacy in weed control and grain yield enhancement.

     

Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris

Background:The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Los1 (loss of supression) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends RLS. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods:A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 ScFv expression were compared between the parent and mutant strains.Results:The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.Conclusion:The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains. Key Words: Aging, Longevity, Pichia pastoris, Recombinant proteins     

The effectiveness of seed priming with synthetic zinc-amino acid chelates in comparison with soil-applied ZnSO4 in improving yield and zinc availability of wheat grain

The effect of soil applied zinc (Zn) sulfate and seed priming with Zn-glutamine [Zn(Gln)₂], Zn-glycine [Zn(Gly)₂], Zn-arginine [Zn(Arg)₂], and Zn-histidine [Zn(His)₂] on yield and grain nutritional quality of two bread (Triticum aestivum L. cvs. Back Cross and Kavir) and a durum wheat genotype (Triticum durum L. cv. Durum) was investigated. Seed priming with [Zn(Gly)₂] and [Zn(Gln)₂] increased grain yield of wheat over soil applied Zn-sulfate treatment by 46 and 14%, respectively. Seed priming with [Zn(Gln)₂], [Zn(Arg)₂], and [Zn(His)₂] significantly increased grain protein content of wheat in comparison with control treatment. Seed priming with [Zn(Arg)₂] and [Zn(His)₂] resulted in higher Zn and Fe accumulation in wheat grain as compared with the other Zn treatments. Priming seeds with [Zn(Arg)₂] reduced phytic acid to Zn molar ratio of wheat grain. Seed priming with [Zn(Arg)₂] and [Zn(His)₂] might be used as an alternative approach for soil application of Zn-sulfate to overcome Zn deficiency in calcareous soils.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells

Background:Bispecific antibodies represent an important class of mAbs, with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti- CD3 × CD19 bsAb is a bispecific T-cell engager (BiTE) currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods:WPRE sequence was incorporated at the 3’ end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion:Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in HEK-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody. Key Words: Acute lymphoblastic leukemia, Bispecific antibodies, Monoclonal antibody     

Acute erythroid leukemia with multilineage dysplasia in a cat

Dysplastic features of erythroid and megakaryocytic lineages were observed in a cat with acute erythroid leukemia. We demonstrated that flow cytometry analysis of the expression of glycophorin A and CD71 by neoplastic cells can be helpful in the diagnosis of this type of feline leukemia.