Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli Abstract Objectives The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. Results In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of − 5.5 kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. Conclusions In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Autochthonous lactic acid bacteria—presentation of potential probiotics application Abstract Objective The objective of this study was to evaluate the probiotic potential as well as the ability of adhesion and aggregation of natural and autochthonous lactic acid bacteria, isolated from traditionally made cheese. Results Lactic acid bacteria from natural food sources can be promising probiotic candidates and they can be used in natural food preservation or like starter cultures. Tested autochthonous isolates showed tolerance to the simulated gastrointestinal condition as well as the sensitivity to clinically relevant antibiotics, especially to ampicillin (MIC at 0.195 μg mL−1 for lactobacilli and from 0.195 to 3.125 μg mL−1 for lactococci). Among isolates, the highest percentage of adhesion was detected with chloroform, while the adhesion ability of selected isolates to pig intestinal epithelium was in the correlation with the results of adhesion ability with solvents. The auto-aggregation ability of isolates was demonstrated, while co-aggregation with Escherichia coli was strain specific. Conclusion The results indicated the potential probiotic properties of the isolates and give evidence for further investigation and potential application in the dairy industry.

Effects of Pseudomonas alkylphenolica KL28 on immobilization of Hg in soil and accumulation of Hg in cultivated plant Abstract Objective The available content of mercury (Hg) in farmland soil is directly related to the safety of agricultural products. Meanwhile, humans may accumulate high concentrations of Hg through the food chain, resulting in health damage. Regarding the remediation technologies of Hg-contaminated soil, research and development is mainly concentrated on the immobilisation of Hg in soil and efficient extraction by accumulators. Therefore, in this work, the highly Hg-tolerant strain Pseudomonas alkylphenolica KL28 was used to study the removal effect of Hg in a solution, immobilization effect of Hg in soil, and its effect on growth, Hg accumulation and photosynthetic characteristics of Brassica campestris L. Results KL28 could effectively remove Hg2+ in the solution, with the removal ratio of 96.0% at 24 h. This strain could reduce decreases in shoots’ and roots’ dry weights by 31% and 16%, respectively, at a Hg concentration of 20 mg/L. The available Hg in the soil decreased to 4.7–9.4% in 8 days treated with KL28 bacterial solution at a dosage of 100 L/hm2. Meanwhile, with increases in Hg concentrations, Fv/Fm, Y(II), Y(I) and Y(NPQ) in the leaves of B. campestris showed a downward trend while Y(ND) and Y(NO) displayed an upward trend. Under the stress of 20 mg/L Hg2+, KL28 could reduce the Fv/Fm from 11.2 to 6.1%. Conclusions KL28 could effectively remove Hg in the solution, immobilize Hg in soil, promote growth, decrease Hg accumulation and affect photosynthetic characteristics of B. campestris, indicating its potential use in Hg contaminated soils.

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides Abstract Objectives Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system. Results Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef60–84, Nef126–144, Vpr34–47, Vpr60–75, Gp16030–53, Gp160308–323 and P248–151 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32 kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293 T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200 nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293 T cells. Conclusion The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

Automation in cell and gene therapy manufacturing: from past to future Abstract As more and more cell and gene therapies are being developed and with the increasing number of regulatory approvals being obtained, there is an emerging and pressing need for industrial translation. Process efficiency, associated cost drivers and regulatory requirements are issues that need to be addressed before industrialisation of cell and gene therapies can be established. Automation has the potential to address these issues and pave the way towards commercialisation and mass production as it has been the case for ‘classical’ production industries. This review provides an insight into how automation can help address the manufacturing issues arising from the development of large-scale manufacturing processes for modern cell and gene therapy. The existing automated technologies with applicability in cell and gene therapy manufacturing are summarized and evaluated here.

Evidence for chaotropicity/kosmotropicity offset in a yeast growth model Abstract Chaotropes are compounds which cause the disordering, unfolding and denaturation of biological macromolecules. It is the chaotropicity of fermentation products that often acts as the primary limiting factor in ethanol and butanol fermentations. Since ethanol is mildly chaotropic at low concentrations, it prevents the growth of the producing microbes via its impacts on a variety of macromolecular systems and their functions. Kosmotropes have the opposite effect to chaotropes and we hypothesised that it might be possible to use these to mitigate chaotrope-induced inhibition of Saccharomyces cerevisiae growth. We also postulated that kosmotrope-mediated mitigation of chaotropicity is not quantitatively predictable. The chaotropes ethanol and urea, and compatible solutes glycerol and betaine (kosmotrope), and the highly kosmotropic salt ammonium sulphate all inhibited the growth rate of Saccharomyces cerevisiae in the concentration range 5–15%. They resulted in increased lag times, decreased maximum specific growth rates, and decreased final optical densities. Surprisingly, neither the stress protectants nor ammonium sulphate reduced the inhibition of growth caused by ethanol. Whereas, in some cases, compatible solutes and kosmotropes mitigated against the inhibitory effects of urea. However, this effect was not mathematically additive from the quantification of chao-/kosmotropicity of each individual compound. The potential effects of glycerol, betaine and/or ammonium sulphate may have been reduced or masked by the metabolic production of compatible solutes. It may nevertheless be that the addition of kosmotropes to fermentations which produce chaotropic products can enhance metabolic activity, growth rate, and/or product formation.

A kinetic model to optimize and direct the dose ratio of Dsz enzymes in the 4S desulfurization pathway in vitro and in vivo Abstract Objective To enhance the biodesulfurization rate using a kinetic model that directs the ratio of Dsz enzymes. Results This study established a kinetic model that predicted the optimal ratio of Dsz enzymes in the 4S biodesulfurization system to be A:B:C = 1:2:4 and 1:4:2. When BCAD+1A+4B+2C, the conversion rate of dibenzothiophene (DBT) to 2-hydroxybiphenyl (HBP) was close to 100% in vitro. When the gene dose of dszC was increased, the HBP yield of the recombinant strain BL21(DE3)/BCAD + C reached approximately 0.012 mM in vivo, which was approximately 6-fold higher than that of the BCAD strain. Conclusions According to the results predicted by the enzyme kinetic model, maintaining higher concentrations of DszC and DszB in the desulfurization system can effectively improve the desulfurization efficiency.

Experimental biogas production from recycled pulp and paper wastewater by biofilm technology Abstract Objective The main objective of this study is the evaluation of RPPW anaerobic digestion feasibility at laboratory scale under Mesophilic condition. The experiment is conducted using a two-stage biofilm digester of 5 L capacity with mobile support material. Results Anaerobic treatment of wastewater from recycled pulp and paper industry in Morocco was tested using a laboratory-scale anaerobic biofilm digester that operated under mesophilic conditions over a 70-day. Chemical oxygen demand (COD) efficiency, volatile and total solid (VS, TS) elimination of the substrate during the process were: 78%, 52% and 48% respectively. The system was stable throughout its operating cycle with an optimum pH (7.24), alkalinity (1750 mg CaCO3/L) and a volatile fatty acid value (760 mg/L). The experimental daily biogas production measured reaches a value of 5 L/day with a composition of 71% methane, 27.6% carbon dioxide, 0.2 oxygen and 7713 ppm of the H2S. The study results show that the anaerobic biofilm reactor is a suitable technique for recycled pulp and paper wastewater (RPPW) treatment. The reactor shows high performances in terms of process stability, removal efficiency (> 70%) and biogas production. Conclusion Anaerobic digestion is an efficient waste treatment technology that uses natural anaerobic decomposition to reduce the volume of waste while producing biogas. However, research is needed to strengthen microbial metabolism, biochemistry and the functioning of the rector to improve biogas production. The RPPW AD experiment with biofilm digester technology was stable throughout the operation period. The digester knows an overloaded in the last phase of the experiment which leads to an inhibition of biogas production.

Evaluating the impact of suramin additive on CHO cells producing Fc-fusion protein Abstract Objective To examine the effects of suramin in CHO cell cultures in terms of the cell culture performance and quality of the Fc-fusion protein. Results Suramin had positive effects on the CHO cell cultures. The addition of suramin caused an increase in the viable cell density, cell viability, and titer of the Fc-fusion protein. Moreover, suramin had no impact on protein aggregation and enhanced the sialic acid contents of Fc-fusion protein by 1.18-fold. The enhanced sialylation was not caused by the increased nucleotide sugar level but by the inhibition of sialidase activity. The results showed that suramin inhibited apoptosis and had positive impacts on the productivity and quality of Fc-fusion protein. Conclusion The addition of suramin increased the production of Fc-fusion protein and enhanced sialylation when added as a supplement to the media component in CHO cell cultures. This study suggested that suramin could be a beneficial additive during the biological production in terms of the productivity and quality of Fc-fusion protein.

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5′-coding region in Escherichia coli Abstract Objective To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed. Results We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. Conclusion Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).