Study on the use of bovine blood protein hydrolysate as a peptone in microbial culture media.

Livestock blood is a protein-rich waste byproduct produced during meat production processes in slaughterhouses. Its utilization through conversion into value-added products is an intriguing management strategy. In this study, bovine blood was used to obtain the protein hydrolysate for use as a peptone for microbial growth medium. Lyophilized bovine blood was heat treated to make it susceptible to enzymic hydrolysis, and then enzymatically treated with trypsin (bovine pancreas protease) to produce protein hydrolysate. Physico-chemical features were determined for protein hydrolysate and compared to commercial Merck peptone from meat. Amino acid compositions of bovine blood and commercial peptones were subjected to multivariate analysis based on Euclidean similarity matrix using software PAST. Strains of Staphylococcus aureus 25,923, Pseudomonas aeruginosa 27,853, Staphylococcus aureus 6538 P, Enterococcus faecalis 11,700, Escherichia coli 8739, Klebsiella pneumoniae 13,883, Salmonella typhimurium 14,028 and Listeria monocytogenes 13,932 were used as test microbial strains. Growth of bacteria in culture media based on the peptone from bovine protein hydrolysate was compared to that in corresponding reference media based on commercial peptone. The results of these growth tests were comparable. Growth data were depicted and statistically analyzed using R packages ggplot2 and growthcurver, respectively, providing data fitting a standard form of logistic equation.

Bioremediation of a salty petrochemical wastewater containing bisphenol A by a novel indigenous Pseudomonas pseudoalcaligenes.

One novel indigenous halotolerant, Pseudomonas sp, with high potential for bisphenol A (BPA) biodegradation was isolated from an outlet of petrochemical wastewater in Iran. The optimal temperature and pH for degradation of BPA by this strain were 30 °C and 7, respectively. This strain was able to decrease COD (chemical oxygen demand) of basal salt medium containing 300 mg L-1 BPA as sole carbon source and 40 g L-1 NaCl from 655.2 to 109.2 mg L-1 (about 83% decrease) after 36 h. The bacterium degraded 56.3 (19%), 202.43 (67%), 288.86 (96%) and 300 mg L-1 (100%) BPA in basal salt medium containing 300 mg L-1 BPA and 40 g L-1 NaCl within 12, 18, 24 and 36 h, respectively. In addition, this strain could degrade phenol (100 mg L-1) and BPA (300 mg L-1) in salty petrochemical wastewater within 24 h, completely. In batch fermentation of petrochemical wastewater using this strain higher growth and phenol (100 mg L-1), BPA (372 mg L-1) removal within 6 h were achieved. High performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS) analysis revealed several intermediates during the BPA degradation process. These intermediates were identified as 4-hydroxybenzaldehyde, 4-hydroxyacetophenone, 4-hydroxyphenylacetate, M-hydroxymandelic acid, 2-phenylpropane-1,2-diol, 2-phenyl-2-propanol and lactic acid. The possible BPA-biodegradation pathway based on the identified metabolites and in agreement with recorded pathway in KEGG database was proposed. Preliminary 16S rDNA sequence analysis and subsequent genetically characterization through comprehensive genomic analysis identified the strain as Pseudomonas pseudoalcaligenes strain YKJ.

Impact of the Transfersome Delivered Human Growth Hormone on the Dermal Fibroblast Cells.

OBJECTIVE: Transfersomes are highly flexible vesicles that are capable of passing through pores smaller than their own sizes due to their elastochemical characteristics, and thus play a key role in drug delivery to the skin. METHODS: In this study, we used transdermal delivery of growth hormone-encapsulated transferosomes (F1 and F2) as antiaging strategy, with the resulting effects being subsequently evaluated. The size, distribution and zeta potential of the particles, together with the in vitro skin permeation and biological activity of the growth hormone loaded onto the transfersomes were measured. RESULTS: The data demonstrated that treatment of fibroblasts with encapsulated hGH increased cell migration, proliferation and collagen I and III gene expression. According to our results, the maximum amount of growth hormone that passes through the skin during a 24 h time period was 489.54 and 248.46 ng/cm3, for the F1 and F2 transfersomes, respectively. In addition, it was determined that F1 formula as the more efficient carrier, showed no toxicity against cells. With regard to fibroblasts, as one of the most important cells involved in collagen synthesis, skin aging and wound healing, concentrations of growth hormone encapsulated in transferosomes that had an effect on fibroblast growth and division, were determined. The results demonstrated that effective concentrations of the encapsulated growth hormone increased the expression of collagen I and collagen III genes. CONCLUSION: Furthermore, analyzing the rate of fibroblast cell migration showed that migration increased significantly at 700 ng/ml growth hormone concentrations, as compared to that of the control.

Hyaluronic acid production enhancement via genetically modification and culture medium optimization in Lactobacillus acidophilus.

Hyaluronic acid (HA) is a natural polymer with various molecular weights that specify multiple biological roles. Traditionally, HA is obtained from animal waste and conventional pathogenic streptococci. However, there are challenges in these processes such as the presence of exotoxins, hyaluronidase, and viral contamination. In order to reduce these problems, this study was conducted to produce HA using recombinant bacterium that is generally recognized as safe (GRAS), and thereafter increase production through experimental design. At first, some lactic acid bacteria were screened and evaluated for HA production. Accordingly, among the selected bacteria, Lactobacillus acidophilus PTCC1643 produced about 0.25 g HA/L in the 48th hour of cultivation, and was thus selected as an alternative host for heterologous HA production. An expression vector containing HA synthase genes was transformed into L. acidophilus by electroporation. Consequently, HA production increased to 0.4 g/L. Eventually, response surface method (RSM) was used, which increased HA production to 1.7 g/L. This is approximately 7-fold higher than that produced at first. The resulting HA was characterized by FTIR spectroscopy and its molecular weight was estimated using agarose gel electrophoresis. In conclusion, L. acidophilus could be a safe, effective, and novel HA producer with industrial potential and commercial prospects.

Signaling network of lipids as a comprehensive scaffold for omics data integration in sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and progressive inflammatory condition that has been linked to the dysregulation of many metabolic pathways including lipid biosynthesis. How lipid metabolism could affect disease progression in smokers with COPD remains unclear. We cross-examined the transcriptomics, proteomics, metabolomics, and phenomics data available on the public domain to elucidate the mechanisms by which lipid metabolism is perturbed in COPD. We reconstructed a sputum lipid COPD (SpLiCO) signaling network utilizing active/inactive, and functional/dysfunctional lipid-mediated signaling pathways to explore how lipid-metabolism could promote COPD pathogenesis in smokers. SpLiCO was further utilized to investigate signal amplifiers, distributers, propagators, feed-forward and/or -back loops that link COPD disease severity and hypoxia to disruption in the metabolism of sphingolipids, fatty acids and energy. Also, hypergraph analysis and calculations for dependency of molecules identified several important nodes in the network with modular regulatory and signal distribution activities. Our systems-based analyses indicate that arachidonic acid is a critical and early signal distributer that is upregulated by the sphingolipid signaling pathway in COPD, while hypoxia plays a critical role in the elevated dependency to glucose as a major energy source. Integration of SpLiCo and clinical data shows a strong association between hypoxia and the upregulation of sphingolipids in smokers with emphysema, vascular disease, hypertension and those with increased risk of lung cancer.

Rational design of hyper-glycosylated interferon beta analogs: a computational strategy for glycoengineering.

Glycoengineering has been successfully used to improve the physicochemical and pharmaceutical properties of therapeutics. One aspect of glycoengineering is to introduce new N-linked glycosylation consensus sequences (Asn, X, Thr/Ser) into desirable positions in the peptide backbone by mutational insertion to generate proteins with increased sialic acid content. In the current work, human interferon beta (huIFN-ß) was used as a model to identify the potential positions for the addition of new N-glycosylation sites. A computational strategy was employed to predict the structural distortions and functional alterations that might be caused by the change in amino acid sequence. Accordingly, three-dimensional (3D) structures of the designed huIFN-ß analogs were generated by comparative modeling. Molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility profile of the structures. Subsequently, for the purpose of glycoengineering huIFN-ß, analogs with 3D structures more similar to the wild-type huIFN-ß and exposed Asn residue in the new N-glycosylation site were identified. These modeling procedures indicated that the addition of the new N-glycosylation site in the loop regions had lower constraining effects on the tertiary structure of the protein. This computational strategy can be applied to avoid alterations in the 3D structure of proteins caused by changes in the amino acid sequences, when designing novel hyper-glycosylated therapeutics. This in turn reduces labor-intensive experimental analyses of each analog.

Cloning and Expression of Gumboro VP2 Antigen in Aspergillus niger.

BACKGROUND: Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger). METHODS: Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG-protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. RESULTS: A number of pyrG (+) positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. CONCLUSION: In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry.

Influenza virus hemagglutinin: a model for protein N-glycosylation in recombinant Escherichia coli.

BACKGROUND: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. OBJECTIVES: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. METHODS: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. RESULTS: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. CONCLUSION: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.

Heat-induced production of human growth hormone by high cell density cultivation of recombinant Escherichia coli.

The temperature-induced, over-expression of the human growth hormone gene in a recombinant E. coli during high cell density cultivation is reported. Human growth hormone (hGH) production and stability were tested under different heat shock conditions. Cell densities were 25 and 60 g l(-1) in a pH-stat fed-batch mode in defined and complex medium, respectively, and the fermentation time was decreased from 41 to 32 h. hGH was produced at 2 g l(-1) in complex medium. By using glycerol as main carbon source in the complex medium with exponential feeding, cell density and hGH production were increased to 100 g l(-1) and 2.7 g l(-1), respectively.