Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors

Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.

     

Brucellosis Causes Alteration in Trace Elements and Oxidative Stress Factors

Brucellosis is regarded as one of the most common diseases among humans and livestock. In the present study, we aimed to assess the effect of this disease on the level of various cations including copper (Cu), manganese (Mn), zinc (Zn), and magnesium (Mg) as well as oxidative stress status in the serum of people suffering from brucellosis. The present case-study was carried out on 40 patients with brucellosis (case) and 20 healthy people (control). Blood specimens were taken from all the people and the level of essential trace elements and oxidative stress status were measured. The serum level of copper in the case group (165.39 ± 43.19 μg/dl) was significantly higher compared with that in the control group (122.12 ± 28.88 μg/dl). Whereas the serum level of zinc was significantly lower in the case group compared with that in the control group (76.47 ± 28.88 vs. 92.85 ± 23.16 μg/dl). The manganese and magnesium serum levels did not differ significantly between the two groups. Furthermore, total antioxidant capacity level was significantly lower in the case group (122.12 ± 28.22 μmol/ml) than that in the control group (3.08 ± 0.12 μmol/ml) and the level of serum malondialdehyde was significantly higher in the case group (7.20 ± 0.23 mmol/ml) than that in the control group (4.0 ± 0.19 mmol/ml). Brucellosis can cause alteration in the serum level of essential trace elements. Moreover, the present study indicated that brucellosis produces oxidative stress in patients.     

Differentiation and Classification of Phytoplasmas Associated with Almond and GF‐677 Witches'‐Broom Diseases using rRNA and rp Genes

Stone fruits are affected by several diseases associated with plant pathogenic phytoplasmas. Previous studies have been shown that phytoplasma agents of almond and GF‐677 witches'‐broom (AlmWB and GWB, respectively) diseases belong to pigeon pea witches'‐broom (16SrIX) phytoplasma group. In this study, partial biological and molecular characterization was used to compare and classify phytoplasma agents of Khafr AlmWB (KAlmWB) and Estahban GWB (EGWB) diseases. Production of different symptoms in periwinkle indicated that agents of KAlmWB and EGWB are differentiable. Expected fragments were amplified from diseased almond and GF‐677 trees in direct PCR using phytoplasma universal primer pairs P1/P7 and rpF1/rpR1 and nested PCR using P1/P7 followed by R16F2n/ R16R2 primer pair. 16S‐rDNA Restriction fragment length polymorphism (RFLP) as well as phylogenetic analysis of rplV‐rpsC and 16S–23S rRNA spacer region sequences classified KAlmWB and EGWB phytoplasmas within 16SrIX‐C (rpIX‐C) and 16SrIX‐B (rpIX‐B) subgroups, respectively.     

Error minimization explains the codon usage of highly expressed genes in Escherichia coli

Different organisms use synonymous codons with different preferences. Several measures have been introduced to compute the extent of codon usage bias within a gene or genome, among which the codon adaptation index (CAI) has been shown to be well correlated with mRNA levels of Escherichia coli. In this work an error adaptation index (eAI) is introduced, which estimates the level at which a gene can tolerate the effects of mistranslations. It is shown that the eAI has a strong correlation with CAI, as well as with mRNA levels, which suggests that the codons of highly expressed genes are selected so that mistranslation would have the minimum possible effect on the structure and function of the related proteins.     

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.     

Kinetics of infected insect cell osmolysis and enhanced protein release using a modified disruption method

We have studied and characterized a cell disruption method to produce a protein extract from recombinant Baculovirus infected insect cells based on osmotic lysis. Cell lysis kinetics were measured during a 24-h incubation in lysis buffer and resulting data sets were curve fitted to a hyperbola, visually similar to the Michaelis–Menten curve, to determine the maximum concentration of released protein and the time required to reach equilibrium. Effect of parameters such as pH, ionic strength and infection phase were evaluated, and based on fittings optimal protein release conditions were obtained for total cell protein as well as the recombinant protein, HPV 16 L1. It was demonstrated that pH and the phase of infection can vastly influence the amount of release while ionic strength only effects the time required to achieve equilibrium in protein release. Osmolysis can be a mild, yet effective method to release recombinant protein with high recovery levels and hence can be used in capacities where stringent criteria regarding contamination with surfactant or non-cytoplasmic contents are observed.     

Theoretical study of electron transfer process between fullerenes and neurotransmitters; acetylcholine,dopamine, serotonin and epinephrine in nanostructures [neurotransmitters].C<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> complexes

Neurotransmitters are the compounds which allow the transmission of signals from one neuron to the next across synapses. They are the brain chemicals that communicate information throughout brain and body. Fullerenes are a family of carbonallotropes, molecules composed entirely of carbon, that take the forms of spheres, ellipsoids, and cylinders. Various empty carbon fullerenes (C_n) with different carbon atoms have been obtained and investigated. Topological indices have been successfully used to construct effective and useful mathematical methods to establish clear relationships between structural data and the physical properties of these materials. In this study, the number of carbon atoms in the fullerenes was used as an index to establish a relationship between the structures of neurotransmitters (NTs) acetylcholine (AC) 1, dopamine (DP) 2, serotonin (SE) 3, and epinephrine (EP) 4 as the well-known redox systems and fullerenes C_n (n = 60, 70, 76, 82, and 86) which create [NT].C_n; A-1 to A-5 up to D-1 to D-5. The relationship between the number of carbon atoms and the free energy of electron transfer (ΔG_et(n); n = 1–4) is assessed using the Rehm-Weller equation for A-1 to A-5 up to D-1 to D-5 supramolecular [NT].C_n complexes. The calculations are presented for the four reduction potentials (^Red.E₁ to ^Red.E₄) of fullerenes C_n. The results were used to calculate the four free energy values of electron transfer (ΔG_et(1) to ΔG_et(4)) of the supramolecular complexes A-1 to A-8 up to D-1 to D-8 for fullerenes C₆₀ to C₁₂₀. The first to fourth free activation energy values of electron transfer and the maximum wavelength of the electron transfers, ΔG^#_et(n) and λ_et (n = 1–4), respectively, were also calculated in this study for A-1 to A-8 up to D-1 to D-8 in accordance with the Marcus theory.     

The Physiological,Biochemical, and Molecular Modifications of Chickpea (Cicer arietinum L.) Seedlings Under Freezing Stress

Journal of Plant Growth Regulation - Fall cultivation of field crops such as chickpea is prone to the risk of freezing stress. It is required to identify the mechanisms through which plants can...