Nanotechnology plays a promising role in biomedical applications, particularly tissue engineering. Recently, the application of magnetic scaffolds and pulsed electromagnetic field (PEMF) exposure has been considered in bone tissue regeneration. In this study, 3rd generation dendrimer-modified superparamagnetic iron oxide nanoparticles (G3-SPIONs) are synthesized comprehensively characterized. Magnetic polycaprolactone (PCL) nanofibers are prepared by incorporating G3-SPIONs within the electrospinning process ,and physicochemical characteristics ,as well as cytocompatibility and cell attachment are assessed. Eventually, the osteogenic differentiation ability of adipocyte-derived mesenchymal stem cells (ADMSCs) cultured on the magnetic scaffold with and without PEMF exposure was investigated by measurement of alkaline phosphatase (ALP) activity and calcium content. The expression of specific bone markers was studied using the Real-time PCR method. According to the results, G3-SPIONs with mean size and zeta potential of 17.95 ± 3.57 nm and 22.7 mV, respectively, show a high saturation magnetization (57.75 emu/g). Adding G3-SPIONs to PCL significantly decrease nanofibers size to 495±144 nm and improves cell attachment and growth. The ADMSCs cultured on the G3-SPION-PCL scaffold in the presence of osteogenic media (OM) and exposure to PEMF expressed the highest Osteocalcin and Runx2 and showed higher calcium content as well as ALP activity. It can be concluded that the synthesized G3-SPION incorporated PCL nanofibers serve as a promising magnetic scaffold for bone regeneration. Also, utilizing the magnetic scaffold in the presence of OM and PEMF provides a synergistic effect toward osteogenic differentiation of ADMSCs. Key Words: Superparamagnetic iron oxide nanoparticles, Dendrimer, Polycaprolactone, Pulsed electromagnetic field, Bone tissue engineering

Several hundred U mL-1 of Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, large-scale preparation and a simple purification process is required. The present study utilized a fed-batch process with a pH-stat and low-glycerol-level-maintain feeding strategy to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). Finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, which represented a 26-fold increase of NK activity by high cell density cultivation compared to the flask culture. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.

The production of purified virus particles with high quality and quantity for vaccine preparation requires scalable purification procedure in downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D -AEC×SEC) is developed for production non-structural protein (NSP) free foot and mouth diseases (FMD) vaccine and the whole procedure is accomplished with 78 % recovery, 85 % virus yield and more than 90 % of residual DNA (rDNA) is removed from the purified vaccine. Due to use AEC as the first column, the injection volume increases four times compare to previous report. Alternatively, a mathematical modeling and simulation approach based on plate model chromatography are developed and matched with the experimental chromatography data to obtain better perception in predicting retention behavior and saving time in downstream scale-up method development. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), transmission electron microscopy (TEM) and biological test provide to the best quality of purified FMD virus.

Electrospinning has been widely used for the development of fibrous scaffolds for cartilage tissue engineering (TE), however their small pores significantly limit cell infiltration throughout the scaffolds, particularly in three-dimensional (3D) designs. In this endeavor, a direct incorporation of chondrocytes into the fibers mesh during the electrospinning presents itself as a promising solution by use of bio-electrospraying. Yet, for this technology to be effectively employed for cartilage TE, it is necessary to assess if chondrocytes are in any way adversely affected. So, in this work, several electrospraying experiments were performed by adjusting various operational parameters to evaluate their influence on chondrocyte viability and function. A high percentage of post-electrosprayed chondrocytes remained viable upon the exposure of an external electric field generated by low needle to collector distances and low applied voltages. No obvious differences were found with non-electrosprayed chondrocytes in terms of viability, morphology and proliferation. The data reported here further suggest that bio-electrospraying under the optimal operational conditions might be a promising alternative to the existent cell seeding techniques, promoting not only cells safe delivery to the scaffold, but also the development of highly cellularized and uniform tissue constructs for cartilage repair.

Celery spices are an important raw material in the production of food and cosmetic, including carveol, carvone and dihydrocarvone. In this study, a new Klebsiella sp. O852 was isolated, which was capable of converting limonene to trans-dihydrocarvone. The yields of trans-dihydrocarvone reached up to 1058 mg/L when the strain O852 was incubated using LB-M medium for 4 h at 36ºC and 150 rpm and the process was monitored for 36 h after adding 1680 mg/L limonene dissolved in ethanol. Limonene was used as a 20% (v/v) solution in the ethanol. Besides, the genome of Klebsiella sp. O852 comprised 20 contigs and 19 scaffolds. The genome size was 5.49 Mb. A total of 5218 protein-encoding genes were predicted, these genes mainly distributed in some metabolism and biosynthesis categories. Finally, several genes involved in trans-dihydrocarvone biosynthesis were further analyzed, identified and verified by quantitative real-time polymerase chain reaction (RT-qPCR) and exogenous expression. These novel genes may find value in the bioconversion of inexpensive raw materials to natural flavors and fragrances.

L-ornithine is a basic amino acid, which shows significant value in food and medicine industries. There is a huge space for L-ornithine production with strains available for metabolic engineering, and it is urgent to develop a high-efficiency engineering strain for industrialization. Here, xylose isomerase and xylulose kinase were introduced into Corynebacterium glutamicum S9114 to establish xylose metabolism pathway, and then xylose became a substitute carbon source of glucose. In addition, the optimization and overexpression of phosphoenolpyruvate carboxylase and pentose transporter have been conducted to promote the synthesis of L-ornithine for the first time. Furthermore, though optimizing the concentration ratio of glucose and xylose (7:3), adding biotin and thiamine hydrochloride, we arrived at the highest L-ornithine yield 41.5g/L in shaking flask fermentation so far. Our results demonstrate that the combination of metabolic engineering and the optimization of fermentation process can make great potential for L-ornithine production by lignocellulose hydrolysate.

.

Laccases are oxidoreductases with the outstanding ability to oxidize phenolic and non-phenolic substrates coupled to the reduction of O2 to H2O. Among them, bacterial enzymes are suitable biocatalysts for application in industrial processes under harsh conditions. However, to be active on high redox potential substrates, bacterial laccases requires of redox mediators: electron carriers between the laccase and other compounds not directly oxidizable by the enzyme. Here we demonstrate that β-(10-phenothiazyl)-propionic acid can be used as an efficient and low-cost redox mediator for decolorization of synthetic dyes by bacterial laccases. Using this laccase-mediator system, more than 80% of Indigo Carmine and Malachite Green decolorization was reached after 1 h or 2 h of incubation, respectively, both at pH 8 and in tap water (pH 6.8). Furthermore, more than 40% of Remazol Brilliant Blue R and 80% of Xylidine ponceau were decolorized after 5 h at pH 8 and 50°C. In addition, we showed this system supports at least 3 decoloration cycles without loss of activity, representing a promising biological process for cost-effective and environmentally friendly decolorization and degradation of synthetic dyes and for other industrial applications of laccases requiring neutral or alkaline pH.

The industrial production of active proteins from E. coli necessitates the refolding of high concentrations of protein over a short period of time. However, it is difficult to simultaneously achieve high concentration and short residence time. The dialysis method can refold high concentrations of proteins, but this process takes a long time. The dilution method can quickly refold proteins, but the resultant proteins are inevitably diluted. In the present study, by designing microchannels in dialysis membranes, which can enlarge the surface area for quickly removing protein denaturant, high concentrations of active carbonic anhydrase---eight times more concentrated than achieved using dilution method---were refolded in 20 minutes, which is orders of magnitude faster than the conventional dialysis method.

Human microbial alterations are associated with environmental stress, nutritional, genetic and triggering de-novo variations. Nevertheless, human gut microbiome at extreme altitude (>5800 m) remains unexplored. We aimed to demonstrate the microbial predominance in individuals with same ethnicity and dietary pattern at extreme altitude with unique challenges like cold, hypoxia, radiation etc. Different analysis pipelines were used for fecal whole genome sequencing at 210m, 3500m, 4420m and 5805m, and 16s rRNA V3-V4 regions amplification sequencing of 19 individuals belonging to the same ethnicity and dietary pattern, for presence of taxonomy & functional potential and confirming the prediction upto the strain level within the same cohort. Principal component analysis, revealed distinct microbiome changes at different altitudes, with varied and higher Bacteroides and Prevotella ratio. There was predominance of genus Prevotella at altitudes 4420m & 5805m than at 210m & 3500m. Appearance of species Prevotella copri strain 61740 was increasing significantly at extreme altitudes, whereas co-occurrence of other bacterial strains had different pattern than Prevotella. The extensive strain level analysis indicated alteration in the metabolic pathways. This study under stressful and hypoxic environment of extreme altitudes, associated microbial variation with altered metabolic pathways, reveals influence of extreme environment on human gut microbiota with predominance of Prevotella.

Production of specialty chemicals increasingly makes use of enzyme catalysts, and Novozym 435 (N435) is among most often applied. However, its polymeric skeleton is unstable in many solvents. In this context, we report results of a systematic study of the biocatalysts, fabricated using highly porous siliceous pellets/enzyme (MH), grafted with octyl (-O), amino (-A) and octyl and amino (-OA) groups, deployed in a rotating bed reactor and tested in hydrolysis and esterification reactions. N435 appeared the most active in both reactions, when activity was related to the catalyst’s mass, mainly owing to very large enzyme load. But its structure degraded in many typical solvents, whereas no such effect was detected in MH-O- and MH-OA-catalysts. MH-O showed the highest specific activity, however, a significant enzyme leaching was observed in a hydrolytic reaction, in contrast to MH-OA. In esterification reaction the MH-O-bound lipase was not only most active but also quite stable.

Flux experiments and fluorescence microscopy were combined and optimized to visualize the membrane surface during biofouling of two mixed cellulose ester membranes. Using flux measurements, the fouling by bovine serum albumin (BSA) was measured in the presence of 1 to 12% labeled BSA. By fitting the relative flux decays to an exponential decay for statistical analysis, the dye in this range of labeled protein was found to not affect the fouling nature of the protein. A 2.5% or 5% labeled protein sample was determined to be the best percent labeled protein for fluorescence imaging the membrane because the beginning of cake formation was observed within 25 min of experimental time. Finally, by fitting the flux data to four different biofouling mechanism equations, we conclude that both membranes, though at different rates, have BSA depositing inside the membrane pores restricting the flow eventually leading to cake formation. The combination of the two techniques allows for further insight into the biofouling mechanism of BSA, and this method can be applied to other biological molecules.

We have constructed a new bioreactor with reciprocal mixing that is better suited for the cultivation of delicate animal cells. In-silico simulation (computational fluid dynamics) suggested both maximum and average shear stresses in the bioreactor with reciprocal mixing to be remarkably lower than in conventional bioreactor with rotary mixing. Although we could not find any difference in growth speed and cell density between the bioreactors with reciprocal and rotary mixing, we did find cell viability in reciprocal-mixing bioreactor to be retained longer than in rotary-paddle bioreactor. This implied that cell culture in a bioreactor with reciprocal mixing could be prolonged for the production of target proteins. Leakage of lactate dehydrogenase activity into the culture medium was suppressed much more in the reciprocal-mixing bioreactor than in the rotary-paddle one. Production of human tissue plasminogen activator in the former was also observed to be much more than in the latter. Therefore, bioreactor with reciprocal mixing was concluded to be better suited for the cultivation of animal cells and efficient production of proteins, such as antibody drugs and various growth factors.

Harvesting of microalgae is essentially the most energy intensive process in commercial algal culture ventures. Developing innovative, cost effective harvesting systems is of paramount need for commercial algal culture ventures. The study thus aimed at investigating the use of eco-friendly Solar Powered Electroflocculation (SPEF) coupled with battery for harvesting marine microalgae using aluminium electrodes. Optimization of various operating parameters like initial algal density, time of operation (5, 10, 15 and 30 min), (initial biomass concentration 0.1, 0.5 and 1.0 gL-1) and electrode distance (35, 55, 75 and 95 cms) were done using direct current (D.C). Best flocculation efficiency (91.31 ± 2.91 %) was obtained using a current density of 37.2 Acm-2, a voltage of 24 V for 15 min at an electrode distance of 95 cm and pH 8.0. The quality of the harvested biomass was ascertained in-terms of biochemical components using Fourier Transform Infrared spectroscopy, total lipid and pigment profile. Due to the low resistance of seawater the energy required for electroflocculation was as low as 0.223 kWh/ton. The energy requirement for the electroflocculation system with or without solar power was also predicted. The results revealed SPEF can be developed as a potential alternative marine microalgal harvesting system for nutraceutical coupled biodiesel production.

A reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly in culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent media, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 x 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 x 12.5 mm, 5µm), using 0.1% TFA and 2% n-propanol as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile as mobile phase B. The column temperature was 80ºC, the flow rate 1 ml/min and the absorbance monitored at 280 nm. The procedures and capabilities of the method were evaluated against the present criteria for linearity, limit of detection (LOD), accuracy, precision, and robustness of the International Conference on Harmonization (ICH) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The methods showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/ml, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Surgery of the entire ear pinna even today presents a challenge to reconstructive surgeons, in the absence of a universally acceptable, quality construct for clinical use. In this article, the authors present a technique to generate a flexible, human-size ear with the aim to meet this limitation for ear reconstructive surgeries. The construct was engineered by using a decellularized goat ear cartilage. This was characterized by hematoxylin-eosin (H/E), diamidino-2-phenylindole (DAPI), Masson’s trichrome (MT), Alcian Blue (AB) staining and Scanning Electron Microscopy (SEM) for extracellular matrix (ECM) analysis. The decellularization protocol followed yielded complete removal of all cellular components without changing the properties of the ECM. In vivo biocompatibility of the ear, pinna showed demonstrable recellularization. Recellularization was tracked using HE, DAPI, MT, AB staining, toluidine staining, SEM, vascular-associated protein (VAP), and CD90+ expressing cells. VAP expression revealed specific vasculogenic pattern (angiogenesis). CD90+ expression reflected the presence of the stromal cell. The graft maintained the properties of ECM and displayed chondrocyte recruitment. In summary, the decellularized goat ear pinna (cartilage) exhibited xenograft biocompatibility, stable mechanical properties, and in vivo chondrocyte recruitment. Subsequently developed tissue-engineered ear pinna offer potential for cartilage flexibility and individualization of ear shape and size for clinical application.

A document by Rafael Oliveira. Click on the document to view its contents.

The optimal growth, maturation and function of bioengineered tissues are mediated by both biochemical and physical cues. We here describe a 3D biomimetic environment directing stem cells towards a chondrogenic phenotype. This system comprises a collagen hydrogel and poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) engineered to protect, carry and release a human Transforming Growth Factor b1 (hTFGb1) payload. PLGA-MCs were prepared using supercritical emulsion extraction technology and integrated into a collagen hydrogel co-seeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs). Testing different concentrations of hTFGb1 supplemented to cell monolayer cultures suggested 10 ng/mL as the most appropriate concentration to promote upregulation of SRY-Related HMG-BOXGene 9 (4-fold) and collagen type II (2-fold) specific markers, at Day 16. A similar growth factor concentration was delivered within the 3D bioengineered environment cultured in a dynamic via a custom perfusion bioreactor. A chondrogenic commitment was obtained as indicated by upregulation of collagen type II (5-fold) and downregulation of collagen types I and III (both 0.1-fold) at Day 16. Histological analysis confirmed the remodeling of the synthetic extracellular matrix in where an enhanced mass exchange was described by FEM analysis of fluid-dynamics and related nutrient mass transfer within the 3D construct. This study supports the use of 3D bioengineered scaffolds cultured in a dynamic environment as a suitable tissue engineered model to study chondrogenic differentiation in vitro and opens perspectives for an injectable collagen-based advanced therapy system.