Stimulation of Neurite Outgrowth Using Autologous NGF Bound at the Surface of a Fibrous Substrate

Peripheral nerve injury still remains a major clinical challenge, since the available solutions lead to dysfunctional nerve regeneration. Even though autologous nerve grafts are the gold standard, tissue engineered nerve guidance grafts are valid alternatives. Nerve growth factor (NGF) is the most potent neurotrophic factor. The development of a nerve guidance graft able to locally potentiate the interaction between injured neurons and autologous NGF would be a safer and more effective alternative to grafts that just release NGF. Herein, a biofunctional electrospun fibrous mesh (eFM) was developed through the selective retrieval of NGF from rat blood plasma. The neurite outgrowth induced by the eFM-NGF systems was assessed by culturing rat pheochromocytoma (PC12) cells for 7 days, without medium supplementation. The biological results showed that this NGF delivery system stimulates neuronal differentiation, enhancing the neurite growth more than the control condition.

Glycolytic flux-signaling controls mouse embryo mesoderm development

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes.

Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate

Extracellular vesicles (EVs) are being increasingly studied owing to its regenerative potential, namely EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs). Those can be used for controlling inflammation, repairing injury, and enhancing tissue regeneration. Differently, the potential of EVs derived from human articular chondrocytes (hACs) to promote cartilage regeneration has not been thoroughly investigated. This work aims to develop an EVs immobilization system capable of selectively bind EVs present in conditioned medium obtained from cultures of hACs or hBM-MSC. For that, an anti-CD63 antibody was immobilized at the surface of an activated and functionalized electrospun nanofibrous mesh. The chondrogenic potential of bound EVs was further assessed by culturing hBM-MSCs during 28 days under basal conditions. EVs derived from hACs cultured under differentiation medium or from chondrogenically committed hBM-MSCs induced a chondrogenic phenotype characterized by marked induction of SOX9, COMP, Aggrecan and Collagen type II, and matrix glycosaminoglycans synthesis. Indeed, both EVs immobilization systems outperformed the currently used chondroinductive strategies. These data show that naturally secreted EVs can guide the chondrogenic commitment of hBM-MSCs in the absence of any other chemical or genetic chondrogenic inductors based in medium supplementation.

Lignin Syngas Bioconversion by Butyribacterium methylotrophicum: Advancing towards an Integrated Biorefinery

Hybrid bio-thermochemical based technologies have the potential to ensure greater feedstock flexibility for the production of bioenergy and bioproducts. This study focused on the bioconversion of syngas produced from low grade technical lignin to C2-/C4-carboxylic acids by Butyribacterium methylotrophicum. The effects of pH, medium supplementation and the use of crude syngas were analyzed. At pH 6.0, B. methylotrophicum consumed CO, CO2 and H2 simultaneously up to 87 mol% of carbon fixation, and the supplementation of the medium with acetate increased the production of butyrate by 6.3 times. In long-term bioreactor experiments, B. methylotrophicum produced 38.3 and 51.1 mM acetic acid and 0.7 and 2.0 mM butyric acid from synthetic and lignin syngas, respectively. Carbon fixation reached 83 and 88 mol%, respectively. The lignin syngas conversion rate decreased from 13.3 to 0.9 NmL/h throughout the assay. The appearance of a grayish pellet and cell aggregates after approximately 220 h was indicative of tar deposition. Nevertheless, the stressed cells remained metabolically active and maintained acetate and butyrate production from lignin syngas. The challenge that impurities represent in the bioconversion of crude syngas has a direct impact on syngas cleaning requirements and operation costs, supporting the pursuit for more robust and versatile acetogens.

Biomass Growth and Composition of Azolla (Azolla Pinnata R. BR.) Supplemented With Inorganic Phosphorus in Outdoor Culture

An experiment was conducted to know the effect of supplemental phosphorus on biomass growth and composition of a floating aquatic fern, Azolla pinnata, cultured in a pit system for 21 days with 4 levels of phosphorus (0, 5, 10, and 15 ppm) supplementation. Water quality parameters of the pits were within a suitable range for A. pinnata culture. It was observed that fresh and dry weights of A. pinnata increased with phosphorus supplementation up to 10 ppm. Doubling time was the fastest when the culture medium was supplemented with 10 ppm of phosphorus. Phosphorus content of A. pinnata was proportional to the phosphorus supplementation in the culture medium. Supplementation of phosphorus also improved the protein and lipid contents of A. pinnata. It was concluded that supplementation of 10 ppm phosphorus to water used for culturing A. pinnata is optimum under outdoor conditions. SAARC J. Agric., 19(1): 177-184 (2021)

Preparation of Aspergillus niger 426 naringinases for debittering citrus juice using of agro-industrial residues

Naringin (4,5,7-trihydroxy flavanone-7-rhamnoglucoside), considered as the main bitter component of grapefruit, requires the use of enzymes to reduce the level of this substance during juice processing. For this reason, it has been the focus of many studies. To increase the production of naringinase by Aspergillus niger cultivated in solid-state fermentation (SSF), it was verified whether the influence of agro-industrial residues as fermentation substrates and, finally, selected the best of the three inducers, or their mixtures to remove the bitterness of grapefruit juice. Cultivation with 2.3 g of grapefruit peel, 2.5 g of rice bran, and 5.2 g of wheat bran and medium supplementation with a mixture of naringin, rutin, and hesperidin in the concentration of (g / L): 2, 5, 4.5, and 3.0, respectively, leading to a maximum activity of 28 U / mL. The results indicate that the sequencing procedure, which allowed the definition of an optimal mixture of components, is a new way for microorganisms to have a high naringinase yield, in particular by SSF, since our data showed a 96% increase in the production of naringinase.

Effect of Polysorbates on the Growth of Rhodotorula Glutinis in Oil Rich Medium

The present study has investigated the effect of oil rich medium supplementation with polysorbates Tween 20, 40 and 80, for the cultivation of red oleaginous yeast Rhodotorula glutinis. R. glutinis has been cultivated in yeast extract peptone glucose modified broth (mYPG) supplemented with 2 % of waste cooking rapeseed oil and three polysorbate types with 0.5 %, 1 %, 2%, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 % and 10 % concentration each. Yeast biomass was measured by the thermogravimetric method at 105 °C each day during 7-day experiment. The oil rich medium supplementation with Tween 20, 40 and 80 at concentrations ranging from 2 % to 10 % significantly increased the biomass of R. glutinis. All three types of the studied polysorbates with 0.5 % and 1 % concentration, did not affect yeast growth and the dry biomass – results were similar to the control sample without polysorbate addition. Between the three types of polysorbates, Tween 20 was selected as the preferable for R. glutinis cultivation with an optimal concentration of 2 %. Cultivation of R. glutinis in oil rich medium with polysorbates Tween 20, 40 and 80, supplementation up to 10 % concentration did not have had an inhibitory effect on the biomass growth.