Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.     

Predominance of terrestrial organic matter in sediments from a cyanobacteria- blooming hypereutrophic lake

Although an understanding of the quantity and quality of sedimentary organic matter (SOM) pools is necessary to design sound environmental management strategies for lacustrine systems, the characterization of organic matter sources and the assessment of their relative contributions to eutrophic and inland lake sediments remain insufficient. In this study, the contribution of potential organic matter sources to sediments in shallow and hypereutrophic lake Taihu, China was assessed on the molecular level using source-specific fatty acid biomarkers. The results indicated that SOM was composed mainly of terrestrial plants with a maximal contribution of 45.3 ± 2.4% to the total organic carbon, which accounted for approximately 66% among the determined organic matter sources. Evidence suggests the terrestrial plants remained in a fresh state in surface sediments: the correlation (R² = 0.62, p < 0.05) between bacterial and terrestrial plant carbon was strong. On the other hand, aquatic plant and bacterial carbon contributed 5–15% to the total organic carbon, which was followed by the faint contribution (<5% of total organic carbon) of algae-derived organic carbon including cyanobacteria, diatoms, and dinoflagellates. The results provided details of the contributions of SOM sources, illustrating the usefulness of fatty acid biomarkers in discriminating organic matter sources within lake environments. Although organic matter sources of sediments varied in spatial and temporal patterns, the strong correlation between terrestrial plant and total organic carbon (R² = 0.60, p < 0.05) indicates that terrestrial plants were the dominant source in lake sediments.     

Sortase A inhibitory metabolites from the roots of Pulsatilla koreana

Three new lignans (3, 4, and 6) along with eight known lignans and phenyl propanoids were isolated from the dried roots of Pulsatilla koreana, an oriental folk medicine. Based upon the results of combined spectroscopic and chemical methods, the structures of new compounds were determined to be lignan glycosides. Included among the known compounds are three compounds (5, 7, and 8) isolated first time from this plant as well as two compounds (2 and 11) previously reported only as synthetic derivatives. These compounds significantly inhibited the action of Sortase A from Streptococcus mutans OMZ65, an isolate from human oral cavity.     

Novel derivatives of usnic acid effectively inhibiting reproduction of influenza A virus

Influenza virus is serious human pathogen leading to high morbidity and mortality all over the world. Due to high rate of mutation, it is able to fast development of drug resistance that makes necessary to search novel antivirals with broad range and alternative targets. In the present study we describe synthesis and anti-viral activity of novel derivatives of usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione). It is shown that anti-viral activity of usnic acid can be increased by side moieties introduction. The modification with chalcones appeared to be the most effective. Our study revealed that (−)-usnic acid exhibited higher antiviral activity than its (+)-enantiomer, but in the pairs of enantiomer derivatives such as enamines, pyrazoles and chalcones, the (+)-enantiomers were more potent inhibitors of the virus. For other groups of compounds the inhibiting activities of the enantiomers were comparable. Further optimization of the structure could therefore result in development of novel anti-influenza compound with alternative target and mechanism of virus-inhibiting action.     

Metabolic Reconfiguration in C. elegans Suggests a Pathway for Widespread Sterol Auxotrophy in the Animal Kingdom

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Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.