Biotechnology

Correction to: FabG: from a core to circumstantial catalyst In the original publication of the article, under section Polyketide synthesis: a pathway similar to fatty acid synthesis, the sentences "Phylogeny of KS domains and proteins of FAS and PKS, inferred by Bayesian estimation. Numbers above branches indicate posterior clade probability values." and "Branch length indicates number of inferred amino acid changes per position." in the first paragraph were included inadvertently.

Biocatalytic synthesis and characterization of sn -1/3 and sn -2 monoacylglycerols Abstract Objectives To investigate the lipase-catalyzed synthesis of high purity sn-1/3 and sn-2 monoacylglycerols (1/3-MAG and 2-MAG) of different fatty acids (FAs). Results The 1/3-MAGs of three FAs (16:0, 17:0, 16:1) were synthesized using lipase-catalyzed esterification of glycerol with FAs. The 2-MAGs were obtained from the ethanolysis of synthetic triacylglycerols using sn-1,3 regiospecific lipase. The effects of lipase types, substrate ratio, temperature, reaction time and lipase load on the MAG conversion were studied. Under the optimal conditions, high purities (96.74%, 95.44%, 92.96%) with acceptable isolated yields (51.00%, 54.28%, 46.00%) were obtained for 1/3-16:0-MAG, 1/3-17:0-MAG, and 1/3-16:1-MAG, respectively. For 2-16:0-MAG, 2-17:0-MAG, and 2-16:1-MAG, the purities were 92.64, 95.04, and 96.48%, with isolated yields of 50.64, 52.16, and 26.12%, respectively. The molecular structures of the synthetic compounds were confirmed by 1H NMR, and MS and the melting points were characterized by DSC. Conclusions High purity MAG isomers can be synthesized via lipase-catalyzed reactions to be building blocks for production of functional lipids, the melting points of which are largely governed by the hydrophobic interactions among the alkanyl chains.

Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli Abstract Objectives Fine-tuning of enzyme expression at low levels is an important challenge for metabolic engineers. Here, theophylline-inducible riboswitch for translational regulation was evaluated. The background expression, translation rate, and time delay for its induction was reported. Results To evaluate the effect of the amount of mRNA on its translation rate, transcription of the riboswitch RNA with red fluorescent protein (RFP) was controlled by the lac system with addition of isopropyl β-d-1-thiogalactopyranoside in Escherichia coli. Regardless of the amount of riboswitch mRNA, the translation of RFP was completely suppressed without theophylline during both growth and stationary phases. Furthermore, a strong positive correlation between theophylline concentration (0 to 1 mM) and specific RFP production rate was observed. The specific RFP production rate with the riboswitch was approximately 2.3% of that without the riboswitch. Furthermore, 60 min of time delay for RFP expression was observed after adding theophylline during the stationary phase. Conclusion Theophylline-inducible riboswitch precisely controls protein translation at low expression levels with significantly low background expression. It can emerge as a powerful tool for fine tuning of enzyme expression.

Pharmacological Notch pathway inhibition leads to cell cycle arrest and stimulates ascl1 and neurogenin2 genes expression in dental pulp stem cells-derived neurospheres Abstract Objective Human dental pulp-derived stem cells (hDPSCs) are becoming an attractive source for cell-based neurorestorative therapies. As such, it is important to understand the molecular mechanisms that regulate the differentiation of hDPSCs toward the neuronal fate. Notch signaling plays key roles in neural stem/progenitor cells (NS/PCs) maintenance and prevention of their differentiation. The aim of this study was to address the effects of Notch signaling inhibition on neurosphere formation of hDPSCs and neuronal differentiation of hDPSCs-neurospheres. Results hDPSCs were isolated from third molar teeth. The cultivated hDPSCs highly expressed CD90 and CD44 and minimally presented CD34 and CD45 surface markers. The osteo/adipogenic differentiation of hDPSCs was documented. hDPSCs were cultured in neural induction medium and N-[N-(3,5-difluorophenacetyl-l-alanyl)]-Sphenylglycine t-butyl ester (DAPT) was applied to impede Notch signaling during transformation into spheres or on the formed neurospheres. Our results showed that the size and number of neurospheres decreased and the expression profile of nestin, sox1 and pax6 genes reduced provided DAPT. Treatment of the formed neurospheres with DAPT resulted in the cleaved Notch1 reduction, G0/G1 arrest and a decline in L-lactate production. DAPT significantly reduced hes1 and hey1 genes, while ascl1 and neurogenin2 expressions augmented. The number of MAP2 positive cells improved in the DAPT-treated group. Conclusions Our findings demonstrated the Notch activity in hDPSCs-neurospheres. DAPT treatment positively regulated proneural genes expression and increased neuronal-like differentiation.

Recent advances in carbohydrate-based cancer vaccines Abstract Cancer is a complex multifactorial disease for which many promising therapeutic strategies such as immunotherapy are emerging. Malignant cells frequently express aberrant cell surface carbohydrates, which differentiate them from normal "healthy" cells. This characteristic presents a window for the development of synthetic carbohydrate antigen-based cancer vaccines which can be recognized by the immune system and can bring about T cell-dependent immune responses. Antibodies generated against the carbohydrate antigens partake in the inactivation of carbohydrate-decorated cancer cells, by slowing down tumor cell growth and inducing cancer cell apoptosis. Novel synthetic strategies for carbohydrate antigens have led to several synthetic cancer vaccine candidates. In the present review, we describe the latest progress in carbohydrate-based cancer vaccines and their clinical evaluation in various cancers.

Precursor-feeding and altered-growth conditions reveal novel blue pigment production by Rubrivivax benzoatilyticus JA2 Abstract Objective To explore the secondary metabolite biosynthetic potential of Rubrivivax benzoatilyticus JA2 using a new metabolite mining strategy. Results Combination of precursor-feeding and altered growth conditions were used to mine new biomolecules. Strain JA2 utilised l-phenylalanine as sole source of nitrogen and showed pigments production only under phenylalanine-amended aerobic cultures. Stable isotope based precursor feeding studies indicated the blue pigment consists of 4-phenyl rings derived from l-phenylalanine. The purified blue pigment displayed characteristic visible-absorption and pH-dependent color variations. Precursor-feeding under altered growth conditions activated the plausible novel aromatic pigment production in strain JA2. Conclusion Our approach unraveled the previously unknown pigment synthesis in strain JA2 and demonstrated the potential of mining strategy in discovering the hidden secondary metabolite repertoire in microorganisms.

FabG: from a core to circumstantial catalyst Abstract Core biochemical pathways such as Fatty-acid synthesis II (FAS II) is ascribed to the synthesis of fatty-acids, biotin and lipoic acid in prokaryotes. It has two dehydrogenases namely, FabG and FabI which interact with the fatty-acid chain bound to Acyl-carrier protein (ACP), a well-studied enzyme which binds to substrates of varying lengths. This protein–protein interaction 'broadens' the active site of these dehydrogenases thus, contributing to their flexible nature. This property is exploited for catalysing numerous chiral synthons, alkanes, long-chain alcohols and secondary metabolites in industries especially with FabG. FASI relegates FASII in eukaryotes making it a 'relic gene pool' and an antibacterial drug target with diverse inhibitor and substrate markush. FabG often substitutes other dehydrogenases for producing secondary metabolites in nature. This redundancy is probably due to gene duplication or addition events possibly making FabG, a progenitor to some of the complex short-chain dehydrogenases used in organisms and industries today.

MiR-499 inhibited hypoxia/reoxygenation induced cardiomyocytes injury by targeting SOX6 Abstract Objective MiR-499 has been reported to be expressed only in cardiomyocytes, and its expression would increase after acute myocardial infarction (AMI). miR-499 plays a role in the process of cardiomyocytes injury induced by hypoxia/reoxygenation (H/R), however, it still remains unclear. Results Hypoxia inhibited miR-499-5p expression and H/R induced apoptosis. SOX6 was a target gene of miR-499-5p, and high expression of miR-499-5p inhibited the expression of SOX6. MiR-499-5p reduced H9c2 cells injury by inhibiting the expression of SOX6, overexpression of which could reverse the effect of miR-499-5p on H9c2 cells. MiR-499-5p inhibited the levels of LDH and MDA, while overexpression of miR-499-5p inhibited H/R-induced cell apoptosis. MiR-499-5p could up-regulate the level of Bcl-2 and down-regulate the expression levels of Bax and caspase-3. However, SOX6 partially reversed these effects of miR-499-5p. Conclusion We proved that miR-499-5p inhibited H/R-induced cardiomyocytes injury by targeting SOX6. Our results suggested that miR-499-5p/SOX6 pathway may present a potential therapeutic target for the treatment of AMI.

Human genome-derived TOP1 matrix attachment region enhances transgene expression in the transfected CHO cells Abstract Objectives To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. Results Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5′ or 3′ ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5′,3′ TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3′ TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. Conclusions The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.

Pseudomonas monteilii PN1: a great potential P -nitrophenol degrader with plant growth promoting traits under drought and saline–alkali stresses Abstract Objectives To evaluate Pseudomonas monteilii strain PN1 for the removal efficiency of P-nitrophenol (PNP) in soils and its growth promotion of maize (Zea mays L.) seedlings under drought and saline–alkali stress. Results PN1 can survive in soils contaminated with PNP dosage between 90 and 155 mg/kg and considerably improved the removal PNP efficiency in soils. Drought and saline–alkali stress reduced maize seedling growth (root length, shoot height and dry or fresh weight) and improved the antioxidant enzyme activities and malondialdehyde (MDA) and proline (PRO) contents. However, maize seedlings treated with PN1 remarkably promoted their growth compared with the control. The reduction in antioxidant enzyme activities and MDA and PRO contents was significant. This result may be correlated to the increased tolerance of maize seedlings to drought and saline–alkali stress. Conclusions Application of P. monteilii PN1 can be an extremely useful approach for the development of bioinoculants in improving plant tolerance to several abiotic stresses and removing PNP in soils to ensure secure crop production.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com