Showing papers in "Biotechnology Letters in 2018"

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

Abstract: Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

Recent developments in chromatographic purification of biopharmaceuticals.

Abstract: Over the last several decades, researchers have time and again proposed use of non-chromatographic methods for processing of biotherapeutic products. However, chromatography continues to be the backbone of downstream processing, particularly at process scale. There are many reasons for this, critical ones being the unparalleled scalability, robustness, and selectivity that process chromatography offers over its peers. It is no surprise then that process chromatography has been a topic of major developments in resin matrix, ligand chemistry, modalities, high throughput process development, process modelling, and approaches for control. In this review, we attempt to summarize major developments in the above-mentioned areas. Greater significance has been given to advancements in the last 5 years (2013-2017).

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Abstract: Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs). Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells. Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

Itaconic acid production in microorganisms

Abstract: Itaconic acid, 2-methylidenebutanedioic acid, is a precursor of polymers, chemicals, and fuels. Many fungi can synthesize itaconic acid; Aspergillus terreus and Ustilago maydis produce up to 85 and 53 g l−1, respectively. Other organisms, including Aspergillus niger and yeasts, have been engineered to produce itaconic acid. However, the titer of itaconic acid is low compared with the analogous major fermentation product, citric acid, for which the yield is > 200 g l−1. Here, we review two types of pathway for itaconic acid biosynthesis as well as recent advances by metabolic engineering strategies and process optimization to enhance itaconic acid productivity in native producers and heterologous hosts. We also propose further improvements to overcome existing problems.

BABY BOOM ( BBM ): a candidate transcription factor gene in plant biotechnology

Abstract: Plants have evolved a number of transcription factors, many of which are implicated in signaling pathways as well as regulating diverse cellular functions. BABY BOOM (BBM), transcription factors of the AP2/ERF family are key regulators of plant cell totipotency. Ectopic expression of the BBM gene, originally identified in Brassica napus, has diverse functions in plant cell proliferation, growth and development without exogenous growth regulators. The BBM gene has been implicated to play an important role as a gene marker in multiple signaling developmental pathways in plant development. This review focuses on recent advances in our understanding of a member of the AP2 family of transcription factor BBM in plant biotechnology including plant embryogenesis, cell proliferation, regeneration, plant transformation and apogamy. Recent discoveries about the BBM gene will inevitably help to unlock the long-standing mysteries of different biological mechanisms of plant cells.

Cinnamic acid attenuates quorum sensing associated virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1

Abstract: Anti-quorum sensing and anti-biofilm efficacy of Cinnamic acid against Pseudomonas aeruginosa was comparatively assessed with respect to potent quorum sensing inhibitor, Baicalein. At sub-lethal concentration, Cinnamic acid effectively inhibited both the production of the QS-dependent virulence factors and biofilm formation in P. aeruginosa without affecting the viability of the bacterium. The phytocompound interfered with the initial attachment of planktonic cells to the substratum thereby causing reduction in biofilm development. In addition, the in vivo study indicated that the test compound protected Caenorhabditis elegans from the virulence factors of P. aeruginosa leading to reduced mortality. The in silico analysis revealed that Cinnamic acid can act as a competitive inhibitor for the natural ligands towards the ligand binding domain of the transcriptional activators of the quorum sensing circuit in P. aeruginosa, LasR and RhlR. The findings suggest that Cinnamic acid may serve as a novel quorum sensing based anti-infective in controlling P. aeruginosa infections.

Examining the sources of variability in cell culture media used for biopharmaceutical production.

Abstract: Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review

Abstract: Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria appropriate organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids, ketones and sugar derivates. Whereas emerging applications include biopolymers, such as bacterial cellulose and fructans, which are getting an increasing interest for the biotechnological industry. However, considering the industrial demand of high performing bioprocesses, the production yield of metabolites obtained by acetic acid bacteria, is still not satisfying. In this paper we review the major acetic acid bacteria industrial applications, considering the current status of bioprocesses. We will also describe new biotechnological advances in order to optimize the industrial production, offering also an overview on future directions.

Marine derived biosurfactants: a vast potential future resource.

Abstract: Surfactants and emulsifiers are surface-active compounds (SACs) which play an important role in various industrial processes and products due to their interfacial properties. Many of the chemical surfactants in use today are produced from non-renewable petrochemical feedstocks, while biosurfactants (BS) produced by microorganisms from renewable feedstocks are considered viable alternatives to petroleum based surfactants, due to their biodegradability and eco-friendly nature. However, some well-characterised BS producers are pathogenic and therefore, not appropriate for scaled-up production. Marine-derived BS have been found to be produced by non-pathogenic organisms making them attractive possibilities for exploitation in commercial products. Additionally, BS produced from marine bacteria may show excellent activity at extreme conditions (temperature, pH and salinity). Despite being non-pathogenic, marine-derived BS have not been exploited commercially due to their low yields, insufficient structural elucidation and uncharacterised genes. Therefore, optimization of BS production conditions in marine bacteria, characterization of the compounds produced as well as the genes involved in the biosynthesis are necessary to improve cost-efficiency and realise the industrial demands of SACs.

Therapeutic applications of CRISPR/Cas9 system in gene therapy

Abstract: Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.

Magnetic immobilization of bacteria using iron oxide nanoparticles

Abstract: Bacterial cell immobilization is a novel technique used in many areas of biosciences and biotechnology. Iron oxide nanoparticles have attracted much attention in bacterial cell immobilization due to their unique properties such as superparamagnetism, large surface area to volume ratio, biocompatibility and easy separation methodology. Adhesion is the basis behind many immobilization techniques and various types of interactions determine bacterial adhesion. Efficiency of bacterial cell immobilization using iron oxide nanoparticles (IONs) generally depends on the physicochemical properties of the IONs and surface properties of bacterial cells as well as environmental/culture conditions. Bacteria exhibit various metabolic responses upon interaction with IONs, and the potential applications of iron oxide nanoparticles in bacterial cell immobilization will be discussed in this work.

Simulation of algal photobioreactors: recent developments and challenges.

Abstract: Widespread cultivation of phototrophic microalgae for sustainable production of a variety of renewable products, for wastewater treatment, and for atmospheric carbon mitigation requires not only improved microorganisms but also significant improvements to process design and scaleup The development of simulation tools capable of providing quantitative predictions for photobioreactor performance could contribute to improved reactor designs and it could also support process scaleup, as it has in the traditional petro-chemical industries However, the complicated dependence of cell function on conditions in the microenvironment, such as light availability, temperature, nutrient concentration, and shear strain rate render simulation of photobioreactors much more difficult than chemical reactors Although photobioreactor models with sufficient predictive ability suitable for reactor design and scaleup do not currently exist, progress towards this goal has occurred in recent years The current status of algal photobioreactor simulations is reviewed here, with an emphasis on the integration of and interplay between sub-models describing hydrodynamics, radiation transport, and microalgal growth kinetics Some limitations of widely used models and computational methods are identified, as well as current challenges and opportunities for the advancement of algal photobioreactor simulation

Biosynthesis of D-lactic acid from lignocellulosic biomass

Abstract: D-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of D-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure D-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial D-lactic acid synthesis will be discussed.

HuR facilitates cancer stemness of lung cancer cells via regulating miR-873/CDK3 and miR-125a-3p/CDK3 axis.

Abstract: To study the roles and mechanisms of HuR in cancer stem cell maintenance of lung cancer. HuR expression was increased in tumor spheres of lung cancer cells. Knockdown of HuR suppressed spheroid formation and size, inhibited the expression of stemness-related marker, Oct4, Nanog and ALDH in lung cancer cells. Importantly, HuR and CDK3 expressions were increased in lung cancer tissues compared with normal adjacent tissues, and positively correlated. Mechanistically, HuR directly bound to CDK3, and increased CDK3 mRNA stability and expression. Additionally, miR-873 or miR-125a-3p attenuated the promotion of HuR on CDK3 expression and lung cancer stemness. Furthermore, HuR facilitated lung cancer stemness dependent on CDK3 expression. miR-873 or miR-125a-3p level was negatively correlated with HuR and CDK3 expression levels in lung cancer tissues. HuR facilitates lung cancer stemness via regulating miR-873/CDK3 and miR-125a-3p/CDK3 axis.

Discovery of an acidic, thermostable and highly NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929.

Abstract: To identify a robust NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. A new NADP+ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 × histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP+ and NAD+, respectively and its Tm value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The KM constants were 49.8, 0.12 and 1.68 mM for formate (with NADP+), NADP+ and NAD+, respectively. An NADP+ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

Development of RNA aptamer that inhibits methyltransferase activity of dengue virus

Abstract: To develop an RNA aptamer specific for the methyltransferase (MTase) of dengue virus (DENV) which is essential for viral genome replication and translation acting directly on N-7 and 2′-O-methylation of the type-I cap structure of the viral RNA. We identified 2′-fluoro-modified RNA aptamers that can specifically bind DENV serotype 2 (DENV2) MTase using systematic evolution of ligands by exponential enrichment technology. We truncated the chosen aptamer into a 45-mer RNA sequence that can bind DENV2 MTase with K d ~ 28 nM and inhibit N-7 methylation activity of the protein. Moreover, the 45-mer truncated aptamer could not only bind with an K d ~ 15.6 nM but also inhibit methylation activity of DENV serotype 3 (DENV3) MTase. The 45-mer aptamer competitively impeded binding of both DENV2 and DENV3 genomic RNA to MTase of each serotype. The selected 45-mer truncated RNA aptamer specifically and avidly bound DENV MTase and competitively inhibited its methylation activity, and thus could be useful for the development of anti-DENV agents.

Equisetin as potential quorum sensing inhibitor of Pseudomonas aeruginosa.

Abstract: To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

Characterization of a mildly alkalophilic and thermostable recombinant Thermus thermophilus laccase with applications in decolourization of dyes.

Abstract: To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli. Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K m) and maximum velocity (Vmax) at 60 °C and pH 6.0 were determined to be 2.4 × 103 µM and 0.04 × 103 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme’s ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS. Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.

Native promoters of Corynebacterium glutamicum and its application in l-lysine production

Abstract: To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering. Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using β-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R 2 = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction. The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.

A two-stage system coupling hydrolytic acidification with algal microcosms for treatment of wastewater from the manufacture of acrylonitrile butadiene styrene (ABS) resin

Abstract: To demonstrate the effectiveness of a novel two-stage system coupling hydrolytic acidification with algal microcosms for the treatment of acrylonitrile butadiene styrene (ABS) resin-manufacturing wastewater. After hydrolytic acidification, the BOD5/COD ratio increased from 0.22 to 0.56, showing improved biodegradability of the wastewater. Coupled with hydrolytic acidification, the algal microcosms showed excellent capability of in-depth removal of COD, NH3–N and phosphorus with removal rates 83, 100, and 89%, respectively, and aromatic pollutants, including benzene, were almost completely removed. The biomass concentration of Chlorella sp. increased from 5 × 106 to 2.1 × 107 cells/ml after wastewater treatment. This two-stage coupling system achieved deep cleaning of the benzene-containing petrochemical wastewater while producing greater algae biomass resources at low cost.

A novel bispecific antibody fusion protein co-targeting EGFR and CD47 with enhanced therapeutic index

Abstract: To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the “off-target” effects caused by CD47 expression on red blood cells. The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy. Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.

Model-based reconstruction of synthetic promoter library in Corynebacterium glutamicum

Abstract: To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum. A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the − 10 and − 35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks. To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

Direct electrospinning of poly(vinyl butyral) onto human dermal fibroblasts using a portable device

Abstract: To demonstrate that uniform poly(vinyl butyral) (PVB) fibres can be safely electrospun onto a monolayer of human dermal fibroblasts using a portable device. PVB in solvent mixtures containing various amounts of ethanol and water was electrospun. Six percent (weight-to-volume ratio) PVB in a 9:1 ethanol:water ratio was the solution with the highest content in water that could be electrospun into consistent fibres with an average diameter of 0.9 μm (± 0.1 μm). Four and five percent PVB solutions created beaded fibres. A 8:2 ethanol:water solution lead to microbead formation while a 7:3 ethanol:water mix failed to fully dissolve. The selected solution was successfully electrospun onto a monolayer of human dermal fibroblasts and the process had no significant effect (p < 0.05) on cell viability compared to the control without fibres. PVB–ethanol–water solutions could be electrospun without damaging the exposed cell layer. However, further work is required to demonstrate the long-term effect of PVB as a wound healing material.

The application of plant in vitro cultures in cannabinoid production

Abstract: Cannabinoids have considerable interest in the pharmaceutical industry. However, the production of medicines from hemp (Cannabis sativa L.) in most countries is restricted by law. Large-scale, field cultivation of hemp is difficult to control. Cannabinoid content in plants is variable and depends on multiple factors. Therefore, alternative methods of production have been investigated. The development of micropropagation techniques is a necessary step for genetic modification. Promising results have been obtained for certain narcotic genotypes. However, micropropagation of fibre types requires further research. Hemp can be genetically modified which may contribute to the breeding of new varieties in the future. Cell suspension cultures and hairy root cultures of hemp have been used to produce cannabinoids but obtaining cannabinoids from callus and cell suspension cultures has proved impossible. Adventitious roots can, however, deliver small amounts of these metabolites but production ceases over time and is too low for industrial applications.

A simple protocol for transfecting human mesenchymal stem cells.

Abstract: Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment. MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.

The evolution of CRISPR/Cas9 and their cousins: hope or hype?

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system allows biologists to edit genomic DNA of any cell in precise and specific way, entailing great potential for crop improvement, drug development and gene therapy. The system involves a nuclease (Cas9) and a designed guide RNA that are involved in wide range of applications such as genome modification, transcriptional modulation, genomic loci marking and RNA tracking. The limitation of the technique, in view of resistance of thymidine-rich genome to Cas9 cleavage, has now been overcome by the use of Cpf1 nuclease. In this review, we present an overview of CRISPR nucleases (Cas9 or Cpf1) with particular emphasis on human genome modification and compare their advantages and limitations. Furthermore, we summarize some of the pros and cons of CRISPR technology particularly in human therapeutics.

Recent advances in enhanced enzyme activity, thermostability and secretion by N-glycosylation regulation in yeast.

Abstract: Yeast has been increasingly used as a host for the expression of enzymes. Compared to other expression systems, the yeast expression system has many advantages including its suitability for large-scale fermentation and its ability to modify enzymes. When expressed in yeast, many recombinant enzymes are N-glycosylated, and this may play an important role in their activity, thermostability and secretion. Although the mechanism underlying this process is not clear, the regulation of N-glycosylation by introducing or eliminating N-glycosylation at specific sites has developed into an important strategy for improving the production or catalytic properties of recombinant enzymes. In this review, we summarize the recent advances in understanding the effects of N-glycosylation on the expression and characteristics of recombinant enzymes, and discuss novel strategies for regulating N-glycosylation in yeast. We hope that this review will help improve the understanding of the expression and the catalytic properties of N-glycosylated proteins.

Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols.

Abstract: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

Yield enhancement strategies of rare pharmaceutical metabolites from endophytes

Abstract: Endophytes are barely untapped as vital sources in the medicine. They are microorganisms which mostly exist in plants. As they are exploited, it is accepted that endophytes can produce active metabolites that possess same function as their hosts such as taxol, podophyllotoxin, hypericin, and azadirachtin. These metabolites have been promising potential usefulness in safety and human health concerns. We are supposed to adopt measures to raise production for the low yield of metabolites. This paper summarizes the latest advances in various bioprocess optimization strategies. These techniques can overcome the limitations associated with rare pharmaceutical metabolite-producing endophytic fungi. These strategies include strain improvement, genome shuffling, medium optimization, fermentation conditions optimization, addition of specific factor, addition of solid sorbent, and co-culturing. It will enable endophytes to produce high and sustainable production of rare pharmaceutical metabolites.

Tenocyte proliferation and migration promoted by rat bone marrow mesenchymal stem cell-derived conditioned medium

Abstract: To investigate the impact of secreted factors of rat bone marrow mesenchymal stem cells (MSCs) on the proliferation and migration of tenocytes and provide evidence for the development of MSC-based therapeutic methods of tendon injury. Rat bone marrow mesenchymal stem cell-derived conditioned medium (MSC-CM) promoted the proliferation of tenocytes within 24 h and decreased the percentage of tenocytes in G1 phase. MSC-CM activated the extracellular signal-regulated kinase1/2 (ERK1/2) signal molecules, while the ERK1/2 inhibitor PD98059 abrogated the MSC-CM-induced proliferation of tenocytes, decreased the fraction of tenocytes in the G1 phase and elevated p-ERK1/2 expression. Furthermore, MSC-CM promoted the migration of tenocytes within 6 h, enhanced the formation of filamentous actin (F-actin) and increased the cellular and nuclear stiffness of tenocytes. MSC-CM promotes tenocyte proliferation by changing cell cycle distribution via the ERK1/2 signaling pathway. MSC-CM-induced tenocyte migration was accompanied by cytoskeletal polymerization and increases in cellular and nuclear stiffness.