Accumulation of β-carotene in Dunaliella salina is highly dependent on light exposure intensity and duration, but quantitative analysis on photon numbers per cell for triggering β-carotene accumulation is not available so far. In this study, experiment results showed that significant β-carotene accumulation occurred with at least 8 hours illumination at 400 µmol photons·m-2·s-1. To quantifying the average number of photons received per cell (APRPC), correlation between light attenuation with light path, biomass concentration, and β-carotene content was built with both Lambert-Beer and Cornet models, and the latter provided a better simulation. With Cornet model, APRPC was calculated and proposed as a parameter for β-carotene accumulation. It was found that once APRPC reached 0.7 µmol photons cell-1, β-carotene accumulation was triggered, and it was saturated at 9.9 µmol photons cell-1. This study showed that APRPC can be used as an important parameter in D. salina cultivation process, to accurately simulate and control β-carotene production.

Phasins are proteins found on the surface of natural polyhydroxyalkanoate (PHA) granules. Due to their high affinity for PHA, they can potentially be used as a fusion partner to immobilize other proteins. In this study, we investigated the immobilization of a lipase onto electrospun polyhydroxybutyrate nanofibers. Due to a superior surface area-to-volume ratio, PHB nanofibers retained much larger amounts of enzyme than conventional immobilization supports. More importantly, when used in combination with a phasin tag, the enzyme immobilized on PHB nanofibers exhibited markedly higher activity and reusability. Our approach combines the advantageous features of nanofibrous materials and the regio-specificity of biomolecular interactions for the efficient use of enzymes.

One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell cultures, is the deregulated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (being both the main nutrients used as a carbon, nitrogen and energy sources) yields to the production and accumulation of large amounts of lactate and ammonia in the culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients are the major causes that triggers the apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line producing an IgG3 (used in the ABO blood testing system) was engineered with BHRF1 protein (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the anti-apoptotic protein Bcl-2, with the aim of protecting the cell line from apoptosis. Surprisingly, besides achieving an effective protection from apoptosis, the expression of BHRF1 modified significantly the metabolism of the hybridoma cell line. The comparison of cell physiology and metabolism analysis of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease on lactate secretion in KB26.5-BHRF1 cells. The flux balance analysis allowed quantifying intracellular fluxes of both cell lines. The main metabolic differences were identified in the glucose consumption and, consequently, the lactate generation. The lactate production flux was reduced by 60% since the need for NADH regeneration in the cytoplasm decreased due to the glucose uptake reduction by more than 50%. In general terms, BHRF1 engineered cell line showed a more efficient metabolism yielding to an increase of the biomass volumetric productivity under identical culture conditions.

The ethanol concentration in batch cultivation with the yeast S. cerevisiae was predicted on-line using a gas sensor array. Head space samples were pumped past the gas sensors array every five minutes for 10 seconds and the voltage changes of the sensors were measured. For the calibration procedure no off-line sampling was used. Instead, a theoretical model of the process has been applied to simulate the ethanol production at any given time. However, the kinetic parameters of the simulation model are unknown at the beginning of the calibration. It will be demonstrated that these kinetic parameters of the theoretical process model can be acquired from the response of the gas sensor array alone. The calculated parameters result in a simulation model that is at least as accurate as a model whose parameters are acquired by least squares fitting to off-line measurements. The root mean square error of calibration as well as the percentage error for validation sets was below 0.2 g/L and 7 %, respectively. The obtained results indicate that, the model-based calibrated gas sensor array can be a cheap alternative to other tools that are used for monitoring yeast cultivations such as spectroscopy based methods.

Cel9B, an endocellulase produced by Thermobifida fusca YX, contains a number of structural domains, including carbohydrate binding modules 2 and 4 (CBM2 and CBM4), a fibronectin-like (Fn3) domain, an Eset domain (an Ig-like domain that may play a role in enzyme folding), a catalytic domain, and a fibronectin-like (Fn3) domain. To elucidate the roles of these domains with respect to Cel9B function, a series of truncation mutants were designed and examined for their binding properties and activities on different substrates. Different binding properties of CBM2 and 4 with a variety of substrates distinguish important roles for these domains and provide insight as to how distinct domains interact with each other during substrate degradation. The results of this study implicate the collective roles of the non-catalytic domains with respect to Cel9B function, and in turn, this information can be incorporated into protein engineering strategies for improved biomass conversion.

3D printing can be of great use, particularly the production of personal medical products and devices such as scaffolds. In this study, the main aim is to develop propolis (Ps) containing wound dressings by making use of 3D printing technology. Different combinations and structures of propolis (Ps) incorporated sodium alginate (SA) scaffolds are developed. The morphological studies show that the porosity of developed scaffolds was optimized when 20% (v/v) of Ps was added in the solution. The pore sizes decreased by increasing Ps concentration up to a certain level due to its adhesive properties. The mechanical, swelling-degradation (weight loss) behaviors and Ps release kinetics were highlighted for the scaffold stability. The antimicrobial assay was employed to test and screen antimicrobial behaviour of Ps against Escherichia coli and Staphylococcus aureus strains. The results show that the Ps added scaffolds have an excellent antibacterial activity because of Ps’s compounds. The in-vitro cytotoxicity test was also applied on the scaffold by using the extract method on the human dermal fibroblasts (HFFF2) cell line. It is clearly found that the control SA and Ps added SA are non-toxic. The 3D printed SA-Ps scaffolds are very effective structures for wound dressing applications with unique properties.

N6-methylated adenosine (m6A) and N1-methylated adenosine (m1A) are two epi-transcriptomic modifications on eukaryotic mRNA which have recently been rediscovered and are generating considerable interest. M6A methylation impacts on all aspects of cellular RNA metabolism and numerous physiological processes. Although less abundant than the m6A epitranscriptomic mark, m1A methylation has recently also attracted interest due to its dynamic nature in response to physiological changes. We investigated the role of the m6A and m1A methylation regulators on the expression of a transgene in Chinese Hamster Ovary (CHO) cells - the host cell of choice in producing biopharmaceutical proteins commercially. Using siRNA-mediated gene depletion and methylation-specific RNA immunoprecipitation with anti-m6A or m1A-antibodies, we show that (i) knock-down of the m6A ‘reader’ YTHDF2 or the m1A ‘eraser’ ALKBH3 dramatically impacts transgene expression; (ii) the effects of YTHDF2 and ALKBH3 depletion on transgene expression are m6A- and m1A-mediated. We conclude that the expression of transgenes in CHO cells can be subjected to regulation by both m6A and m1A regulators. These findings open up the prospect of previously unexplored epi-transcriptomic-based approaches to CHO cell line engineering for improved recombinant protein production.

The cleavage of heparin by heparin lyases showed great potential as a cost-effective and innoxious method for producing heparin with low molecular weight (LMWH). One of the most studied and sought heparin lyase is heparinase I (HepI). However, the industrial use of HepI was largely hampered by its low specific activity and thermal stability. In this article we describe increasing in specific heparinase I activity by stepwise site-directed mutagenesis. Thus after two cycles of mutagenesis, we obtained mutant heparinase I Flavobacterium heparinum with significantly increased specific activity (25%).

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell selection. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.

Heterologous production of 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new ganoderic acid (GA) 3,28-dihydroxy-lanosta-8, 24-dien-26 oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by LC-MS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmid pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR and hygromycin resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418 resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work not only enriches the library of GAs and GA biosynthetic enzymes, but also helps to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum.

Tissue engineering (TE) constitutes a multidisciplinary scientific discipline focused on the construction of artificial tissues to regenerate end-stage organs. The impact of TE has led to a clinical revolution since novel therapies are available to attend several conditions. In this sense, its onset has supposed the communication of innovative discoveries in the age of social and mass media. This study aims to evaluate the global online dimension of TE from 2012 to 2018 by using data from the Web of Science (WoS) and Altmetrics. We have analysed 23,719 documents through descriptive and statistical methodologies. First, the descriptive analysis showed the evolution of TE original articles in five online platforms (Twitter, Patents, Facebook, Mendeley readers and News) and compared the most relevant TE documents ranked by their traditional and alternative metrics of impact. Secondly, we carried out a correlation and factorial analysis and then constructed a linear regression model to define a mathematical equation for the prediction of future TE citations counts from Altmetric scores. The obtained results suggest a growing presence of TE in the online social web and the feasibility in the context of global science to anticipate TE traditional academic impact by using social media

After surgical removal of bone tumors, elimination of the remains of cancer cells along with tissue healing and functionality is a therapeutic goal. Regarding the destructive effect of chemo-/radiotherapy on healthy cells, the development of multimodal scaffolds with simultaneous anticancer and osteo-regenerative potency is of particular importance as regenerative medicine for bone tissue engineering. Our previous study demonstrated that hollow pollen grain (HPG) of Pistacia vera L. offers a unique bone-forming activity and encapsulating capacity that it can be considered as an excellent scaffolding building block in bottom-up bone engineering. In the present study, for the first time, the anticancer potential of Pistacia vera L. HPG was investigated in-vitro using human osteosarcoma cell line MG63. Optical imaging of the HPG-cell interface indicated large focal adhesion due to the HPG unique surface features such as functionality and ornamentation. MTT assay results showed an anti-proliferative effect of HPG on the MG63 cells. Moreover, from the gene expression and DAPI staining analyses, HPG of Pistacia vera L. was found to be an apoptosis-inducing building block against MG63 bone cancer cells. So therefore, such a multifunctional building block with the ability of discriminatory killing human osteosarcoma cell line is proposed to be used after surgery to not only hinder cancer recurrence after surgery but also to stimulate bone healing.

Antimicrobial photodynamic therapy (APDT) is a promising alternative to traditional antibiotics for bacterial infections, which inactivates a broad spectrum of bacteria. However, it has some disadvantages including poor water solubility and easy aggregation of hydrophobic photosensitizers (PS), and poor tissue penetration and cytotoxicity when using UV as light source, leading to photodynamic therapy efficacy. Herein, we develop a novel water-soluble natural PS (sorbicillinoids) obtained by microbial fermentation using filamentous fungus Trichoderma reesei (T. reesei). Sorbicillinoids could effectively generate singlet oxygen (1O2) under UV light irradiation, and ultimately display photoinactivation activity on Gram-positive bacteria, but not Gram-negative ones. Staphylococcus aureus (S. aureus) treated with sorbicillinoids and UV light displayed high levels of intracellular reactive oxygen species (ROS), notable DNA photocleavage, and compromised membrane permeability without overt cell membrane disruption. Moreover, the dark toxicity, phototoxicity or hemolysis activity of sorbicillinoids is negligible, showing its excellent biocompatibility.

Pressure ulcers are commonly associated with microbial infections on the wounds which need an effective wound dressing. However, the silver dressings have shown promising result but they have toxicity and argyria. Hence, this study aimed to develop and characterize chitosan-polyethylene glycol (PEG) nanocomposite hydrogel loaded with phomopsidione as an antimicrobial dressing. The hydrogel being synthesized was analyzed with transmission and scanning electron microscopes. Drug release and mechanical properties were studied having confirmed the functional groups with Fourier transform infrared (FTIR) spectroscopy. Finally, antimicrobial activities were evaluated against the clinical wound pathogens. The developed hydrogel was soft, flexible and elastic, having nanospheres of chitosan-PEG but no sign of aggregation under the electron microscopes. Releasing of phomopsidione from the nanocomposite hydrogel was slow and gradual following the first order of kinetic. On average, 34 μg/mL phomopsidione released per hour and 67.9% active ingredients delivered into the surrounding medium over the study period. Although, the bioactivity activity of the hydrogel was narrow-spectrum, it showed significant results against all Gram-negative bacteria and Candida utilis with 99.99% reduction of microbial growth. The findings reveal that the phomopsidione loaded hydrogel has a great promise to act as an antimicrobial dressing for chronic wounds.

A document by Laipubam Sharma . Click on the document to view its contents.

Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In the present study, expression of Vitreoscilla hemoglobin in the chromosome of Escherichia coli was used as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the fully aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the non-expressing strain. The fed-batch phase was carried out with a change of regime from aerobic to microaerobic conditions. The pDNA yields from biomass increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the non-expressing strain during microaerobic conditions. The ratio of positive/negative plasmid replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the non-expressing strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the flux in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD+ in glycolysis under microaerobic conditions. These results are valuable for faster development of robust pDNA production processes.

Urea hydrolysis is widely used in agriculture, environment and other engineering fields, among which urease contained in beans can catalyze urea hydrolysis. The urea hydrolysis activity of legume plant leaching solution(LPLS) was investigated, including soybeans, black beans, mung beans, red beans as well as soybean hulls, soybean leaves, soybean stems and soybean pods. For the high urea hydrolysis activity and economic efficiency, soybean is most suitable for agricultural engineering and other fields than other beans and soybean-related parts extract. The urea hydrolysis activity increases with the concentration of LPLS, while decreases gradually with reaction time. When the heating temperature reaches 25, 35, 45, 55 and 65 ℃, the urea hydrolysis activity is steady and the enzyme activity is high. Enzyme activity decreases after 65 ° C (i.e.75, 90 ° C). Meanwhile, the soaking time of LPLS has a little effect on the urea hydrolysis activity compared with other factors. These results make a positive contribution to domestic production urease experimental basis.

Background Application of genetically modified non-pathogenic bacteria expressing specific anti-tumor proteins under certain conditions specific to tumors is an effective approach for selective targeting of tumors. We developed here, for the first time, a novel spatiotemporal cancer targeted therapy applying engineered E. coli bacteria with capability of expressing cardiac peptides under hypoxic conditions of tumor. Method E. coli BW25133 was transformed with construction of co-expressing cardiac hormones and GFP. Bacteria bearing constructs were then IV administered in mice bearing tumors and then tumor localization, as well as tumor proliferation, invasion and angiogenesis biomarkers (Ki-67, VEGFR, CD31and MMP9), changes in cytokine profile, suppression of tumor growth and survival were analyzed. Results IV Administered bacteria bearing constructs could specifically localize at tumor site and express cardiac peptides under hypoxic conditions. Administration of bacteria significantly enhanced survival rate, suppressed tumor progression and lowered expression levels of MMP-9, VEGFR2, CD31and Ki67 as potent markers for angiogenesis, tumor proliferation and metastasis. Furthermore, applied bacteria resulted in significant reduction in the expression of IL-1β, IL-6, GC-SF, IL-12 and TNF-α proinflammatory cytokines, whereas increasing IL-10, IL-17A and INF-γ cytokines. Conclusion Overall, administration of E. coli bearing cardiac hormone expression construct could effectively suppress tumor growth, angiogenesis, invasion and metastasis while enhancing survival rate in mice model of breast cancer.