Avian transgenesis has served as a suitable approach to generate bioreactors for the manufacturing of recombinant proteins. Production in chicken cells comes with significant advantages over other systems including providing the human-like glycosylation on target proteins. In this regard, the oviduct-specific ovalbumin promoter has been one of the ideal candidates to drive the expression of transgenes. Previous plasmid-based studies on the regulatory sequences of ovalbumin promoter have led researchers to exploit ovalbumin regulatory elements out of their native genomic context (ex situ) to direct transgene expression in the transgenic chicken bioreactors. Although the inherent limitations on the ex situ use of ovalbumin promoter have promoted the use of native ovalbumin promoter for the expression of a transgene, generation of transgenic chicken is relatively difficult, inefficient, and time-consuming. To overcome these obstacles, in this study we show that CRISPR-mediated deletion of some distal ovalbumin promoter sequences in a non-oviduct cell can lead to the significant expression of the ovalbumin gene, and also a knocked-in reporter, in an estrogen-independent manner. These findings overcome the limitation of cloned promoters, where the promoter regulatory sequences have to be taken out of their cis context and also their native spatial nuclear organization into a plasmid.

The synthesis of starch-based physical hydrogels in combination with chitosan and polyvinyl alcohol, and their potential co-application with chitosan nanoparticles was evaluated. The potential of starch-chitosan hydrogel obtained by physical/chemical method for tissue engineering uses was also studied in a mouse wound healing model. Although the microscopical structure of each synthesized hydrogel suggests a possible biological application, starch-polyvinyl alcohol hydrogel exhibited rigid behavior with minor channel diameters, a lower swelling rate (less than 300%), and negatively affected cell viability in a cytotoxicity assay. Starch-chitosan hydrogel obtained by chemical crosslinking with glutaraldehyde demonstrated the higher swelling rate (about 1100%), cell viability values over 80%, and a homogeneous tri-dimensional structure; along with an excellent interaction with chitosan nanoparticles. This type of hydrogel was selected for an in vivo experiment, showing significant differences in wound healing process against a non-treated control, in terms of inflammation, exudate production and tissue recovering.

Stem cell (SC) differentiation towards somatic cells has proven to be an effective technique in the understanding and progression of regenerative medicine. Despite improvements, concerns regarding the efficiency of differentiation and the differences between SC products and their in vivo counterparts must be addressed. Biomaterials that mimic endogenous growth conditions represent one recent method used to improve the quality and efficiency of SC differentiation. Here, we aim to use bioinformatics approaches to accomplish two aims: 1) determine the effect of different biomaterials on SC growth and differentiation, and 2) understand the effect of cell of origin on the differentiation potential of multipotent SCs. First, we demonstrate that the dimensionality (2D versus 3D) and the degradability of biomaterials affects the way that the cells are able to grow and differentiate at the transcriptional level. Additionally, the particular cell of origin is an important factor in determining the response of SCs to same biomaterial transcriptionally. Our data demonstrates the ability of bioinformatics to understand novel molecular mechanisms and context by which SCs are most efficiently able to differentiate. These results and strategies may suggest proper combinations of biomaterials and SCs to achieve high differentiation efficiency and functionality of desired cell types.

The hemibiotrophic oomycetes are significant threats to a wide range of Cucurbitaceae species, causing substantial losses of plant productions. Particularly, Phytophthora melonis evokes severe symptoms, thus dramatically limiting yield in cucumber. However, information about cucumber–P. melonis interaction is still limited. This study explored changes in the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), superoxide dismutase (SOD), and polyphenol oxidase (PPO) in cucumber roots of two resistant genotypes (Soheil and Ramezz), one moderately resistant genotype (Baby) and three highly susceptible genotypes (Extrem, Mini 6-23 and Yalda), over the time courses of 7, 14 and 21 days after inoculation (DAI). The results indicated that the activities of defence‐related enzymes differed between the resistant and highly susceptible genotypes. Although, the defense-related enzymatic activities were elevated sharply in the resistant and moderately resistant genotypes after inoculation, but no significant correlations were present between the activity trends of PPO, SOD and CAT and resistance characteristics. Moreover, no significant changes in enzyme activities were found in the control plants, non-inoculated plants of the six genotypes during the testing period. Altogether, the resistance of cucumber to P. melonis is related to POX and PAL activities, but does not show relationship with PPO, SOD and CAT activities. Studying the physiological metabolic pathways of POX and PAL appears to be an important direction in research to elucidate resistance to P. melonis in cucumber genotypes.

Based on the analysis of CpxP genes among Escherichia coli strains, CpxP gene-targeting short guide RNA (sgRNA) was designed and inserted into the pGL3-MGP-RNA. The donor sequences (MG-HR) for homologous repair were designed and cloned by PCR. MG-HR and pGL3-MGP-RNA were transformed into E. coli MG1655 (pCas9). The CpxP gene expression cassette was amplified by PCR and subcloned into pBBR1MCS-2. Then the pBBR-CpxP was independently transformed into E. coli MG1655. The results of motility experiment suggest that CpxP gene had a significant effect on the movement ability of E. coli strain. The CpxP protein had a significant inhibition of bacterial activity. The lastest 81 CpxP proteins sequences were selected and analyzed by multi-sequence alignment and molecular cluster. The CpxP proteins were roughly divided into three categories. Our results suggest that the CpxP protein was involved in bacterial motility, infection and pathogenicity.

Continuous improvements of cell-free synthesis (CFS) systems have generated interest in adopting the technology for the manufacture of biologics. This paper provides an evaluation of the manufacturing cost-effectiveness of CFS for a range of commercial scenarios. The evaluation was performed using an advanced techno-economic engine (TEE) built in Python. The TEE is programmed in an object-oriented environment capable of simulating a plethora of process flowsheets and predicting size and cost metrics for the process and the facility. A case study was formulated to compare the economics of whole bioprocesses based on either a CFS system or a mammalian cell system (CHO) for the manufacture of an antibody drug conjugate (ADC) at different commercial product demand levels (100 – 1000kg/year). The analysis demonstrated the potential of CFS for the commercial manufacture of biologics and identified key cost drivers related with the system. The CFS system showed approximately a two-fold increase in the cost of goods compared to CHO with a significant cost attributed to the in-house manufacture of the bacterial cell extract, necessary for the CFS reaction step in the process. A sensitivity and target analysis highlighted the impetus for further process improvements especially in the titre for the CFS process to become more competitive against well-established systems.

Background: Increasing the use of tobacco by the younger generation has increased in oral cavity tumours. Surgical treatment is radical and results in severe functional morbidity. Using computer-aided designing technology, surgical and rehabilitative planning can be better. We present here our concept of a biomechanical 3D tongue model and its clinical utility in the management of tongue tumours. Methods: Patients diagnosed with Carcinoma of Tongue were included. These patients underwent a pre-operative Magnetic Resonance Imaging (MRI) of the head and neck region at our center. These patients were informed about the use of a 3D biomechanical patient-specific model for treatment planning and execution. Using Materialise Mimics Innovation Suite 19 DICOM data was imported, visualized, edited and segmented. Flashforge ‘Creator-pro’3D Printer was used for 3D Printing. Fused Deposition Modeling (FDM) technology was used to print the tumour and uninvolved tongue in two different colors for easy identification. These patients underwent surgery with the 3D model serving as a guide for margins. Results: Two patients with stage III squamous cell carcinoma of tongue underwent the surgery based on the plan evolved from the 3D model. All the surgical margins were clear. The model helped address the discordance between patient expectations and surgical outcomes. We found that the model aided the reconstructive surgeon to plan the flap volume better and this translated into better rehabilitative outcomes. Discussion and Conclusion: The 3D biomechanical tongue model is a novel concept and may aid in improving our overall treatment outcomes.

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