Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. We have assessed the α-casein (CSN1S1) gene as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but much lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

The stiffness of the tumor microenvironment (TME) is dynamic and drives metabolic reprogramming in cancer cells as a consequence of tumor progression. To demonstrate the possibility to modulate the mechano-metabolomic profile of breast cancers by tuning the mechanical property and dimensionality of extracellular matrices (ECMs), we cultured triple-negative MDA-MB-231 and luminal MCF-7 cells on 2D and in 3D hydrogels based on tyramine functionalized hyaluronic acid (HTA). Using high-throughput metabolomics analyses, we established that we can differentially regulate breast cancer mechano-metabolome. The stiff hydrogels resulted in upregulated lipid and amino acid metabolism along with increasing malignancy and chemoresistancy. Reprogramming in glucose metabolism is primarily observed in cells seeded on 2D hydrogels, whereas modifications in amino acid metabolism is predominant in cells embedded in 3D stiff hydrogels. These findings suggest that matrix stiffness and dimensions have decisive roles in reprogramming breast cancer metabolome, which is the hallmark of breast cancer development and progression.

Recently, nanomedicine have been successfully applied in the cancer therapy. However, how to precisely control the drug release from nanomedicine in tumor tissue and overcome the hypoxic microenvironment of tumor tissue is still an important challenge in the development of nanomedicine. In this work, a new type of drug-loaded nanoparticles P(AAm-co-AN)-AuNRs@CeO2-DOX (PA-DOX) was prepared by combining high-efficiency photothermal reagents, critical up-conversion temperature polymer layer and anti-cancer drug doxorubicin (DOX) for the treatment of liver cancer. In this system, CeO2 can decompose hydrogen peroxide to H2O2 and O2 alleviate the anaerobic microenvironment of liver cancer cells. As a photothermal reagent, AuNRs@CeO2 can convert near-infrared light into heat energy to achieve local heat to kill cancer cells and ablate solid tumors. In addition, the elevated temperature would enable the polymer layer to undergo a phase transition to release more DOX to achieve a controlled release mechanism, which will open up a new horizon for clinical cancer treatment

The ApxII toxin and outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) are vital vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease in the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli (E. coli). However, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml in E. coli by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The four most potent expression vectors were screened, and the expression of three vaccine antigens Oml1, Oml7, and ApxII in these four bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates led to improved production of Oml1, Oml7, and ApxII. Finally, the production yields reached unprecedented levels of 2.43 g/L, 2.59 g/L, and 1.21 g/L, respectively, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study established a highly efficient bicistronic T7 expression system and achieved the hyper-production of PCP vaccine proteins. This bicistronic T7 expression system could be a valuable tool for the improved production of other proteins, especially recombinant vaccines, in E. coli.

The study of cellular metabolism is often hindered by limitations in the amount of exper- imental data available. Therefore computational methods that exploit maximally the possible measurements, and are able to extract relevant predictions from a minimum of information are always welcome. Maximum Entropy (ME) inference has been succesfully applied to genome-scale models of cellular metabolism in various cell culture contexts, yielding insights into biologically relevant properties which are not accessible to traditional optimization based methods. Recent data-driven studies have suggested that in chemostat cultures, the growth rate and uptake rates of limiting nutrients are the most informative parameters about the metabolism. In this work, we propose the thesis that chemostat dynamics typically drives the culture towards maximally restricted metabolic states. A practical consequence is that experimental values of limiting up- take rates can be replaced by more readily available measurements of metabolite concentrations in the feed media and the steady state cell concentration. We show how these results can be justified from a mechanistic perspective by studying simulations of a simplified model where we test the quality of the inference, and unveil the mechanisms defining the performance of our approach. We then apply our method to E. coli experimental data. We evaluate the effects of heterogeneity in chemostat cultures and its potential impact on flux inference quality of ME and optimization based strategies. Additionally, we evaluate the quality of the inference comparing M E to alternative formulations that rest on a Flux Balance Analysis (F BA).

Not only carbon dioxide (CO2) but also air pollutants, such as sulfur oxides (SOx) and nitrogen oxides (NOx), are present in flue gas, and their reasonable and effective utilization is conducive to reducing the cost of microalgal biomass production. By utilizing simulated flue gas, the absorption characteristics of different components in transfer units were explored. The results showed that the presence of SO2 decreased the absorptivity of CO2, which reduced the concentration of the available carbon source for microalgal cells in the culture medium at the same pH value. Moreover, the presence of high-concentration oxygen (O2) in flue gas could improve the absorptivity of nitric oxide (NO). Scenedesmus dimorphus was cultured by using sulfur- or nitrogen-deficient culture media. The results showed that SOx and NOx in flue gas did not significantly influence the growth and biochemical compositions of microalgal cells when these gases were dissolved in water. Based on the above results and the metabolic kinetics of microalgal cells for nutrient elements, an adjustment strategy for the initial gas source when culturing microalgae with flue gas is proposed: (1) flue gas should be partially desulfurized so that the SOx and CO2 concentrations after desulfurization satisfy a certain relationship with the components; and (2) denitrification should not be performed because flue gas can be oxidized before utilization to increase the rate of utilization of NO.

The production of vaccines in plant cells, termed as plant-made pharmaceuticals or molecular farming, is a promising technology. Compared to mammalian cell lines, like HEK293 and CHO as established platform, plants can be grown cost effectively on a large-scale without necessitating any sophisticated technologies. An innovative application of this alternative system is the production of vaccines in edible tissues that can be consumed orally to deliver protein antigen without any further processing. In this project we reported stable expression of TM-1 protein of MG as vaccine candidate antigen against Chronic Respiratory Disease (CRD) in wheat seeds tissues. The molecular and Immunoblotting analysis confirmed the integration of recombinant protein of 41.8 kDa with expression level of 1.03mg/g DW in endosperm tissues. When orally delivered, the plant made vaccine were highly effective in terms of developing antibodies in chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited an immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrated that plant-made vaccines are not only safe but also similarly effective to commercially available vaccines.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproduction activity in different types of livestock. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, none approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU.106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG not only demonstrated biological activity in bovine cattle but also this bioactivity appeared to be higher than PMSG, since 140 IU of reCG were needed to exert the same biologic effect in an ovulation synchronization protocol compared to 400 IU of PMSG. The results obtained show that the developed strategy represents an attractive option to produce reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

The implementation of human pluripotent stem cells (hiPSCs) in cell therapy has an extraordinary potential but faces many practical challenges, including costs associated with growth media and factors. There is an immediate need to establish an optimized culture platform to direct the differentiation of hiPSCs into germ layers in a defined nutritional microenvironment to generate cost-effective and robust therapeutics. The aim of this study was to identify the optimal nutritional environment by mimicking the in vivo concentrations of three key factors (glucose, pyruvate and oxygen) during the spontaneous differentiation of hiPSCs derived from cord blood, which greatly differ from the in vitro expansion and differentiation scenarios. Moreover, we hypothesized that the high glucose, pyruvate and oxygen concentrations found in typical growth media could inhibit the differentiation of certain lineages. A design of experiments (DoE) was used to investigate the interaction between these three variables during the spontaneous differentiation of hiPSCs. We found that lower oxygen and glucose concentrations enhance the expression of mesodermal (Brachyury, KIF1A) and ectodermal (Nestin, B-Tubulin) markers. Our findings present a novel approach for efficient directed differentiation of hiPSCs through the manipulation of media components whilst simultaneously avoiding the usage of growth factors thus reducing costs.

Yeast has been well-used as a typical microbial platform to make fermented fine chemicals. However, various stress conditions severely restrict the production costs and benefits. One effective way to resolve such bottlenecks is to engineer transcription factors (TFs) to enhance strain tolerance and production efficiency through remodeling the transcript levels of different stress resistant genes. Here, we focus on the recent advances in the mechanisms of yeast adaptive responses upon stresses of heat, acetic acid and oxidants and classify them into different modules within yeast cells. In particular, novel strategies for the enhancement of both tolerance and yield by TFs engineering are examined. In addition, the applications of artificial transcription factor (ATFs)-based fabricating in metabolic fluxes optimization and quantitative evaluation are discussed. Lastly, we discuss challenges and potential solutions in exploiting TFs engineering and for A bio-based economy products.

Results of the preparation of biodegradable porous scaffolds using an original modification of a wet phase inversion method were presented. Influence of gelatin nano-non-woven as a non-classic pore precursor and polyvinylpyrrolidone, Pluronic® as classic pore precursors on the structure of obtained scaffolds was analyzed. It was shown that the addition of gelatin nano-non-wovens enables the preparation of scaffolds, which allow for the growth of cells (size, distribution, and shape of pores). Mechanical properties of the obtained cell scaffolds were determined. The influence of pore precursors on mass absorption of scaffolds against isopropanol and plasma was investigated. Interaction of scaffolds with a T-lymphocyte line (Jurkat) and with fibroblasts (L929) was investigated. Obtained scaffolds are not cytotoxic and can be used as implants, e.g. the regeneration of cartilage tissue.

Background. 2018 Guideline from the College of American Pathologists(CAP), the International Association for the Study of Lung Cancer (IASLC) , and the Association for Molecular Pathology(AMP) established a benchmark turnaround time (TAT), according to which the results were supposed to be available to the treating oncologist within 10 working days. Methods. In this study, we established and validated a new protocol for pathological diagnosis and treatment pathway that was tested in the Henan Cancer Hospital, China. Results. Compared with the routine pathway, the new path adopts a parallel approach to conventional pathology and molecular pathology, which significantly shortens the TAT to 4-6 days. Self-designed DNA biology collector “BIOCO” tool, designed by our team to collect specimens that are discarded when the wax block is trimmed, can effectively save pathological samples, avoid cross-contamination and reduce the period extension caused by re-sampling. Most importantly, its accuracy and effectiveness are consistent with conventional collection methods. Conclusions. Therefore, the new diagnosis and treatment pathway based on the BIOCO collector can be used as a practical approach to the molecular diagnostic platform of the hospital pathology department.

Stem cell-derived extracellular vesicles (EVs) have shown great promise in the field of regenerative medicine and tissue engineering. Recently, human bone marrow-derived mesenchymal stem cell (BMSC)-derived EVs have been considered for bone tissue engineering applications. In this study, we evaluated the osteogenic capability of placental stem cell (PSC)-derived EVs and compared them to the well-characterized BMSC-derived EVs. EVs were extracted from three designated time points (0, 7, and 21 days) after osteogenic differentiation. The results showed that the PSC-derived EVs had much higher protein and lipid concentrations than EVs derived from BMSCs. The extracted EVs were characterized by observing their morphology and size distribution before utilizing next-generation sequencing to determine their miRNA profiles. It was identified that 306 miRNAs within the EVs, of which 64 were significantly expressed in PSC-derived EVs that related to osteogenic differentiation. In vitro osteogenic differentiation study indicated that the late-stage (21-day extracted) derived EVs higher osteogenic enhancing capability when compared to the early-stage derived EVs. We demonstrated that EVs derived from PSCs could be a new source of EVs for bone tissue engineering applications.

A document by Mitch Raith. Click on the document to view its contents.

This study aimed to evaluate the antitumor potential and cytotoxicity induction mechanisms of green synthesized AgCl-NPs and Ag/AgCl-NPs through image-based high content analysis method. The antitumor potential of AgCl-NPs and Ag/AgCl-NPs was evaluated in breast cancer BT-474 and MDA-MB-436 cell lines treated with 0-40 μg/mL AgCl-NPs or 0-12.5 μg/mL Ag/AgCl-NPs. Normal human retinal pigment epithelial 1 (RPE-1) cells were used for comparison. The growth rate of the RPE-1 cells treated with AgCl-NPs or Ag/AgCl-NPs was little affected, and no significant changes in cell viability were observed. In these cells, the nanoparticle treatments did not induce lysosomal damage, changes in reactive oxygen species (ROS) production or a reduction in the mitochondrial membrane potential. Moreover, the percentage of apoptotic cells was minimally affected, reaching a maximum of 3.51% of the population. The level of BT-474 and MDA-MB-436 cell proliferation was markedly decreased, and cell viability was reduced by 64.19 and 46.19% after treatment with AgCl-NPs and reduced by 98.36 and 82.29% after treatment with Ag/AgCl-NPs. The cells also showed a significant increase in ROS production and loss of mitochondrial membrane potential, which culminated in an increase in the percentage of apoptotic cells. BT-474 cells also presented lysosomal damage when treated with the highest concentrations of both nanoparticle types, and actin polymerization was observed after exposure to Ag/AgCl-NPs. Together, the results obtained show overall cytotoxic effects of both AgCl-NPs and Ag/AgCl-NPs towards breast cancer cells with negligible effects against healthy cells, which suggests their promising anticancer and biomedical applications.

The contamination of heavy metals (e.g., Hg, Pb, Cd and As) poses great risks to environment and human health. Rapid and simple detection of heavy metals of considerable toxicity in low concentration levels is an important task in biological and environmental analysis. Among the many convenient detection methods for heavy metals, DNA-inspired gold nanoparticles (AuNPs) (DNA-AuNPs) have been becoming a well-established approach, in which assembly/disassembly of AuNPs serves for colorimetric signaling of the recognition event between DNA and target heavy metals at the AuNP interface. This review focuses on the recent efforts of employing DNA to manipulate the interfacial properties of AuNPs, and summarizes the recent advances in the colorimetric detection of heavy metals. Beginning with the introduction of the fundamental aspects of DNA and AuNPs, three main strategies of constructing DNA-AuNPs with DNA binding-responsive interface are discussed, namely, crosslinking, electrostatics, and base pair stacking. Then, recent achievements in colorimetric biosensing of heavy metals based on the interfacial manipulation of DNA-AuNPs are surveyed and compared. Finally, conclusions and perspectives on the futuristic directions in the field are provided, including the evaluation of the pros and cons of the three strategies, with an expectation of providing a reference for researchers who works in the related field. It is also suggested that manipulation of the interface of DNA-AuNPs represent enormous potential in the pursuit of colorimetric biosensors for a range of analytes of interest.

Breast cancer is a heterogeneous pathology. There are subgroups with different molecular characteristics, and they must be identified to direct the therapy and to have a clear prognosis. HER2/neu (human epidermal growth receptor factor 2/neuro glioblastoma derived oncogene homolog) receptor overexpression is a characteristic that must be evaluated in each population. We apply the monoclonal antibody technique in the indigenous population of Peru, we also correlate the presence of the receptor with global survival. It’s an experimental, prospective and analytical study. Twenty three samples were evaluated, obtained from patients with adenocarcinoma of the breast. The HER2/neu expression was determined by immunohistochemical technique (monoclonal antibodies). The results were verified 10 cases were positive for overexpression (43.47%). The negative cases were 13 (56.52%). The overall survival at 3 years was 69.90% for the Her2/neu positive and 85.00% for negative, showed statistically significant differences (p=0.017). Finally, it was feasible to apply the monoclonal antibody technique and to statistically correlate HER2/neu overexpression with TNM clinical stage, predicting less survival when present. Applying an immunohistochemical technique is feasible in native Peruvian women. The main benefit is to apply biological therapy with monoclonal antibodies, according to their molecular profile.

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from donor tissue reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2) extraction can significantly improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, decellularization of complex 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, type of co-solvent, values of pressure and temperature insight the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the efficiency of decellularization using approaches based on various combinations of these parameters. The protocols were based on combinations of treatment in alkaline, ethanol or detergent solutions with scCO2 extraction at different modes. Based on a histological analysis, we have selected an optimal protocol for the decellularization of ovine aortic root employing preconditioning in a detergent solution. The positive effects of scCO2 on the decellularization extent, cytotoxicity and histoarchitecture of the tissue were demonstrated.