scholarly journals In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vitro Recombination ◽  
In Vivo Recombination ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
Heterologous Pathway ◽  
Β Carotene

Abstract Background : Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganisms is still limited by the poor efficiency of the heterologous pathway. Results: To address the bottleneck of astaxanthin yield in microbes, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modules of β-carotene ketolase ( crtW ) and hydroxylase ( crtZ ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05 mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination. Conclusions: Recombination of heterologous modules by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high-throughput screening of heterologous pathways by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW , but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathways.

scholarly journals In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vivo Recombination ◽  
Environmental Friendly ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
The Cost ◽  
Heterologous Pathway

Abstract Background: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway.Results: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologousβ-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11~8.51 folds and 3.0~9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg/g DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains.Conclusions: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

In vitro and In vivo recombination of heterologous modular for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
Chemical Synthesis ◽  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vivo Recombination ◽  
The Poor ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
Heterologous Pathway

Abstract Bacground: Astaxanthin is a kind of tetraterpene with strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganism is still limited by the poor efficiency of heterologous pathway.Results: To address the bottleneck of astaxanthin yield in microbe, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modular ofβ-carotene ketolase (crtW) and hydroxylation (crtZ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination.Conclusions: Recombination of heterologous modular by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high throughput screening of heterologous pathway by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW, but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathway.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals In Vitro Probitotic Evaluation of Saccharomyces boulardii with Antimicrobial Spectrum in a Caenorhabditis elegans Model

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1428
Author(s):  
Ramachandran Chelliah ◽  
Eun-Ji Kim ◽  
Eric Banan-Mwine Daliri ◽  
Usha Antony ◽  
Deog-Hwan Oh
Keyword(s):  
Pancreatic Enzyme ◽  
Saccharomyces Boulardii ◽  
Antimicrobial Spectrum ◽  
Antimicrobial Efficiency ◽  
Heat Stable ◽  
Yeast Strains ◽  
Probiotic Yeast ◽  
Probiotic Strains

In the present study, we screened for potential probiotic yeast that could survive under extreme frozen conditions. The antimicrobial and heat-stable properties of the isolated yeast strains Saccharomyces boulardii (S. boulardii) (KT000032, KT000033, KT000034, KT000035, KT000036, and KT000037) was analyzed and compared with commercial probiotic strains. The results revealed that the tested S. boulardii KT000032 strain showed higher resistance to gastric enzymes (bile salts, pepsin, and pancreatic enzyme) at low pH, with broad antibiotic resistance. In addition, the strain also showed efficient auto-aggregation and co-aggregation abilities and efficient hydrophobicity in the in-vitro and in-vivo C. elegens gut model. Further, the KT000032 strain showed higher antimicrobial efficiency against 13 different enteropathogens and exhibited commensal relationships with five commercial probiotic strains. Besides, the bioactive compounds produced in the cell-free supernatant of probiotic yeast showed thermo-tolerance (95 °C for two hours). Furthermore, the thermo-stable property of the strains will facilitate their incorporation into ready-to-eat food products under extreme food processing conditions.

scholarly journals TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

2020 ◽  
Author(s):  
Hua Sang ◽  
Jiali Liu ◽  
Fang Zhou ◽  
Xiaofang Zhang ◽  
Jingwei Zhang ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Therapeutic Efficacy ◽  
High Throughput Screening ◽  
Early Stage ◽  
Cellular Level ◽  
Drug Conjugates ◽  
Therapeutic Outcomes ◽  
Lysosomal Proteolysis

<p></p><p>Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by <i>in vitro</i> viability assays and <i>in vivo</i> tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly <i>in vivo</i> experiments.</p><br><p></p>

scholarly journals A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo

2021 ◽  
Vol 22 (22) ◽  
pp. 12502
Author(s):  
Shoji Kokubo ◽  
Shinobu Ohnuma ◽  
Megumi Murakami ◽  
Haruhisa Kikuchi ◽  
Shota Funayama ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Cells ◽  
High Throughput Screening ◽  
Atp Hydrolysis ◽  
Pheophorbide A ◽  
Chemical Library ◽  
Hct 116 ◽  
Potential Inhibitors

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

In vitro antihemolytic effect, in vivo anti inflammatory and In vitro antioxidant activity of Anchusa azurea Mill

2021 ◽  
Vol 20 ◽  
Author(s):  
Boussoualim Naouel ◽  
Trabsa Hayat ◽  
Krache Imane ◽  
Ouhida Soraya ◽  
Arrar Lekhmissi ◽  
...  
Keyword(s):  
Methanolic Extract ◽  
Folk Medicine ◽  
Oxidative Degradation ◽  
Negative Control ◽  
Dependent Manner ◽  
Anti Inflammatory ◽  
Β Carotene ◽  
Inflammatory Effect

Background: Anchusa azurea Mill. (AA) is a medicinal plant largely used traditionally in folk medicine in Algeria, it is locally named: hamham. It is effective in the treatment of various diseases. Objectives: The aim of the present study is to determine the antioxidant, anti-inflammatory and anti-hemolytic effects of phenolic fractions from Anchusa azurea Mill. Methods: In this study, various extracts from Anchusa azurea Mill. (AA) using solvents with increasing polarity were prepared. The quantification of polyphenols and flavonoids was determined. The anti-radical activity of the different extracts was evaluated using DPPH and by measuring the inhibition of the oxidative degradation of β-carotene. The In vitro antihemolytic effect of the plant extracts is determined (CrE, ChE, AcE and AqE). For each extract, four concentrations were tested: 10.59, 21.18, 42.37, 84.74 µg/ml. Vitamin C is used as a standard. Free-radical attack was measured by measuring the HT50 (Half-Hemolysis Time). The anti-inflammatory effect using PMA on mice of the methanolic extract (CrE) was evaluated. Results: The quantification of polyphenols and flavonoids showed that ethyl acetate extract (AcE) contains a higher amount of polyphenols. However, chloroform extract (ChE) presents a higher amount of flavonoids. AcE showed an important scavenging activity using the DPPH radical (IC50= 68.35 µg/ml). The results showed that AcE also exhibited very great inhibition on the oxidation of β-carotene/linoleic acid (84.33%). All extracts increased the HT50 values (Half-Hemolysis Time) in a dose-dependent manner. The three highest concentrations (21.18, 42.37 and 84.74 µg / ml) of ChE caused a very significant delay (p ≤ 0.001) of hemolysis compared to the negative control and the positive control "VIT C". The anti-inflammatory effect using PMA on mice showed that the methanolic extract (CrE) of AA reduced the weight of the ear edema. Conclusions: This plant has a strong pharmacological power, which supports its traditional medicinal use.

scholarly journals A Novel Assay for Profiling GBM Cancer Model Heterogeneity and Drug Screening

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 702 ◽  
Author(s):  
Christian T. Stackhouse ◽  
James R. Rowland ◽  
Rachael S. Shevin ◽  
Raj Singh ◽  
G. Yancey Gillespie ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Therapeutic Response ◽  
Specific Gene ◽  
Cancer Model ◽  
In Vivo Models ◽  
Cancer Models ◽  
Model Independent ◽  
Orthotopic Xenografts

Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology and in gauging therapeutic response. It remains to be seen whether or not cancer model-specific genes may be directly or indirectly, through changes to tumor microenvironment, manipulated to improve the concordance of in vitro derivative models with in vivo models yielding better prediction of therapeutic response.

scholarly journals β-carotene Suppresses Colon Cancer by Regulating M2 Macrophages and Tumor-associated Fibroblasts in Vitro and in Vivo (P02-015-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Yerin Kim ◽  
Na Youn Lee ◽  
Yoo Sun Kim ◽  
Yuri Kim
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Macrophage Polarization ◽  
M2 Macrophage ◽  
Invasion And Migration ◽  
And Migration ◽  
Emt Markers ◽  
Β Carotene

Abstract Objectives Tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs) are consisted of tumor microenvironment (TME), which are involved in cancer progression and metastasis. Interactions within TME induce M2 macrophage phenotype, TAMs, and activate TAFs. β-carotene (BC) is a well-known antioxidant and showed protective effects on several diseases, including cancers. The object of this study is to investigate the anti-colorectal cancer (CRC) effects of BC by controlling macrophage polarization and fibroblast activation. Methods TAMs were induced by treating with phorbol-12-myristate-13-acetate (PMA) and interleukin-4 (IL-4) in U937 cells and TAFs were induced by treating with transforming growth factor-β1 (TGF-β1) in CCD-18Co cells. To understand the effect of TME on cancer cells, HCT116 colon cancer cells were co-cultured with TAM or TAF conditioned media. The effects of BC on the expressions of cancer stem cells (CSCs) markers, epithelial-mesenchymal transition (EMT) markers along with invasion and migration were investigated. To confirm these results, the azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colitis-associated CRC mice model was used. Results BC decreased M2 macrophage polarization with activating IL-6/STAT3 signaling pathways and suppressed the expressions of fibroblast activation markers and EMT markers. In addition, BC inhibited the expressions of TME-induced CSCs markers and EMT and suppressed cell invasion and migration. Furthermore, BC supplementation suppressed tumorigenesis and the expressions of M2 macrophage-associated markers, including CD206, Arg1, and Ym-1 as well as CSCs markers in vivo. Conclusions BC suppressed CRC by regulating TAMs and TAFs in vitro and in vivo, which indicated the potential therapeutic effects of BC on inflammatory diseases. Funding Sources This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and Brain Korea 21 Plus.

scholarly journals Matrix Drug Screen Identifies Synergistic Drug Combinations to Augment SMAC Mimetic Activity in Ovarian Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3784
Author(s):  
Anne M. Noonan ◽  
Amanda Cousins ◽  
David Anderson ◽  
Kristen P. Zeligs ◽  
Kristen Bunch ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Single Agent ◽  
Recurrent Ovarian Cancer ◽  
Drug Combinations ◽  
Smac Mimetic ◽  
Synergistic Drug Combinations

Inhibitor of apoptosis (IAP) proteins are frequently upregulated in ovarian cancer, resulting in the evasion of apoptosis and enhanced cellular survival. Birinapant, a synthetic second mitochondrial activator of caspases (SMAC) mimetic, suppresses the functions of IAP proteins in order to enhance apoptotic pathways and facilitate tumor death. Despite on-target activity, however, pre-clinical trials of single-agent birinapant have exhibited minimal activity in the recurrent ovarian cancer setting. To augment the therapeutic potential of birinapant, we utilized a high-throughput screening matrix to identify synergistic drug combinations. Of those combinations identified, birinapant plus docetaxel was selected for further evaluation, given its remarkable synergy both in vitro and in vivo. We showed that this synergy results from multiple convergent pathways to include increased caspase activation, docetaxel-mediated TNF-α upregulation, alternative NF-kB signaling, and birinapant-induced microtubule stabilization. These findings provide a rationale for the integration of birinapant and docetaxel in a phase 2 clinical trial for recurrent ovarian cancer where treatment options are often limited and minimally effective.

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