scholarly journals Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

2017 ◽  
Author(s):  
Richard Kelwick ◽  
Luca Ricci ◽  
Soo Mei Chee ◽  
David Bell ◽  
Alexander J. Webb ◽  
...  
Keyword(s):  
Industrial Waste ◽  
Low Cost ◽  
Efficient Production ◽  
Biosynthetic Pathways ◽  
Whey Permeate ◽  
Coa Transferase ◽  
Clostridium Propionicum ◽  
Atp Generation

ABSTRACTThe polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising polyhydroxyalkanoates production.

scholarly journals Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Richard Kelwick ◽  
Luca Ricci ◽  
Soo Mei Chee ◽  
David Bell ◽  
Alexander J Webb ◽  
...  
Keyword(s):  
Industrial Waste ◽  
Gas Chromatography Mass Spectrometry ◽  
Efficient Production ◽  
Biosynthetic Pathways ◽  
Whey Permeate ◽  
In Vivo Assays ◽  
Production Processes ◽  
Clostridium Propionicum

Abstract The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimize several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimizing PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customized our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimize in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by approximately 50%. In cell-free transcription–translation prototyping reactions, gas chromatography–mass spectrometry quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ± 0.39 µM), C104 ΔPhaC_C319A (4.62 ± 1.31 µM) and C101 ΔPhaC_C319A (2.65 ± 1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription–translation reactions produced greater yields of 3HB (32.87 ± 6.58 µM), and these reactions were also used to characterize a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimizing PHAs production.

scholarly journals Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Lingyu Yang ◽  
Dehai Xian ◽  
Xia Xiong ◽  
Rui Lai ◽  
Jing Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Low Cost ◽  
Natural Agents ◽  
Molecular Damage ◽  
And Control

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerousin vitroandin vivostudies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs.

Development of Natural Bioactive Alkaloids: Anticancer perspective

2021 ◽  
Vol 21 ◽  
Author(s):  
Ashish Patel ◽  
Ravi Vanecha ◽  
Jay Patel ◽  
Divy Patel ◽  
Umang Shah ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Low Cost ◽  
Chemical Compounds ◽  
Cost Effective ◽  
Drug Discovery And Development ◽  
Anticancer Properties ◽  
Antimetastatic Activity ◽  
New Chemical Entities

: Cancer is a frightful disease that still poses a 'nightmare' worldwide, causing millions of casualties annually due to one of the human race's most significant healthcare challenges that requires a pragmatic treatment strategy. However, plants and plant-derived products revolutionize the field as they are quick, cleaner, eco-friendly, low-cost, effective, and less toxic than conventional treatment methods. Plants are repositories for new chemical entities and have a promising cancer research path, supplying 60% of the anticancer agents currently used. Alkaloids are important chemical compounds that serve as a rich reservoir for drug discovery and development. However, some alkaloids derived from natural herbs display anti-proliferation and antimetastatic activity on different forms of cancer, both in vitro and in vivo. Alkaloids have also been widely formulated as anticancer medications, such as camptothecin and vinblastine. Still, more research and clinical trials are required before final recommendations can be made on specific alkaloids. This review focuses on the naturally-derived bioactive alkaloids with prospective anticancer properties based on the information in the literature.

scholarly journals xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

2018 ◽  
Author(s):  
Avi Z. Rosenberg ◽  
Carrie Wright ◽  
Karen Fox-Talbot ◽  
Anandita Rajpurohit ◽  
Courtney Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Small Rna ◽  
Mirna Expression ◽  
Low Cost ◽  
Expression Patterns ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Cell Type

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

scholarly journals Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Shashank Chetty ◽  
S. Praneetha ◽  
Sandeep Basu ◽  
Chetana Sachidanandan ◽  
A. Vadivel Murugan
Keyword(s):  
Large Scale ◽  
Near Infrared ◽  
Organic Dyes ◽  
Low Cost ◽  
Fluorescence Emission ◽  
Zebrafish Embryos ◽  
Fluorescence Bioimaging ◽  
Rapid Labeling

Abstract Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 μg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 μg/mL. Hence, we propose that the rapid, low-cost, large-scale “sustainable” MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI).

Phytochemicals as Potential Inhibitors of Advanced Glycation End Products: Health Aspects and Patent Survey

2021 ◽  
Vol 12 ◽  
Author(s):  
Annayara C. F. Fernandes ◽  
Jeane B. Melo ◽  
Vanize M. Genova ◽  
Ádina L. Santana ◽  
Gabriela Macedo
Keyword(s):  
Phenolic Compounds ◽  
Inflammatory Responses ◽  
Low Cost ◽  
Advanced Glycation Endproducts ◽  
Advanced Glycation ◽  
Glycation Endproducts ◽  
Synthetic Drugs ◽  
Peanut Skins

Background: Glycation is a chemical reaction that synthesize advanced glycation endproducts (AGEs). The AGEs irreversibly damage macromolecules present in tissues and organs, leading to the impairment of biological functions. For instance, the accumulation of AGEs induces oxidative stress and consequently inflammatory responses in human body, leading to the on set/worsening of diseases, including obesity, asthma, cognitive impairment, and cancer. There is a current demand on natural and low-cost sources of antiglycant agents. As a result, food phytochemicals presented promising results to inhibit glycation and consequently, the formation of AGEs. Objective: Here, we describe the mechanism of glycation on the worsening of diseases, the methods os detection, and the current findings on the use of phytochemicals (phenolic compounds, phytosterols, carotenoids, terpenes and vitamins) as natural therapeuticals to inhibit health damages via inhibition of AGEs in vitro and in vivo. Methods: This manuscript reviewed publications available in the PubMed and Science Direct databases dated from the last 20 years on the uses of phytochemicals to inhibit the AGEs in vitro and in vivo. Also, recent patents on the use of anti-glycant drugs were reviewed using the Google Advanced Patents database. Results and Discussion: Phenolic compounds have been mostly studied to inhibit AGEs. Food phytochemicals derived from agroindustry wastes, including peanut skins, and the bagasses derived from citrus and grapes are promising antiglycant agents via scavenging of free radicals, metal ions, the suppression of metabolic pathways that induces inflammation, the activation of pathways that promote antioxidant defense, the blocking of AGE connection with the receptor for advanced glycation endproducts (RAGE). Conclusion: Phytochemicals derived from agroindustry are promising anti-glycants, which can be included to replace synthetic drugs for AGE inhibition, and consequently to act as a therapeutical strategy to prevent and treat diseases caused by AGEs, including diabetes, ovarian cancer, osteoporosis, and Alzheimer’s disease.

Smart delivery of lethal poly-peptide composite for effective cancer therapy

2022 ◽  
Author(s):  
Xin Yuan ◽  
Yingzhou Qin ◽  
Qingmei Tian ◽  
Cuijuan Liu ◽  
Xiangzhou Meng ◽  
...  
Keyword(s):  
Half Life ◽  
Low Cost ◽  
Drug Loading ◽  
Nsclc Cell Line ◽  
Peptide Drug ◽  
Loading System ◽  
Penetration Ability ◽  
Cleavable Peptide

Abstract In the past decade, multifunctional peptides have attracted increasing attention in the biomedical field. Peptides possess many impressive advantages, such as high penetration ability, low cost, and etc. However, the short half-life and instability of peptides limit their application. In this study, a poly-peptide drug loading system (called HKMA composite) was designed based on the different functionalities of four peptides. The peptide compositions of HKMA composite from N-terminal to C-terminal were HCBP1, KLA, MMP-2-cleavable peptide and ABD. The targeting and lethality of HKMA to NSCLC cell line H460 sphere cells and the half-life of the system were measured in vivo. The results showed that the HKMA composite had a long half-life and specific killing effect on H460 sphere cells in vitro and in vivo. Our result proposed smart peptide drug loading system and provided a potential methodology for effective cancer treatment.

scholarly journals A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1304
Author(s):  
Robson Amaral ◽  
Maike Zimmermann ◽  
Ai-Hong Ma ◽  
Hongyong Zhang ◽  
Kamilla Swiech ◽  
...  
Keyword(s):  
Drug Resistance ◽  
In Vitro Culture ◽  
Cancer Cells ◽  
Low Cost ◽  
Maintenance Cost ◽  
Tumor Spheroids ◽  
Patient Derived Xenograft ◽  
Pdx Models

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient’s outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 μm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.

scholarly journals Requirements for Efficient Production and Transduction of Human Immunodeficiency Virus Type 1-Based Vectors

1999 ◽  
Vol 73 (3) ◽  
pp. 1828-1834 ◽  
Author(s):  
Mehdi Gasmi ◽  
Jacqueline Glynn ◽  
Ming-Jie Jin ◽  
Douglas J. Jolly ◽  
Jiing-Kuan Yee ◽  
...  
Keyword(s):  
High Titer ◽  
Regulatory Element ◽  
Target Cells ◽  
Efficient Production ◽  
Replication Competent Virus ◽  
Constitutive Transport Element ◽  
Hiv 1

ABSTRACT A number of human immunodeficiency type 1 (HIV-1)-based vectors have recently been shown to transduce nondividing cells in vivo as well as in vitro. However, if these vectors are to be considered for eventual clinical use, a major consideration is to reduce the probability of unintended generation of replication-competent virus. This can be achieved by eliminating viral genetic elements involved in the generation of replication-competent virus without impairing vector production. We have designed a system to transiently produce HIV-1-based vectors by using expression plasmids encoding Gag, Pol, and Tat of HIV-1 under the control of the cytomegalovirus immediate-early promoter. Our data show that the best vector yield is achieved in the presence of the Rev/Rev-responsive element (RRE) system. However, the constitutive transport element of Mason-Pfizer monkey virus can substitute for RRE and Rev at least to some extent, whereas the posttranscriptional regulatory element of human hepatitis B virus appeared to be inefficient. In addition, we show that high-titer virus preparations can be obtained in the presence of sodium butyrate, which activates the expression of both the packaging construct and the vector genome. Finally, our results suggest that efficient infectivity of vectors defective in the accessory proteins Vif, Vpr, Vpu, and Nef depends on the nature of the target cells.

scholarly journals Power spectral density-based nearinfrared sub-band detection for noninvasive blood glucose prediction in both in-vitro and in-vivo studies

2018 ◽  
Vol 11 (06) ◽  
pp. 1850035
Author(s):  
Ibrahim Akkaya ◽  
Erman Selim ◽  
Mert Altintas ◽  
Mehmet Engin
Keyword(s):  
Blood Glucose ◽  
Spectral Density ◽  
Power Spectral Density ◽  
Optical System ◽  
Low Cost ◽  
Linear Regression Analysis ◽  
In Vivo Studies ◽  
Power Spectral

Diabetes is a widespread and serious disease and noninvasive measurement has been in high demand. To address this problem, a power spectral density-based method was offered for determining glucose sensitive sub-bands in the nearinfrared (NIR) spectrum. The experiments were conducted using phantoms of different optical properties in-vitro conditions. The optical bands 1200–1300[Formula: see text]nm and 2100–2200[Formula: see text]nm were found feasible for measuring blood glucose. After that, a photoplethysmography (PPG)-based low cost and portable optical system was designed. It has six different NIR wavelength LEDs for illumination and an InGaAs photodiode for detection. Optical density values were calculated through the system and used as independent variables for multiple linear regression analysis. The results of blood glucose levels for 24 known healthy subjects showed that the optical system prediction was nearly 80% in the A zone and 20% in the B zone according to the Clarke Error Grid analysis. It was shown that a promising easy-use, continuous, and compact optical system had been designed.

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