scholarly journals Human Paraoxonase 1 as a Pharmacologic Agent: Limitations and Perspectives

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Priyanka Bajaj ◽  
Rajan K. Tripathy ◽  
Geetika Aggarwal ◽  
Abhay H. Pande
Keyword(s):  
Large Scale ◽  
Ex Vivo ◽  
Expression System ◽  
Active Form ◽  
Hydrolytic Activity ◽  
Paraoxonase 1 ◽  
Scale Production ◽  
Pharmacologic Agent ◽  
Wide Range

Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of substrates. The enzyme shows anti-inflammatory, antioxidative, antiatherogenic, ant-diabetic, antimicrobial, and organophosphate (OP)-detoxifying properties. However, there are certain limitations regarding large-scale production and use of h-PON1 as a therapeutic candidate. These include difficulties in producing recombinant h-PON1 (rh-PON1) using microbial expression system, low hydrolytic activity of wild-type h-PON1 towards certain substrates, and low storage stability of the purified enzyme. This review summarizes the work done in our laboratory to address these limitations. Our results show that (a) optimized polynucleotide sequence encoding rh-PON1 can express the protein in an active form inE. coliand can be used to generate variant of the enzyme having enhanced hydrolytic activity, (b)in vitrorefolding of rh-PON1 enzyme can dramatically increase the yield of an active enzyme, (c) common excipients can be used to stabilize purified rh-PON1 enzyme when stored under different storage conditions, and (d) variants of rh-PON1 enzyme impart significant protection against OP-poisoning in human blood (ex vivo) and mouse (in vivo) model of OP-poisoning. The rh-PON1 variants and their process of production discussed here will help to develop h-PON1 as a therapeutic candidate.

scholarly journals Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sadaf Vahdat ◽  
Sara Pahlavan ◽  
Elena Mahmoudi ◽  
Maryam Barekat ◽  
Hassan Ansari ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Large Scale ◽  
Culture Medium ◽  
Gene Expression Pattern ◽  
Extended Period ◽  
Scale Production ◽  
Chemical Screening ◽  
Wide Range ◽  
Cell Sources

Abstract Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

scholarly journals In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2

2020 ◽  
Author(s):  
Magnus A. G. Hoffmann ◽  
Collin Kieffer ◽  
Pamela J. Bjorkman
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Viral Infections ◽  
Expression System ◽  
Scale Production ◽  
Specific Expression ◽  
In Vitro Characterization ◽  
Hiv 1

AbstractEngineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

Nanofiber-Based Expansion and Differentiation Technology for High-Volume Ex Vivo Production of Reticulocytes for P. Vivax Malaria Research

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2357-2357
Author(s):  
Hong Wang ◽  
Adam M Sorkin ◽  
Ramasamy Sakthivel
Keyword(s):  
Cord Blood ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Rapid Development ◽  
High Volume ◽  
In Vitro Models ◽  
Scale Production ◽  
Hematopoietic Stem

Abstract Abstract 2357 Infection by Plasmodium Vivax (P. Vivax) is the most common cause of Sleeping Malaria. P. Vivax and other plasmodia have grown increasingly resistant to antimalarial drugs. Introduced by mosquito bite, P. vivax sporozoites enter circulation and preferentially penetrate reticulocytes by attaching to the Fya and Fyb Duffy antigen/chemokine receptor (DARC) via PvRBP-1 and PvRBP-2 proteins located at their apical poles. Once in a reticulocyte, the parasite begins to reproduce asexually, releasing of thousands of merozoites into circulation. At this point, merozoites can also enter the liver and triggering relapses months or years later. The emergence of drug-resistant strains of p. vivax has stimulated development of new vaccines and treatments, but progress has been slowed by the dearth of reliable screening platforms. Many vaccine candidates have been developed to act upon vivax merozoites by preventing binding of PvRBP-1 and 2 to DARC, thereby arresting reproduction. However, there is a distinct lack of in vitro models to evaluate candidates that employ this mechanism. We are addressing this issue with a novel ex vivo expansion and differentiation technology for large-scale production of DARC expressing reticulocytes for in vitro P. vivax infection studies. This technology comprises an expansion system that can produce high yields of hematopoietic precursors (CD133+/CD34+ cells) from a variety of sources (marrow, peripheral blood, and cord blood), and a differentiation system to produce a relatively pure population of enucleated erythrocytes. In this study, we have refined the polyethersulfone (PES) nanofiber-based culturing system containing growth factors and cytokines in a serum-free media, to expand hematopoietic stem and progenitor cells (HSPC) ex vivo. This expansion technology allows rapid 200-fold ex vivo proliferation within 7 days of umbilical cord blood derived CD133+/CD34+ HSPCs from a DARC+ donor. Following expansion, over 50% of these cells retained HSPC phenotype (expression of CD34+). We have subsequently demonstrated that feeder layer free three-step differentiation of nanofiber-expanded cells using cytokines results in a population containing predominately enucleated reticulocyte-like cells. At 21 days of differentiation, cells had expanded 50-fold. Around 41% of cells were enucleated reticulocytes. These cells expressed glycophorin-A, a major sialoglycoprotein present on the human erythrocyte membrane. ∼28% of cells were CD36+, and ∼70% were CD71+ indicating an erythroid lineage. These results suggest that this technology can produce a population of DARC+ reticulocytes that is ∼5,000-fold greater than the starting population of HSPCs. We are partnering with leading malaria vaccine researchers to demonstrate that these reticulocytes can be parasitized by p. vivax. We believe that this will provide a unique platform to jumpstart research of malaria parasites and enable rapid development of effective vaccines. Further development of this technology may also have significant implications for large-scale ex vivo production of erythrocytes for general use. Reticulocyte-like cells and expelled nuclei during differentiation of nanofiber-expanded HSPC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Addressing Challenges to Enhance the Bioactives ofWithania somniferathrough Organ, Tissue, and Cell Culture Based Approaches

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Pritika Singh ◽  
Rupam Guleri ◽  
Amrita Angurala ◽  
Kuldeep Kaur ◽  
Kulwinder Kaur ◽  
...  
Keyword(s):  
Cell Culture ◽  
Secondary Metabolites ◽  
Culture System ◽  
Large Scale ◽  
Distinct Advantage ◽  
Scale Production ◽  
Genetic Base ◽  
Wide Range ◽  
Organ Tissue

Withania somniferais a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues.In vitropropagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential ofin vitrocell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years.In vitroculture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

Exosomes in Therapy: Engineering, Pharmacokinetics and Future Applications

2018 ◽  
Vol 20 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Claudia Arenaccio ◽  
Chiara Chiozzini ◽  
Flavia Ferrantelli ◽  
Patrizia Leone ◽  
Eleonora Olivetta ◽  
...  
Keyword(s):  
Large Scale ◽  
Ex Vivo ◽  
Biological Fluids ◽  
Therapeutic Interventions ◽  
Scale Production ◽  
Membrane Composition ◽  
Therapeutic Molecules ◽  
Key Aspects

Background:Eukaryotic cells release vesicles of different sizes under both physiological and pathological conditions. On the basis of the respective biogenesis, extracellular vesicles are classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are considered tools for innovative therapeutic interventions, especially when engineered with effector molecules. The delivery functions of exosomes are favored by a number of typical features. These include their small size (i.e., 50-200 nm), the membrane composition tightly similar to that of producer cells, lack of toxicity, stability in serum as well as other biological fluids, and accession to virtually any organ and tissue including central nervous system. However, a number of unresolved questions still affects the possible use of exosomes in therapy. Among these are the exact identification of both in vitro and ex vivo produced vesicles, their large-scale production and purification, the uploading efficiency of therapeutic macromolecules, and the characterization of their pharmacokinetics. </P><P> Objective: Here, we discuss two key aspects to be analyzed before considering exosomes as a tool of delivery for the desired therapeutic molecule, i.e., techniques of engineering, and their in vivo biodistribution/ pharmacokinetics. In addition, an innovative approach aimed at overcoming at least part of the obstacles towards a safe and efficient use of exosomes in therapy will be discussed.Conclusion:Several biologic features render exosomes an attractive tool for the delivery of therapeutic molecules. They will surely be a part of innovative therapeutic interventions as soon as few still unmet technical hindrances will be overcome.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals Xylanolytic Bacillus species for xylooligosaccharides production: a critical review

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Rozina Rashid ◽  
Muhammad Sohail
Keyword(s):  
Large Scale ◽  
Extracellular Enzymes ◽  
Membrane Separation ◽  
Well Being ◽  
Bacillus Species ◽  
Scale Production ◽  
Food Ingredients ◽  
Large Scale Production ◽  
Wide Range ◽  
Biological Impacts

AbstractThe capacity of different Bacillus species to produce large amounts of extracellular enzymes and ability to ferment various substrates at a wide range of pH and temperature has placed them among the most promising hosts for the industrial production of many improved and novel products. The global interest in prebiotics, for example, xylooligosaccharides (XOs) is ever increasing, rousing the quest for various forms with expanded productivity. This article provides an overview of xylanase producing bacilli, with more emphasis on their capacity to be used in the production of the XOs, followed by the purification strategies, characteristics and application of XOs from bacilli. The large-scale production of XOs is carried out from a number of xylan-rich lignocellulosic materials by chemical or enzymatic hydrolysis followed by purification through chromatography, vacuum evaporation, solvent extraction or membrane separation methods. Utilization of XOs in the production of functional products as food ingredients brings well-being to individuals by improving defense system and eliminating pathogens. In addition to the effects related to health, a variety of other biological impacts have also been discussed.

scholarly journals A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Tao Yue ◽  
Da Zhao ◽  
Duc T. T. Phan ◽  
Xiaolin Wang ◽  
Joshua Jonghyun Park ◽  
...  
Keyword(s):  
Large Scale ◽  
Circulatory System ◽  
Vertical Direction ◽  
Microfluidic System ◽  
Vital Role ◽  
Microvascular Networks ◽  
Biological Studies ◽  
Wide Range ◽  
High Flexibility

AbstractThe vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body. In this paper, a novel modular microfluidic system with a vertical two-layered configuration is developed to generate large-scale perfused microvascular networks in vitro. The two-layer polydimethylsiloxane (PDMS) configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly. This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities. The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction, serving as the interface between the medium channels and tissue chambers. Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels. Various large-scale microvascular networks were generated and quantified in terms of vessel length and density. Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers. This platform enables the culturing of interconnected, large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications, including basic biological studies and drug screening.

scholarly journals Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3577
Author(s):  
Julia Gerstmeier ◽  
Anna-Lena Possmayer ◽  
Süleyman Bozkurt ◽  
Marina E. Hoffmann ◽  
Ivan Dikic ◽  
...  
Keyword(s):  
Vitamin D3 ◽  
Ex Vivo ◽  
Active Form ◽  
Treatment Resistance ◽  
Serum Levels ◽  
Disease Recurrence ◽  
Primary Brain Tumor ◽  
High Rate ◽  
Therapeutic Doses

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor, with a very high rate of recurrence and a median survival of 15 months after diagnosis. Abundant evidence suggests that a certain sub-population of cancer cells harbors a stem-like phenotype and is likely responsible for disease recurrence, treatment resistance and potentially even for the infiltrative growth of GBM. GBM incidence has been negatively correlated with the serum levels of 25-hydroxy-vitamin D3, while the low pH within tumors has been shown to promote the expression of the vitamin D3-degrading enzyme 24-hydroxylase, encoded by the CYP24A1 gene. Therefore, we hypothesized that calcitriol can specifically target stem-like glioblastoma cells and induce their differentiation. Here, we show, using in vitro limiting dilution assays, quantitative real-time PCR, quantitative proteomics and ex vivo adult organotypic brain slice transplantation cultures, that therapeutic doses of calcitriol, the hormonally active form of vitamin D3, reduce stemness to varying extents in a panel of investigated GSC lines, and that it effectively hinders tumor growth of responding GSCs ex vivo. We further show that calcitriol synergizes with Temozolomide ex vivo to completely eliminate some GSC tumors. These findings indicate that calcitriol carries potential as an adjuvant therapy for a subgroup of GBM patients and should be analyzed in more detail in follow-up studies.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

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