scholarly journals Automatic production of double coated alginate microcapsules: Analysis of the experimental parameters by design of experiments

2007 ◽  
Vol 13 (3) ◽  
pp. 117-126 ◽  
Author(s):  
Stefania Mazzitelli ◽  
Giovanni Luca ◽  
Azzurra Tosi ◽  
Cosimo Balestra ◽  
Lorenzo Capretto ◽  
...  
Keyword(s):  
Design Of Experiments ◽  
Large Scale ◽  
Scale Production ◽  
Pumping Rate ◽  
Automatic Production ◽  
Novel Approach ◽  
Experimental Parameters ◽  
Calcium Alginate Gel ◽  
Neonatal Porcine Islets

This paper describes a design of experiments (DoE) approach, of an automatic procedure for the microencapsulation of isolated neonatal porcine islets (NPI). Monodisperse droplets were prepared by a vibrational nozzle, using an alginate/polyornithine encapsulation procedure, that has been developed and validated in our laboratory to encapsulate pancreatic islets. Different experimental parameters were considered such as: frequency of vibration, amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. The produced calcium-alginate gel microbeads displayed an excellent morphology as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology and viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

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.

Confocal Microscopy in Ecophysiological Studies of Algae: A Door to Understanding Autofluorescence in Red Algae

2021 ◽  
pp. 1-9
Author(s):  
Teresa Coronado-Parra ◽  
Mónica Roldán ◽  
Marina Aboal
Keyword(s):  
Laser Scanning ◽  
Large Scale ◽  
Nitrogen Concentration ◽  
Monochromatic Light ◽  
Environmental Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Scale Production ◽  
Light Intensities

Alga in the genus Chroothece have been reported mostly from aquatic or subaerial continental environments, where they grow in extreme conditions. The strain Chroothece mobilis MAESE 20.29 was exposed to different light intensities, red and green monochromatic light, ultraviolet (UV) radiation, high nitrogen concentrations, and high salinity to assess the effect of those environmental parameters on its growth. Confocal laser scanning microscopy (CLSM) was used as an “in vivo” noninvasive single-cell method for the study. The strain seemed to prefer fairly high light intensities and showed a significant increase in allophycocyanin (APC) and chlorophyll a [photosystem I (PSI) and photosystem II (PSII)] fluorescence with 330 and 789 μM/cm2/s intensities. Green monochromatic light promoted a significant increase in the fluorescence of APC and chlorophyll a (PSI and PSII). UV-A significantly decreased phycocyanin and increased APC, while UV-A + B showed a greater decreasing effect on c-Phycocyanin but did not significantly change concentrations of APC. The increase in nitrogen concentration in the culture medium significantly and negatively affected all pigments, and no effect was observed with an increase in salinity. Our data show that CLSM represents a very powerful tool for ecological research of microalgae in small volumes and may contribute to the knowledge of phycobiliproteins in vivo behavior and the parameters for the large-scale production of these pigments.

Liposomes

2017 ◽  
pp. 52-87
Author(s):  
Mangal Shailesh Nagarsenker ◽  
Megha Sunil Marwah
Keyword(s):  
Quality Control ◽  
Large Scale ◽  
Clinical Success ◽  
Scale Production ◽  
Large Scale Production ◽  
Diagnostic Applications ◽  
Set Up ◽  
Insight Into ◽  
Characterisation Techniques

The science of liposomes has expanded in ambit from bench to clinic through industrial production in thirty years since the naissance of the concept. This chapter makes an attempt to bring to light the impregnable contributions of great researchers in the field of liposomology that has witnessed clinical success in the recent times. The journey which began in 1965 with the observations of Bangham and further advances made en route (targeting/stealthing of liposomes) along with alternative and potential liposome forming amphiphiles has been highlighted in this chapter. The authors have also summarised the conventional and novel industrially feasible methods used to formulate liposomes in addition to characterisation techniques which have been used to set up quality control standards for large scale production. Besides, the authors have provided with an overview of primary therapeutic and diagnostic applications and a brief insight into the in vivo behaviour of liposomes.

scholarly journals Production of Phytotoxic Metabolite Using Biphasic Fermentation System from Strain C1136 of Lasiodiplodia pseudotheobromae, a Potential Bioherbicidal Agent

2017 ◽  
Vol 9 (3) ◽  
pp. 371-377
Author(s):  
Charles Oluwaseun ADETUNJI ◽  
Julius Kola OLOKE ◽  
Gandham PRASAD ◽  
Moses ABALAKA ◽  
Emenike Onyebum IROKANULO
Keyword(s):  
Large Scale ◽  
Wild Strain ◽  
Fermentation Medium ◽  
Scale Production ◽  
Conventional Farming ◽  
Large Scale Production ◽  
Phytotoxic Metabolite ◽  
Biphasic Fermentation

Formulation of effective and environmental friendly bioherbicides depends on the type of fermentation medium used for the production of phytotoxic metabolites. The effect of biomass, colony forming unit and the phytotoxic metabolite produced from the biphasic fermentation was carried out, while the phytotoxic metabolite was  tested in vivo and in-vitro on Echinochola crus-galli and dicotyledonous Chromolaena odorata. The mutant strain of Lasiodiplodia pseudotheobromae C1136 (Lp90) produced the highest amount of conidia and the largest necrotic area on the two tested weeds when compared to its wild strain in the different biphasic media combinations. The study revealed that the biphasic system containing PDB + rice produced the highest bioherbicidal activities. Therefore, the phytotoxic metabolites from strain C1136 are suggested for large scale production of bioherbicides for the management of weeds in conventional farming to improve yield and enhance food security.

scholarly journals Blakeslea trispora Photoreceptors: Identification and Functional Analysis

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Wei Luo ◽  
Chao Xue ◽  
Yuzheng Zhao ◽  
Huili Zhang ◽  
Zhiming Rao ◽  
...  
Keyword(s):  
Large Scale ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Blakeslea Trispora ◽  
Scale Production ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Flavin Binding

ABSTRACT Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora. Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa. The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli. The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora. IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides. The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

scholarly journals Cloning and Characterization of a Novel Subunit of Protein Serine/Threonine Phosphatase 4 from Mesangial Cells

2001 ◽  
Vol 12 (12) ◽  
pp. 2601-2608 ◽  
Author(s):  
Takehiko Wada ◽  
Toshio Miyata ◽  
Reiko Inagi ◽  
Masaomi Nangaku ◽  
Masako Wagatsuma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Cultured Cells ◽  
Mesangial Cells ◽  
Spindle Pole ◽  
Regulatory Subunit ◽  
Glomerular Diseases ◽  
Regulatory Subunits ◽  
Novel Approach

ABSTRACT. Mesangial cells play an important role in maintaining glomeruli structure and function and in the pathogenesis of glomerular diseases. With a novel approach using a rapid large-scale DNA sequencing strategy and computerized data processing, a new human gene, PP4Rmegwas cloned. The full-length cDNA clone of human PP4Rmegcoded for a novel 950-amino acid protein, which was similar to a subunit of protein serine/threonine phosphatase 4 (PP4). Recombinant PP4Rmegproduced in COS-7 cells bound to the catalytic subunit of PP4. PP4Rmegis therefore structurally and functionally related to the recently reported regulatory subunit of PP4, PP4R1. Amino acid sequence analysis of rat PP4Rmeghomologue revealed that the sequences were well conserved between human and rat (86.3% identity). Northern blot analyses of human tissues and cultured cells demonstrated that the regulatory subunits were expressed abundantly in human cultured mesangial cells, although their expression was relatively ubiquitous.In situhybridization studies in normal human renal tissues confirmed their expression in glomeruliin vivo. The expression was upregulated in glomeruli of anti-Thy1 glomerulonephritis rats before mesangial proliferation. These data demonstrate that PP4Rmegis a novel regulatory subunit of PP4, which is expressed ubiquitously but abundantly in mesangial cells. Its pathophysiologic role in mesangial cells and glomerulus remains unknown. As PP4 is an essential protein for nucleation, growth, and stabilization of microtubules at centrosomes/spindle pole bodies during cell division, PP4Rmegmay play a role in regulation of mitosis in mesangial cells.

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Activated Platelet P-selectin

1997 ◽  
Vol 77 (04) ◽  
pp. 755-759 ◽  
Author(s):  
Jianming Gu ◽  
Yue Liu ◽  
Lijun Xia ◽  
Haiying Wan ◽  
Peixia Li ◽  
...  
Keyword(s):  
Large Scale ◽  
Variable Region ◽  
Expression Vectors ◽  
Chimeric Antibody ◽  
Scale Production ◽  
Fab Fragment ◽  
Variable Regions ◽  
E Coli ◽  
Large Scale Production

SummaryA murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector. pHENl-SZ51 Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG γICHI and Cκ genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Igγl and κ. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then cotransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/1 of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

scholarly journals Bacteriocins, Potent Antimicrobial Peptides and the Fight against Multi Drug Resistant Species: Resistance Is Futile?

Antibiotics ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 32 ◽  
Author(s):  
Meade ◽  
Slattery ◽  
Garvey
Keyword(s):  
Large Scale ◽  
Proteolytic Enzymes ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Food Preservation ◽  
Scale Production ◽  
Major Drawback ◽  
Resistant Species ◽  
Medical Sector

Despite highly specialized international interventions and policies in place today, the rapid emergence and dissemination of resistant bacterial species continue to occur globally, threatening the longevity of antibiotics in the medical sector. In particular, problematic nosocomial infections caused by multidrug resistant Gram-negative pathogens present as a major burden to both patients and healthcare systems, with annual mortality rates incrementally rising. Bacteriocins, peptidic toxins produced by bacteria, offer promising potential as substitutes or conjugates to current therapeutic compounds. These non-toxic peptides exhibit significant potency against certain bacteria (including multidrug-resistant species), while producer strains remain insusceptible to the bactericidal peptides. The selectivity and safety profile of bacteriocins have been highlighted as superior advantages over traditional antibiotics; however, many aspects regarding their efficacy are still unknown. Although active at low concentrations, bacteriocins typically have low in vivo stability, being susceptible to degradation by proteolytic enzymes. Another major drawback lies in the feasibility of large-scale production, with these key features collectively limiting their current clinical application. Though such limitations require extensive research, the concept of expanding bacteriocins from food preservation to human health opens many fascinating doors, including novel drug delivery systems and anticancer treatment applications.

scholarly journals Coordinating the in vivo processes for minicircle production - a novel approach to large scale manufacturing

2018 ◽  
Author(s):  
Peter Mayrhofer ◽  
Hana Jug ◽  
Aleš Štrancar ◽  
Alexandre Di Paolo ◽  
Laurent Jost ◽  
...  
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Process Development ◽  
Scale Up ◽  
Hydrophobic Interaction Chromatography ◽  
Viral Gene ◽  
Novel Approach ◽  
Dna Production ◽  
Minicircle Dna

ABSTRACTSafety as well as efficiency issues in connection with bacterial backbone sequences should be carefully considered when designing new DNA vaccines or non-viral gene therapy approaches. Bacterial backbone sequences like antibiotic resistance markers or regulatory bacterial elements constitute biological safety risks and reduce the overall efficiency of the DNA agent. To overcome these problems the minicircle technology has been developed. But, despite all the obvious advantages, minicircles have so far not replaced their problem laden conventional counterpart in gene transfer applications what can be contributed to efficiency issues in large scale manufacturing. In this article we describe the combined efforts of experts in the field of minicircle development, large scale biomanufacturing and downstream process development to provide a new approach. The Recombination Based Plasmid Separation (RBPS) Technology, which has already solved crucial problems associated with minicircle-DNA production, has been developed further for this purpose. A novel parental plasmid exploiting advanced in vivo process coordination for restriction and subsequent degradation of miniplasmid-DNA will be introduced. Furthermore we describe the scale-up of minicircle-DNA production by fermentation in combination with high performance downstream processes including purification by ion exchange and hydrophobic interaction chromatography on monolithic material.

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