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1
Faucher, Mélanie, Thibaud R. Geoffroy, Jacinthe Thibodeau, Sami Gaaloul, and Laurent Bazinet. "Semi-Industrial Production of a DPP-IV and ACE Inhibitory Peptide Fraction from Whey Protein Concentrate Hydrolysate by Electrodialysis with Ultrafiltration Membrane." Membranes 12, no. 4 (April 9, 2022): 409. http://dx.doi.org/10.3390/membranes12040409.
Повний текст джерелаАнотація:
The separation by electrodialysis with ultrafiltration membranes (EDUF), at a semi-industrial scale, of a new whey protein hydrolysate obtained from a whey protein concentrate was assessed. After 6 h of treatment, more than 9 g of peptides were recovered in the peptide recovery fraction, for a recovery yield of 5.46 ± 0.56% and containing 18 major components. Among these components, positively charged peptides, such as ALPMHIR + PHMIR, LIVTQTMK and TKIPAVF, were present, and their relative abundances increased by nearly 1.25 X and up to 7.55 X. The presence of these peptides may be promising, as ALPMHIR has a strong activity against angiotensin-converting enzyme (ACE), and LIVTQTMK has structural properties that could interfere with dipeptidyl peptidase-IV (DPP-IV). Many neutral peptides were also recovered alongside those. Nevertheless, the inhibitory activity against DPP-IV and ACE increased from 2 X and 4 X, respectively, in the peptide recovery fraction compared to the initial hydrolysate, due to the improved content in bioactive peptides. Thus, this new hydrolysate is well-suited for the large-scale production of a peptide fraction with high bioactivities. Furthermore, what was achieved in this work came close to what could be achieved for the industrial production of a bioactive peptide fraction from whey proteins.
2
Cienfuegos-Jiménez, Oscar, Abril Morales-Hernández, Olivia A. Robles-Rodríguez, Sergio Bustos-Montes, Kevin A. Bañuelos-Alduncin, Aurora R. Cortés-Castillo, Hugo D. Barreto-Hurtado, Luis Carrete-Salgado, and Iván A. Marino-Martínez. "High-yield production and purification of the fusion pH-responsive peptide GST-pHLIP in <i>Escherichia coli</i> BL21." AIMS Molecular Science 9, no. 4 (2022): 136–44. http://dx.doi.org/10.3934/molsci.2022008.
Повний текст джерелаАнотація:
<abstract> <p>The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An <italic>Escherichia coli</italic> (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.</p> </abstract>
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Tai, Hsueh-Ming, Ming-Feng You, Chia-Hua Lin, Tsung-Yu Tsai, Chieh-Yu Pan, and Jyh-Yih Chen. "Scale-up production of and dietary supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus." PLOS ONE 16, no. 6 (June 24, 2021): e0253661. http://dx.doi.org/10.1371/journal.pone.0253661.
Повний текст джерелаАнотація:
Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.
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Schmidt, Signe Tandrup, Dennis Christensen, and Yvonne Perrie. "Applying Microfluidics for the Production of the Cationic Liposome-Based Vaccine Adjuvant CAF09b." Pharmaceutics 12, no. 12 (December 19, 2020): 1237. http://dx.doi.org/10.3390/pharmaceutics12121237.
Повний текст джерелаАнотація:
Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20–40% v/v), [Lipid] (DDA and MMG, 6–12 mg/mL) and dimethyl sulfoxide [DMSO] (0–10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.
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Mardanova, Eugenia S., and Nikolai V. Ravin. "Plant-produced Recombinant Influenza A Vaccines Based on the M2e Peptide." Current Pharmaceutical Design 24, no. 12 (July 5, 2018): 1317–24. http://dx.doi.org/10.2174/1381612824666180309125344.
Повний текст джерелаАнотація:
Background: Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems. Methods: : Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2. <P> Results: Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies. Conclusion: Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity.
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Joglekar, Alok, Michael Troy Leonard, John Jeppson, Michael T. Bethune, and David Baltimore. "T Cell Antigen Discovery using Signaling and Antigen-presenting Bifunctional Receptors (SABRs)." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 181.9. http://dx.doi.org/10.4049/jimmunol.200.supp.181.9.
Повний текст джерелаАнотація:
Abstract Checkpoint inhibitors, cancer vaccines, and adoptive cell therapy exploit T cell mediated immune responses to cancers. Discovering the exact antigens targeted by T cell responses is important for their efficacy. Antigen discovery for ‘orphan’ T cells or TCRs has been a challenging prospect due to high number of possible pMHC specificities. Several current approaches to decipher antigen specificities require prior knowledge of antigen sequences, are unable to scale up, or require production of soluble TCRs. To overcome these drawbacks, we have developed chimeric receptors called Signaling and Antigen-presenting Bifunctional Receptors (SABRs) that allow identification of antigen-presenting cells. SABRs present display pMHC on their extracellular domain, which is recognized by an orphan TCR. Upon recognition, SABRs initiate signaling in the presenting cell using a CD3zeta signaling domain. We transduced reporter cells with SABRs presenting HLA-A2-restricted epitopes from MelanA and NY-ESO-1, and co-incubated them with target cells expressing their cognate TCRs, which resulted in signal transduction only upon correct pMHC-TCR pairing, allowing the presenting cells to express GFP. Second, we showed that SABRs displaying independently expressed peptide and MHC could function similarly. These receptors could present pulsed peptides or endogenously expressed proteins, allowing the uncoupling of peptide and MHC, while retaining their signaling capability. We are currently testing the use of SABR-based antigen libraries to identify novel antigenic specificities targeted by T cells in cancers, infectious diseases, and autoimmune diseases.
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Xu, Jun, Qinglin Dong, Ye Yu, Baolong Niu, Dongfeng Ji, Muwang Li, Yongping Huang, Xin Chen, and Anjiang Tan. "Mass spider silk production through targeted gene replacement in Bombyx mori." Proceedings of the National Academy of Sciences 115, no. 35 (August 6, 2018): 8757–62. http://dx.doi.org/10.1073/pnas.1806805115.
Повний текст джерелаАнотація:
Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.
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Nguyen, Sy Le Thanh, Dinh Thi Quyen, and Hong Diep Vu. "Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/324705.
Повний текст джерелаАнотація:
The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK fromS. pyogenessDT7 overexpressed inE. coli, purification, and biochemical characterization. A gene coding for the SK was cloned fromS. pyogenessDT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed inE. coliBL21 DE3/pESK under the control of the strong promotertacinduced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinantE. coliBL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.
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Babut, Thomas, Mona Semsarilar, Marc Rolland, and Damien Quemener. "Nano-Fibrous Networks from Co-Assembly of Amphiphilic Peptide and Polyelectrolyte." Polymers 13, no. 22 (November 18, 2021): 3983. http://dx.doi.org/10.3390/polym13223983.
Повний текст джерелаАнотація:
Organize the matter on an increasingly small scale is sought in order to increase the performance of materials. In the case of porous materials, such as filtration membranes, a compromise must be found between the selectivity provided by this nanostructuring and a permeability in particular linked to the existing pore volume. In this work, we propose an innovative waterborne approach consisting in co-assembling peptide amphiphiles (PA) which will provide nanostructuring and polyelectrolytes which will provide them with sufficient mechanical properties to sustain water pressure. C16-V3A3K3G-NH2 PA nanocylinders were synthesized and co-assembled with poly(sodium 4-styrenesulfonate) (PSSNa) into porous nano-fibrous network via electrostatic interactions. The ratio between C16-V3A3K3G-NH2 and PSSNa was studied to optimize the material structure. Since spontaneous gelation between the two precursors does not allow the material to be shaped, various production methods have been studied, in particular via tape casting and spray-coating. Whereas self-supported membranes were mechanically weak, co-assemblies supported onto commercial ultrafiltration membranes could sustain water pressure up to 3 bars while a moderate permeability was measured confirming the existence of a percolated network. The produced membrane material falls into the ultrafiltration range with a pore radius of about 7.6 nm.
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Schroeder, Sarah, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, et al. "Abstract 3555: Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3555. http://dx.doi.org/10.1158/1538-7445.am2022-3555.
Повний текст джерелаАнотація:
Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancies worldwide. 45% of patients are diagnosed at a late tumor stage associated with poor survival. For metastatic, unresectable or recurrent (m/uR) HNSCC, immune checkpoint inhibition (ICI) was recently approved as a novel therapeutic option showing significant survival benefits compared to standard chemotherapy-based treatment. However, response to ICI is still limited to a small number of patients calling for further improvement of T cell-based immunotherapies. Peptide-based approaches, which rely on the specific immune recognition of tumor-associated human leukocyte antigen (HLA) presented peptides, represent promising and low side effect treatment options. Peptide vaccination has been shown to enhance and induce long-term anti-tumoral immune responses and even clinical responses in HNSCC patients. However, current vaccines are either monovalent, based on patient-individual tumor-specific mutations or restricted to a single HLA allotype and therefore neither widely applicable nor suitable for reliable studies and large-scale production. In this study, using mass spectrometry (MS) -based immunopeptidome analysis of a large cohort of HNSCC patient (n = 30) tumor and adjacent benign samples, we established a tumor-associated off-the-shelf peptide warehouse for broadly applicable personalized therapies. The malignant dataset, comprising 91651 HLA ligands, was compared to adjacent benign and various benign tissues (www.hla-ligand-atlas.org) to identify tumor-exclusive antigens. Further antigen selection was based on allotype-specific high frequent presentation. In total, 23 frequently presented and tumor-exclusive HNSCC-associated peptides were selected for six of the most common HLA class I allotypes (A*01, A*02, A*24, B*15, B*35, B*40) covering >75% of the world population, as well as five HLA class II presented peptides binding various different HLA class II allotypes. Immunogenicity was validated by IFN-γ ELISPOT screening for spontaneous preexisting T cell responses targeting the respective peptides as well as by in vitro priming experiments of naïve T cells in HNSCC patients and healthy volunteers. Furthermore, immunopeptidome analyses identified these antigens in patient plasma samples providing first evidence for “liquid biopsy” immunopeptidome analysis without the need of primary tumor tissue. A phase I study evaluating safety, immunogenicity as well as first efficacy of this warehouse-based vaccine in combination with ICI in HNSCC patients is currently being set up, with personalized peptide selection based on individual HLA-allotype and MS analysis of patient tumor/plasma sample. In conclusion, we here designed a peptide warehouse that enables a polyvalent and widely applicable but still personalized peptide vaccination in HNSCC patients. Citation Format: Sarah Schroeder, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, Lena Muehlenbruch, Yacine Maringer, Paul-Stefan Mauz, Martin Sailer, Julia Philipp, Sven Becker, Thomas Breuer, Helmut R. Salih, Hans-Georg Rammensee, Hubert Löwenheim, Juliane S. Walz. Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3555.
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Chaturvedi, Rajiv, Tanya Soboleva, Linda Johnson, Anthony Parsons, and Susanne Rasmussen. "Systems level modelling of metabolism in fungal endophytes - implications for the symbiosis with ryegrass." NZGA: Research and Practice Series 13 (January 1, 2007): 203–6. http://dx.doi.org/10.33584/rps.13.2006.3049.
Повний текст джерелаАнотація:
We used constraint based stoichiometric modelling of metabolic fluxes in EpichloÑ' festucae (FL1), a targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae, and the symbiotic association of these endophytic fungi and their host Lolium perenne. SidF encodes an excreted ironchelating siderophore and the sidF knockouts (KO) are impaired in their ability to take up iron. After constructing the metabolic network at a genome scale, we applied constraints on enzymatic reactions that require iron as co-factor to study the variations in metabolic network capabilities of the siderophore mutant versus wildtype, in culture and in planta. We compared fluxes calculated for the production of amino acids with observed concentrations of these amino acids in planta. We report a counter-intuitive result from considering metabolism on a systems level in our models. Keywords: stoichiometric metabolic network modelling, flux balance analysis, symbiosis, Neotyphodium lolii, Lolium perenne, EpichloÑ' festucae.
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Hanh, Do Thi, Nguyen Anh Minh, Nguyen Van Cuu, Phung Thi Thanh Huong, Pham Xuan Hoi, and Nguyen Duy Phuong. "Generation of Synthetic Peptide-Specific Antibody for the Development of A Southern Rice Black-Streaked Dwarf Virus Diagnostic Test." Vietnam Journal of Agricultural Sciences 4, no. 3 (October 29, 2021): 1176–84. http://dx.doi.org/10.31817/vjas.2021.4.3.08.
Повний текст джерелаАнотація:
Southern rice black-streaked dwarf virus (SRBSDV) causes severe epidemical disease on rice with the infected area up to millions of hectares in South China and North and Central of Vietnam. So far, there are no effective, cheap, quick, and practicable methods for diagnosing SRBSDV. The conventional RT-PCR technique is the most popular method for detecting SRBSDV with high accuracy. However, it is hard to apply this method for large-scale SDBSDV diagnosis because of the requirements of expensive reagents and instruments, as well as complex procedures. Meanwhile, SRBSDV diagnostic techniques based on antigen detection have outstanding advantages due to their low cost, easy manipulation, and wide application possibility. Today, there are still no commercially available specific antibodies to SRBSDV. In a previous study, to develop the SRBSDV diagnostic technique by the ELISA technique, a SRBSDV specific antibody was generated by a recombinant P10 envelope protein (66kDa), which has a titer of 1:5,000. In this study, we continued to study the production of SRBSDV specific polyclonal antibodies from small antigen–rich peptides from the SRBSDV P10 envelope protein. The resulting purified antibody can specifically bind to the P10 protein and at the diluted concentration of 1:100,000 it can detect SRBSDV in infected rice samples via the dot-blot technique. Our research results open up new opportunities for proactive antibodies to develop a SRBSDV membrane rapid diagnostic kit.
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ZBIKOWSKA, Halina M., Nadia SOUKHAREVA, Reza BEHNAM, Henryk LUBON, David HAMMOND та Serguei SOUKHAREV. "Uromodulin promoter directs high-level expression of biologically active human α1-antitrypsin into mouse urine". Biochemical Journal 365, № 1 (1 липня 2002): 7–11. http://dx.doi.org/10.1042/bj20020643.
Повний текст джерелаАнотація:
We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human α1-antitrypsin (α1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) α1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14μg/ml in the F0-generation mice, and reached up to 65μg/ml in the F1 generation. The transgenically produced rh α1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant α1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.
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Tipper, Donald J., and Eva Szomolanyi-Tsuda. "Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection." Journal of Immunology Research 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/2743292.
Повний текст джерелаАнотація:
Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposesβ-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use.Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.
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Bukhari, Syed Nisar Hussain, Amit Jain, Ehtishamul Haq, Moaiad Ahmad Khder, Rahul Neware, Jyoti Bhola, and Moslem Lari Najafi. "Machine Learning-Based Ensemble Model for Zika Virus T-Cell Epitope Prediction." Journal of Healthcare Engineering 2021 (October 1, 2021): 1–10. http://dx.doi.org/10.1155/2021/9591670.
Повний текст джерелаАнотація:
Zika virus (ZIKV), the causative agent of Zika fever in humans, is an RNA virus that belongs to the genus Flavivirus. Currently, there is no approved vaccine for clinical use to combat the ZIKV infection and contain the epidemic. Epitope-based peptide vaccines have a large untapped potential for boosting vaccination safety, cross-reactivity, and immunogenicity. Though many attempts have been made to develop vaccines for ZIKV, none of these have proved to be successful. Epitope-based peptide vaccines can act as powerful alternatives to conventional vaccines due to their low production cost, less reactogenic, and allergenic responses. For designing an effective and viable epitope-based peptide vaccine against this deadly virus, it is essential to select the antigenic T-cell epitopes since epitope-based vaccines are considered safe. The in silico machine-learning-based approach for ZIKV T-cell epitope prediction would save a lot of physical experimental time and efforts for speedy vaccine development compared to in vivo approaches. We hereby have trained a machine-learning-based computational model to predict novel ZIKV T-cell epitopes by employing physicochemical properties of amino acids. The proposed ensemble model based on a voting mechanism works by blending the predictions for each class (epitope or nonepitope) from each base classifier. Predictions obtained for each class by the individual classifier are summed up, and the class with the majority vote is predicted upon. An odd number of classifiers have been used to avoid the occurrence of ties in the voting. Experimentally determined ZIKV peptide sequences data set was collected from Immune Epitope Database and Analysis Resource (IEDB) repository. The data set consists of 3,519 sequences, of which 1,762 are epitopes and 1,757 are nonepitopes. The length of sequences ranges from 6 to 30 meter. For each sequence, we extracted 13 physicochemical features. The proposed ensemble model achieved sensitivity, specificity, Gini coefficient, AUC, precision, F-score, and accuracy of 0.976, 0.959, 0.993, 0.994, 0.989, 0.985, and 97.13%, respectively. To check the consistency of the model, we carried out five-fold cross-validation and an average accuracy of 96.072% is reported. Finally, a comparative analysis of the proposed model with existing methods has been carried out using a separate validation data set, suggesting the proposed ensemble model as a better model. The proposed ensemble model will help predict novel ZIKV vaccine candidates to save lives globally and prevent future epidemic-scale outbreaks.
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Walther, Frans J., Monik Gupta, Larry M. Gordon, and Alan J. Waring. "A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities." Gates Open Research 2 (July 10, 2018): 13. http://dx.doi.org/10.12688/gatesopenres.12799.2.
Повний текст джерелаАнотація:
Background: Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied ‘B-YL’, a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Methods: Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. Results: CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent in vitro surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. Conclusions: ‘Sulfur-free’ B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL’s possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS.
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Comoli, Patrizia, Marco W. Schilham, Sabrina Basso, Tamara van Vreeswijk, Rita Maccario, Maarten J. D. van Tol, Franco Locatelli, and Louise A. Veltrop-Duits. "Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides." Blood 110, no. 11 (November 16, 2007): 3273. http://dx.doi.org/10.1182/blood.v110.11.3273.3273.
Повний текст джерелаАнотація:
Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.
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Blyth, Emily, Leighton E. Clancy, Upinder Sandher, and David J. Gottlieb. "BK Virus Specific Cytotoxic T Cells Expanded for Clinical Use Exhibit Multiple Cytokine Functions and Individual Variation in Antigen Specificity." Blood 114, no. 22 (November 20, 2009): 2437. http://dx.doi.org/10.1182/blood.v114.22.2437.2437.
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Abstract Abstract 2437 Poster Board II-414 Introduction: BK virus (BKV) is a polyomavirus that is ubiquitous in humans, infecting over 85% of normal individuals. After initial infection it persists in a latent state in the urothelium from whence it can reactivate causing disease in immunocompromised patients. BKV is a cause of haemorrhagic cystitis after allogeneic SCT and is emerging as one of the major causes of graft loss after renal transplant. Current treatment is limited to reduction of immunosuppression as possible. Aim: To develop a method for production of a T cell product with BKV specificity from normal donors for use in adoptive immunotherapy post hemopoietic stem cell transplantation. Methods: Peripheral blood mononuclear cells (PBMC) or monocyte derived dendritic cells (mo-DC) were pulsed with mixes of overlapping peptides covering the 5 BKV proteins (VP1, VP2, VP2 isoform 3, large T antigen (LTA) and small T antigen (sTA)). Mo-DC were produced by isolating monocytes by adherence to plastic and culturing for 7 days in GM-CSF and IL-4 containing media. On day 6 mo-DC were matured by the addition of TNF. T cells were stimulated on day 1 and 7 with peptide pulsed PBMC or mo-DC and cultured for 21 days with increasing doses of IL-2 from day 7. The cellular product was then analysed for phenotype, BKV specificity and functionality by examining cytokine production and cytotoxicity. Results: Cellular proliferation was seen in all of 10 normal donors with a mean increase of 6.4 fold in total cell number. All cellular products were >84% CD3 positive (Mean 96%, SEM 1.3) with CD4 and CD8 ratio varying significantly between individual donors (CD4 range 9.7 to 97.5%, mean 70.79; CD8 range 0.8 to 77.0%, mean 23.3). Cells were of memory phenotype, being CD28+ (mean 86.1%, SEM 6.5), CD45RO+ (mean 84.1%, SEM 5.9) and a variable proportion were of central memory phenotype (CD62L+ mean 21.6%, range 6.9 to 55.0). Cytokine responses to stimulation with BKV peptides could be elicited in 5 of 6 evaluable donors. Multiple cytokines were produced by the responding cells: IFN-γ (mean 29.9% of CD3 cells, range 4.5 to 78.8), TNF (mean 19.9%, range 2.7 to 63) and IL-2 (mean 12.8%, range 1.2 to 37.8). Cytokine responses were seen in both CD4 and CD8 cells and showed significant individual variation. VP1 and LTA specific cells dominated most cultures while a smaller percentage of the cells were specific for VP2, VP2 isoform 3 and sTA. CD8 specificity was mainly confined to a single protein whereas CD4 responses tended to be of lower magnitude but broader specificity. Cultures exhibited cytotoxic activity with the lysis of BKV antigen coated target cells in a pattern that correlated with the presence of CD8 positive cytokine producing cells (up to 78.9% specific lysis at effector to target ratio of 20:1). Discussion: The clinical utility of this product remains to be determined. Potential uses include prophylaxis and therapy of reactivation of BK virus after hemopoietic stem cell or renal transplantation. This method for large-scale expansion of BKV specific CTL could be utilised for analysis of BKV targeted immune responses and epitope identification. Disclosures: No relevant conflicts of interest to declare.
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Farias, Ticiane Carvalho, Thaiza Serrano Pinheiro de Souza, Ana Elizabeth Cavalcante Fai, and Maria Gabriela Bello Koblitz. "Critical Review for the Production of Antidiabetic Peptides by a Bibliometric Approach." Nutrients 14, no. 20 (October 14, 2022): 4275. http://dx.doi.org/10.3390/nu14204275.
Повний текст джерелаАнотація:
The current bibliometric review evaluated recent papers that researched dietary protein sources to generate antidiabetic bioactive peptides/hydrolysates for the management of diabetes. Scopus and PubMed databases were searched to extract bibliometric data and, after a systematic four-step process was performed to select the articles, 75 papers were included in this review. The countries of origin of the authors who published the most were China (67%); Ireland (59%); and Spain (37%). The journals that published most articles on the subject were Food Chemistry (n = 12); Food & Function (n = 8); and Food Research International (n = 6). The most used keywords were ‘bioactive peptides’ (occurrence 28) and ‘antidiabetic’ (occurrence 10). The most used enzymes were Alcalase® (17%), Trypsin (17%), Pepsin, and Flavourzyme® (15% each). It was found that different sources of protein have been used to generate dipeptidyl peptidase IV (DPP-IV), α-amylase, and α-glucosidase inhibitory peptides. In addition to antidiabetic properties, some articles (n = 30) carried out studies on multifunctional bioactive peptides, and the most cited were reported to have antioxidant and antihypertensive activities (n = 19 and 17, respectively). The present review intended to offer bibliometric data on the most recent research on the production of antidiabetic peptides from dietary proteins to those interested in their obtention to act as hypoglycemic functional ingredients. The studies available in this period, compiled, are not yet enough to point out the best strategies for the production of antidiabetic peptides from food proteins and a more systematic effort in this direction is necessary to allow a future scale-up for the production of these possible functional ingredients.
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Clancy, Leighton Edward, Emily Blyth, Barbara Withers, Jane Burgess, Renee Simms, Chun Kei Kris Ma, Kenneth P. Micklethwaite, and David Gottlieb. "Therapeutic Infusion of Partially HLA-Matched Third-Party Virus-Specific T Cells in HSCT Patients with Refractory Viral Infection." Blood 124, no. 21 (December 6, 2014): 3835. http://dx.doi.org/10.1182/blood.v124.21.3835.3835.
Повний текст джерелаАнотація:
Abstract Introduction Adoptive transfer of donor derived virus specific T cells (VST) can be effective therapy for infections in allogeneic HSCT recipients. However, this is not a practical strategy to treat acute infections due to the time required to prepare products and potential unavailability of transplant donors. To overcome this, treatment with cryopreserved partially HLA-matched T cells from third-party donors is being investigated. A recent report described disease resolution using cells matched at only one or two HLA alleles (Leen et al., (2013) Blood 121(26):5113-23). This less stringent requirement for matching would allow a small bank of cells to provide most patients with a therapeutic product. We describe the establishment of a virus specific T cell bank in Australia with centralized manufacturing by the Westmead Hospital BMT laboratory. The bank has been used to treat patients in multiple states in a Phase I clinical trial to treat patients who have failed antiviral pharmacotherapy. Aim To assess the safety and feasibility of treatment with partially HLA-matched VSTs derived from third-party donors for refractory cytomegalovirus (CMV), Epstein-Barr Virus (EBV), or adenoviral (AdV) infection in allogeneic HSCT patients. Methods We generated a bank of cryopreserved VSTs from peripheral blood or G-CSF mobilized stem cell product of healthy donors. Products were generated by co-culturing PBMC with dendritic cells loaded with overlapping peptides covering CMV pp65, AdV hexon or EBV BZLF1, LMP2A and EBNA1 proteins. Cultures were re-stimulated once with peptide loaded DC and cultured for 14 days with IL-2. Products were assessed for phenotype, sterility and specificity by MHC multimer staining where applicable or production of interferon-gamma in response to peptides by flow cytometry. Patients with persistent viral reactivation/infection after 2 weeks of standard therapy were eligible to receive up to 4 fortnightly infusions of 2x107 cells/m2partially HLA matched (minimum 1/6) CMV, EBV, or AdV specific T cells, and were followed for up to 12 months. Results T cell products were expanded from 25 donors to create a bank of 177 bags of VSTs (75 CMV, 47 AdV and 55 EBV). CMV specific products were predominantly T cells (mean 95.8±3%) with a higher proportion of CD8+ compared to CD4+ T cells (mean 66.6±23.9% versus 20.1±6.2%). Specificity was mapped by MHC multimer staining to epitopes restricted to common HLA types including HLA-A*0101, HLA-A*0201, HLA-A*2402, HLA-B*0702 and HLA-B*3501. AdV specific T cells had a higher proportion of CD4+ T cells (mean 64.6±23.8% versus 34.2±20.1% CD8 T cells). Specificity was mapped to CD8 epitopes restricted to HLA-A*0101 and HLA-A*2402 as well as 10 CD4 T cell epitopes restricted to three HLA-DRB1 alleles (DRB1*0301, DRB1*0701, DRB1*1501). EBV specific products contained a mix of CD8+ and CD4+T cells (mean 38.9%±18% AND 42.5±23.1% respectively). The antigen specificity of EBV products showed high variability between donors. Dominant responses to known MHC class I restricted epitopes were infrequent though responses were mapped to HLA-A*0201 and HLA-A*2402 restricted LMP2A epitopes, a HLA-B*0801 restricted BZLF1 epitope and a HLA-B*0702 restricted EBNA1 epitope. Based on HLA frequency analysis in the Australian recipient population we estimate 94%, 89% and 74% of patients would have access to a CMV, AdV and EBV specific product respectively with the current bank. To date nine patients have received VSTs, with median follow-up of 5.5 months (0-12 months). All patients had treatment resistant CMV after a median of 26 days (19-116 days) prior therapy. Six patients received a single infusion and 3 patients received 2, 3 and 4 infusions respectively. HLA matching ranged from 2-4/6 HLA match. There were no instances of 24hr infusion related toxicity. Follow-up data is available for 7 patients. One patient with chronic hepatitis C developed abnormal liver function tests 3 months post-infusion. One patient died from presumed progressive CMV disease 6 months post-enrolment. Five patients achieved a best response of CMV PCR negativity (2 with complete resolution of CMV-colitis). One patient has shown >50% reduction in CMV copy number over 3 weeks. Conclusion The infusion of third party CMV specific T cells is a promising therapy that offers the advantage of rapid availability, centralized manufacturing and relatively low cost per dose when produced on a large scale. Disclosures No relevant conflicts of interest to declare.
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Khairnar, Sakshi V., Pritha Pagare, Aditya Thakre, Aswathy Rajeevan Nambiar, Vijayabhaskarreddy Junnuthula, Manju Cheripelil Abraham, Praveen Kolimi, Dinesh Nyavanandi, and Sathish Dyawanapelly. "Review on the Scale-Up Methods for the Preparation of Solid Lipid Nanoparticles." Pharmaceutics 14, no. 9 (September 6, 2022): 1886. http://dx.doi.org/10.3390/pharmaceutics14091886.
Повний текст джерелаАнотація:
Solid lipid nanoparticles (SLNs) are an alternate carrier system to liposomes, polymeric nanoparticles, and inorganic carriers. SLNs have attracted increasing attention in recent years for delivering drugs, nucleic acids, proteins, peptides, nutraceuticals, and cosmetics. These nanocarriers have attracted industrial attention due to their ease of preparation, physicochemical stability, and scalability. These characteristics make SLNs attractive for manufacture on a large scale. Currently, several products with SLNs are in clinical trials, and there is a high possibility that SLN carriers will quickly increase their presence in the market. A large-scale manufacturing unit is required for commercial applications to prepare enough formulations for clinical studies. Furthermore, continuous processing is becoming more popular in the pharmaceutical sector to reduce product batch-to-batch differences. This review paper discusses some conventional methods and the rationale for large-scale production. It further covers recent progress in scale-up methods for the synthesis of SLNs, including high-pressure homogenization (HPH), hot melt extrusion coupled with HPH, microchannels, nanoprecipitation using static mixers, and microemulsion-based methods. These scale-up technologies enable the possibility of commercialization of SLNs. Furthermore, ongoing studies indicate that these technologies will eventually reach the pharmaceutical market.
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Echelard, Yann, Daniel P. Pollock, Catherine De Coupade, Aurélie Groseil Olivier, Frédérique Brune, LiHow Chen, Nicholas C. Masiello, et al. "TG20, a Transgenically-Derived Anti-CD20 Monoclonal Antibody, Exhibits Enhanced Cytotoxicity Against Cells with Low Levels of CD20." Blood 118, no. 21 (November 18, 2011): 2730. http://dx.doi.org/10.1182/blood.v118.21.2730.2730.
Повний текст джерелаАнотація:
Abstract Abstract 2730 CD20 is a cell-surface glycoprotein that is highly expressed on most B-cells, tightly restricted to the B-cell lineage, and not expressed on either precursor lymphoid cells or the majority of plasma cells. These characteristics make CD20 an appealing target for mAb therapy of B-cell malignancies and B-cell dependent autoimmune conditions, because antibody production is maintained during therapy and B-cell regeneration post-CD20 mAb treatment is facilitated. Among several marketed anti-CD20 mAbs, rituximab is the prototype; a chimeric human–mouse type-1 antibody that has proven efficacy in a wide variety of mature B-cell malignancies, including CD20+ B-cell lymphoma, and has also been approved for the treatment of refractory rheumatoid arthritis. However, patients do not always respond to this therapy, and it has been reported that close to 60% of follicular lymphoma patients previously treated with chemotherapy, while initially responsive, become resistant upon repeat treatment with rituximab monotherapy. Furthermore, the high cost of treatment with rituximab and other anti-CD20 mAbs severely curtails their availability to patients in emerging economies, as well as their use post treatment for maintenance therapy. Transgenic production offers an easily scalable system for the cost-effective manufacturing of large amounts of complex therapeutic proteins. The regulatory approvals of ATryn® (recombinant antithrombin), first by the EMA (August 2006) and by the FDA (February 2009) have provided a strong regulatory validation of this production platform. Produced in the milk of transgenic goats, TG20, a novel chimeric IgG1 strongly binding a specific discontinuous epitope on CD20 (KD = 1–2 × 10−8 M), is being developed as a second-generation anti-CD20 mAb with enhanced cytotoxic activity. Using regulatory sequences from the caprine beta-casein gene to target expression of the TG20 heavy- and light-chains to the lactating mammary gland, 7 independent lines of transgenic goats were generated by somatic cell nuclear transfer. Induced lactation studies indicated that these lines expressed TG20 in their milk at levels varying from 2 g/liter up to ∼ 10 g/liter. Following genetic characterization (Southern blotting, FISH analysis) and expression analysis (protein and peptide mapping, glycosylation profiling, amino-acid sequence analysis), one line was selected for expansion and further development. In-vitro studies comparing TG20 to rituximab showed a significantly increased ability to activate effector cells expressing the FcgRIIIa receptor (CD16a). Not surprisingly, this translates to a significantly increased ability to activate NK cells and to induce target lysis. This increased ADCC has been demonstrated both with CD20+ Raji cells and with primary B-cells isolated from patients with B-CLL (TG20 EC50 = 3.6 ng/ml vs. rituximab EC50 = 59.4 ng/ml). The ability of TG20 to activate the complement system was assessed using human serum as complement and WIL2-S cells as target exhibiting a > 50% increase in CDC activity vs. rituximab (TG20 = 170% CDC activity vs. rituximab = 100%). Finally, studies conducted in Cynomolgus monkeys were performed to assess pharmacokinetics and in-vivo depletion activities of TG20. In conclusion, TG20 is a highly active anti-CD20 mAb showing interesting characteristics in terms of cytotoxicity that makes it a promising agent for indications in which rituximab is poorly active. Furthermore, the use of the cost-efficient transgenic production platform for the large-scale production of this antibody drug candidate may allow expanded access of anti-CD20 therapy to those who currently cannot afford these expensive treatments, especially in emerging market countries. Disclosures: Echelard: GTC Biotherapeutics, Inc.: Employment. Pollock:GTC Biotherapeutics, Inc.: Employment. De Coupade:LFB Biotechnologies: Employment. Groseil Olivier:LFB Biotechnologies: Employment. Brune:LFB Biotechnologies: Employment. Chen:GTC Biotherapeutics, Inc.: Employment. Masiello:GTC Biotherapeutics, Inc.: Employment. Williams:GTC Biotherapeutics, Inc.: Employment. Gavin:GTC Biotherapeutics, Inc.: Employment. Chtourou:LFB Biotechnologies: Employment. Meade:GTC Biotherapeutics, Inc.: Employment.
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Kuter, Davi d. J., Ghulam Mufti, Barbara Bain, Robert Hasserjian, and Mark Rutstein. "Evaluation of Bone Marrow Reticulin Formation in Romiplostim-Treated Adult Patients with Chronic Immune Thrombocytopenic Purpura (ITP)." Blood 112, no. 11 (November 16, 2008): 3416. http://dx.doi.org/10.1182/blood.v112.11.3416.3416.
Повний текст джерелаАнотація:
Abstract Introduction: Romiplostim is an investigational Fc-peptide fusion protein (peptibody) that stimulates platelet production by a mechanism similar to endogenous thrombopoietin and is being investigated for its ability to treat patients with chronic ITP. Some degree of reticulin deposition is often a normal finding in bone marrow, and increased reticulin has been detected in patients treated with thrombopoietin mimetics (Kuter et al, Br J Haem 2007). We analyzed bone marrow biopsy samples from ITP patients at baseline and after romiplostim treatment for the presence and degree of reticulin. Methods: Baseline and post-treatment samples were analyzed from two sets of bone marrow data: (1) a prospective study in which both baseline (pre-treatment) and follow-up (post-treatment) samples were taken, and (2) a retrospective study of spontaneously reported observations of reticulin occurring across all romiplostim clinical trials. Assessments were made from aspirate smears, core biopsies, reticulin stains, trichrome stains, and written reports. Only patients with evaluable baseline and post-treatment samples are included in this report. Reticulin was graded according to the following scale: 0 (absent), 1 (fine fibers), 2 (diffuse fine fiber network), 3 (diffuse fiber network with scattered coarse fibers), and 4 (areas of collagen). Grade 0–2 reticulin can be found in bone marrow from healthy individuals, and reticulin grades 1 to 2 have been described in the bone marrow of 66% of ITP patients (Mufti et al, ASH 2006). Results: Six of 10 prospective study patients had both evaluable baseline and follow-up samples. Reticulin grades in all 6 samples at baseline were 0–1. Only 1 patient demonstrated an increase in reticulin (from 0–1 to 1–2 after 3 months of romiplostim). Higher degrees of reticulin deposition (grades >2) were not observed, and trichrome staining demonstrated absence of collagen in all 6 cases. Baseline and follow-up bone marrow samples were available in 5 of 9 retrospective study patients, including one patient from the prospective study in whom reticulin was also spontaneously reported following romiplostim administration. In these 5 patients, the baseline reticulin grade was 0 to 1 in all cases and increased after treatment in all but one case. Reticulin typically decreased soon after discontinuation of romiplostim. Two patients were exposed to drug doses exceeding those used in current clinical studies (≥ 10 μg/kg). One patient showed minimal collagen deposition (reticulin grade 4) that was absent in a further follow-up sample after treatment discontinuation. Conclusion: Increased reticulin was observed in the bone marrow of some romiplostim-treated patients and typically decreased soon after drug withdrawal. There was no evidence that romiplostim exposure led to development of chronic idiopathic myelofibrosis or other clonal disorders in this small sample of patients. Table 1. Reticulin scores in bone marrow samples from ITP patients before and following romiplostim therapy Reticulin Assessment (Weeks after initiating romiplostim) Follow-up during treatment Follow-up after treatment discontinuation Age, Years Prior Splenectomy, Y/N Max Dose, μg/kg Baseline (prior to romiplostim treatment) 1 2 3 1 2 A Reticulin in this patient was also spontaneously reported and included in the retrospective analysis B A focal area of possible collagen deposition was seen on trichrome stain. Prospective study cases 70 Y 7 0–1 1–2 (wk 15) - - - - 43 Y 2 0–1 0–1 (wk 37) - - - - 42 Y 4 0–1 1 (wk 13) - - - - 55 Y 3 0–1 0–1 (wk 34) - - - - 83 N 2 0–1 0–1 (wk 13) - - - - 53 Y 7 0–1A 0–1A (wk 35) - - - - Retrospective study cases of spontaneously reported reticulin 40 Y 9 0–1, focal 2 3 (wk 5) - - 1–2 (wk 17) - 31 Y 18 0 2–3 (wk 26) - - 1–2 (wk 34) 1 (wk 46) 58 Y 15 0–1 1–3B (wk 31) 2–3 (wk 51) 1–2 (wk 67) - - 37 Y 9 1 4 (wk 26) - - 1–2 (wk 38) -
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Yang, Xianwen, Ping Wang, Xujie Zhao, Huahua Zhu, Sai-Juan Chen, Ji Zhang, and Kankan Wang. "PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia." Blood 116, no. 21 (November 19, 2010): 3866. http://dx.doi.org/10.1182/blood.v116.21.3866.3866.
Повний текст джерелаАнотація:
Abstract Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.
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Jahnke, Justin P., Deborah A. Sarkes, Jessica L. Liba, James J. Sumner, and Dimitra N. Stratis-Cullum. "Improved Microbial Fuel Cell Performance by Engineering E. coli for Enhanced Affinity to Gold." Energies 14, no. 17 (August 30, 2021): 5389. http://dx.doi.org/10.3390/en14175389.
Повний текст джерелаАнотація:
Microorganism affinity for surfaces can be controlled by introducing material binding motifs into proteins such as fimbrial tip and outer membrane proteins. Here, controlled surface affinity is used to manipulate and enhance electrical power production in a typical bioelectrochemical system, a microbial fuel cell (MFC). Specifically, gold-binding motifs of various affinity were introduced into two scaffolds in Escherichia coli: eCPX, a modified version of outer membrane protein X (OmpX), and FimH, the tip protein of the fimbriae. The behavior of these strains on gold electrodes was examined in small-scale (240 µL) MFCs and 40 mL U-tube MFCs. A clear correlation between the affinity of a strain for a gold surface and the peak voltage produced during MFC operation is shown in the small-scale MFCs; strains displaying peptides with high affinity for gold generate potentials greater than 80 mV while strains displaying peptides with minimal affinity to gold produce potentials around 30 mV. In the larger MFCs, E. coli strains with high affinity to gold exhibit power densities up to 0.27 mW/m2, approximately a 10-fold increase over unengineered strains lacking displayed peptides. Moreover, in the case of the modified FimH strains, this increased power production is sustained for five days.
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Operti, Maria Camilla, Alexander Bernhardt, Jeanette Pots, Vladimir Sincari, Eliezer Jager, Silko Grimm, Andrea Engel, et al. "Translating the Manufacture of Immunotherapeutic PLGA Nanoparticles from Lab to Industrial Scale: Process Transfer and In Vitro Testing." Pharmaceutics 14, no. 8 (August 13, 2022): 1690. http://dx.doi.org/10.3390/pharmaceutics14081690.
Повний текст джерелаАнотація:
Poly(lactic-co-glycolic acid) (PLGA) nanoparticle-based drug delivery systems are known to offer a plethora of potential therapeutic benefits. However, challenges related to large-scale manufacturing, such as the difficulty of reproducing complex formulations and high manufacturing costs, hinder their clinical and commercial development. In this context, a reliable manufacturing technique suitable for the scale-up production of nanoformulations without altering efficacy and safety profiles is highly needed. In this paper, we develop an inline sonication process and adapt it to the industrial scale production of immunomodulating PLGA nanovaccines developed using a batch sonication method at the laboratory scale. The investigated formulations contain three distinct synthetic peptides derived from the carcinogenic antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) together with an invariant natural killer T-cell (iNKT) activator, threitolceramide-6 (IMM60). Process parameters were optimized to obtain polymeric nanovaccine formulations with a mean diameter of 150 ± 50 nm and a polydispersity index <0.2. Formulation characteristics, including encapsulation efficiencies, release profiles and in vitro functional and toxicological profiles, are assessed and statistically compared for each formulation. Overall, scale-up formulations obtained by inline sonication method could replicate the colloidal and functional properties of the nanovaccines developed using batch sonication at the laboratory scale. Both types of formulations induced specific T-cell and iNKT cell responses in vitro without any toxicity, highlighting the suitability of the inline sonication method for the continuous scale-up of nanomedicine formulations in terms of efficacy and safety.
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Mertz, Michael, and Kathrin Castiglione. "Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process." International Journal of Molecular Sciences 22, no. 13 (July 1, 2021): 7134. http://dx.doi.org/10.3390/ijms22137134.
Повний текст джерелаАнотація:
Hollow vesicles made from a single or double layer of block-copolymer molecules, called polymersomes, represent an important technological platform for new developments in nano-medicine and nano-biotechnology. A central aspect in creating functional polymersomes is their combination with proteins, especially through encapsulation in the inner cavity of the vesicles. When producing polymersomes by techniques such as film rehydration, significant proportions of the proteins used are trapped in the vesicle lumen, resulting in high encapsulation efficiencies. However, because of the difficulty of scaling up, such methods are limited to laboratory experiments and are not suitable for industrial scale production. Recently, we developed a scalable polymersome production process in stirred-tank reactors, but the statistical encapsulation of proteins resulted in fairly low encapsulation efficiencies of around 0.5%. To increase encapsulation in this process, proteins were genetically fused with hydrophobic membrane anchoring peptides. This resulted in encapsulation efficiencies of up to 25.68%. Since proteins are deposited on the outside and inside of the polymer membrane in this process, two methods for the targeted removal of protein domains by proteolysis with tobacco etch virus protease and intein splicing were evaluated. This study demonstrates the proof-of-principle for production of protein-functionalized polymersomes in a scalable process.
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Wang, Shounan, Peng Zhang, Yibin Xue, Qiaojuan Yan, Xue Li, and Zhengqiang Jiang. "Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides." Foods 10, no. 12 (November 30, 2021): 2949. http://dx.doi.org/10.3390/foods10122949.
Повний текст джерелаАнотація:
Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.
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Papadopoulou, Anastasia, Panayotis Kaloyannidis, Maria Alvanou, Joanne Kalogeropoulou, Achilles Anagnostopoulos, and Evangelia Yannaki. "An Optimized, Large Scale Generation and Validation of Aspergillus-Specific T Lymphocytes for the Management of Invasive Aspergillosis in Immunocompromised Patients." Blood 126, no. 23 (December 3, 2015): 4293. http://dx.doi.org/10.1182/blood.v126.23.4293.4293.
Повний текст джерелаАнотація:
Abstract Invansive aspergillosis (IA) represents a leading cause of morbidity and mortality in patients with hematological malignancies and hematopoietic stem cell (HSC)/solid organ transplant recipients. The limitations of the contemporary antifungal treatment include the lack of efficacy in several cases, the drug-associated toxicity, the emergence of uncommon or drug-resistant molds and the added financial burden in transplant care. Adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) constitutes an alternative and promising therapeutic approach against IA, however, the complex and costly production limits its broader application. Consequently, and in contrast to the remarkable clinical progress with virus-specific T cells, clinical development of fungus-specific T cell therapy is still in its infancy; only one clinical trial of adoptive immunotherapy for IA has been conducted up to date, showing that recipients of Asp-ST clones after haploidentical HSC transplantation cleared IA more often than conventionally treated patients. We developed and validated a novel, rapid, simplified and minimally laborious process of generating and scaling up functionally active Asp-STs, from a single blood draw, in only 10 days. A total of 40x106 peripheral blood mononuclear cells (PBMCs) derived from 30-40ml of peripheral blood of normal donors were exposed to either an Aspergillus fumigatus lysate (n=10) or a pool of Aspergillus fumigatus overlapping peptides of the glycosidase Crf1, the 1,3-beta-glucanosyltransferase gel1 and the serine hydroxymethyltransferase SHMT, all of which are considered potential immunogenic targets (n=5). To determine the most potent stimulus for the generation of Asp-STs, dual, lysate-derived and peptide-derived, Asp-ST cell lines were produced from four donor samples and compared in terms of antigen specificity. Pulsed PBMCs were cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Cell lines produced after stimulation with either Aspergillus lysate or peptide pools had a similar fold-expansion reaching a mean absolute number of 230±81x106 cells (range 131-363x106) and 223±19x106 cells (range 197-244x106), respectively. The cell lines derived from either condition were polyclonal, comprised predominantly of CD4+ cells (75±10% and 74±10%, respectively) and CD8+ cells as well (17±9% and 15±2%, respectively), and expressed central (CD45RA-/CD62L+: 44±20% and 60±5%, respectively) and effector memory markers (CD45RA-/CD62L-: 36±20% and 21±2%, respectively). To address whether these highly expanded T cells are functional and specific against the targeted mold, each cell line was pulsed with its initial stimuli, either Asp lysate or peptide pool, and the secretion of interferon-gamma (IFN-γ) was measured by Elispot. For peptide-derived Asp-STs, specificity was assessed both, collectively for all and individually for each peptide. All lines (5/5) pulsed with peptide pools but only three of 10 cell clones pulsed with Asp lysate presented activity against Asp [lysate: 72±39 Spot forming cells (SFC)/5x105 cells; all 3 peptides: 297±81 SFC/5x105 cells]. Interestingly, not only all donor cell lines derived from pooled peptides elicited strong IFN-γ response, but 4/5 were inducible to stimulation by each peptide separately and 1/5 by Crf1, at comparable levels (Crf1: 109±59 SFC/5x105 cells, Gel1: 113±58 SFC/5x105 cells, SHMT: 121±34 SFC/5x105 cells), indicating that T cells specific for these antigens are commonly present in healthy subjects. To directly compare the stimulatory capacity of Asp lysate versus peptide pools, 4 donor samples were tested for their ability to produce specific clones against both conditions. Interestingly, all 4 donor clones showed response against peptides but only 1 of 4 against the lysate, suggesting that the Asp proteins Crf1, Gel1 and SHMT induce stronger Th1 responses than Asp lysate. In conclusion, we established and validated an optimized, rapid and simple process - which can be easily GMP-adapted- of scaling up Asp-STs, at clinically relevant numbers. Since the effective management of IA in immunocompromised patients still remains a desirable target, our proposal provides a promising therapeutic approach for the management of IA. Disclosures No relevant conflicts of interest to declare.
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Ruiz, Carlos A., Susana G. Rossi, and Richard L. Rotundo. "Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits." Journal of Biological Chemistry 290, no. 34 (July 2, 2015): 20774–81. http://dx.doi.org/10.1074/jbc.m115.653741.
Повний текст джерелаАнотація:
The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.
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Pereira, Ana Margarida, André da Costa, Simoni Campos Dias, Margarida Casal, and Raul Machado. "Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-like Fusion Tag." Pharmaceuticals 14, no. 10 (September 23, 2021): 956. http://dx.doi.org/10.3390/ph14100956.
Повний текст джерелаАнотація:
Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.
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Wu, Chih-Lung, Ya-Han Chih, Hsin-Ying Hsieh, Kuang-Li Peng, Yi-Zong Lee, Bak-Sau Yip, Shih-Che Sue, and Jya-Wei Cheng. "High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli." Biomedicines 10, no. 6 (June 8, 2022): 1351. http://dx.doi.org/10.3390/biomedicines10061351.
Повний текст джерелаАнотація:
Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.
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Sen Gupta, Anirban, Aditya Girish, Ketan Jolly, Maria de la Fuente, Xu Han, Marvin T. Nieman, and Arielle Recchione. "Intravenous Nanomedicine for Targeted Delivery of Thrombin to Augment Hemostasis." Blood 138, Supplement 1 (November 5, 2021): 1029. http://dx.doi.org/10.1182/blood-2021-153708.
Повний текст джерелаАнотація:
Abstract Non-compressible uncontrolled hemorrhage remains a major cause of mortality from traumatic injuries. Additionally, patients with congenital, disease-associated or drug-induced hemostatic dysfunctions, may often be at risk of excessive bleeding. Therefore, treatments that render rapid hemostasis are clinically significant in potentially saving lives. The clinical gold standard for this is the transfusion of whole blood (WB) or blood components (e.g. controlled ratios of platelets, RBCs, and plasma), as evidenced by several clinical studies (e.g. PROPPR, PROMMTT and PAMPer). However, the availability of such blood products is donor-dependent, their shelf-life is limited due to contamination risks, and, their portability and storage is often challenging. While extensive research efforts are currently being focused on addressing these challenges, e.g. using low titer Group O whole blood, cold-storage and freeze-drying of platelets and plasma, in vitro generation of platelets from iPSCs etc., a parallel research focus has emerged in designing biomaterials-based I.V.-administrable technologies (nanoparticles, polymers etc.) that can provide specific functional attributes of hemostasis while allowing donor-independent manufacturing, scale-up, and on-demand availability. Prominent examples of these are 'synthetic platelet' (SynthoPlate) nanoparticles that recapitulate platelet's binding interactions with von Willebrand Factor (vWF), collagen and active platelet integrin GPIIb-IIIa, flexible platelet-like particles (PLP) that bind fibrin to recapitulate platelet's biomechanical properties, fibrinogen function-mimicking nanoparticles that amplify the aggregation of active platelets, peptide-modified synthetic polymers (e.g. PolySTAT, HAPPI etc.) that render clot stabilization etc. In this framework, we present the design and evaluation of I.V.-administrable unique platelet-inspired nanoparticles that render injury site-targeted, enzyme-responsive direct delivery of thrombin, to site-specifically augment fibrin generation for hemostasis. Our design is inspired by platelets' crucial hemostatic mechanisms of : (i) rapidly accumulating at the injury site to form a plug and (ii) serving as a coagulation amplifier via presenting anionic phospholipids on the activated platelet surface to render tenase and prothrombinase factor assemblies leading to thrombin (FIIa) burst, which can then site-specifically convert fibrinogen to fibrin. Thrombin delivery to augment hemostasis is clinically well-accepted, as exemplified by products like Tisseel where thrombin and fibrinogen are co-delivered by syringe directly at wound site. Researchers have also studied thrombin-loaded topical dressings and topical administration of thrombin-loaded particles on wounds to mitigate bleeding, but these cannot be used intravenously. A recent interesting study has explored encapsulation of thrombin-loaded nanoparticles inside actual platelets with the idea of the particles being released (analogous to granule secretion) upon platelet activation, but this was only demonstrated in vitro because optimizing this complex strategy for consistent in vivo function may be challenging. Our approach circumvents these challenges by: (i) loading consistent amount of thrombin in I.V.-administrable lipid nanoparticles (LNPs), (ii) directly targeting the thrombin-loaded LNPs (TLNPs) to the injury site via specific binding to vWF and collagen, and (iii) releasing the loaded thrombin via particle destabilization by the action of injury site-specific enzyme phospholipase A2 for in situ fibrin production. We evaluated the TLNPs in vitro in human blood and plasma where hemostatic defects were created by platelet depletion and anticoagulant treatment. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM) based studies of clot characteristics and BioFlux microfluidics based real-time imaging of fibrin generation under simulated vascular flow conditions, confirmed the ability of TLNPs to restore fibrin generation in hemostatic dysfunction settings. Subsequently, the in vivo feasibility of these TLNPs was tested in a mouse tail-clip bleeding model where a combination of platelet depletion plus anticoagulant treatment was used to render significant hemostatic defect. TLNPs were able to effectively reduce tail-bleeding in mice. Figure 1 Figure 1. Disclosures Sen Gupta: Haima Therapeutics: Other: Co-founder, Patents & Royalties: US 9107845, US 9107963.
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Bussel, James B., Gregory Cheng, Mansoor N. Saleh, Balkis Meddeb, Christine Bailey, Nicole L. Stone, and Manuel Aivado. "Safety and Efficacy of Long-Term Treatment with Oral Eltrombopag for Chronic Idiopathic Thrombocytopenic Purpura." Blood 112, no. 11 (November 16, 2008): 3432. http://dx.doi.org/10.1182/blood.v112.11.3432.3432.
Повний текст джерелаАнотація:
Abstract INTRODUCTION: Eltrombopag (PROMACTA®/REVOLADE®; GlaxoSmithKline, Collegeville, PA) is the first oral, small molecule, non-peptide thrombopoietin receptor agonist under investigation for the treatment of thrombocytopenia due to various causes, including idiopathic thrombocytopenic purpura (ITP). Chronic ITP is characterized by autoantibody-induced platelet destruction and reduced platelet production, leading to chronically low peripheral platelet counts. Eltrombopag treatment has previously demonstrated a significant increase in platelet counts and a reduction in clinically relevant bleeding symptoms in 2 placebo-controlled trials evaluating a total of >200 patients with chronic ITP after up to 6 weeks of treatment. EXTEND is an ongoing open-label, phase III extension study to assess the long-term safety and efficacy of oral eltrombopag in ITP patients that have previously completed an eltrombopag trial. METHODS: Patients with previously treated, chronic ITP who completed a prior eltrombopag study were eligible to participate in EXTEND. Eltrombopag treatment was initiated at 50 mg once daily and then adjusted in order to maintain platelet counts ≥ 50,000/μL and <400,000/μL, with doses between 75 mg once daily and 25 mg once daily or less often than once daily, if necessary. Patients who achieved platelet counts ≥ 50,000/μL during treatment with eltrombopag were considered responders. Bleeding events were prospectively evaluated using the WHO Bleeding Scale: Grade 0 = no bleeding, Grade 1 = mild bleeding, Grade 2 = moderate bleeding, Grade 3 = gross bleeding and Grade 4 = debilitating blood loss. RESULTS: At the time of this analysis, 207 patients (median age, 50 years; 67% female) had received eltrombopag on this study. At baseline, 33% were receiving concomitant ITP medication and 40% were splenectomized. The majority of patients (70%) enrolled with baseline platelet counts <30,000/μL, followed by 18% and 12% with baseline platelet counts from ≥ 30,000/μL to ≤ 50,000/μL, and >50,000/μL, respectively. The duration of eltrombopag treatment ranged from 3 to 523 days. Seventy-nine percent (159/201) of patients achieved a platelet count ≥ 50,000/μL, and 24% (18/75) of patients who had received eltrombopag for at least 25 weeks maintained platelet counts ≥ 50,000/μL continuously for ≥ 25 weeks. Patients responded to eltrombopag regardless of splenectomy status (non-splenectomized: 78%, splenectomized: 81%) and use of baseline concomitant ITP medications (no baseline ITP medications: 79%, baseline ITP medications: 80%). Median platelet counts remained ≥ 50,000/μL throughout the observation period of the study (Figure 1) with only 3 exceptions, when the median platelet counts remained >40,000/μL. At baseline, 59% of patients reported bleeding symptoms (WHO Grades 1–4) compared with approximately 30% at months 1, 3, and 6. Adverse events (AEs) were reported in 150 patients (72%) while on therapy, the majority of which were mild to moderate. Headache (15%) was the most commonly reported on-therapy AE, followed by upper respiratory tract infection (13%), diarrhea (10%), and nasopharyngitis (9%). Six thromboembolic events were reported during the study. No clinically relevant effects of eltrombopag on patient bone marrow were detected. Thirty-nine serious AEs were reported by 17 patients (8%) while on therapy +1 day. Four deaths were reported in the study (2 deaths on therapy and 2 deaths >30 days after the last dose of eltrombopag); none were considered related to study medication. CONCLUSION: Oral eltrombopag is effective at raising platelet counts and decreasing bleeding symptoms during long-term treatment, regardless of splenectomy status or the use of baseline ITP medications. Eltrombopag is well tolerated during long-term treatment in patients with previously treated chronic ITP. Figure 1. Median platelet counts.a BL, median baseline value. aDotted line indicates 50,000 platelets/μL. Figure 1. Median platelet counts.a BL, median baseline value. aDotted line indicates 50,000 platelets/μL.
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Jawan, Roslina, Sahar Abbasiliasi, Joo Shun Tan, Shuhaimi Mustafa, Murni Halim, and Arbakariya B. Ariff. "Influence of Culture Conditions and Medium Compositions on the Production of Bacteriocin-Like Inhibitory Substances by Lactococcus lactis Gh1." Microorganisms 8, no. 10 (September 23, 2020): 1454. http://dx.doi.org/10.3390/microorganisms8101454.
Повний текст джерелаАнотація:
Antibacterial peptides or bacteriocins produced by many strains of lactic acid bacteria have been used as food preservatives for many years without any known adverse effects. Bacteriocin titres can be modified by altering the physiological and nutritional factors of the producing bacterium to improve the production in terms of yield and productivity. The effects of culture conditions (initial pH, inoculum age and inoculum size) and medium compositions (organic and inorganic nitrogen sources; carbon sources) were assessed for the production of bacteriocin-like inhibitory substances (BLIS) by Lactococcus lactis Gh1 in shake flask cultures. An inoculum of the mid-exponential phase culture at 1% (v/v) was the optimal age and size, while initial pH of culture media at alkaline and acidic state did not show a significant impact on BLIS secretion. Organic nitrogen sources were more favourable for BLIS production compared to inorganic sources. Production of BLIS by L. lactis Gh1 in soytone was 1.28-times higher as compared to that of organic nitrogen sources ((NH4)2SO4). The highest cell concentration (XmX = 0.69 ± 0.026 g·L−1) and specific growth rate (μmax = 0.14 h−1) were also observed in cultivation using soytone. By replacing carbon sources with fructose, BLIS production was increased up to 34.94% compared to BHI medium, which gave the biomass cell concentration and specific growth rate of 0.66 ± 0.002 g·L−1 and 0.11 h−1, respectively. It can be concluded that the fermentation factors have pronounced influences on the growth of L. lactis Gh1 and BLIS production. Results from this study could be used for subsequent application in process design and optimisation for improving BLIS production by L. lactis Gh1 at larger scale.
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Chen, Hua You, Xiang Hui Qi, Xu Geng, Qing Gang Xu, Jing Wang, and Zi Rong Wu. "Expression, Purification and Characterization of the Recombinant Hirudin Variant iii in the Bacillus Subtilis." Advanced Materials Research 343-344 (September 2011): 753–63. http://dx.doi.org/10.4028/www.scientific.net/amr.343-344.753.
Повний текст джерелаАнотація:
Hirudin is the most potent natural inhibitor of thrombin and a powerful anticoagulant. Large-scale production of recombinant hirudin is desirable for therapy. In this study, the gene encoding hirudin variant III was redesigned and synthesized by usingBacillus subtilispreferred codons, and the recombinant hirudin variant III (rHV3) was overexpressed inB. subtilisDB403 with strong anticoagulation activity for the first time. The hirudin activity from the supernatant of culture with optimized expression conditions could reach 210 ATU/ml. The protein in culture supernatant was precipitated by trichloroacetic acid, then desalted by ultrafiltration and purified by anion exchange chromatography. Strong anion Q F.F. performed better than weak anion DEAE F.F. The proper pH and conductivity was determined at pH 8 and 6 ms/cm, respectively. The maximum applied sample was 240 ATU/ml to medium of strong anion Q F.F. This optimized procedure was employed in strong anion exchange HiPrep 16/10Q with the 90% recovery rate and 70.2% purity. After gel filtration, the purity of rHV3 checked by HPLC could reach 95.1%, and the recovery rate was 93% for this step. The purified recombinant rHV3 showed a single band in SDS-PAGE. The rHV3 was stable at 100 °C and acidity condition, but was unstable under the condition of both heating and alkalinity. In conclusion, theses studies suggests thatB.subtilismight be useful for the production of biologically active medicine peptides in secretion facilitating purification procedures, and that this isolation method was suitable for scale-up purification process at a low cost.
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Nath, Arijit, Attila Csighy, Burak Attila Eren, David Tjandra Nugraha, Klára Pásztorné-Huszár, Attila Tóth, Krisztina Takács, et al. "Bioactive Peptides from Liquid Milk Protein Concentrate by Sequential Tryptic and Microbial Hydrolysis." Processes 9, no. 10 (September 22, 2021): 1688. http://dx.doi.org/10.3390/pr9101688.
Повний текст джерелаАнотація:
Recently, bioactive peptides as a health-promoting agent have come to the forefront of health research; however, industrial production is limited, possibly due to the lack of the required technological knowledge. The objective of the investigation was to prepare bioactive peptides with hypoallergenic properties from liquid milk protein concentrate (LMPC), through sequential enzymatic and microbial hydrolysis. LMPC was produced from ultra-heat-treated (UHT) skimmed cow’s milk using a nanofiltration membrane. The effect of the concentration of trypsin (0.008–0.032 g·L−1) on the hydrolysis of LMPC was studied. Subsequently, the hydrolysis of tryptic-hydrolyzed LMPC (LMPC-T) with lactic acid bacteria was performed, and the effect of glucose in microbial hydrolysis was studied. Aquaphotomic analysis of the hydrolysis of LMPC was performed using the spectral range of 1300–1600 nm (near-infrared spectra). Changes in antioxidant capacity, anti-angiotensin-converting enzyme activity, and antibacterial activity against Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes were noted after the sequential tryptic and microbial hydrolysis of LMPC. Allergenicity in LMPC was reduced, due to sequential hydrolysis with 0.016 g·L−1 of trypsin and lacteal acid bacteria. According to the aquaphotomic analysis result, there was a dissociation of hydrogen bonds in compounds during the initial period of fermentation and, subsequently, the formation of compounds with hydrogen bonds. The formation of compounds with a hydrogen bond was more noticeable when microbial hydrolysis was performed with glucose. This may support the belief that the results of the present investigation will be useful to scale up the process in the food and biopharmaceutical industries.
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Schuster, Bernhard, and Uwe B. Sleytr. "Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules." Journal of The Royal Society Interface 11, no. 96 (July 6, 2014): 20140232. http://dx.doi.org/10.1098/rsif.2014.0232.
Повний текст джерелаАнотація:
Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems.
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Fawzya, Yusro Nuri, Safira M. Nursatya, Rini Susilowati, and Ekowati Chasanah. "Characteristics of Fish Protein Hydrolysate from Yellowstripe Scad (Selaroides leptolepis) Produced by a Local Microbial Protease." E3S Web of Conferences 147 (2020): 03017. http://dx.doi.org/10.1051/e3sconf/202014703017.
Повний текст джерелаАнотація:
Fish protein hydrolysate (FPH) containing small protein or peptides and amino acids has a great attention related to the provision of high protein foods to overcome the problem of malnutrition. This research was purposed to prepare FPH from yellowstripe scad (Selaroides leptolepis) by using a local microbial protease from Bacillus subtilis BII-1. Hydrolysis process was done in a laboratory scale (500 g minced fish) at 55oC for 6 h. The liquid hydrolysate was then spray dried using whey protein and maltodextrin at a concentration of 20 and 30% for each filler. The treatment of whey protein powder produced FPHs with higher protein content (31.71-33.97% db) and slightly yellowish in color compared to maltodextrin (11.88-16.66% db). Their foaming capacity and stability were 20-100% and 15% in 5-10 min, respectively. However, FPHs prepared with maltodextrin had no foaming capacity. The hydrolysates from both treatments had low water and oil absorption with the value less than 3 mL/g hydrolysate. A trial on scaling up production using 30 kg fish, showed that optimization or adjustment should be carried out due to the high amount and high protein content of the residual products.
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Melini, Francesca, Valentina Melini, Francesca Luziatelli, Anna Grazia Ficca, and Maurizio Ruzzi. "Health-Promoting Components in Fermented Foods: An Up-to-Date Systematic Review." Nutrients 11, no. 5 (May 27, 2019): 1189. http://dx.doi.org/10.3390/nu11051189.
Повний текст джерелаАнотація:
Fermented foods have long been produced according to knowledge passed down from generation to generation and with no understanding of the potential role of the microorganism(s) involved in the process. However, the scientific and technological revolution in Western countries made fermentation turn from a household to a controlled process suitable for industrial scale production systems intended for the mass marketplace. The aim of this paper is to provide an up-to-date review of the latest studies which investigated the health-promoting components forming upon fermentation of the main food matrices, in order to contribute to understanding their important role in healthy diets and relevance in national dietary recommendations worldwide. Formation of antioxidant, bioactive, anti-hypertensive, anti-diabetic, and FODMAP-reducing components in fermented foods are mainly presented and discussed. Fermentation was found to increase antioxidant activity of milks, cereals, fruit and vegetables, meat and fish. Anti-hypertensive peptides are detected in fermented milk and cereals. Changes in vitamin content are mainly observed in fermented milk and fruits. Fermented milk and fruit juice were found to have probiotic activity. Other effects such as anti-diabetic properties, FODMAP reduction, and changes in fatty acid profile are peculiar of specific food categories.
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Schneider-Ohrum, Kirsten, Corinne Cayatte, Yi Liu, Zhaoti Wang, Alivelu Irrinki, Floro Cataniag, Nga Nguyen, et al. "Production of Cytomegalovirus Dense Bodies by Scalable Bioprocess Methods Maintains Immunogenicity and Improves Neutralizing Antibody Titers." Journal of Virology 90, no. 22 (August 31, 2016): 10133–44. http://dx.doi.org/10.1128/jvi.00463-16.
Повний текст джерелаАнотація:
ABSTRACTWith the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.IMPORTANCEDevelopment of a vaccine to prevent congenital HCMV infection remains a high priority. Vaccination with human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccination strategy that mimics natural infection. The standard approach for purification of virus particles has been to use a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. In the study described here, we employed an approach that combines treatment with an antiviral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.
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Leni, G., L. Soetemans, J. Jacobs, S. Depraetere, N. Gianotten, L. Bastiaens, A. Caligiani, and S. Sforza. "Protein hydrolysates from Alphitobius diaperinus and Hermetia illucens larvae treated with commercial proteases." Journal of Insects as Food and Feed 6, no. 4 (August 11, 2020): 393–404. http://dx.doi.org/10.3920/jiff2019.0037.
Повний текст джерелаАнотація:
Insect proteins have been proposed as a promising alternative for feed and food formulations. In the present work protease-assisted extraction was studied as a way to separate and extract proteins from two different insect species: Alphitobius diaperinus (AD) and Hermetia illucens (HI). The proteolytic activity of seven enzymes (papain, pancreatin, dispase I, pepsin, protease from Bacillus licheniformis, bromelain and trypsin) was evaluated determining the protein extraction yield, the degree of hydrolysis (DH) and the released free amino acids (FAA). Both insects represent an interesting source of proteins, not only for their amount (more than 40% on dry matter) but also for the nutritional value, with essential amino acid profile exceeding the requirements proposed for human nutrition. Enzyme-assisted protein extraction, performed at laboratory scale, gave for HI an average yield of extraction of 71±8% and for AD 67±6%. Hydrolysates produced from HI gave a DH% ranging between 3 to 18%, whereas hydrolysates produced from AD yielded a DH% between 7 to 23%. The protein hydrolysates were composed by peptides and FAA (which accounted for more than 30% of the extracted protein fraction), which were released according to their abundance in initial protein. A moderate correlation between the DH% and the total amount of FAA was found, except for AD hydrolysed with trypsin and HI with papain. Based on these results, the production of hydrolysates was preliminary scaled up in a proof-of-concept experiment, focusing on the most promising insect-enzyme combination. The final product resulted to be rich in protein (60% on dry matter). This work support enzymatic hydrolysis as an effective method to extract and isolate proteins from insects, with minimal sample preparation, tailoring their composition, preserving the nutritional quality, decreasing the risk of allergic reactions and making them more accessible for their future use as feed/food supplements.
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Li, Xia, Meifeng Li, Junling Guo, Xian Liu, Xuepin Liao, and Bi Shi. "Collagen peptide provides Streptomyces coelicolor CGMCC 4.7172 with abundant precursors for enhancing undecylprodigiosin production." Journal of Leather Science and Engineering 3, no. 1 (July 15, 2021). http://dx.doi.org/10.1186/s42825-021-00059-y.
Повний текст джерелаАнотація:
Abstract Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources. Undecylprodigiosin (UP) is a promising antibiotic, but the direct feeding of pure precursor amino acids makes it costly for large-scale production. Here, collagen peptide (CP), a renewable animal-derived biomass contains abundant precursor amino acids of UP. CP can act as carbon and nitrogen source for the growth of Streptomyces coelicolor CGMCC 4.7172. The plant biomasses including soybean meal, wheat bran, and malt extract were unsuitable for UP prodution. However, 365.40 µg/L UP was detected after 24 h in the media containing CP, and its highest concentration reached 1198.01 µg/L. UP was also detected in the media containing meat hydrolysates of domestic animals, but its initial production time was delayed, and final concentration was lower than that in the medium containing CP only. Compared the fermentation performances of CP and other proteins, CP has a special superiority for UP production. These results revealed that UP biosynthesis may be dependent on amino acid availability of substrates and CP is beneficial for UP production because of its specific amino acid composition. Graphical abstract
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Yurkova, Maria S., Elchin G. Sadykhov, and Alexey N. Fedorov. "Production of a toxic polypeptide as a fusion inside GroEL cavity." Scientific Reports 10, no. 1 (December 2020). http://dx.doi.org/10.1038/s41598-020-78094-8.
Повний текст джерелаАнотація:
AbstractThe system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.
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Doerr, Frederik J. S., Lee J. Burns, Becky Lee, Jeremy Hinds, Rebecca L. Davis-Harrison, Scott A. Frank, and Alastair J. Florence. "Peptide Isolation via Spray Drying: Particle Formation, Process Design and Implementation for the Production of Spray Dried Glucagon." Pharmaceutical Research 37, no. 12 (December 2020). http://dx.doi.org/10.1007/s11095-020-02942-5.
Повний текст джерелаАнотація:
Abstract Purpose Spray drying plays an important role in the pharmaceutical industry for product development of sensitive bio-pharmaceutical formulations. Process design, implementation and optimisation require in-depth knowledge of process-product interactions. Here, an integrated approach for the rapid, early-stage spray drying process development of trehalose and glucagon on lab-scale is presented. Methods Single droplet drying experiments were used to investigate the particle formation process. Process implementation was supported using in-line process analytical technology within a data acquisition framework recording temperature, humidity, pressure and feed rate. During process implementation, off-line product characterisation provided additional information on key product properties related to residual moisture, solid state structure, particle size/morphology and peptide fibrillation/degradation. Results A psychrometric process model allowed the identification of feasible operating conditions for spray drying trehalose, achieving high yields of up to 84.67%, and significantly reduced levels of residual moisture and particle agglomeration compared to product obtained during non-optimal drying. The process was further translated to produce powders of glucagon and glucagon-trehalose formulations with yields of >83.24%. Extensive peptide aggregation or degradation was not observed. Conclusions The presented data-driven process development concept can be applied to address future isolation problems on lab-scale and facilitate a systematic implementation of spray drying for the manufacturing of sensitive bio-pharmaceutical formulations.
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Li, Tao, Chenyi Zhan, Gege Guo, Zhaoxing Liu, Ning Hao, and Pingkai Ouyang. "Tofu processing wastewater as a low-cost substrate for high activity nattokinase production using Bacillus subtilis." BMC Biotechnology 21, no. 1 (October 7, 2021). http://dx.doi.org/10.1186/s12896-021-00719-1.
Повний текст джерелаАнотація:
Abstract Background Even though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level. Results The peptide component in TPW is a crucial factor in the production of nattokinase. Box–Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively. Conclusions By replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.
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Molavi, Fatima, Mohammad Barzegar-Jalali, and Hamed Hamishehkar. "Changing the daily injection of glatiramer acetate to a monthly long acting product through designing polyester-based polymeric microspheres." BioImpacts, August 13, 2022. http://dx.doi.org/10.34172/bi.2022.23733.
Повний текст джерелаАнотація:
Introduction: Glatiramer acetate (GA) is a newly emerged therapeutic peptide to reduce the frequency of relapses in multiple sclerosis (MS). Despite its good performance in controlling MS, it is not widely used due to daily or biweekly subcutaneous injections due to rapid degradation and body clearance. Therefore, implant design with sustained release leads to prolonged biological effects by gradually increasing drug exposure and protecting GA from rapid local degradation. Methods: Different emulsion methods, PLGA type, surfactant concentration, drug/polymer ratio, drying processes, stirring method, and other variables in preliminary studies modified the final formulation. The release kinetics were studied through mechanistic kinetic models such as zero-order, Weibull, Higuchi, etc. In this study, all challenges for easy scale-up, methodological detail, and a simple, feasible setup in mass production were discussed. Results: The optimized formulation was obtained by 1:6 drug/PLGA, 0.5% w/w polyvinyl alcohol, and 0.75% w/w NaCl in the external aqueous phase, 1:10 continuous phase to dispersed phase ratio, and without any surfactant in the primary emulsion. The final freeze-dried particles presented a narrow distributed size of 1-10 µm with 7.29% ± 0.51 drug loading and zero-order release behavior with appropriate regression correlation (R2 98.7), complete release, and only 7.1% initial burst release. Conclusion: Therefore, to achieve improvement in patient compliance through better and longer efficacy, designing the parenteral sustained release microspheres (MPSs) of this immune modulator is a promising approach that should be considered.
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Müller, Carolin, Chika L. Igwe, Wolfgang Wiechert, and Marco Oldiges. "Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay." Microbial Cell Factories 20, no. 1 (September 30, 2021). http://dx.doi.org/10.1186/s12934-021-01672-6.
Повний текст джерелаАнотація:
Abstract Background The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. Results The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. Conclusions GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.
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Borges, Sandra, Clara Piccirillo, Francesca Scalera, Rui Martins, Ana Rosa, José António Couto, André Almeida, and Manuela Pintado. "Valorization of porcine by-products: a combined process for protein hydrolysates and hydroxyapatite production." Bioresources and Bioprocessing 9, no. 1 (March 21, 2022). http://dx.doi.org/10.1186/s40643-022-00522-6.
Повний текст джерелаАнотація:
AbstractThe meat industry generates large amounts of by-products that are costly to be treated and discarded ecologically; moreover, they could be used to extract high added-value compounds. In this work, we present an innovative combined process which allowed the parallel extraction of both organic and mineral compounds; more specifically protein hydrolysates and single-phase hydroxyapatite were obtained. The protein hydrolysates, extracted through an enzymatic hydrolysis with alcalase, showed a degree of hydrolysis of 53.3 ± 5.1%; moreover, they had a high protein content with peptides with molecular weight lower than 1.2 kDa. Their antioxidant activities, measured with ABTS and ORAC tests, were 21.1 ± 0.5 mg ascorbic acid equivalent/g of dry extract and 87.7 ± 6.3 mg Trolox equivalent/g of dry extract, respectively. Single-phase hydroxyapatite, obtained with a simple calcination at 700 °C on the residues of the hydrolysis process, showed a Ca/P ratio close to the stoichiometric one (1.65 vs. 1.67) and presented a nanometric structure. This study reports a simple and feasible process for the valorization of porcine by-products in a large-scale up generating products with potential applications for environment remediation, biomedicine, nutrition and catalysis/bioenergy. Graphic Abstract
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Sulheim, Snorre, Fredrik A. Fossheim, Alexander Wentzel, and Eivind Almaas. "Automatic reconstruction of metabolic pathways from identified biosynthetic gene clusters." BMC Bioinformatics 22, no. 1 (February 23, 2021). http://dx.doi.org/10.1186/s12859-021-03985-0.
Повний текст джерелаАнотація:
Abstract Background A wide range of bioactive compounds is produced by enzymes and enzymatic complexes encoded in biosynthetic gene clusters (BGCs). These BGCs can be identified and functionally annotated based on their DNA sequence. Candidates for further research and development may be prioritized based on properties such as their functional annotation, (dis)similarity to known BGCs, and bioactivity assays. Production of the target compound in the native strain is often not achievable, rendering heterologous expression in an optimized host strain as a promising alternative. Genome-scale metabolic models are frequently used to guide strain development, but large-scale incorporation and testing of heterologous production of complex natural products in this framework is hampered by the amount of manual work required to translate annotated BGCs to metabolic pathways. To this end, we have developed a pipeline for an automated reconstruction of BGC associated metabolic pathways responsible for the synthesis of non-ribosomal peptides and polyketides, two of the dominant classes of bioactive compounds. Results The developed pipeline correctly predicts 72.8% of the metabolic reactions in a detailed evaluation of 8 different BGCs comprising 228 functional domains. By introducing the reconstructed pathways into a genome-scale metabolic model we demonstrate that this level of accuracy is sufficient to make reliable in silico predictions with respect to production rate and gene knockout targets. Furthermore, we apply the pipeline to a large BGC database and reconstruct 943 metabolic pathways. We identify 17 enzymatic reactions using high-throughput assessment of potential knockout targets for increasing the production of any of the associated compounds. However, the targets only provide a relative increase of up to 6% compared to wild-type production rates. Conclusion With this pipeline we pave the way for an extended use of genome-scale metabolic models in strain design of heterologous expression hosts. In this context, we identified generic knockout targets for the increased production of heterologous compounds. However, as the predicted increase is minor for any of the single-reaction knockout targets, these results indicate that more sophisticated strain-engineering strategies are necessary for the development of efficient BGC expression hosts.