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Article ; Online: Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Contreras, María Angélica / Serrano-Rivero, Yunier / González-Pose, Alaín / Salazar-Uribe, Julieta / Rubio-Carrasquilla, Marcela / Soares-Alves, Matheus / Parra, Natalie C / Camacho-Casanova, Frank / Sánchez-Ramos, Oliberto / Moreno, Ernesto

Molecules (Basel, Switzerland)

2023 Volume 28, Issue 9

Abstract: Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages ... ...

Abstract	Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 10
MeSH term(s)	Animals ; Peptide Library ; Single-Domain Antibodies ; Vascular Endothelial Growth Factor A/genetics ; Antigens ; Cell Surface Display Techniques
Chemical Substances	Peptide Library ; Single-Domain Antibodies ; Vascular Endothelial Growth Factor A ; Antigens
Language	English
Publishing date	2023-04-25
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules28093708
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Article ; Online: First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile.

Hugues, Florence / Cabezas, Ignacio / Garigliany, Mutien / Rivas, Felipe / Casanova, Tomás / González, Eddy E / Sánchez, Oliberto / Castillo, Raúl / Parra, Natalie C / Inostroza-Michael, Oscar / Moreno, Lucila / Hernández, Cristián E / Toledo, Jorge R

Virology journal

2023 Volume 20, Issue 1, Page(s) 205

Abstract: Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located ... ...

Abstract	Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.
MeSH term(s)	Animals ; Cattle ; Chile/epidemiology ; Phylogeny ; Diarrhea Viruses, Bovine Viral/genetics ; 5' Untranslated Regions ; Diarrhea
Chemical Substances	5' Untranslated Regions
Language	English
Publishing date	2023-09-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2160640-7
ISSN	1743-422X ; 1743-422X
ISSN (online)	1743-422X
ISSN	1743-422X
DOI	10.1186/s12985-023-02170-4
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Article: Isolation, characterization, and immunomodulatory activity evaluation of probiotic strains from colostrum and canine milk.

Quilodrán-Vega, Sandra Rayén / Muñoz-Flores, Carolina / Pino, Ana / Buldres, Paula / Sandoval, Felipe / Aguirre, Alex / Portillo, Brígida / Parra, Natalie / Altamirano, Claudia / Albarracín, Leonardo / Villena, Julio / Toledo, Jorge R

Frontiers in veterinary science

2023 Volume 10, Page(s) 1266064

Abstract: Background: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, ...

Abstract	Background: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, including their resistance to gastrointestinal conditions, inhibitory effect against pathogens, and intestinal adhesion. Methods: The immunomodulatory effects of the strains were also analyzed in Results: The TUCO-16 and TUCO-17 strains induced a significant increase in the expression of TNF-α, IL-8, and TLR2 in canine macrophages. The oral administration of TUCO-16 and TUCO-17 strains to mice significantly augmented their resistance to pathogenic Conclusion: Both strains, TUCO-16 and TUCO-17, are potential probiotic candidates for improving intestinal health in dogs, particularly for their ability to inhibit the growth of Gram-negative pathogens common in gastrointestinal infections and modulate the animal's immune response. Further studies are required to effectively demonstrate the beneficial effects of TUCO-16 and TUCO-17 strains in dogs.
Language	English
Publishing date	2023-11-23
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2834243-4
ISSN	2297-1769
ISSN	2297-1769
DOI	10.3389/fvets.2023.1266064
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Article: Immunomodulatory role of vasoactive intestinal peptide and ghrelin in

Muñoz-Flores, Carolina / Roa, Francisco J / Saavedra, Paulina / Fuentealba, Pablo / Starck, María F / Ortega, Leonardo / Montesino, Raquel / Valenzuela, Ariel / Astuya, Allisson / Parra, Natalie / González-Chavarría, Iván / Sánchez, Oliberto / Toledo, Jorge R / Acosta, Jannel

Heliyon

2023 Volume 9, Issue 12, Page(s) e23215

Abstract: Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous ... ...

Abstract	Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In
Language	English
Publishing date	2023-12-02
Publishing country	England
Document type	Journal Article
ZDB-ID	2835763-2
ISSN	2405-8440
ISSN	2405-8440
DOI	10.1016/j.heliyon.2023.e23215
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Article ; Online: Effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen.

Gutiérrez-Reinoso, Miguel A / Arreseigor, Cesar J / Driedger, Brian / Cabezas, Ignacio / Hugues, Florence / Parra, Natalie C / Sánchez, Oliberto / Toledo, Jorge R / Garcia-Herreros, Manuel

Animal reproduction science

2023 Volume 252, Page(s) 107226

Abstract: Superovulation is a drug-based method used in cattle to stimulate the ovarian folliculogenesis and the number of oocytes and transferable embryos. The present study aimed to test the effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on ... ...

Abstract	Superovulation is a drug-based method used in cattle to stimulate the ovarian folliculogenesis and the number of oocytes and transferable embryos. The present study aimed to test the effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on ovarian response and in vivo embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen. Forty healthy Holstein heifers subjected to a superovulation (SOV) protocol by using FSH-p or bscrFSH were divided randomly into four groups: a) FSH-p inseminated with unsorted semen (USP; n = 10), b) FSH-p inseminated with sex-sorted semen (SSP; n = 10), c) bscrFSH inseminated with unsorted semen (USR; n = 10), and d) bscrFSH inseminated with sex-sorted semen (SSR; n = 10). Ultrasonography was carried out on Day 8 (estrus) and Day 15 (embryo collection) to evaluate the ovarian structures [follicles (FL), corpora lutea (CL), and non-ovulated follicles (NOFL)]. Embryonic-derived parameters were scored on Day 15 [total structures collected (TS), unfertilised oocytes (UFOs), total embryos (TEs), transferable embryos (TFEs), freezable embryos (FEs), and degenerated embryos (DEs)]. No differences were observed regarding ovarian structures (FL and NOFL) irrespective of SOV protocol or group assessed (P > 0.05). CL increased in bscrFSH-derived SOV protocol (P < 0.05). On Day 15, the embryonic-derived parameters TEs, TFEs, and FEs decreased in SSP/SSR compared to USP/USR (P < 0.05). Differences were observed regarding UFOs, with a greater number in SSP and SSR (P = 0.01). In conclusion, the bscrFSH-derived SOV protocol showed improved results compared to FSH-p-derived SOV protocol regarding ovarian (CL) and embryo-derived (TFE) parameters irrespective of the type of semen used.
MeSH term(s)	Animals ; Cattle ; Female ; Embryo, Mammalian ; Follicle Stimulating Hormone/pharmacology ; Insemination, Artificial/veterinary ; Semen/physiology ; Superovulation
Chemical Substances	Follicle Stimulating Hormone (9002-68-0)
Language	English
Publishing date	2023-03-28
Publishing country	Netherlands
Document type	Journal Article ; Randomized Controlled Trial, Veterinary
ZDB-ID	429674-6
ISSN	1873-2232 ; 0378-4320
ISSN (online)	1873-2232
ISSN	0378-4320
DOI	10.1016/j.anireprosci.2023.107226
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Article ; Online: Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Manrique-Suárez, Viana / Macaya, Luis / Contreras, Maria Angélica / Parra, Natalie / Maura, Rafael / González, Alaín / Toledo, Jorge R / Sánchez, Oliberto

Proteins

2021 Volume 89, Issue 11, Page(s) 1508–1521

Abstract: Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα ...

Abstract	Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.
MeSH term(s)	Animals ; Anti-Inflammatory Agents/chemistry ; Anti-Inflammatory Agents/metabolism ; Anti-Inflammatory Agents/pharmacology ; Blood-Brain Barrier/metabolism ; CHO Cells ; Cell Line ; Cell Survival/drug effects ; Cricetulus ; Endothelial Cells/cytology ; Endothelial Cells/drug effects ; Endothelial Cells/metabolism ; Endotoxins/antagonists & inhibitors ; Endotoxins/metabolism ; Endotoxins/toxicity ; Gene Expression ; Humans ; Low Density Lipoprotein Receptor-Related Protein-1/genetics ; Low Density Lipoprotein Receptor-Related Protein-1/metabolism ; Mice ; Models, Biological ; Models, Molecular ; Peptides/genetics ; Peptides/metabolism ; Protein Binding ; Protein Conformation ; Protein Engineering/methods ; Receptors, Tumor Necrosis Factor, Type II/genetics ; Receptors, Tumor Necrosis Factor, Type II/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Recombinant Fusion Proteins/pharmacology ; Tumor Necrosis Factor-alpha/antagonists & inhibitors ; Tumor Necrosis Factor-alpha/metabolism ; Tumor Necrosis Factor-alpha/toxicity ; Vascular Endothelial Growth Factor A/genetics ; Vascular Endothelial Growth Factor A/metabolism
Chemical Substances	Angiopep-2 ; Anti-Inflammatory Agents ; Endotoxins ; LRP1 protein, human ; Low Density Lipoprotein Receptor-Related Protein-1 ; Peptides ; Receptors, Tumor Necrosis Factor, Type II ; Recombinant Fusion Proteins ; Tumor Necrosis Factor-alpha ; VEGFA protein, human ; Vascular Endothelial Growth Factor A
Language	English
Publishing date	2021-07-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	806683-8
ISSN	1097-0134 ; 0887-3585
ISSN (online)	1097-0134
ISSN	0887-3585
DOI	10.1002/prot.26173
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Article ; Online: Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Vásquez-Suárez, Aleikar / Muñoz-Flores, Carolina / Ortega, Leonardo / Roa, Francisco / Castillo, Carolina / Romero, Alex / Parra, Natalie / Sandoval, Felipe / Macaya, Luis / González-Chavarría, Iván / Astuya, Allisson / Starck, María Francisca / Villegas, Milton F / Agurto, Niza / Montesino, Raquel / Sánchez, Oliberto / Valenzuela, Ariel / Toledo, Jorge R / Acosta, Jannel

Fish & shellfish immunology

2024 Volume 146, Page(s) 109373

Abstract: Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 ... ...

Abstract	Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as T
MeSH term(s)	Animals ; Toll-Like Receptor 5/genetics ; Toll-Like Receptor 5/metabolism ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Salmo salar/genetics ; Salmo salar/metabolism ; HMGB1 Protein ; Molecular Docking Simulation ; Peptides/pharmacology ; Flagellin/pharmacology
Chemical Substances	Toll-Like Receptor 5 ; NF-kappa B ; HMGB1 Protein ; Peptides ; Flagellin (12777-81-0)
Language	English
Publishing date	2024-01-23
Publishing country	England
Document type	Journal Article
ZDB-ID	1067738-0
ISSN	1095-9947 ; 1050-4648
ISSN (online)	1095-9947
ISSN	1050-4648
DOI	10.1016/j.fsi.2024.109373
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Article ; Online: New insight into the biological activity of

Frontiers in immunology

2024 Volume 15, Page(s) 1191966

Abstract: NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In ... ...

Abstract	NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of
MeSH term(s)	Animals ; Antimicrobial Peptides/metabolism ; Antimicrobial Peptides/pharmacology ; Fish Diseases/immunology ; Fish Diseases/microbiology ; Fish Proteins/metabolism ; Fish Proteins/pharmacology ; Immunity, Innate ; Proteolipids/metabolism ; Proteolipids/pharmacology ; Salmo salar/immunology ; Signal Transduction
Chemical Substances	Antimicrobial Peptides ; Fish Proteins ; NK-lysin ; Proteolipids
Language	English
Publishing date	2024-04-09
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2024.1191966
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Article ; Online: Corrigendum to "In vivo modification of the goat mammary gland glycosylation pathway" [New Biotechnol 61 (2021) 11-21].

Leiva-Carrasco, María J / Jiménez-Chávez, Silvana / Harvey, David J / Parra, Natalie C / Tavares, Kaio C / Camacho, Frank / González, Alain / Sánchez, Oliberto / Montesino, Raquel / Toledo, Jorge R

New biotechnology

2022 Volume 68, Page(s) 108

Language	English
Publishing date	2022-02-04
Publishing country	Netherlands
Document type	Published Erratum
ZDB-ID	2400836-9
ISSN	1876-4347 ; 1876-4347
ISSN (online)	1876-4347
ISSN	1876-4347
DOI	10.1016/j.nbt.2022.01.005
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Article ; Online: Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells.

Bravo, Felipe E / Parra, Natalie C / Camacho, Frank / Acosta, Jannel / González, Alaín / Toledo, Jorge R / Sanchez, Oliberto

Scientific reports

2020 Volume 10, Issue 1, Page(s) 12840

Abstract: Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, ... ...

Abstract	Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.
MeSH term(s)	Biotechnology/methods ; Clone Cells ; Flow Cytometry/methods ; Follicle Stimulating Hormone/biosynthesis ; Follicle Stimulating Hormone/genetics ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Mutagenesis, Insertional/methods ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transduction, Genetic ; Transgenes/genetics
Chemical Substances	Recombinant Proteins ; Green Fluorescent Proteins (147336-22-9) ; Follicle Stimulating Hormone (9002-68-0)
Language	English
Publishing date	2020-07-30
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-69709-1
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