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BACKGROUND/OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, was declared a public health emergency in early 2020. The infection initiates when the receptor-binding domain (RBD) of the viral spike protein binds to human angiotensin-converting enzyme 2 (ACE2). Despite the success of vaccination efforts, the emergence of new variants highlights the ongoing need for treatments targeting these evolving strains. In silico methods previously identified peptides BP2, BP9, and BP11 as being capable of disrupting the RBD-ACE2 interaction, though their efficacy has not been experimentally validated until now. METHODS: In this study, these peptides were recombinantly produced in the yeast Komagataella phaffii, and the activity was assessed in vitro using binding assays with multiple RBD variants and the inhibition of the RBD-ACE2 interaction. RESULTS: The production yield for BP2, BP9, and BP11 was 14.34, 4.01, and 1.35 mg per culture liter, respectively. Noteworthy, the three BPs interacted with the RBD of SARS-CoV-2 variants of concern, with BP2 showing higher recognition. Finally, the BPs showed an RBD/hACE2 interaction blocking capacity with IC50 values between 1.03 and 5.35 nM, with BP2 showing the lowest values among the evaluated peptides. CONCLUSIONS: These results demonstrate that BP2, specifically, is a promising candidate for the development of novel therapeutic interventions targeting SARS-CoV-2 and other coronaviruses that use hACE2 for cellular entry.
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Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.
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Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.
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Acetilglucosaminidasa , Mucopolisacaridosis III , Humanos , Mucopolisacaridosis III/tratamiento farmacológico , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/patología , Acetilglucosaminidasa/metabolismo , Acetilglucosaminidasa/antagonistas & inhibidores , Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Sitio Alostérico/efectos de los fármacos , Regulación Alostérica/efectos de los fármacosRESUMEN
BACKGROUND: Bovine herpes virus (BoHV 1 and BoHV-5) are the causative agents of infectious bovine rhinotracheitis (IBR). IBR is responsible for important economic losses in the cattle industry. The envelope glycoprotein B (gB) is essential for BoHV infection of cattle's upper respiratory and genital tract. gB is one of the main candidate antigens for a potential recombinant vaccine since it induces a strong and persistent immune response. RESULTS: In this study, gB of BoHV-1 and BoHV-5 was characterized in terms of function, structure, and antigenicity through bioinformatics tools. gB showed conserved sequence and structure, so, both domains named PH Like 1 and 2 domains of each virus were selected for the design of a bivalent vaccine candidate. The immunoinformatic study showed that these two domains have epitopes recognizable by B and T lymphocytes, followed by this, the cDNA domains from BoHV-1/5 gB (Domains-gB) were transformed into the yeast Komagataella phaffii GS115 (previously known as Pichia pastoris). A recombinant protein with molecular weight of about 110 kDa was obtained from the culture media. The vaccine candidate protein (Domains-gB) was recognized by a monoclonal antibody from a commercial ELISA kit used for IBR diagnostic, which may suggest that the epitopes are conserved of the entire infectious virus. CONCLUSION: Overall, it was shown that the recombinant domains of BoHV-1/5 gB have antigenic and immunogenic properties similar to the native gB. This vaccine candidate is promising to be used in future studies to assess its immunogenicity in an animal model.
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Alphaherpesvirinae , Enfermedades de los Bovinos , Rinotraqueítis Infecciosa Bovina , Animales , Bovinos , Epítopos , Anticuerpos Monoclonales , Biología Computacional , Rinotraqueítis Infecciosa Bovina/prevención & control , Glicoproteínas , Enfermedades de los Bovinos/prevención & controlRESUMEN
Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.
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Condroitinsulfatasas , Mucopolisacaridosis , Mucopolisacaridosis IV , Nanopartículas , Condroitinsulfatasas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Óxido Ferrosoférrico/uso terapéutico , Edición Génica , Glicosaminoglicanos , Humanos , Mucopolisacaridosis/genética , Mucopolisacaridosis/terapia , Mucopolisacaridosis IV/genética , Mucopolisacaridosis IV/terapiaRESUMEN
The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.
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Gangliosidosis GM2 , Enfermedad de Tay-Sachs , Desoxirribonucleasa I/metabolismo , Fibroblastos/metabolismo , Proteína Activadora de G (M2) , Gangliósido G(M2)/genética , Gangliósido G(M2)/metabolismo , Gangliosidosis GM2/genética , Gangliosidosis GM2/metabolismo , Gangliosidosis GM2/terapia , Edición Génica , Globósidos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidasa A/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Liposomas/metabolismo , Enfermedad de Tay-Sachs/genética , Enfermedad de Tay-Sachs/metabolismo , Enfermedad de Tay-Sachs/terapia , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Sphingolipids (SLs) are lipids derived from sphingosine, and their metabolism involves a broad and complex network of reactions. Although SLs are widely distributed in the body, it is well known that they are present in high concentrations within the central nervous system (CNS). Under physiological conditions, their abundance and distribution in the CNS depend on brain development and cell type. Consequently, SLs metabolism impairment may have a significant impact on the normal CNS function, and has been associated with several disorders, including sphingolipidoses, Parkinson's, and Alzheimer's. This review summarizes the main SLs characteristics and current knowledge about synthesis, catabolism, regulatory pathways, and their role in physiological and pathological scenarios in the CNS.
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Esfingolipidosis , Esfingolípidos , Sistema Nervioso Central/metabolismo , Humanos , Metabolismo de los Lípidos , Esfingolipidosis/metabolismo , Esfingolípidos/metabolismoRESUMEN
Resumen Introducción: Las investigaciones sobre violencia en la pareja se han centrado principalmente en el estudio de las parejas heterosexuales y son escasos los datos sobre la violencia de parejas del mismo sexo en el noviazgo. Objetivo: Analizar la violencia intragénero entre parejas homosexuales en universitarios de Bucaramanga. Materiales y métodos: Investigación cuantitativa con muestreo tipo bola de nieve mediante el cual se obtuvo una población de 132 participantes mayores de 18 años. Se aplicó el instrumento pre-validado Lista de Chequeo de Experiencias de Maltrato en la Pareja. Los datos se analizaron con el software SPSS, versión 23, mediante la prueba X 2 y ANOVA Unidireccional, considerando un α=0,05. Resultados: El 91,7% de los participantes fue violentado por lo menos con una de las conductas de estudio, la violencia predominante en las parejas fue la psicológica, seguida de la violencia emocional, la violencia física, la violencia sexual y, por último, la violencia económica. Se encontraron diferencias significativas para algunos ítems del instrumento entre hombres y mujeres. Conclusiones: La violencia psicológica tuvo mayor presencia en los participantes.
Abstract Introduction: Research on partner violence has mainly focused on studies of heterosexual couples, while data on same-sex dating violence are scarce. Objective: To analyze intra-gender violence in homosexual university couples in Bucaramanga. Materials and methods: A quantitative research with a snowball sampling approach was applied to obtain a population of 132 participants who were older than 18 years of age. The pre-validated instrument Check List for Partner Abuse Experience was used. Data were analyzed with SPSS software (version 23), using the X2 test and one-way ANOVA, considering an α=0.05. Results: 91.7% of participants experience violence with at least one of the studied behaviors. Psychological violence was the most predominant form in the studied couples, followed by emotional, physical, sexual, and economic violence. Significant differences between males and females were found for some items of the instrument. Conclusions: Psychological violence was the most frequent in the study's participants.
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Violencia , Minorías Sexuales y de Género , EstudiantesRESUMEN
GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.
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Hexosaminidasas , Enfermedad de Sandhoff , Fibroblastos , Humanos , Lisosomas , Saccharomycetales , Enfermedad de Sandhoff/tratamiento farmacológico , Enfermedad de Sandhoff/genéticaRESUMEN
GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.
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Proteína Activadora de G (M2)/genética , Gangliosidosis GM2/patología , beta-N-Acetilhexosaminidasas/genética , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapéutico , Barrera Hematoencefálica , Ensayos Clínicos como Asunto , Dieta Cetogénica , Gangliósido G(M2)/metabolismo , Gangliosidosis GM2/genética , Gangliosidosis GM2/metabolismo , Gangliosidosis GM2/terapia , Terapia Genética , Humanos , Mutación , Pirimetamina/uso terapéutico , Trasplante de Células MadreRESUMEN
Lysosomal storage disorders (LSDs) are a group of monogenic diseases characterized by progressive accumulation of undegraded substrates into the lysosome, due to mutations in genes that encode for proteins involved in normal lysosomal function. In recent years, several approaches have been explored to find effective and successful therapies, including enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, hematopoietic stem cell transplantation, and gene therapy. In the case of gene therapy, genome editing technologies have opened new horizons to accelerate the development of novel treatment alternatives for LSD patients. In this review, we discuss the current therapies for this group of disorders and present a detailed description of major genome editing technologies, as well as the most recent advances in the treatment of LSDs. We will further highlight the challenges and current bioethical debates of genome editing.
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Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/genética , Lisosomas/genética , Animales , Edición Génica/métodos , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Proteínas/genéticaRESUMEN
Lysosomal storage diseases (LSDs) are a group of about 50 inborn errors of metabolism characterized by the lysosomal accumulation of partially or non-degraded molecules due to mutations in proteins involved in the degradation of macromolecules, transport, lysosomal biogenesis or modulators of lysosomal environment. Significant advances have been achieved in the diagnosis, management, and treatment of LSDs patients. In terms of approved therapies, these include enzyme replacement therapy (ERT), substrate reduction therapy, hematopoietic stem cell transplantation, and pharmacological chaperone therapy. In this review, we summarize the Colombian experience in LSDs thorough the evidence published. We identified 113 articles published between 1995 and 2019 that included Colombian researchers or physicians, and which were mainly focused in Mucopolysaccharidoses, Pompe disease, Gaucher disease, Fabry disease, and Tay-Sachs and Sandhoff diseases. Most of these articles focused on basic research, clinical cases, and mutation reports. Noteworthy, implementation of the enzyme assay in dried blood samples, led to a 5-fold increase in the identification of LSD patients, suggesting that these disorders still remain undiagnosed in the country. We consider that the information presented in this review will contribute to the knowledge of a broad spectrum of LSDs in Colombia and will also contribute to the development of public policies and the identification of research opportunities.
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The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system.
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Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Colombia/epidemiología , Humanos , Errores Innatos del Metabolismo/epidemiología , Enfermedades Raras/diagnóstico , Enfermedades Raras/epidemiologíaRESUMEN
Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.
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Iduronato Sulfatasa/genética , Iduronato Sulfatasa/metabolismo , Lisosomas/enzimología , Pichia/genética , Animales , Biomasa , Reactores Biológicos , Western Blotting , Células CHO , Codón , Cricetulus , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Fermentación , Células HEK293 , Semivida , Humanos , Concentración de Iones de Hidrógeno , Iduronato Sulfatasa/aislamiento & purificación , Oxígeno/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Introducción. La temida complicación de la tiroidectomía es la parálisis de las cuerdas vocales secundaria a lesiones del nervio laríngeo recurrente. En este estudio se analiza una técnica de reconstrucción para estas lesiones neurales. Objetivo. Describir los resultados funcionales de la reconstrucción inmediata de las lesiones del nervio laríngeo recurrente con la técnica de Horsley. Material y métodos. Se llevó a cabo un estudio prospectivo entre enero del 2000 y diciembre del 2015, en pacientes con sección del nervio laríngeo recurrente y reconstrucción de Horsley, en el cual se evalúan: a) los índices del análisis acústico de voz [tiempo máximo de fonación, perturbación involuntaria de la frecuencia (jitter), perturbación de la amplitud (shimmer) y frecuencia fundamental], b) los hallazgos estroboscópicos, y c) el índice de discapacidad vocal. El análisis estadístico se hizo con la prueba exacta de Fisher y con el programa SPSS™. Resultados. Se practicaron 1.547 tiroidectomías y se produjeron 10 secciones del nervio laríngeo recurrente (0,64 %): dos (0,12 %) inadvertidas (p=0,0001) y 8 (0,51 %) advertidas por infiltración tumoral. En los exámenes de la calidad de voz, se encontraron: frecuencias fundamentales bajas con medias de 104,79 ± 0,29 Hz en hombres (valor de referencia, VR=141,74) y de 208,12 ± 22,72 Hz en mujeres (VR=241,08), que se correlaciona con un jitter de 1,39 ± 0,99 % (VR=1,04); y también, disminución del tiempo máximo de fonación (media=10,9 ± 3,07 s). El índice de percepción de calidad de la voz fue de discapacidad leve de la voz (22,7 ± 11,8). La estroboscopia mostró cierre completo de la glotis en nueve pacientes (90 %) (p=0,005), con una posición adecuada de los cartílagos aritenoides, en siete. Conclusiones. La tasa de lesión inadvertida del nervio laríngeo recurrente en el Hospital Militar Central es de 0,12 %. La técnica de Horsley tiene unos resultados funcionales satisfactorios en el 90 % de los casos
Background. The most feared complication of thyroidectomy is the vocal cord palsy secondary to injury of the recurrent laryngeal nerve. In this study we analize the Horsley technique for reconstruction for this surgical injury. Objective. The aim of this study was to describe the functional outcomes of the reconstruction of the recurrent laryngeal nerve by the Horsley technique. Materials and methods. A prospective study including patients with section of the recurrent laryngeal nerve and the use of the of the Horsley technique for reconstruction was carried out in the period January 2000 to December 2015. The outcomes evaluated were: a) acoustic voice analysis indexes (maximum phonation time, involuntary disturbance of frequency (jitter), disturbance of amplitude (shimmer), and fundamental frequency); b) stroboscopic findings; and c) vocal disability index. The Fisher's exact test and the SPSS™ program were used for the statistical analysis. Results.The study included 1,547 thyroidectomies with 10 complete sections of the recurrent laringeal nerve (0.64%), 2 unnoticed injuries (0.12%) (p=0,0001), and 8 injuries identified intraoperatively in patients with tumor infiltration. In the voice quality test we found: low fundamental frequencies with median values of 104.79 ± 0, Hz in the male population (reference value, RV=141,74) and 208,12 ± 22,72 Hz in the female population (RV=241,08), wich correlates with a jitter of 1,39 ± 0,99% (RV=1,04) and with a decrease in maximum phonation time (median=10,9 ±3,07s). Index of perception of voice quality was mild voice disability (22,7 ± 11,8). Stroboscopy showed complete clossure of glottis in 9 patients (90%) (p=0,005), with an adequate position of the arytenoid cartilages in 7 patients. Conclusions. The rate of unnoticed injuries of recurrent laringeal nerve at Central Military Hospital in Bogotá, Colombia, is 0.12%. The Horsley reconstruction technique demonstrated satisfactory functional results in 90% of cases
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Humanos , Tiroidectomía , Nervio Laríngeo Recurrente , Traumatismos del Nervio Laríngeo Recurrente , Parálisis de los Pliegues VocalesRESUMEN
Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L-1. The enzyme extract showed optimum pH and temperature of 5.0-6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min-1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.
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Abstract β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant α-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.
Resumen Las β-hexosaminidasas (Hex) son enzimas diméricas involucradas en la degradación lisosomal de glicolípidos y glicanos. Estas enzimas están formadas por las subunidades α- y/o β-codificadas por los genes HEXA and HEXB respectivamente. Las mutaciones de estos genes conducen a las enfermedades de Tay Sachs o Sandhoff, que son desórdenes neurodegenerativos causados por la acumulación de glicolípidos no degradados. Aunque las Hex derivadas de tejido han sido ampliamente caracterizadas, la información disponible sobre las p-hexosaminidasas recombinantes es limitada. En este estudio se produjeron Hex recombinantes lisosomales (rhHex-A, rhHex-B y rhHex-S) en la levadura metilotrófica Pichia pastoris GS115. Las actividades específicas más altas de las enzimas fueron 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Estos resultados fueron 25 a 50 veces más altos que los obtenidos de leucocitos humanos normales. Las proteínas se purificaron y se caracterizaron a diferentes condiciones de pH y temperatura. Todas las proteínas fueron estables a pH ácido y a 4°C y 37°C. A 45°C la rhHex-S se inactivó completamente, mientras que rhHex-A y rhHex-B mostraron alta estabilidad. Este estudio demuestra el potencial de P. pastoris GS115 para la producción de enzimas lisosomales poliméricas y presenta la caracterización de distintas β-hexosaminidasas recombinantes producidas en un único hospedero.
Resumen As β-hexosaminidases (Hex) são enzimas diméricas envolvidas na degradação lisossomal de glicolipídeos e glicanos. Essas enzimas são formadas por subunidades a- e/ou p-codificadas pelos genes HEXA e HEXB, respectivamente. As mutações nesses genes causam a doença de Sandhoff ou Tay Sachs, que são desordens neurodegenerativas causadas pela acumulação de glicolipídeos não degradados. Embora Hex derivadas de tecido hajam sido caracterizadas extensivamente, as informações disponíveis sobre as p-hexosaminidases recombinantes são limitadas. Esse estudo produziu Hex recombinantes lisossomais (rhHex-A, rhHex-B e rhHex-S) na levedura metilotrófica Pichia pastoris GS115. As atividades específicas mais altas das enzimas foram 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Esses resultados foram 25 a 50 vezes mais altos do que os obtidos a partir de leucócitos humanos normais. As proteínas foram purificadas e caracterizadas em diferentes condições de pH e temperatura. Todas as proteínas foram estáveis a pH ácido e a 4°C e 37°C. A 45°C a rhHex-S foi completamente inativada, enquanto rhHex rhHex-A e B se mostraram altamente estáveis. Esse estudo demonstra o potencial de P. pastoris GS115 para a produção de enzimas lisossomais poliméricas e apresenta a caracterização de diferentes p-hexosaminidases recombinantes produzidas em único hospedeiro.
RESUMEN
Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.
Asunto(s)
Condroitinsulfatasas/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Células Cultivadas , Condroitinsulfatasas/química , Condroitinsulfatasas/genética , Condroitinsulfatasas/aislamiento & purificación , Endocitosis , Estabilidad de Enzimas , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Pichia/genética , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sulfatasas/genética , Sulfatasas/metabolismo , TemperaturaRESUMEN
Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.