A close-up view of the Hunter syndrome.

The Lysosomal Storage disease known as Mucopolysaccharidosis type II, is caused by mutations affecting the iduronate-2-sulfatase required for heparan and dermatan sulfate catabolism. The central nervous system (CNS) is mostly and severely affected by the accumulation of both substrates. The complexity of the CNS damage observed in MPS II patients has been limitedly explored. The use of mass spectrometry (MS)-based proteomics tools to identify protein profiles may yield valuable information about the pathological mechanisms of Hunter syndrome. In this further study, we provide a new comparative proteomic analysis of MPS II models by using a pipeline consisting of the identification of native protein complexes positioned selectively by using a specific antibody, coupled with mass spectrometry analysis, allowing us to identify changes involving in a significant number of new biological functions, including a specific brain antioxidant response, a down-regulated autophagic, the suppression of sulfur catabolic process, a prominent liver immune response and the stimulation of phagocytosis among others.

Characterization and expression of domains of Alphaherpesvirus bovine 1/5 envelope glycoproteins B in Komagataella phaffi.

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.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

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.

Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.

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.

Sphingolipids and their role in health and disease in the central nervous system.

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.

Effect of two preservation methods on the viability and enzyme production of a recombinant Komagataella phaffii (Pichia pastoris) strain.

The methylotrophic yeast Komagataella phaffii, previously known as Pichia pastoris, has been reported as a host for producing human recombinant lysosomal enzymes intended for enzyme replacement therapy. K. phaffii has advantages such as easy genetic handling, rapid growth, cost-effective mediums, and the ability to develop mammalian-like post-translational modifications. To maintain cell viability and enzyme activity over time, it is important to consider the bioprocess optimization and the proper selection and preservation of clones. In this study, we evaluated the effect of glycerol and skim milk in cryopreservation at -80 °C, as well as the use of skim milk or its combination with NaCl, disaccharides or sorbitol in freeze-drying on the cell viability and activity of a recombinant lysosomal enzyme (i.e., human ß-hexosaminidase-A) produced in K. phaffii GS115 strain. The results showed that cryopreservation with glycerol and skim milk, as well as freeze-drying using disaccharides and sorbitol with skim milk, maintained the viability above 80%. Although variations in enzyme activity among treatments were found, the use of disaccharides had a positive effect on the enzymatic activity levels. This is the first report of the evaluation of two suitable methods to preserve a recombinant K. phaffii strain, preventing the loss of viability and maintaining the activity of the recombinant protein.

Violencia intragénero entre parejas homosexuales en universitarios de Bucaramanga, Colombia / Intra-gender violence in homosexual university students from Bucaramanga, Colombia

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.

Human recombinant lysosomal ß-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

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.

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

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.

Lysosomal storage diseases: current therapies and future alternatives.

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.

A perspective on research, diagnosis, and management of lysosomal storage disorders in Colombia.

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.

Therapeutic Options for Mucopolysaccharidoses: Current and Emerging Treatments.

Mucopolysaccharidoses (MPS) are inborn errors of metabolism produced by a deficiency of one of the enzymes involved in the degradation of glycosaminoglycans (GAGs). Although taken separately, each type is rare. As a group, MPS are relatively frequent, with an overall estimated incidence of around 1 in 20,000-25,000 births. Development of therapeutic options for MPS, including hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT), has modified the natural history of many MPS types. In spite of the improvement in some tissues and organs, significant challenges remain unsolved, including blood-brain barrier (BBB) penetration and treatment of lesions in avascular cartilage, heart valves, and corneas. Newer approaches, such as intrathecal ERT, ERT with fusion proteins to cross the BBB, gene therapy, substrate reduction therapy (SRT), chaperone therapy, and some combination of these strategies may provide better outcomes for MPS patients in the near future. As early diagnosis and early treatment are imperative to improve therapeutic efficacy, the inclusion of MPS in newborn screening programs should enhance the potential impact of treatment in reducing the morbidity associated with MPS diseases. In this review, we evaluate available treatments, including ERT and HSCT, and future treatments, such as gene therapy, SRT, and chaperone therapy, and describe the advantages and disadvantages. We also assess the current clinical endpoints and biomarkers used in clinical trials.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme. In this study, we further studied the therapeutic potential of prGALNS through the characterization of the N-glycosylation structure, in vitro cell uptake and keratan sulfate reduction, and in vivo biodistribution and generation of anti-prGALNS antibodies. Taken together, these results represent an important step in the development of a P. pastoris-based platform for production of a therapeutic GALNS for MPS IVA enzyme replacement therapy.

Neural stem cells for disease modeling and evaluation of therapeutics for Tay-Sachs disease.

BACKGROUND: Tay-Sachs disease (TSD) is a rare neurodegenerative disorder caused by autosomal recessive mutations in the HEXA gene on chromosome 15 that encodes ß-hexosaminidase. Deficiency in HEXA results in accumulation of GM2 ganglioside, a glycosphingolipid, in lysosomes. Currently, there is no effective treatment for TSD. RESULTS: We generated induced pluripotent stem cells (iPSCs) from two TSD patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). The TSD neural stem cells exhibited a disease phenotype of lysosomal lipid accumulation. The Tay-Sachs disease NSCs were then used to evaluate the therapeutic effects of enzyme replacement therapy (ERT) with recombinant human Hex A protein and two small molecular compounds: hydroxypropyl-ß-cyclodextrin (HPßCD) and δ-tocopherol. Using this disease model, we observed reduction of lipid accumulation by employing enzyme replacement therapy as well as by the use of HPßCD and δ-tocopherol. CONCLUSION: Our results demonstrate that the Tay-Sachs disease NSCs possess the characteristic phenotype to serve as a cell-based disease model for study of the disease pathogenesis and evaluation of drug efficacy. The enzyme replacement therapy with recombinant Hex A protein and two small molecules (cyclodextrin and tocopherol) significantly ameliorated lipid accumulation in the Tay-Sachs disease cell model.

Research, diagnosis and education in inborn errors of metabolism in Colombia: 20 years' experience from a reference center.

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.

Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris.

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.

Premature Thelarche.

Premature thelarche is a benign condition that affects young girls and may be interpreted as a sign of central precocious puberty (CPP). Parental concern is common when breast development is noted in a young girl. It is important to differentiate premature thelarche from CPP, as the latter is a more serious disorder that may affect final adult height and menarcheal age, and may have psychological implications as well. Distinguishing between the two conditions clinically may help the patients avoid unnecessary testing. Pediatricians can play a pivotal role by providing reassurance to families and helping alleviate parental anxiety. This article reviews the clinical presentation of premature thelarche, its usual course, and implications. [Pediatr Ann. 2018;47(1):e12-e15.].

Técnica de Horsley para la reconstrucción del nervio laríngeo / Reconstruction of the laryngeal nerve by yhe Horsley technique

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

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus.

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.

Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS).

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.