Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

Distinct human stem cell subpopulations drive adipogenesis and fibrosis in musculoskeletal injury.

Fibroadipogenic progenitors (FAPs) maintain healthy skeletal muscle in homeostasis but drive muscle degeneration in chronic injuries by promoting adipogenesis and fibrosis. To uncover how these stem cells switch from a pro-regenerative to pro-degenerative role we perform single-cell mRNA sequencing of human FAPs from healthy and injured human muscles across a spectrum of injury, focusing on rotator cuff tears. We identify multiple subpopulations with progenitor, adipogenic, or fibrogenic gene signatures. We utilize full spectrum flow cytometry to identify distinct FAP subpopulations based on highly multiplexed protein expression. Injury severity increases adipogenic commitment of FAP subpopulations and is driven by the downregulation of DLK1. Treatment of FAPs both in vitro and in vivo with DLK1 reduces adipogenesis and fatty infiltration, suggesting that during injury, reduced DLK1 within a subpopulation of FAPs may drive degeneration. This work highlights how stem cells perform varied functions depending on tissue context, by dynamically regulating subpopulation fate commitment, which can be targeted improve patient outcomes after injury.

Analysis of Building Accessibility Using Inertial and Optical Sensors.

The inclusive use of urban spaces necessitates detailed knowledge of the accessibility of public buildings or places where educational, health or administrative services are provided. Despite the improvements already made in many cities regarding architectural work, further changes to public buildings and other spaces, such as old buildings or areas of historical importance, are still required. To study this problem, we developed a model based on photogrammetric techniques and the use of inertial and optical sensors. The model allowed us to perform a detailed analysis of urban routes in the surroundings of an administrative building, by means of mathematical analysis of pedestrian routes. It was applied to the specific case of people with reduced mobility and included analysis of the building accessibility as well as detection of suitable transit routes, deterioration of the road surfaces and the presence of architectural obstacles on the route.

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome.

Background: Andersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel. Methods and Results: We identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations. Conclusion: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias.

Gender and Racial Diversity Among the Head Medical and Athletic Training Staff of Women's Professional Sports Leagues.

Background: Despite increased awareness for promoting diversity, orthopaedics remains one of the least diverse specialties. Studying health care providers in women's professional sports provides a unique opportunity to analyze gender and racial diversity. Hypotheses: There would be low female and minority representation across the various women's professional sports leagues. There would be an increased number of female head certified athletic trainers (ATCs) when compared with head team physicians (HTPs). Study Design: Cross-sectional study. Methods: We evaluated the perceived race and sex of designated HTPs and ATCs in the Women's National Basketball Association, National Women's Soccer League, and National Women's Hockey League. Type of doctorate degree, specialty, and years in practice were also collected. Kappa (κ) coefficient measurements were used to determine interobserver agreement on race. Categorical and continuous variables were analyzed using chi-square and t tests, respectively. Results: There were significantly more female ATCs than female HTPs (74.1% vs 37.5%; P = .01). Minority representation between HTPs and ATCs was not significantly different (20.8% vs 40.7%; P = .13). Black HTPs (12.5%) and Black ATCs (22.2%) composed the largest proportion among the minority groups. There was high interobserver agreement of perceived race across HTPs (κ = 1.0) and ATCs (κ = 0.95). Conclusion: Although there were more female ATCs than HTPs in women's professional sports leagues, both cohorts lack perceived racial diversity. These data suggest an opportunity for diversification in medical and training staff of women's professional sports.

Current Perceptions of Diversity Among Head Team Physicians and Head Athletic Trainers: Results Across US Professional Sports Leagues.

BACKGROUND: Discrepancies in race, ethnicity, and sex among health care providers and their patients have been shown to affect the patient-provider relationship as well as the quality of care. Currently, minority and female representation among orthopaedic surgeons remains low. Given the large proportion of minority athletes and their degree of public visibility, professional sports serves as an important arena within which to analyze the diversity of health care providers. PURPOSE: To describe and evaluate the current level of diversity of head team physicians (HTPs) and head athletic trainers (ATCs), primarily in terms of race and sex, within men's professional sports leagues in the United States. STUDY DESIGN: Cross-sectional study. METHODS: Five major US professional sports leagues were evaluated: National Basketball Association, National Football League, National Hockey League, Major League Soccer, and Major League Baseball. Publicly available data were collected to identify the HTPs and head ATCs for each team within these leagues. Two independent observers analyzed photographs and names of these individuals to determine his or her perceived race and sex, with disagreements being resolved by a third independent observer. Other physician data collected included graduate degree(s), specialty, and number of years in practice. Kappa coefficients (κ) were employed to evaluate interobserver reliability. Chi-square, Fisher exact, and t tests were used for statistical comparisons across leagues. RESULTS: The κ values for perceived race were 0.85 for HTPs and 0.89 for head ATCs, representing near-perfect interobserver agreement. Minorities comprised 15.5% of HTPs and 20.7% of ATCs (P = .24). Women comprised 3.9% of HTPs and 1.3% of head ATCs (P = .017). The majority of HTPs were orthopaedic surgeons with medical doctorates. Female HTPs had significantly fewer years in practice compared with male HTPs (15.0 ± 4.9 vs 23.1 ± 9.6; P = .04). CONCLUSION: The lead physicians and athletic training providers for men's professional sports teams demonstrated low rates of minority and female representation, denoting a highly visible area for discussing the role of increased diversity in health care.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

Paraspinal muscle degeneration and regenerative potential in a Murine model of Lumbar Disc Injury.

BACKGROUND CONTEXT: The association between low back pain and lumbar disc degeneration is not fully characterized. One potentially overlooked factor of this process is the lumbar multifidus, which plays a role in segmental stabilization and locomotion. Previous reports have shown multifidus degeneration to be associated with disc degeneration. The goal of this study was to develop a mouse model of advanced lumbar disc degeneration to recapitulate the pathology of the human multifidus in patients with lumbar disc degeneration and low back pain. METHODS: C57BL/6 mice underwent a left anterolateral approach to the lumbar spine and disc puncture with a micro scalpel at L5/6 and L6/S1. Mice underwent behavioral analysis and functional gait testing. A subset of mice underwent 14T T2-weighted MRI to assess disc degeneration and paraspinal muscle quality. At 6 and 15 weeks, mice were sacrificed, and the multifidus muscles were harvested and divided into proximal and distal segments relative to disc injury. Histological analysis was performed to assess muscle degeneration, fiber type, and macrophage density. Fibroadipogenic progenitors (FAPs) were isolated for gene expression analysis with qPCR. RESULTS: MRI demonstrated decreased intervertebral disc signal and paraspinal muscle atrophy at 6 weeks, with progressive degeneration and atrophy at 15 weeks. Disc injury resulted in delayed functional recovery and impaired gait. Histology demonstrated progressive multifidus fibrofatty degeneration between 6 and 15 weeks. CD68+ macrophage density was increased at 6 weeks but not 15 weeks. FAPs exhibited increased fibrotic and adipogenic gene expression at 6 weeks compared to sham with less of a difference in gene expression by 15 weeks. CONCLUSIONS: We have developed a mouse model of disc injury-mediated paraspinal muscle degeneration that recapitulates features of degenerative muscle pathology observed in patients with lumbar disc degeneration, and highlights the role of FAPs in mediating fibrofatty muscle degeneration after disc injury.

Rotator cuff tear degeneration and the role of fibro-adipogenic progenitors.

The high prevalence of rotator cuff tears poses challenges to individual patients and the healthcare system at large. This orthopedic injury is complicated further by high rates of retear after surgical repair. Outcomes following repair are highly dependent upon the quality of the injured rotator cuff muscles, and it is, therefore, crucial that the pathophysiology of rotator cuff degeneration continues to be explored. Fibro-adipogenic progenitors, a major population of resident muscle stem cells, have emerged as the main source of intramuscular fibrosis and fatty infiltration, both of which are key features of rotator cuff muscle degeneration. Improvements to rotator cuff repair outcomes will likely require addressing the muscle pathology produced by these cells. The aim of this review is to summarize the current rotator cuff degeneration assessment tools, the effects of poor muscle quality on patient outcomes, the role of fibro-adipogenic progenitors in mediating muscle pathology, and how these cells could be leveraged for potential therapeutics to augment current rotator cuff surgical and rehabilitative strategies.

Neurocirugía transicional: ¿una nueva subespecialidad? / Transitional neurosurgery: a new subspecialty?

Introducción: El proceso de transición de un paciente de la etapa pediátrica a la adulta es un proceso dinámico, complejo y planificado, que incluye la transferencia propiamente dicha de una institución pediátrica a una adulta. El aumento de la sobrevida de niños y adolescentes con patologías crónicas, la falta de acuerdos formales entre instituciones de salud y la falta de enfoque multidisciplinario de estos casos son los principales problemas a tener en cuenta. El objetivo del presente trabajo es describir y proponer una respuesta a las situaciones y dificultades que se encuentran en la actualidad en la salud pública durante el proceso de transición de pacientes pediátricos con patología neuroquirúrgica crónica y de pacientes adultos con patología congénita. Para tal fin se deben considerar factores sociales, económicos y comunicacionales. Material y Método: Estudio observacional, transversal y retrospectivo de pacientes con patología neuroquirúrgica transicional desde el 01 de enero de 2017 al 31 de diciembre de 2018. En total se revisaron las historias clínicas de 47 pacientes del Hospital "El Cruce". Resultados: De los 47 pacientes observados, con un rango etario entre 17 y 42 años, realizamos una división de éstos en 2 grupos: GRUPO 1: pacientes adultos operados en la edad pediátrica que requieren un seguimiento crónico de su patología de origen; y GRUPO 2: pacientes adultos con patología congénita. En el GRUPO 1, observamos 38 pacientes (24 masculinos y 14 femeninos) de los cuales 24 fueron operados y 14 no operados (solo seguimiento clínico). Dentro de este grupo, el 63% de los pacientes (n=29) presentó como su patología de base para su seguimiento la hidrocefalia. En el GRUPO 2, observamos a 9 pacientes (2 masculinos y 7 femeninos) de los cuales 7 fueron operados y 2 no operados. Todos los pacientes de este último grupo presentaron como diagnóstico de base un disrafismo espinal. Conclusión: Se debe lograr una transición planificada para el bienestar de los jóvenes con necesidades especiales de atención de salud. Actualmente no hay acuerdos interinstitucionales formales para el seguimiento y atención de estos pacientes.Consideramos que existe un grupo de pacientes que se beneficiarían con la creación de una nueva subespecialidad neuroquirúrgica: la neurocirugía transicional. La misma debería desarrollarse en hospitales generales, de alta complejidad, donde coincidan en el servicio de neurocirugía de adultos, neurocirujanos con formación pediátrica

Introduction: The transition process of a patient from pediatric to adult stage is a dynamic, complex, and planned process which, strictly speaking, includes the transfer from a pediatric to an adult institution. The increased rate of survival of children and adolescents with chronic pathologies, lack of formal agreements between health institutions and lack of multidisciplinary approach to these cases are the main issues to consider. The purpose of this paper is to describe and provide a response to situations and difficulties that currently exist in the public health during the transition process of pediatric patients with chronic neurosurgical pathology and adult patients with congenital pathology. For this purpose, communication, social, and economic factors must be considered. Methods: Observational, cross-sectional, and retrospective study of patients with transitional neurosurgical pathology from January 1, 2017, to December 31, 2018. In total, 47 patient's medical records were reviewed from "El Cruce" Hospital. Results: A total of 47 patients, with an age range between 17 and 42 years, were observed. We divided our study population into 2 groups: GROUP 1: adult patients who have been operated in pediatric age and require chronic follow-up of their origin pathology; and GROUP 2: adult patients with congenital pathology. In GROUP 1, we observed 38 patients (24 male and 14 female) of whom 24 were performed neurosurgery, and 14 were not. Within this group, 63% of the patients (n = 29) presented hydrocephalus as their basic pathology for monitoring. In GROUP 2, we observed 9 patients (2 male and 7 female) of whom 7 were operated and 2 were not. All patients in this last group presented spinal dysraphism as their basic diagnosis. Conclusion: A planned transition for the well-being of young patients with special health care needs must be achieved. Currently, there are no formal institutional agreements for the monitoring and care of these patients. We believe that there is a group of patients who would benefit from the creation of a new neurosurgical sub-specialty: transitional neurosurgery. It should be developed in high complexity general hospitals, where neurosurgeons with pediatric training could be part of a general neurosurgery department.

Novel Simulation Model with Pulsatile Flow System for Microvascular Training, Research, and Improving Patient Surgical Outcomes.

BACKGROUND: Simulation allows surgical trainees to acquire surgical skills in a safe environment. With the aim of reducing the use of animal experimentation, different alternative nonliving models have been pursued. However, one of the main disadvantages of these nonliving models has been the absence of arterial flow, pulsation, and the ability to integrate both during a procedure on a blood vessel. In the present report, we have introduced a microvascular surgery simulation training model that uses a fiscally responsible and replicable pulsatile flow system. METHODS: We connected 30 human placentas to a pulsatile flow system and used them to simulate aneurysm clipping and vascular anastomosis. RESULTS: The presence of the pulsatile flow system allowed for the simulation of a hydrodynamic mechanism similar to that found in real life. In the aneurysm simulation, the arterial flow could be evaluated before and after clipping the aneurysm using a Doppler ultrasound system. When practicing anastomosis, the use of the pulsatile flow system allowed us to assess the vascular flow through the anastomosis, with verification using the Doppler ultrasound system. Leaks were manifested as "blood" pulsatile ejections and were more frequent at the beginning of the surgical practice, showing a learning curve. CONCLUSIONS: We have provided a step-by-step guide for the assembly of a replicable and inexpensive pulsatile flow system and its use in placentas for the simulation of, and training in, performing different types of anastomoses and intracranial aneurysms surgery.

Risk factors for periprosthetic joint infection after total knee arthroplasty.

INTRODUCTION: Periprosthetic joint infection (PJI) is the most serious and feared complication in total knee arthroplasty (TKA) and can have catastrophic consequences. The number of total knee arthroplasties is increasing, so infections could also be greater in the future. The aim of this study is to identify the most relevant risk factors associated with infection after a total knee arthroplasty. METHODS: This is a case-control study of patients who underwent total knee arthroplasty at the University Hospital of Salamanca. We included 66 TKA PJI patients and 66 control TKA patients. Demographic and clinical variables were collected. A descriptive and inferential analysis was performed by logistic regression and attributable risk fraction assessed. RESULTS: Prolonged operative time (> 90') and tourniquet time (> 60') were the most relevant risk factors described (OR 40.77, AFE 0.97, p > 0.001 and OR 37.14, AFE 0.97, p < 0.001, respectively). The use of non-antibiotic-laded cement (OR 3.62), obesity (BMI > 30, OR 8.86), diabetes (OR 2.33), high ASA grade (III-IV, OR 15.30), and blood transfusion requirement (OR 4.60) were also statistically significant risk factors for TKA PJI. CONCLUSIONS: Our study provides evidence concerning that operative time, tourniquet time, cement type, diabetes, obesity, ASA grade, and blood transfusion requirement as independently associated risk factors for TKA PJI. Modifiable risk factors were specifically relevant, so we should be able to reduce the infection rate.

Author Correction: Combined quantification of procalcitonin and HLA-DR improves sepsis detection in surgical patients.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase.

BACKGROUND: The recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market. RESULTS: The gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium. CONCLUSIONS: A K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.

Combined quantification of procalcitonin and HLA-DR improves sepsis detection in surgical patients.

Early recognition of sepsis is a key factor to improve survival to this disease in surgical patients, since it allows prompt control of the infectious source. Combining pro-inflammatory and immunosupression biomarkers could represent a good strategy to improve sepsis detection. Here we evaluated the combination of procalcitonin (PCT) with gene expression levels of HLA-DRA to detect sepsis in a cohort of 154 surgical patients (101 with sepsis and 53 with no infection). HLA-DRA expression was quantified using droplet digital PCR, a next-generation PCR technology. Area under the receiver operating curve analysis (AUROC) showed that the PCT/HLA-DRA ratio outperformed PCT to detect sepsis (AUROC [CI95%], p): PCT: 0.80 [0.73-0.88], <0.001; PCT/HLA-DRA: 0.85 [0.78-0.91], <0.001. In the multivariate analysis, the ratio showed a superior ability to predict sepsis compared to that of PCT (OR [CI 95%], p): PCT/HLA-DRA: 7.66 [1.82-32.29], 0.006; PCT: 4.21 [1.15-15.43] 0.030. Multivariate analysis was confirmed using a new surgical cohort with 74 sepsis patients and 21 controls: PCT/HLA-DRA: 34.86 [1.22-995.08], 0.038; PCT: 5.52 [0.40-75.78], 0.201. In conclusion, the combination of PCT with HLA-DRA is a promising strategy for improving sepsis detection in surgical patients.

Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring.

Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

Structural features of Aspergillus niger ß-galactosidase define its activity against glycoside linkages.

ß-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of ß-galactosides. Among them, the Aspergillus niger ß-galactosidase (AnßGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnßGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnßGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides production than the Aspergillus oryzae ß-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 ß-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design. DATABASE: Structural data are available in PDB database under the accession numbers 5IFP (native form), 5IHR (in complex with 6GalGlu), 5IFT (in complex with 3GalGlu), 5JUV (in complex with 6GalGal), 5MGC (in complex with 4GalLac), and 5MGD (in complex with 6GalLac).

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

Kluyveromyces lactis ß-galactosidase (Kl-ß-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-ß-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-ß-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-ß-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-ß-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-ß-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.