Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins.

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide-homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.

Increased Selectivity of Novozym 435 in the Asymmetric Hydrolysis of a Substrate with High Hydrophobicity Through the Use of Deep Eutectic Solvents and High Substrate Concentrations.

The effects of the reaction medium and substrate concentration were studied on the selectivity of Novozym 435 using the asymmetric hydrolysis of dimethyl-3-phenylglutarate as a model reaction. Results show that the use of choline chloride ChCl:urea/phosphate buffer 50% (v/v) as a reaction medium increased the selectivity of Novozym 435 by 16% (e.e = 88%) with respect to the one in 100% phosphate buffer (e.e = 76%). Best results were obtained when high substrate concentrations (well above the solubility limit, 27-fold) and ChCl:urea/phosphate buffer 50% (v/v) as reaction medium at pH 7 and 30 °C were used. Under such conditions, the R-monoester was produced with an enantiomeric purity of 99%. Novozym 435 was more stable in ChCl:urea/phosphate buffer 50% (v/v) than in phosphate buffer, retaining a 50% of its initial activity after 27 h of incubation at pH 7 and 40 °C. Results suggest that the use of deep eutectic solvents (ChCl:urea/phosphate buffer) in an heterogeneous reaction system (high substrate concentration) is a viable and promising strategy for the synthesis of chiral drugs from highly hydrophobic substrates.

A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation.

The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (µ) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same µ at either 22 or 30 °C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10-10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.

Isquemia arterial aguda trombótica de los miembros inferiores / Acute thrombotic arterial ischemia of the lower limbs

Introducción: La enfermedad arterial periférica es una de las afecciones más prevalentes. Resulta habitual su coexistencia con la enfermedad vascular en otras localizaciones. El diagnóstico precoz tiene importancia para mejorar la calidad de vida del paciente y reducir el riesgo de eventos secundarios mayores, como el infarto agudo de miocardio o el ictus. Objetivo: Caracterizar el comportamiento de la isquemia arterial aguda trombótica en miembros inferiores en pacientes que ingresaron en el Instituto Nacional de Angiología y Cirugía Vascular. Métodos: Se realizó un estudio observacional, descriptivo y retrospectivo, con el fin de determinar factores pronósticos en la evolución final del tratamiento quirúrgico de la isquemia arterial aguda trombótica en miembros inferiores, en pacientes que ingresaron en los servicios de Arteriología y Angiopatía Diabética del Instituto Nacional de Angiología y Cirugía Vascular durante un período de cuatro años. Resultados: El grupo de edades más afectado estuvo entre 40 y 59 años, con un predomino del sexo masculino. El hábito de fumar fue el factor de riesgo vascular más frecuente; y la amputación mayor, el procedimiento quirúrgico más empleado, por lo que el patrón oclusivo femoropoplíteo resultó el más prevalente. Conclusiones: Predominaron el sexo masculino, el hábito de fumar, la amputación mayor y el patrón oclusivo femoropoplíteo(AU)

Introduction: Peripheral arterial disease is one of the most prevalent conditions. Its coexistence with vascular disease in other locations is common. Early diagnosis is important to improve the patient's quality of life and reduce the risk of major secondary events, such as acute myocardial infarction or stroke. Objective: To characterize the behavior of acute thrombotic arterial ischemia in lower limbs in patients admitted to the National Institute of Angiology and Vascular Surgery. Methods: An observational, descriptive and retrospective study was conducted in order to determine prognostic factors in the final evolution of surgical treatment of acute thrombotic arterial ischemia in the lower limbs in patients admitted to the Arteriology and Diabetic Angiopathy services of the National Institute of Angiology and Vascular Surgery for a period of four years. Results: The most affected age group was the one of 40 to 59 years, with a predominance of males. Smoking was the most frequent vascular risk factor; and major amputation, the most used surgical procedure, so the femoropopliteal occlusive pattern was the most prevalent. Conclusions: Male sex, smoking habit, major amputation and femoropopliteal occlusive pattern predominated(AU)

Aneurisma venoso yuxta-anastomótico en fístula arterio-venosa para hemodiálisis / Juxtaanastomotic venous aneurysm in arteriovenous fistula for hemodialysis

Introducción: A pesar del gran avance técnico que representan las fístulas arterio-venosas internas para la hemodiálisis, estas no están exentas de complicaciones que comprometen su durabilidad. Entre ellas se encuentran los aneurismas venosos yuxta-anastomóticos. Objetivo: Presentar el tratamiento quirúrgico utilizado para reparar los aneurismas venosos yuxta-anastomóticos. Presentación del caso: Paciente de 54 años, de género femenino y color de piel negra. Presentó antecedentes de padecer insuficiencia renal crónica de 12 años de evolución, con transplante renal fallido, la cual tuvo un aneurisma venoso yuxta-anastomótico, localizado en una fístula arterio-venosa húmero-cefálica en miembro superior izquierdo, que fue confirmado por eco-doppler. Se emplearon como tratamiento quirúrgico la aneurismectomía y el injerto por sustitución con prótesis vascular expandible de poli-tetrafluoretileno. Finalmente, se preservó la fístula arterio-venosa, así como su permeabilidad. Conclusiones: La evolución fue satisfactoria en cuanto a su durabilidad y utilización como vía de acceso para la hemodiálisis(AU)

Introduction: Despite the great technical advance represented by internal arterio-venous fistulas for hemodialysis, these are not exempt from complications that compromise their durability. Among them are juxtaanastomotic venous aneurysms. Objective: Present the surgical treatment used to repair juxtaanastomotic venous aneurysms. Case presentation: 54-year-old patient, female and black skin color. She presented a history of chronic renal failure of 12 years of evolution, with failed kidney transplant, which had a juxtaanastomotic venous aneurysm, located in a humerus-cephalic arterio-venous fistula in the left upper limb, which was confirmed by Doppler echo. Aneurysmectomy and replacement grafting with expandable polytetrafluorethylene vascular prostheses were used as surgical treatment. Finally, the arterio-venous fistula was preserved, as well as its permeability. Conclusions: The evolution was satisfactory in terms of durability and use as an access route for hemodialysis(AU)

Factores de riesgo cardiovasculares asociados a enfermedad arterial periférica de miembros inferiores en sus estadios iniciales / Cardiovascular risk factors associated with peripheral arterial disease of lower limbs in their initial stages

Introducción: La enfermedad arterial periférica en la actualidad se considera una verdadera epidemia. Se estima que puede afectar al 10 por ciento de los individuos mayores de 55 años. El principal problema de la enfermedad radica en su infravaloración diagnóstica y terapéutica, debido a la modificación de los factores de riesgo, el uso de fármacos antiplaquetarios y el tratamiento de los síntomas. Objetivo: Identificar los factores de riesgo cardiovasculares en pacientes ambulatorios con enfermedad arterial periférica de los miembros inferiores en sus estadios iniciales. Métodos: Se realizó un estudio descriptivo-retrospectivo de corte transversal en una muestra de 57 pacientes con diagnóstico de enfermedad arterial periférica de miembros inferiores en sus estadios iniciales, atendidos en consulta externa de Arteriología durante dos años. Las variables estudiadas fueron: clínicas y de laboratorio, y algunos de los factores de riesgo cardiovasculares como: edad, sexo, diabetes mellitus, hábito de fumar, hiperlipemia y micro albuminuria. Resultados: Se encontró un predominio del sexo masculino (57,8 por ciento). El 100 por ciento de los pacientes fumaban. Se evidenció la presencia de variables de laboratorio elevadas, lo que justificó la presencia de factores de riesgo cardiovasculares desde etapas tempranas de la enfermedad. Conclusiones: Se logró identificar a los pacientes ambulatorios en sus estadios iniciales y la presencia de algunos factores de riesgo cardiovasculares en etapas tempranas de la enfermedad(AU)

Introduction: Peripheral arterial disease is currently considered a true epidemic. Estimates indicate that it can affect 10 percent of individuals over 55 years of age. The main problem of the disease is its diagnostic and therapeutic underestimation, due to the modification of risk factors, the use of antiplatelet drugs, and the treatment of symptoms. Objective: To identify the cardiovascular risk factors in ambulatory patients with peripheral arterial disease of lower limbs in its initial stages. Methods: A cross-sectional descriptive-retrospective study was carried out in a sample of 57 patients with diagnosis of peripheral arterial disease of lower limbs in its initial stages and who attended an outpatient department of arteriology for two years. The variables studied included clinical ones and of laboratory, as well as some cardiovascular risk factors, such as age, sex, diabetes mellitus, smoking, hyperlipidemia, and microalbuminuria. Results: A predominance of the male sex was found (57.8 percent). 100 percent of the patients smoked. The presence of elevated laboratory variables was evidenced, a fact consistent with the presence of cardiovascular risk factors from the early stages of the disease. Conclusions: In the initial stages of the disease, ambulatory patients were identified, as well as some cardiovascular risk factors in early stages of the disease(AU)

Disección de la aorta abdominal a propósito de un caso / Abdominal aortic dissection apropos of a case

RESUMEN La disección de la aorta abdominal tiene una baja incidencia, se produce a partir de una laceración, desgarro o rotura intimal, con la consecuente separación longitudinal de las capas de la pared y la aparición de una falsa luz con flujo en su interior. Se presenta el caso de un hombre de 67 años de edad con antecedentes de hipertensión arterial, sin tratamiento regular, que acudió al cuerpo de guardia del Hospital Salvador Allende (La Habana, Cuba) por presentar dolor abdominal con irradiación a fosa ilíaca izquierda y espalda, de una semana de duración, que no aliviaba con los analgésicos habituales. Se le realizó ultrasonido abdominal y se encontró una dilatación aneurismática de la aorta abdominal, con signos de disección hacia la ilíaca derecha; por lo que se realizó angiotomografía que confirmó el diagnóstico. Se realizó baipás aorto-ilíaco con fenestración en aorta abdominal y anastomosis término-terminal en ambas arterias ilíacas.

ABSTRACT Abdominal aortic dissection has a low incidence. It may happen when a small tear or rupture occurs in the tunica intima, dividing the wall layers and forming a false channel, or lumen with blood flow inside. We present the case of a 67-year-old man with a history of high blood pressure, with no regular treatment, who sought care at the Hospital Salvador Allende (Havana, Cuba) as he presented with a week-long abdominal pain radiating to the left iliac fossa and back, which was not relieved by the usual analgesics. An abdominal ultrasound was performed which found an aneurysmal dilatation of the abdominal aorta, with signs of dissection towards the right iliac artery; therefore, computed tomography angiography (CTA) was performed and the diagnosis was confirmed. He underwent aorto-iliac bypass with abdominal-aortic-fenestration and end-to-end anastomosis in both iliac arteries.

Tratamiento endovascular del aneurisma de la aorta abdominal con endoprótesis percutáneas en pacientes cubanos / Endovascular treatment of abdominal aortic aneurysm with percutaneous endoprosthesis in Cuban patients

Introducción: Los aneurismas de aorta abdominal resultan dilataciones arteriales a dicho nivel. Su ruptura constituye uno de los principales riesgos y provoca la muerte del paciente, de ahí la importancia de tratarlos a tiempo. Las opciones actuales de tratamiento son la cirugía abierta y la reparación aneurismática endovascular; esta última representa la primera línea de tratamiento, por constituir una técnica mínimamente invasiva con bajas tasas de morbilidad y mortalidad. Objetivo: Evaluar el tratamiento endovascular del aneurisma de la aorta abdominal con endoprótesis percutáneas en pacientes cubanos. Métodos: Entre enero y abril de 2018 se realizó un estudio prospectivo, descriptivo y cualitativo en 6 pacientes del sexo masculino con aneurisma de aorta abdominal, seleccionados aleatoriamente, que cumplieron los criterios de uso de endoprótesis vasculares y estuvieron de acuerdo con realizar el proceder. Se colocaron 6 dispositivos en una sala de angiografía, y se utilizó en los pacientes anestesia general y guía fluoroscópica. Resultados: De forma satisfactoria fue posible el tratamiento endovascular por vía percutánea de los 6 pacientes seleccionados; solo 1 presentó una complicación menor y fue dado de alta a las 72 horas y el resto fue egresado en menos de 24 horas. Se logró implementar por vez primera en el país el sistema de cierre percutáneo Proglide sin complicaciones. Conclusiones: El tratamiento endovascular por vía percutánea de aneurismas de aorta abdominal con endoprótesis vasculares es una alternativa eficaz para la cirugía convencional en pacientes que cumplen los criterios de uso del dispositivo(AU)

Introduction: Abdominal aortic aneurysms are arterial dilations in that level. Their rupture is one of the main risks and it causes death in patients; that is why the importance of treating them early. Among the current treatment options are open surgery and endovascular aneurismal reparation, being the last one in the first line of treatment since it is a minimal invasive technique with low rates of morbidity and mortality. Objective: To evaluate the endovascular treatment of abdominal aortic aneurysm with percutaneous endoprosthesis in Cuban patients. Methods: It was conducted from January to April 2018 a retrospective, descriptive and qualitative study in 6 male patients with aneurysm of the abdominal aorta, selected randomly and who met the criteria of vascular endoprosthesis use and agreed with the performance of the procedure. Six devices were placed in an angiography room and it was used general anaesthetic and fluoroscopic guidance. Results: It was possible in a satisfactory way the endovascular treatment by percutaneous way in the six patients selected; just one presented a minor complication and he was discharged after 72 hours; and the rest were discharged in less than 24 hours. It was implemented for the first time in the country without complications the system of percutaneous closure called Proglide. Conclusions: Endovascular treatment by percutaneous way of abdominal aortic aneurysms with vascular endoprosthesis is an efficient alternative for conventional surgery in patients that met the criteria of use of the device(AU)

Improved catalytic properties of immobilized lipases by the presence of very low concentrations of detergents in the reaction medium.

The addition of a very small concentration of a detergent (in many instances under the critical micellar concentration (cmc)) has been found to greatly increase the activity of immobilized lipases, using those from Pseudomonas fluorescens (PFL) and Candida antarctica (isoform B) as model enzymes. However, the detergents may also have a negative effect on enzyme activity; in fact, for all enzyme preparations and substrates the activity/detergent concentration curve reached a maximum value and started to decrease, in many instances even under the initial value. The concentration and nature of the detergent (SDS, CTAB, Triton X-100, or X-45) that permitted the maximum hyperactivation was different depending on the substrate. The best hyperactivation values promoted by the presence of detergent were over a 20-fold factor. The presence of detergents permitted the inhibition of lipases by irreversible covalent inhibitors (e.g., 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) (AEBSF) while the enzyme, in the absence of detergent, is not inhibited by these irreversible inhibitors. This suggested that the main effect of the detergents is to shift the conformational equilibrium of lipases toward the open form. Moreover, the presence of detergents also permitted to improve the enantioselectivity exhibited by the immobilized lipases in some cases. For example, the enantioselectivity of PFL-glyoxyl agarose increased from 40 to more than 100 in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester by using 0.1% CTAB.

Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance.

Lipases from Candida rugosa (CRL) and lipase isoforms A and B from Candida antarctica (CAL-A and CAL-B) were adsorbed on aminated supports in the presence of detergents to have individual lipase molecules. Then, one fraction was washed to eliminate the detergent, and both preparations were treated with glutaraldehyde. The presence of detergent during the cross-linking of the lipases to the support permitted an increase in the recovered activity (in some instances, even by a 10-fold factor). This activity was higher even than that exhibited by the just adsorbed lipases, suggesting that it was not a result of some protective effect of the detergent in the enzyme activity during glutaraldehyde chemical modification. Moreover, the enantioselectivity of the different enzyme preparations was very different if the glutaraldehyde was offered in the presence or in the absence of detergent, in some cases increasing the E value (even by a 7-fold factor in the case of CAL-A in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester), in other cases even inverting the enantio preference (e.g., in the case of CRL). The irreversible chemical inhibition of the enzyme that was immobilized and cross-linked with glutaraldehyde in the presence of detergents was more rapid than that in the other preparations (by more than a 10-fold factor). This experiment reveals an exposition degree of the active serine in the preparation cross-linked with the support in the presence of detergent that is higher than that in the other preparations. The results suggested that different enzyme structures were "stabilized" by the glutaraldehyde treatment if performed in the presence or in the absence of detergent, and that, in the presence of detergent, a form of the lipase with the serine residue more exposed to the medium and much more active could be obtained. This strategy seems to be of general use to improve the lipase activity to be used in macroaqueous media.

Henoch-Schõnlein purpura in adults: a case series from a multidisciplinary study group

BACKGROUND: Henoch-Schõnlein purpura (HSP) is a systemic vasculitis involving small vessels with the deposition of immune complexes containing IgA. It has been extensively studied in children, but in adults, its natural history is much less known. OBJECTIVES: to report a series of patients with HSP presenting in their adulthood. PATIENTS AND METHODS: the Minas Gerais Vasculitis Study Groupïs Members (MGVSG) were invited to report patients with HSP who appeared in their adulthood. A standardized retrospective chart review was done. RESULTS: eleven patients, two male and nine female, age 39.4 +/- 20.1 yearsold were studied. Nine patients presented purpura, seven presented arthritis, four patients had gastrointestinal involvement and ten patients (91 percent) presented glomerulonephritis (GN). Eight patients were subjected to renal biopsies. Six of them presented endocapillary proliferative GN and only two of them had minimal mesangial proliferation. In the other three patients, HSP was confirmed by skin biopsies. All patients received steroids, in five of them steroids were combined with other immunosuppressive agents. After a follow-up of 39.0 +/- 64.6 months, four patients (36 percent) presented impairment of renal function, but only one (9 percent) developed end stage renal disease and was successfully appeared to renal transplantation. At the end of follow-up, seven patients (64 percent) are in complete remission and four in partial remission. CONCLUSION: in adulthood, HSP represents a distinct clinical syndrome with a higher frequency of renal involvement and more severe systemic vasculitis. Nevertheless, the final outcome in this series was as good as reported in children, maybe due to aggressive immunosuppressive therapy.

INTRODUÇÃO: púrpura de Henoch-Schõnlein (PHS) é uma vasculite sistêmica que acomete vasos de pequeno calibre com depósitos de imunocomplexos contendo IgA. Esta vasculite já foi extensamente estudada em crianças, mas sua história natural em adultos é menos conhecida. OBJETIVOS: descrever uma série de pacientes adultos com PHS. PACIENTES E MÉTODOS: membros do Grupo Mineiro de Estudo das Vasculites foram convidados a reportar pacientes com PHS apresentando-se na idade adulta. Foi realizada revisão padronizada de prontuários. RESULTADOS: 11 pacientes, dois homens e nove mulheres, com idade + desvio padrão (DP) de 39,4 +/- 20,1 anos foram estudados. Nove pacientes apresentaram púrpura; sete apresentaram artrite; quatro apresentaram envolvimento gastrintestinal e dez (91 por cento) pacientes apresentaram glomerulonefrite (GN). Oito pacientes foram submetidos à biópsia renal. Seis deles apresentaram GN endocapilar proliferativa e dois apresentaram apenas proeminência mesangial mínima. Em outros três pacientes, o diagnóstico de PHS foi confirmado através de biópsia de pele. Todos os pacientes receberam corticóide, em cinco associado a outros agentes imunossupressores. Após seguimento (média + DP) 39,0 + 64,6 meses, quatro pacientes (36 por cento) apresentaram comprometimento da função renal, mas apenas um (9 por cento) desenvolveu insuficiência renal terminal e foi submetido a transplante renal com sucesso. Após o seguimento, sete (64 por cento) estão em remissão completa e quatro em remissão parcial. CONCLUSÃO: em adultos, PHS representa uma entidade clínica distinta, com maior freqüência de envolvimento renal e vasculite sistêmica mais grave. Entretanto, o prognóstico nesta série foi tão bom quanto em crianças, talvez pela terapia imunossupressiva mais vigorosa.