RESUMEN
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.
Asunto(s)
Lipasa/metabolismo , Manosa/metabolismo , Aspergillus niger/enzimología , Cromatografía Liquida , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Lipasa/química , Manosa/química , Espectrometría de MasasRESUMEN
INTRODUCTION: The concept of high-risk patients suggests that such patients will experience a higher rate of postoperative complications and worse short- and long-term outcomes, and should therefore benefit from the use of endovascular techniques for aortic abdominal aneurysm (AAA) repair. The primary goal of this study was to assess the relevance of the different high-risk criteria, defined by the French health agency Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS) in a single-centre continuous series. Secondary goals were to retrospectively compare the incidence of postoperative complications and short- and long-term survival in three groups of patients. MATERIALS AND METHODS: Between January 1999 and December 2006, details of all the patients undergoing elective surgery for AAA in our hospital were recorded into a prospective registry (n=626). Three groups were considered according to the level of risk and type of repair defined by the AFSSAPS: endovascular aortic aneurysm repair (EVAR) high-risk (HR) (at least one high-risk factor and EVAR, n=138), open HR (at least one high-risk factor and open repair, n=134) and open low-risk (LR) (no high-risk factors and open repair, n=344). None of the low-risk patients were treated using an endovascular approach. The demographics, preoperative risk factors, intra-, postoperative data and short- and long-term survival were compared between the groups. Interrelations among the set of high-risk criteria for mortality were calculated using multiple correspondence analysis (MCA). RESULTS: The distribution of high-risk criteria was similar in both high-risk groups, except for age, heart failure and hostile abdomen, which were significantly more frequent in EVAR HR. Operation time, blood loss and length of stay in an intensive care unit and hospital were significantly lower in the EVAR HR group. The 30-day mortality and survival rates at 5 years were 5.4 and 59.4% for EVAR HR, 3.7 and 70.4% for open HR and 2.3 and 83.7% for open LR, respectively, with no significant difference between the three groups for the mortality, but a significant higher survival at 5 years for the open LR versus both high-risk groups. CONCLUSION: The high-risk AFSSAPS criteria were not predictive of postoperative mortality and should not be used to determine the choice of treatment technique. Other criteria therefore need to be established to determine whether open or EVAR repair should be used.
Asunto(s)
Aneurisma de la Aorta Abdominal/cirugía , Implantación de Prótesis Vascular , Procedimientos Quirúrgicos Vasculares , Adulto , Anciano , Anciano de 80 o más Años , Aneurisma de la Aorta Abdominal/mortalidad , Implantación de Prótesis Vascular/efectos adversos , Implantación de Prótesis Vascular/mortalidad , Distribución de Chi-Cuadrado , Femenino , Francia/epidemiología , Mortalidad Hospitalaria , Humanos , Estimación de Kaplan-Meier , Tiempo de Internación , Masculino , Persona de Mediana Edad , Selección de Paciente , Guías de Práctica Clínica como Asunto , Sistema de Registros , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Procedimientos Quirúrgicos Vasculares/efectos adversos , Procedimientos Quirúrgicos Vasculares/mortalidadRESUMEN
A cross-sectional study in Itagua, Paraguay tested 192 people for the presence, intensity and species of hookworm infection. Fifty-nine percent of these individuals were found to be infected. Intensity of infection was determined on 92% of infected individuals by quantitative egg counts. The high intensity hookworm infections, which cause the greatest morbidity, were clustered between the ages of five and 14 years. No differences were seen between genders. The species of hookworm was determined for parasites reared from 72% of infected individuals. Both Necator americanus and Ancylostoma duodenale were identified, although the former species predominated. We conclude that hookworm infection continues to be a public health problem in Paraguay, particularly among children and adolescents who suffer from high intensity infections. A. duodenale continues to persist in the Western Hemisphere and has not been completely displaced by N. americanus.