INTERFERENCIA DE HEPARINA EN LA DETERMINACIÓN DE PROTEÍNAS TOTALES POR EL MÉTODO DE BRADFORD / Heparin interference in the Bradford protein quantification assay

La determinación de proteínas totales es una técnica de rutina utilizada en la industria biofarmacéutica durante los procesos de purificación de derivados plasmáticos y/o producción de proteínas recombinantes; y la elección del método analítico es un paso crítico para la obtención de resultados confiables. En este trabajo se demostró la interferencia de heparina (Hep), un compuesto comúnmente utilizado como excipiente en la formulación de Concentrados de Factores de la Coagulación (CFC), en la determinación de proteínas por el método de Bradford, cuya interferencia no está descripta como tal en los insertos de los kits comerciales de Bradford ni en la Farmacopea Europea. Esta interferencia fue demostrada tanto en soluciones de Albúmina Sérica Bovina (BSA) fortificadas con Hep, como en muestras de CFC. Para una solución de BSA 40 mg %, la concentración límite de Hep que no interfiere en la determinación de proteínas totales fue de 1,6 UI/ml. Cabe destacar que, si se trabaja a un nivel menor de concentración de proteínas, una proteína diferente o mezclas complejas de proteínas, se debe evaluar el grado de interferencia para cada caso particular, pudiéndose utilizar los valores obtenidos en este trabajo como referencia. Alternativamente, se evaluó la neutralización de la Hep con Polybrene (Poly), previo a la determinación de proteínas por Bradford. En el caso de soluciones de BSA, el efecto interferente se revirtió logrando una correcta cuantificación de proteínas, mientras que en las muestras de CFC, la neutralización con Poly no fue efectiva. Por lo tanto, para considerar el uso de Poly, previo a la aplicación del método de Bradford, debe evaluarse cada caso particular según las características de la muestra proteica en estudio. Si esto no es posible, se recomienda la utilización de un método alternativo, por ejemplo, el colorimétrico que utiliza ácido bicinconínico.

Protein quantification in biological samples is a routine assay used in the biopharmaceutical industry during the purification procedures of plasma-derived products and/or recombinant proteins. Thus, choosing the method for this purpose is a critical step to obtaining reliable results. In this work, it was shown that Heparin (Hep), a compound commonly used as an excipient in the formulation step of Coagulation Factor Concentrates (CFC), interferes in protein quantification by the Bradford assay. This interference was proved both in solutions of Hep-fortified Bovine Serum Albumin (BSA) and in CFC samples. For a 40 mg % BSA solution, the Hep concentration which shows no interference in protein quantification is 1.6 IU/ml. It should be noted that a specific analysis must be carried out if working at a lower protein concentration, with a different protein, or with complex protein solutions, so the values obtained in this work can be used as a reference. Alternatively, Hep neutralization with Polybrene (Poly) was evaluated before protein determination by Bradford. In the case of BSA solutions, the interference was reversed and correct protein quantification was achieved, whereas in the CFC samples, the results were unsatisfactory. Until now, Hep interference in the Bradford assay has not been described in the scientific literature, nor listed in the inserts of the commercial kits for protein quantification by this method. It was concluded that for protein samples containing Hep, the choice of an alternative method, such as the one that uses bicinchoninic acid, is the most convenient option to obtain reliable results

Factors associated with in-hospital mortality and readmission in a cohort of patients treated with noninvasive ventilation during emergency department or out-of-hospital emergency care: the VentilaMadrid study. / Factores asociados a la mortalidad intrahospitalaria y reingreso en una cohorte tratada con ventilación no invasiva en urgencias extrahospitalarias y hospitalarias: estudio VentilaMadrid.

OBJECTIVES: To describe clinical, outcome, and risk factors in a cohort of patients treated with noninvasive ventilation (NIV) in a hospital emergency department (ED) or by out-of-hospital emergency medical services (OHEMSs). MATERIAL AND METHODS: Multicenter, prospective cohort study enrolling consecutive patients with acute pulmonary edema and/or exacerbated chronic obstructive pulmonary disease who were treated with NIV between November 2018 and November 2020 in a hospital ED or OHEMS setting in Madrid. We recorded baseline data, variables related to the acute episode, and outcome variables, including in-hospital mortality and 30-day readmission. RESULTS: A total of 317 patients were included; 132 (41.6%) were treated in an OHEMS setting and 185 (58.4%) in a hospital ED. Forty-seven (16.3%) in-hospital deaths occurred, and 78 patients (28.8%) were readmitted within 30 days. Mortality in the hospital ED and OHEMS subsamples did not differ, but the patients who received NIV in an OHEMS setting had a lower 30-day readmission rate. On multivariate analysis, in-hospital mortality was associated with prior dependence in activities of daily living in the multivariate analysis (odds ratio [OR], 2.4; 95% CI, 1.11-5.27) and a low-moderate score on the Simplified Acute Physiology Score II (SAPS II) versus a high-very high one (OR, 2.69; 95% CI, 1.26-5.77). Mortality after OHEMS ventilation was associated with discontinuance of NIV during transfer (OR, 8.57; 95% CI, 2.19-33.60). Readmission within 30 days was associated with group (in-hospital ED application of NIV) (OR, 3.24; 95% CI, 2.62-6.45) and prior dependence (OR, 2.08; 95% CI, 1.02-4.22). CONCLUSION: Patients treated in the hospital ED and OHEMS setting have similar baseline characteristics, although acute episodes were more serious in the OHEMS group. No significant differences were found related to in-hospital mortality. Higher mortality was associated with dependence, a SAPS II score greater than 52, and discontinuance of NIV. Readmission was associated with dependence and NIV treatment in the hospital ED setting.

OBJETIVO: Describir las características clínicas, evolutivas y los factores pronóstico de una cohorte de pacientes tratados con ventilación no invasiva (VNI) en servicios de urgencias extrahospitalarios (SUEH) y hospitalarios (SUH). METODO: Estudio de cohortes multicéntrico, prospectivo con inclusión consecutiva de pacientes con edema agudo de pulmón o agudización de enfermedad pulmonar obstructiva crónica tratados con VNI entre noviembre 2018 y noviembre de 2020 en SUEH y SUH de la Comunidad de Madrid. Se recogieron características basales, del episodio agudo, así como variables de resultado incluyendo la mortalidad hospitalaria y el reingreso a 30 días. RESULTADOS: Se incluyeron 317 pacientes, 132 (41,6%) en SUEH y 185 (58,4%) en SUH. Hubo 47 muertes intrahospitalarias (16,3%) y 78 reingresos a los 30 días (28,8%). No hubo diferencias en la mortalidad, pero el grupo VNI-SUEH tuvo menor reingreso a 30 días. En el análisis multivariado la mortalidad intrahospitalaria se asoció con la dependencia previa (OR = 2,4; IC 95%: 1,11-5,27) y el SAPS-II bajo-moderado frente al alto-muy alto (OR = 2,69; IC 95%: 1,26-5,77). En la cohorte extrahospitalaria, la mortalidad intrahospitalaria se asoció con la retirada de la VNI en la transferencia del paciente (OR = 8,57; IC 95%: 2,19-33,60). Los reingresos a los 30 días se asociaron con inicio de VNI en el hospital (OR = 3,24; IC 95%: 2,62-6,45) y dependencia previa (OR = 2,08; IC 95%: 1,02-4,22). CONCLUSIONES: Los pacientes de ambos grupos, SUH y SUEH, tienen un perfil clínico basal similar, aunque con mayor gravedad del episodio en el grupo SUEH. No se encontraron diferencias estadísticamente significativas en la mortalidad intrahospitalaria. Se asociaron a una mayor mortalidad la dependencia, la escala SAPS-II > 52 y la retirada de la VNI. El reingreso se asoció con la dependencia y pertenecer al grupo SUH.

Factores asociados a la mortalidad intrahospitalaria y reingreso en una cohorte tratada con ventilación no invasiva en urgencias extrahospitalarias y hospitalarias: estudio VentilaMadrid / Factors associated with in-hospital mortality and readmission in a cohort of patients treated with noninvasive ventilation during emergency department or out-of-hospital emergency care: the VentilaMadrid study

Objetivo. Describir las características clínicas, evolutivas y los factores pronóstico de una cohorte de pacientes trata- dos con ventilación no invasiva (VNI) en servicios de urgencias extrahospitalarios (SUEH) y hospitalarios (SUH). Método. Estudio de cohortes multicéntrico, prospectivo con inclusión consecutiva de pacientes con edema agudo de pulmón o agudización de enfermedad pulmonar obstructiva crónica tratados con VNI entre noviembre 2018 y noviembre de 2020 en SUEH y SUH de la Comunidad de Madrid. Se recogieron características basales, del episodio agudo, así como variables de resultado incluyendo la mortalidad hospitalaria y el reingreso a 30 días. Resultados. Se incluyeron 317 pacientes, 132 (41,6%) en SUEH y 185 (58,4%) en SUH. Hubo 47 muertes intrahospi talarias (16,3%) y 78 reingresos a los 30 días (28,8%). No hubo diferencias en la mortalidad, pero el grupo VNI-SUEH tuvo menor reingreso a 30 días. En el análisis multivariado la mortalidad intrahospitalaria se asoció con la dependen- cia previa (OR = 2,4; IC 95%: 1,11-5,27) y el SAPS-II bajo-moderado frente al alto-muy alto (OR = 2,69; IC 95%: 1,26- 5,77). En la cohorte extrahospitalaria, la mortalidad intrahospitalaria se asoció con la retirada de la VNI en la transferencia del paciente (OR = 8,57; IC 95%: 2,19-33,60). Los reingresos a los 30 días se asociaron con inicio de VNI en el hospital (OR = 3,24; IC 95%: 2,62-6,45) y dependencia previa (OR = 2,08; IC 95%: 1,02-4,22). Conclusiones. Los pacientes de ambos grupos, SUH y SUEH, tienen un perfil clínico basal similar, aunque con mayor gravedad del episodio en el grupo SUEH. No se encontraron diferencias estadísticamente significativas en la mortalidad intrahospitalaria. Se asociaron a una mayor mortalidad la dependencia, la escala SAPS-II > 52 y la retirada de la VNI. El reingreso se asoció con la dependencia y pertenecer al grupo SUH.

Objective. To describe clinical, outcome, and risk factors in a cohort of patients treated with noninvasive ventilation (NIV) in a hospital emergency department (ED) or by out-of-hospital emergency medical services (OHEMSs). Methods. Multicenter, prospective cohort study enrolling consecutive patients with acute pulmonary edema and/or exacerbated chronic obstructive pulmonary disease who were treated with NIV between November 2018 and November 2020 in a hospital ED or OHEMS setting in Madrid. We recorded baseline data, variables related to the acute episode, and outcome variables, including in-hospital mortality and 30-day readmission. Results. A total of 317 patients were included; 132 (41.6%) were treated in an OHEMS setting and 185 (58.4%) in a hospital ED. Forty-seven (16.3%) in-hospital deaths occurred, and 78 patients (28.8%) were readmitted within 30 days. Mortality in the hospital ED and OHEMS subsamples did not differ, but the patients who received NIV in an OHEMS setting had a lower 30-day readmission rate. On multivariate analysis, in-hospital mortality was associated with prior dependence in activities of daily living in the multivariate analysis (odds ratio [OR], 2.4; 95% CI, 1.11– 5.27) and a low-moderate score on the Simplified Acute Physiology Score II (SAPS II) versus a high-very high one (OR, 2.69; 95% CI, 1.26–5.77). Mortality after OHEMS ventilation was associated with discontinuance of NIV during transfer (OR, 8.57; 95% CI, 2.19–33.60). Readmission within 30 days was associated with group (in-hospital ED application of NIV) (OR, 3.24; 95% CI, 2.62–6.45) and prior dependence (OR, 2.08; 95% CI, 1.02–4.22). [...]

Cross-sectional study of the healthcare provided by a medicalized helicopter from Extremadura (Spain).

OBJECTIVE: To analyze geographic dispersion, demographic factors, clinical features of patients attended and response times of a helicopter emergency medical service in a region of Spain. METHODS: According to the principles of the Declaration of Helsinki and to the standards of Good Clinical Practice, a cross-sectional observational study from January 2014 to December 2016 was carried out. Socio-demographic, clinical and transfer related variables were analyzed from an anonymized database loaned by the service. RESULTS: 642 missions were flown (on average 0.59 flights per day). The patients were 65.6% males (they were significantly younger than women were) and 79% older than 45. Primary transfers composed 68.8% of cases. Medical pathologies were the most prevalent (74.9%), followed by traumatic pathologies (19.8%). Cardiovascular pathologies took up 68.8% of attended medical pathologies and 51.56% of the total of recorded cases. The accident rate was 20.25% and the response time in 75% of cases was under 32 min. Twenty deceased patients were registered (70% males). CONCLUSIONS: This study reports for the international community the features of several Spanish HEMS and profile of patients attended. Adult men are a common profile and cardiovascular issues have replaced traumatic issues as main healthcare demand. However, it should be confirmed because it could be due to differences in local profile of patients. Men had higher risk of road traffic and occupational accidents as well as a higher mortality than women did. Socio-demographical and organizational aspects have been provided that could be taken into consideration to implement new air emergency services.

Optimization and validation of simultaneous analyses of ecgonine, cocaine, and seven metabolites in human urine by gas chromatography-mass spectrometry using a one-step solid-phase extraction.

The presence of ecgonine in urine has been proposed as an appropriate marker of cocaine use. Only a few methods have been published for their determination along with cocaine and the rest of its metabolites. Due to their high polarity and consequent solubility in water, these have low recoveries, which is why it is necessary to increase the sensitivity, by the formation of hydrochloric salts or multiderivatization of the analytes or by performing two solid-phase extractions (SPEs), considerably increasing the time and cost of the analysis. This work describes a fast and fully validated procedure for the simultaneous detection and quantification of ecgonine, ecgonine-methyl-ester, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, cocaethylene, cocaine, norcocaine, and norcocaethylene in human urine (500 µL) using one SPE and simple derivatization. Separation and quantification were achieved by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) in selected-ion monitoring mode. Quantification was performed by the addition of deuterated analogs as internal standards. Calibration curves were linear in the adopted ranges, with determination coefficients higher than 0.99. The lower limits of quantification ranged from 2.5 to 10 ng/mL. The intra- and inter-day precision, calculated in terms of relative standard deviation, were 1.2%-14.9% and 1.8%-17.9%, respectively. The accuracy, in terms of relative error, was within a ± 16.4% interval. Extraction efficiency ranged from 84% to 103%. Compared with existing methods, the procedure described herein is fast, since only one SPE is required, and cost-effective. In addition, this method provides a high recovery for ecgonine, resulting in a better alternative to the previously published methods.

Evolutionary adaptation of an RNA bacteriophage to the simultaneous increase in the within-host and extracellular temperatures.

Bacteriophages are the most numerous biological entities on Earth. They are on the basis of most ecosystems, regulating the diversity and abundance of bacterial populations and contributing to the nutrient and energy cycles. Bacteriophages have two well differentiated phases in their life cycle, one extracellular, in which they behave as inert particles, and other one inside their hosts, where they replicate to give rise to a progeny. In both phases they are exposed to environmental conditions that often act as selective pressures that limit both their survival in the environment and their ability to replicate, two fitness traits that frequently cannot be optimised simultaneously. In this study we have analysed the evolutionary ability of an RNA bacteriophage, the bacteriophage Qß, when it is confronted with a temperature increase that affects both the extracellular and the intracellular media. Our results show that Qß can optimise its survivability when exposed to short-term high temperature extracellular heat shocks, as well as its replicative ability at higher-than-optimal temperature. Mutations responsible for simultaneous adaptation were the same as those selected when adaptation to each condition proceeded separately, showing the absence of important trade-offs between survival and reproduction in this virus.

Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qß.

RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qß, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior.

Changes in protein domains outside the catalytic site of the bacteriophage Qß replicase reduce the mutagenic effect of 5-azacytidine.

UNLABELLED: The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qß replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qß replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE: In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qß replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qß. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qß is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

Adaptation to fluctuating temperatures in an RNA virus is driven by the most stringent selective pressure.

The frequency of change in the selective pressures is one of the main factors driving evolution. It is generally accepted that constant environments select specialist organisms whereas changing environments favour generalists. The particular outcome achieved in either case also depends on the relative strength of the selective pressures and on the fitness costs of mutations across environments. RNA viruses are characterized by their high genetic diversity, which provides fast adaptation to environmental changes and helps them evade most antiviral treatments. Therefore, the study of the adaptive possibilities of RNA viruses is highly relevant for both basic and applied research. In this study we have evolved an RNA virus, the bacteriophage Qß, under three different temperatures that either were kept constant or alternated periodically. The populations obtained were analyzed at the phenotypic and the genotypic level to characterize the evolutionary process followed by the virus in each case and the amount of convergent genetic changes attained. Finally, we also investigated the influence of the pre-existent genetic diversity on adaptation to high temperature. The main conclusions that arise from our results are: i) under periodically changing temperature conditions, evolution of bacteriophage Qß is driven by the most stringent selective pressure, ii) there is a high degree of evolutionary convergence between replicated populations and also among populations evolved at different temperatures, iii) there are mutations specific of a particular condition, and iv) adaptation to high temperatures in populations differing in their pre-existent genetic diversity takes place through the selection of a common set of mutations.

Evolution at increased error rate leads to the coexistence of multiple adaptive pathways in an RNA virus.

BACKGROUND: When beneficial mutations present in different genomes spread simultaneously in an asexual population, their fixation can be delayed due to competition among them. This interference among mutations is mainly determined by the rate of beneficial mutations, which in turn depends on the population size, the total error rate, and the degree of adaptation of the population. RNA viruses, with their large population sizes and high error rates, are good candidates to present a great extent of interference. To test this hypothesis, in the current study we have investigated whether competition among beneficial mutations was responsible for the prolonged presence of polymorphisms in the mutant spectrum of an RNA virus, the bacteriophage Qß, evolved during a large number of generations in the presence of the mutagenic nucleoside analogue 5-azacytidine. RESULTS: The analysis of the mutant spectra of bacteriophage Qß populations evolved at artificially increased error rate shows a large number of polymorphic mutations, some of them with demonstrated selective value. Polymorphisms distributed into several evolutionary lines that can compete among them, making it difficult the emergence of a defined consensus sequence. The presence of accompanying deleterious mutations, the high degree of recurrence of the polymorphic mutations, and the occurrence of epistatic interactions generate a highly complex interference dynamics. CONCLUSIONS: Interference among beneficial mutations in bacteriophage Qß evolved at increased error rate permits the coexistence of multiple adaptive pathways that can provide selective advantages by different molecular mechanisms. In this way, interference can be seen as a positive factor that allows the exploration of the different local maxima that exist in rugged fitness landscapes.

Identification of mutations conferring 5-azacytidine resistance in bacteriophage Qß.

RNA virus replication takes place at a very high error rate, and additional increases in this parameter can produce the extinction of virus infectivity. Nevertheless, RNA viruses can adapt to conditions of increased mutagenesis, which demonstrates that selection of beneficial mutations is also possible at higher-than-standard error rates. In this study we have analysed the evolutionary behaviour of bacteriophage Qß populations when replication proceeds in the presence of the mutagenic nucleoside analogue 5-azacytidine (AZC). We have obtained a virus population with reduced capacity to accumulate mutations in the presence of AZC and able to avoid extinction under conditions that are lethal for the wild type virus. Adapted populations fix a substitution in the readthrough protein gene and incorporate several mutations in the replicase gene that, despite having selective value, remain polymorphic after a large number of transfers in the presence of AZC.