Diagnosis, genetic characterization and clinical follow up of mitochondrial fatty acid oxidation disorders in the new era of expanded newborn screening: A single centre experience.

INTRODUCTION: Mitochondrial fatty acid oxidation disorders (FAODs) are a heterogeneous group of hereditary autosomal recessive diseases included in newborn screening (NBS) program in Italy. The aim of this study was to analyse FAODs cases, identified either clinically or by NBS,for clinical and genetic characterization and to evaluate a five years' experience of NBS, in the attempt to figure out the complexity of genotype-phenotype correlation and to confirm the clinical impact of NBS in our centre experience. MATERIALS AND METHODS: We analysed FAODs patients diagnosed either by NBS or clinically, followed since February 2014 to April 2019 at the Regional Screening Centre and Inherited Metabolic Diseases Unit of Verona. Diagnosis was confirmed by plasma acylcarnitines, urinary organic acids, enzymatic and genetic testing. For not clear genotypes due to the presence of variants of uncertain significance, in silico predictive tools have been used as well as enzymatic activity assays. Patients underwent clinical, nutritional and biochemical follow up. RESULTS: We diagnosed 30 patients with FAODs. 20 by NBS: 3 CUD, 6 SCADD, 5 MCADD, 4 VLCADD, 2 MADD. Overall incidence of FAODs diagnosed by NBS was 1:4316 newborns. No one reported complications during the follow up period. 10 patients were diagnosed clinically: 2 CUD, 2 CPT2D, 1 VLCADD, 5 MADD. Mean age at diagnosis was 29.3 years. Within this group, complications or symptoms were reported at diagnosis, but not during follow-up. 12 mutations not previously reported in literature were found, all predicted as pathogenic or likely pathogenic. DISCUSSION AND CONCLUSIONS: Our study highlighted the great phenotypic variability and molecular heterogeneity of FAODs and confirmed the importance of a tailored follow up and treatment. Despite the short duration of follow up, early identification by NBS prevented diseases related complications and resulted in normal growth and psycho-motor development as well.

New developments in online OUR monitoring and its application to animal cell cultures.

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance the productivity of bioprocesses through intensification of culture process. Fed-batch and perfusion culturing strategies are considered the most attractive choices, but the application of these processes requires the availability of reliable online measuring systems for the estimation of cell density and metabolic activity. This manuscript reviews the methods (and the devices used) for monitoring of the oxygen consumption, also known as oxygen uptake rate (OUR), since it is a straightforward parameter to estimate viable cell density and the physiological state of cells. Furthermore, as oxygen plays an important role in the cell metabolism, OUR has also been very useful to estimate nutrient consumption, especially the carbon (glucose and glutamine) and nitrogen (glutamine) sources. Three different methods for the measurement of OUR have been developed up to date, being the dynamic method the golden standard, even though DO and pH perturbations generated in the culture during each measurement. For this, many efforts have been focused in developing non-invasive methods, such as global mass balance or stationary liquid mass balance. The low oxygen consumption rates by the cells and the high accuracy required for oxygen concentration measurement in the gas streams (inlet and outlet) have limited the applicability of the global mass balance methodology in mammalian cell cultures. In contrast, stationary liquid mass balance has successfully been implemented showing very similar OUR profiles compared with those obtained with the dynamic method. The huge amount of studies published in the last years evidence that OUR have become a reliable alternative for the monitoring and control of high cell density culturing strategies with very high productivities.

High-dose benznidazole in a 62-year-old Bolivian kidney transplant recipient with Chagas central nervous system involvement.

There is little published data on benznidazole dosing, or levels in cerebrospinal fluid. In this report, we describe the clinical course of an immunosuppressed patient with Chagas central nervous system involvement. He was treated successfully with larger benznidazole doses than are recommended, in order to reach therapeutically effective concentrations in the brain.

A new strategy for fed-batch process control of HEK293 cell cultures based on alkali buffer addition monitoring: comparison with O.U.R. dynamic method.

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance productivity of bioprocesses through different strategies. This is why fed-batch and perfusion cultures are considered more attractive choices than batch processes. In this context, the availability of reliable online measuring systems for cell density and metabolic activity estimation will help the application of these processes. The present work focuses on the comparison of two different monitoring tools for indirect estimation of biomass concentration in a HEK293 cell cultures producing IFN-γ: on one side, the oxygen uptake rate (O.U.R.) determination, by means of application of the dynamic method measurement which is already a widely used tool and, on the other side, a new robust online monitoring tool based on the alkali buffer addition used to maintain the pH set point. Both strategies allow a proper monitoring of cell growth and metabolic activity, with precise identification of the balanced cell growth and the most important action in the process, as is the media feeding. The application of these monitoring systems in fed-batch processes allows extending the growth of HEK293 cells, which in turn results in higher final cell concentrations compared with Batch strategy (7 · 106 cells mL-1), achieving 14 · 106 cells mL-1 for the fed-batch based on O.U.R. and 19 · 106 cells mL-1 for the fed-batch based on the alkali addition. Product titter is also increased in respect of the batch strategy (3.70 mg L-1), resulting in 8.27 mg L-1 when fed-batch was based on O.U.R. and 11.49 mg L-1 when it was based on the alkali buffer strategy. Results prove that fed-batch strategy based on the alkali buffer addition is a robust online monitoring method that has shown its great potential to optimize the feeding strategy in HEK293 fed-batch cultures.

Effect of continuous feeding of CO2 and pH in cell concentration and product titers in hIFNγ producing HEK293 cells: Induced metabolic shift for concomitant consumption of glucose and lactate.

Although pH control at physiological levels is generally considered as the optimal culture condition, in some cases other strategies should be taken into account for their beneficial effects on process performance. pH and CO2 levels are chemical variables that have a major impact in cell growth and product titers in cell culture since their effect on key metabolic routes. HEK293 cells expressing recombinant hIFNγ showed different metabolic behavior when cultured in shake flask compared to pH-controlled bioreactors, in which a decrease in cell density and product titer were observed. This yield loss observed in bioreactor cultures could be reverted by adding 1% CO2 to air inlet flow in a non-controlled pH bioprocess. With this strategy, a significant outcome of 4-fold increase in terms of maximum cell density and 2-fold increase in volumetric concentration of recombinant protein (hIFNγ) when compared to the pH-controlled culture in bioreactor (standard culture conditions) has been obtained. Results evidenced the importance of pH and CO2 concentration in this case, in order to reproduce the behavior observed in optimization experiments performed in shake flasks. Thus, it was demonstrated that not always constant controlled variable setpoint (like pH or CO2 addition) becomes the best bioprocess performance strategy.

[Environmental-analytical risk factors influence on the phenotype of amyotrophic lateral sclerosis in a rural setting]. / Influencia de los factores ambientales-analiticos sobre el fenotipo de esclerosis lateral amiotrofica en un medio rural.

INTRODUCTION: There are discrepancies in the different studies that attempt to correlate the risk factors of developing amyotrophic lateral sclerosis (ALS) and the impact on their prognosis. Some of these factors are intrinsic to the rural livelihood. Therefore, we propose to study if any of these influence the onset and/or prognosis of the disease in the health region of Lleida, a predominantly rural area. PATIENTS AND METHODS: Variables related to general factors, clinical, environmental and laboratory were collected and analyzed at the time of diagnosis of ALS in 38 patients and were retrospectively related to the onset of the disease and its survival. RESULTS: The family history of ALS (p < 0.02) and elevated CK (p < 0.0001) were associated with increased survival. Smoking (p < 0.04), physical work (p < 0.03), low creatinine (p < 0.03), elevated CK (p = 0.0005) were associated with an early onset of the disease. The bulbar onset form was significantly related to a late onset of the disease (p < 0,01). Total cholesterol and PCR did not influence the onset or course of ALS. There is a non-significant trend at statistical level in favor of moderate physical exercise being associated with a later onset, while intense exercise at an early onset of ALS. CONCLUSIONS: The results suggest that there are a number of factors that influence the development and prognosis of ALS, some of which are more prevalent in rural areas, such as physical work.

TITLE: Influencia de los factores ambientales-analiticos sobre el fenotipo de esclerosis lateral amiotrofica en un medio rural.Introduccion. Existen discrepancias en los diversos estudios que intentan correlacionar los factores de riesgo de desarrollar esclerosis lateral amiotrofica (ELA) y el impacto en su pronostico. Algunos de dichos factores son intrinsecos al medio de vida rural. Por ello, proponemos estudiar si alguno de estos influye sobre el inicio o el pronostico de la enfermedad en la region sanitaria de Lleida, un ambito predominantemente rural. Pacientes y metodos. Se recogieron y analizaron variables relacionadas con factores generales, clinicos, ambientales y de laboratorio en el momento del diagnostico de ELA en 38 pacientes, y se relacionaron retrospectivamente con el inicio de la enfermedad y su supervivencia. Resultados. Los antecedentes familiares de ELA (p < 0,02) y la creatincinasa elevada (p < 0,0001) se asociaron a mayor supervivencia. El tabaquismo (p < 0,04), el trabajo fisico (p < 0,03), la creatinina baja (p < 0,03) y la creatincinasa elevada (p = 0,0005) se asociaron a un inicio precoz de la enfermedad de modo estadisticamente significativo. La forma de inicio bulbar se relaciono significativamente con un inicio tardio de la enfermedad (p < 0,01). El colesterol total y la proteina C reactiva no influyeron en el inicio ni en el curso de la ELA en nuestra muestra. Existe una tendencia estadisticamente no significativa a favor de que el ejercicio fisico moderado se asocia con un inicio mas tardio, mientras que el ejercicio intenso se asocia con un inicio precoz de la ELA. Conclusiones. Los resultados apuntan a que existe una serie de factores que influye en el desarrollo y pronostico de la ELA, y alguno de estos es mas prevalente en el ambito rural, como el trabajo fisico.

Valoración de la precarga y la respuesta cardiovascular al aporte de volumen / Assessment of cardiovascular preload and response to volume expansion

El aporte de volumen es de especial interés en los pacientes ingresados en cuidados intensivos con inestabilidad hemodinámica en los que buscamos optimizar el gasto cardiaco .La predicción de la respuesta a esta expansión de volumen, evaluando el grado de precarga dependencia nos permitiría realizar una reanimación guiada evitando los efectos deletéreos del volumen. Actualmente, disponemos de parámetros tanto estáticos como dinámicos que identifican esta precarga-dependencia en diferentes escenarios. En este capítulo definiremos los conceptos de precarga y precarga-dependencia para luego describir cada uno de los parámetros hemodinámicos conocidos para poder predecir la respuesta a volumen, tanto en pacientes con ventilación mecánica como en respiración espontánea (AU)

Volume expansion is used in patients with hemodynamic insufficiency in an attempt to improve cardiac output. Finding criteria to predict fluid responsiveness would be helpful to guide resuscitation and to avoid excessive volume effects. Static and dynamic indicators have been described to predict fluid responsiveness under certain conditions. In this review we define preload and preload-responsiveness concepts. A description is made of the characteristics of each indicator in patients subjected to mechanical ventilation or with spontaneous breathing (AU)