Reconstitution of early paclitaxel biosynthetic network.

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

Reconstitution of Early Paclitaxel Biosynthetic Network.

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. This diverts metabolic flux away from the paclitaxel precursor, taxadien-5α-ol, thus previous attempts of reconstitution have not yielded sufficient material for characterization, regardless of the heterologous host. Here, we structurally characterized four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression, levels of these proposed byproducts decrease with a concomitant increase in the accumulation of taxadien-5α-ol by four-fold. This engineered system enabled the reconstitution of a six step biosynthetic pathway to produce isolatable 5α,10ß-diacetoxy-taxadien-13α-ol. Furthermore, we showed that this pathway may function as a metabolic network rather than a linear pathway. The engineering of the paclitaxel biosynthetic network demonstrates that Taxus genes can coordinatively function for the biosynthetic production of key early stage paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

Complex scaffold remodeling in plant triterpene biosynthesis.

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

First Outpatient Clinical Trial of a Full Closed-Loop Artificial Pancreas System in South America.

BACKGROUND: The first two studies of an artificial pancreas (AP) system carried out in Latin America took place in 2016 (phase 1) and 2017 (phase 2). They evaluated a hybrid algorithm from the University of Virginia (UVA) and the automatic regulation of glucose (ARG) algorithm in an inpatient setting using an AP platform developed by the UVA. The ARG algorithm does not require carbohydrate (CHO) counting and does not deliver meal priming insulin boluses. Here, the first outpatient trial of the ARG algorithm using an own AP platform and doubling the duration of previous phases is presented. METHOD: Phase 3 involved the evaluation of the ARG algorithm in five adult participants (n = 5) during 72 hours of closed-loop (CL) and 72 hours of open-loop (OL) control in an outpatient setting. This trial was performed with an own AP and remote monitoring platform developed from open-source resources, called InsuMate. The meals tested ranged its CHO content from 38 to 120 g and included challenging meals like pasta. Also, the participants performed mild exercise (3-5 km walks) daily. The clinical trial is registered in ClinicalTrials.gov with identifier: NCT04793165. RESULTS: The ARG algorithm showed an improvement in the time in hyperglycemia (52.2% [16.3%] OL vs 48.0% [15.4%] CL), time in range (46.9% [15.6%] OL vs 50.9% [14.4%] CL), and mean glucose (188.9 [25.5] mg/dl OL vs 186.2 [24.7] mg/dl CL) compared with the OL therapy. No severe hyperglycemia or hypoglycemia episodes occurred during the trial. The InsuMate platform achieved an average of more than 95% of the time in CL. CONCLUSION: The results obtained demonstrated the feasibility of outpatient full CL regulation of glucose levels involving the ARG algorithm and the InsuMate platform.

Dynamical Models in Neuroscience From a Closed-Loop Control Perspective.

Modifying neural activity is a substantial goal in neuroscience that facilitates the understanding of brain functions and the development of medical therapies. Neurobiological models play an essential role, contributing to the understanding of the underlying brain dynamics. In this context, control systems represent a fundamental tool to provide a correct articulation between model stimulus (system inputs) and outcomes (system outputs). However, throughout the literature there is a lack of discussions on neurobiological models, from the formal control perspective. In general, existing control proposals applied to this family of systems, are developed empirically, without theoretical and rigorous framework. Thus, the existing control solutions, present clear and significant limitations. The focus of this work is to survey dynamical neurobiological models that could serve for closed-loop control schemes or for simulation analysis. Consequently, this paper provides a comprehensive guide to discuss and analyze control-oriented neurobiological models. It also provides a potential framework to adequately tackle control problems that could modify the behavior of single neurons or networks. Thus, this study constitutes a key element in the upcoming discussions and studies regarding control methodologies applied to neurobiological systems, to extend the present research and understanding horizon for this field.

Non-pharmaceutical intervention to reduce COVID-19 impact in Argentina.

This work is focused on the multilevel control of the population confinement in the city of Buenos Aires and its surroundings due to the pandemic generated by the COVID-19 outbreak. The model used here is known as SEIRD and two objectives are sought: a time-varying identification of the infection rate and the inclusion of a controller. A control differential equation has been added to regulate the transitions between confinement and normal life, according to five different levels. The plasma treatment from recovered patients has also been considered in the control algorithm. Using the proposed strategy the ICU occupancy is reduced, and as a consequence, the number of deaths is also decreased.

Reporte de altos requerimientos de insulina en pacientes críticos pediátricos con COVID-19. Experiencia con monitoreo remoto continuo de glucosa / Report of high insulin requirements in pediatric critically ill patients with COVID-19. Experience with continuous remote glucose monitoring

Los pacientes en estado crítico con COVID-19 sufren hiperglucemias sostenidas de difícil manejo. A esto se suma el desafío de minimizar la exposición al contagio. En el presente artículo analizamos la evolución metabólica de dos pacientes pediátricos con COVID-19 admitidos en unidad de cuidados intensivos (UCI) para pacientes COVID-19 del Hospital "Prof. Dr. Juan P. Garrahan" de la Ciudad Autónoma de Buenos Aires, Argentina, que requirieron tratamiento con insulina endovenosa y cuya glucemia fue monitoreada de manera remota con la plataforma InsuMate® desarrollada en la Universidad Nacional de La Plata. Los pacientes requirieron tasas de infusión de insulina en dosis marcadamente mayores que las de otros pacientes críticos que impresionaron relacionadas con los valores de marcadores de inflamación. La infusión pudo ajustarse con cuatro monitoreos diarios de glucosa y las métricas obtenidas con el monitor de glucosa. El uso del sistema de monitoreo remoto continuo de glucosa permitió disminuir la frecuencia de monitoreo glucémico durante el tratamiento.

Critically ill patients with COVID-19 suffer from sustained hyperglycemia that is difficult to manage. Added to this is the challenge of minimizing exposure to contagion. In this article we analyze the metabolic evolution of two pediatric patients with COVID-19 admitted to the intensive care unit (ICU) for COVID-19 patients at the Hospital "Prof. Dr. Juan P. Garrahan "from the Autonomous City of Buenos Aires, Argentina, who required treatment with intravenous insulin and whose blood glucose was remotely monitored with the InsuMate® platform developed at the National University of La Plata. The patients required insulin infusion rates in doses markedly higher than those of other critically ill patients, who were impressively related to the values of inflammation markers. The infusion could be adjusted with four daily glucose monitors and the metrics obtained with the glucose monitor. The use of the continuous remote glucose monitoring system made it possible to decrease the frequency of glycemic monitoring during treatment.

Rerouting plant terpene biosynthesis enables momilactone pathway elucidation.

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).

Determination of Glyphosate in Water from a Rural Locality in México and Its Implications for the Population Based on Water Consumption and Use Habits.

Glyphosate is a broad-spectrum herbicide widely used worldwide. Indeed, it is the herbicide most applied to all Mexican crops. Due to the overuse and poor disposal of the waste, this herbicide can reach the aquatic environments such as groundwater and surface water. Thus, there is a clear need to implement monitoring and surveillance programs for evaluating and controlling the exposure to this herbicide in rural populations. The goal of this study was to quantify the presence of glyphosate in different water bodies (groundwater, surface and drinking water) as well as to identify the uses and managements of water resources by rural communities to evaluate the potential human exposure to glyphosate in the Tenampulco region of the Mexican state of Puebla. Measurements were performed by a rapid and cost-effective ELISA-based method in groundwater and surface water from various sampling sites of the Tenampulco region. Glyphosate was detected in all groundwater samples to be below the maximum limit for glyphosate in water in Mexico. Nevertheless, these results indicate an exposure of glyphosate in these agricultural communities and the need to establish a monitoring program.

Synthesis of Spirocyclic 1-Pyrrolines from Nitrones and Arynes through a Dearomative [3,3']-Sigmatropic Rearrangement.

A dearomative [3,3']-sigmatropic rearrangement that converts N-alkenylbenzisoxazolines into spirocyclic pyrroline cyclohexadienones has been developed by using the dipolar cycloaddition of an N-alkenylnitrone and an aryne to access these unusual transient rearrangement precursors. This cascade reaction affords spirocyclic pyrrolines that are inaccessible through dipolar cycloadditions of exocyclic cyclohexenones and provides a fundamentally new approach to novel spirocyclic pyrroline and pyrrolidine motifs that are common scaffolds in biologically-active molecules. Diastereoselective functionalization processes have also been explored to demonstrate the divergent synthetic utility of the unsaturated spirocyclic products.

Control-Oriented Model With Intra-Patient Variations for an Artificial Pancreas.

In this work, a low-order model designed for glucose regulation in Type 1 Diabetes Mellitus (T1DM) is obtained from the UVA/Padova metabolic simulator. It captures not only the nonlinear behavior of the glucose-insulin system, but also intra-patient variations related to daily insulin sensitivity ( SI) changes. To overcome the large inter-subject variability, the model can also be personalized based on a priori patient information. The structure is amenable for linear parameter varying (LPV) controller design, and represents the dynamics from the subcutaneous insulin input to the subcutaneous glucose output. The efficacy of this model is evaluated in comparison with a previous control-oriented model which in turn is an improvement of previous models. Both models are compared in terms of their open- and closed-loop differences with respect to the UVA/Padova model. The proposed model outperforms previous T1DM control-oriented models, which could potentially lead to more robust and reliable controllers for glycemia regulation.

Genetically Different Isolates of the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Induce Differential Responses to Stress in Cassava.

Water scarcity negatively impacts global crop yields and climate change is expected to greatly increase the severity of future droughts. The use of arbuscular mycorrhizal fungi (AMF) can potentially mitigate the effects of water stress in plants. Cassava is a crop that feeds approximately 800 million people daily. Genetically different isolates of the AMF R. irregularis as well as their clonal progeny have both been shown to greatly alter cassava growth in field conditions. Given that cassava experiences seasonal drought in many of the regions in which it is cultivated, we evaluated whether intraspecific variation in R. irregularis differentially alters physiological responses of cassava to water stress. In a first experiment, conducted in field conditions in Western Kenya, cassava was inoculated with two genetically different R. irregularis isolates and their clonal progeny. All cassava plants exhibited physiological signs of stress during the dry period, but the largest differences occurred among plants inoculated with clonal progeny of each of the two parental fungal isolates. Because drought had not been experimentally manipulated in the field, we conducted a second experiment in the greenhouse where cassava was inoculated with two genetically different R. irregularis isolates and subjected to drought, followed by re-watering, to allow recovery. Physiological stress responses of cassava to drought differed significantly between plants inoculated with the two different fungi. However, plants that experienced higher drought stress also recovered at a faster rate following re-watering. We conclude that intraspecific genetic variability in AMF significantly influences cassava physiological responses during water stress. This highlights the potential of using naturally existing variation in AMF to improve cassava tolerance undergoing water stress. However, the fact that clonal progeny of an AMF isolate can differentially affect how cassava copes with natural drought stress in field conditions, highlights the necessity to understand additional factors, beyond genetic variation, which can account for such large differences in cassava responses to drought.

Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3ß-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3ß-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

Artificial Pancreas: Evaluating the ARG Algorithm Without Meal Announcement.

BACKGROUND: Either under standard basal-bolus treatment or hybrid closed-loop control, subjects with type 1 diabetes are required to count carbohydrates (CHOs). However, CHO counting is not only burdensome but also prone to errors. Recently, an artificial pancreas algorithm that does not require premeal insulin boluses-the so-called automatic regulation of glucose (ARG)-was introduced. In its first pilot clinical study, although the exact CHO counting was not required, subjects still needed to announce the meal time and classify the meal size. METHOD: An automatic switching signal generator (SSG) is proposed in this work to remove the manual mealtime announcement from the control strategy. The SSG is based on a Kalman filter and works with continuous glucose monitoring readings only. RESULTS: The ARG algorithm with unannounced meals (ARGum) was tested in silico under the effect of different types of mixed meals and intrapatient variability, and contrasted with the ARG algorithm with announced meals (ARGam). Simulations reveal that, for slow-absorbing meals, the time in the euglycemic range, [70-180] mg/dL, increases using the unannounced strategy (ARGam: 78.1 [68.6-80.2]% (median [IQR]) and ARGum: 87.8 [84.5-90.6]%), while similar results were found with fast-absorbing meals (ARGam: 87.4 [86.0-88.9]% and ARGum: 87.6 [86.1-88.8]%). On the other hand, when intrapatient variability is considered, time in euglycemia is also comparable (ARGam: 81.4 [75.4-83.5]% and ARGum: 80.9 [77.0-85.1]%). CONCLUSION: In silico results indicate that it is feasible to perform an in vivo evaluation of the ARG algorithm with unannounced meals.

Volume-Conducted Origin of the Field Potential at the Lateral Habenula.

Field potentials (FPs) are easily reached signals that provide information about the brain's processing. However, FP should be interpreted cautiously since their biophysical bases are complex. The lateral habenula (LHb) is a brain structure involved in the encoding of aversive motivational values. Previous work indicates that the activity of the LHb is relevant for hippocampal-dependent learning. Moreover, it has been proposed that the interaction of the LHb with the hippocampal network is evidenced by the synchronization of LHb and hippocampal FPs during theta rhythm. However, the origin of the habenular FP has not been analyzed. Hence, its validity as a measurement of LHb activity has not been proven. In this work, we used electrophysiological recordings in anesthetized rats and feed-forward modeling to investigate biophysical basis of the FP recorded in the LHb. Our results indicate that the FP in the LHb during theta rhythm is a volume-conducted signal from the hippocampus. This result highlight that FPs must be thoroughly analyzed before its biological interpretation and argues against the use of the habenular FP signal as a readout of the activity of the LHb.

Artificial Pancreas: Clinical Study in Latin America Without Premeal Insulin Boluses.

BACKGROUND: Emerging therapies such as closed-loop (CL) glucose control, also known as artificial pancreas (AP) systems, have shown significant improvement in type 1 diabetes mellitus (T1DM) management. However, demanding patient intervention is still required, particularly at meal times. To reduce treatment burden, the automatic regulation of glucose (ARG) algorithm mitigates postprandial glucose excursions without feedforward insulin boluses. This work assesses feasibility of this new strategy in a clinical trial. METHODS: A 36-hour pilot study was performed on five T1DM subjects to validate the ARG algorithm. Subjects wore a subcutaneous continuous glucose monitor (CGM) and an insulin pump. Insulin delivery was solely commanded by the ARG algorithm, without premeal insulin boluses. This was the first clinical trial in Latin America to validate an AP controller. RESULTS: For the total 36-hour period, results were as follows: average time of CGM readings in range 70-250 mg/dl: 88.6%, in range 70-180 mg/dl: 74.7%, <70 mg/dl: 5.8%, and <50 mg/dl: 0.8%. Results improved analyzing the final 15-hour period of this trial. In that case, the time spent in range was 70-250 mg/dl: 94.7%, in range 70-180 mg/dl: 82.6%, <70 mg/dl: 4.1%, and <50 mg/dl: 0.2%. During the last night the time spent in range was 70-250 mg/dl: 95%, in range 70-180 mg/dl: 87.7%, <70 mg/dl: 5.0%, and <50 mg/dl: 0.0%. No severe hypoglycemia occurred. No serious adverse events were reported. CONCLUSIONS: The ARG algorithm was successfully validated in a pilot clinical trial, encouraging further tests with a larger number of patients and in outpatient settings.

Delirium during the first evaluation of children aged five to 14 years admitted to a paediatric critical care unit.

OBJECTIVES: To describe the prevalence and characteristics of delirium during the initial evaluation of critically ill patients aged 5-14 years. METHOD/DESIGN: This is a cross-sectional descriptive study in a critical care unit. For six months, all patients were evaluated within the first 24-72 hours or when sedation permitted the use of the paediatric confusion assessment method for the intensive care unit (PCAM-ICU) and the Delirium Rating Scale-Revised-98 items #7 and #8 to determine motor type. We report the characteristics of PCAM-ICU delirium (at least three of the required items scored positive) and of subthreshold score cases (two positive items). RESULTS: Of 77 admissions, 15 (19.5%) had delirium, and 11 (14.2%) were subthreshold. A total of 53.3% of delirium and 45.5% of subthreshold cases were hypoactive. The prevalence of delirium and subthreshold PCAM-ICU was 83.3% and 16.7% in mechanically ventilated children. The most frequent combination of PCAM-ICU alterations in subthreshold cases was acute onset-fluctuation with altered alertness. The main nursing diagnoses were related to reduced cellular respiration. CONCLUSIONS: Delirium is common in critically ill children. It is necessary to assess whether certain nursing diagnoses imply an increase in delirium. Longitudinal studies of subthreshold PCAM-ICU cases are needed to understand their importance better.

Language Barriers Impact Access to Services for Children with Autism Spectrum Disorders.

Racial and ethnic disparities in accessing health care have been described in children with autism spectrum disorder (ASD). In a retrospective chart review of 152 children with ASD, children of parents whose primary language was English were significantly more likely to have both social skills and communication goals within their individualized education plan (IEP) compared to children of parents whose primary language was not English. Additionally, children of primary English speakers received significantly more hours of direct services from their state disability program. After controlling for demographic covariates, findings suggest that language barriers may negatively affect parents' abilities to access health care services for their child with ASD. Acculturation factors must therefore be considered when analyzing disparities in autism.

Comportamiento de los parámetros hemodinámicos evaluados por el monitoreo ambulatorio de presión arterial de 24 horas / Behavior of hemodynamic parameters assessed with 24-hour ambulatory blood pressure monitoring

Resumen Objetivo: Describir el comportamiento de los parámetros hemodinámicos en un grupo de individuos sometidos al estudio de monitoreo ambulatorio de la presión arterial. Materiales y métodos: Estudio observacional descriptivo sobre el comportamiento de los parámetros hemodinámicos en el estudio de monitoreo ambulatorio de la presión arterial en un grupo de pacientes atendidos en dos instituciones de Medellín. Resultados: Se analizaron un total de 108 pacientes atendidos en el período comprendido entre septiembre de 2012 y octubre de 2013. Los promedios de la presión arterial sistólica y presión arterial diastólica en 24 horas fueron de 128.34 ± 12.79 mmHg y 75.87 ± 8.74 mm Hg, respectivamente. La media de la presión del pulso fue de 52.47 ± 11.12 mmHg. Las presiones arteriales medias diurna y nocturna fueron de 96,23 ± 9,5 mmHg y 85.19 ± 10,23 mmHg, respectivamente. La frecuencia cardiaca fue de 71.4 ± 10.03 latidos por minuto, para la carga sistólica la media fue de 39,38 ± 27,80% y para la carga diastólica fue de 26,26 ± 24,32%. Conclusiones: El monitoreo ambulatorio de la presión arterial es considerado actualmente como el método más confiable y exacto para el diagnóstico y evaluación de la hipertensión arterial. Permite no solo el diagnóstico y seguimiento del comportamiento de las cifras tensionales en los hipertensos, sino la evaluación de otras variables, como es mostrado en este estudio, con un comportamiento de los resultados muy similar a otros estudios locales.

Abstract Objetive: To describe the behavior of hemodynamic parameters in a group of individuals undergoing ambulatory blood pressure monitoring. Material and methods: Descriptive, observational study on the behavior of hemodynamic parameters in ambulatory blood pressure monitoring of a group of patients assisted in two institutions in Medellín. Results: A total of 108 patients were analysed, assisted between September 2012 and October 2013. Average 24-hour systolic and diastolic blood pressure values were 128.34 ± 12.79 mmHg and 75.87 ± 8,74 mmHg, respectively. Average pulse pressure was 52.47 ± 11.12 mmHg. Day and night blood pressure values were 96.23 ± 9.5 mmHg and 85.19 ± 10.23 mmHg, respectively. Heart rate was 71.4 ± 10.03 beats per minute, it was 39.38 ± 27.80% for the systolic load and 26.26 ± 24.32% for the diastolic load. Conclusions : Ambulatory monitoring of blood pressure is considered nowadays to be the most reliable and exact method for diagnosing and assessing arterial hypertension. Not only does it allow diagnosis and control of the behavior of blood pressure levels in hypertensive patients, but also assesses other variables, like the ones shown in this study, with results being similar to those of other local studies.