Synthetic biology tools for environmental protection.

Synthetic biology transforms the way we perceive biological systems. Emerging technologies in this field affect many disciplines of science and engineering. Traditionally, synthetic biology approaches were commonly aimed at developing cost-effective microbial cell factories to produce chemicals from renewable sources. Based on this, the immediate beneficial impact of synthetic biology on the environment came from reducing our oil dependency. However, synthetic biology is starting to play a more direct role in environmental protection. Toxic chemicals released by industries and agriculture endanger the environment, disrupting ecosystem balance and biodiversity loss. This review highlights synthetic biology approaches that can help environmental protection by providing remediation systems capable of sensing and responding to specific pollutants. Remediation strategies based on genetically engineered microbes and plants are discussed. Further, an overview of computational approaches that facilitate the design and application of synthetic biology tools in environmental protection is presented.

Estudio exploratorio para la validación de la Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) en la población con esclerosis múltiple remitente recurrente del Hospital San Juan de Dios en Santiago de Chile / Exploratory study for the validation of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) in the population with recurrent remitting multiple sclerosis of the San Juan de Dios Hospital in Santiago, Chile

La evaluación cognitiva en personas adultas con esclerosis múltiple (EM) es un área fundamental a tener en cuenta en el proceso de intervención, debido a la alta prevalencia de deterioro cognitivo. En la actualidad, se ha recomendado la evaluación cognitiva por medio de la BICAMS (del inglés Brief International Cognitive Assessment for MS), que es una batería específica para evaluar a personas con EM, pero que no cuenta con validación en nuestro país. El presente estudio tiene como objetivo identificar el impacto de algunos factores clínicos (meses de evolución de la enfermedad y nivel de discapacidad) y personales (sexo, años de escolaridad y edad) que influyen en las medidas cognitivas de la BICAMS, a fin de contar con información relevante y precisa en un futuro proceso de validación. La muestra estuvo constituida por 38 personas con Esclerosis Múltiple Remitente Recurrente (EMRR). Los resultados mostraron que de los cinco factores clínicos observados, solo edad y sexo influyeron de manera significativa sobre los puntajes de las tres pruebas de la BICAMS. Por lo tanto, la validación de esta batería para la población chilena debiera incluir y/o controlar ambas variables de edad y sexo.

The evaluation of cognitive aspects among individuals with multiple sclerosis (MS) is key when considering intervention, because of high prevalence of cognitive impairments. At present, cognitive evaluation has been recommended by means of BICAMS (Brief International Cognitive Assessment for MS), which is a battery specifically constructed to assess individuals with MS. However, the battery has not been validated in Chile.The present study aims atdetermining the impact of clinical factors (months since condition's diagnosis and severeness level) and individual factors (sex, age, and years of schooling), which is expected to be accurate and valuable input for future validation processes. Sample consisted of 38 people with remittent-recurrent multiple sclerosis (RRMS). Results showed that only age and gender do significantly impact cognitive performance on all of three BICAMs subtests. Therefore, when validating this battery for Chilean individuals, both age and gender should be included and or controlled.

Engineering Saccharomyces cerevisiae for the Overproduction of ß-Ionone and Its Precursor ß-Carotene.

ß-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment ß-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current ß-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of ß-carotene and its subsequent conversion to ß-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on ß-carotene accumulation, and later, on ß-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For ß-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest ß-carotene-producing strains, reaching up to 33 mg/L of ß-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing ß-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Build Your Bioprocess on a Solid Strain-ß-Carotene Production in Recombinant Saccharomyces cerevisiae.

Robust fermentation performance of microbial cell factories is critical for successful scaling of a biotechnological process. From shake flask cultivations to industrial-scale bioreactors, consistent strain behavior is fundamental to achieve the production targets. To assert the importance of this feature, we evaluated the impact of the yeast strain design and construction method on process scalability -from shake flasks to bench-scale fed-batch fermentations- using two recombinant Saccharomyces cerevisiae strains capable of producing ß-carotene; SM14 and ßcar1.2 strains. SM14 strain, obtained previously from adaptive evolution experiments, was capable to accumulate up to 21 mg/gDCW of ß-carotene in 72 h shake flask cultures; while the ßcar1.2, constructed by overexpression of carotenogenic genes, only accumulated 5.8 mg/gDCW of carotene. Surprisingly, fed-batch cultivation of these strains in 1L bioreactors resulted in opposite performances. ßcar1.2 strain reached much higher biomass and ß-carotene productivities (1.57 g/L/h and 10.9 mg/L/h, respectively) than SM14 strain (0.48 g/L/h and 3.1 mg/L/h, respectively). Final ß-carotene titers were 210 and 750 mg/L after 80 h cultivation for SM14 and ßcar1.2 strains, respectively. Our results indicate that these substantial differences in fermentation parameters are mainly a consequence of the exacerbated Crabtree effect of the SM14 strain. We also found that the strategy used to integrate the carotenogenic genes into the chromosomes affected the genetic stability of strains, although the impact was significantly minor. Overall, our results indicate that shake flasks fermentation parameters are poor predictors of the fermentation performance under industrial-like conditions, and that appropriate construction designs and performance tests must be conducted to properly assess the scalability of the strain and the bioprocess.

Expanding Metabolic Capabilities Using Novel Pathway Designs: Computational Tools and Case Studies.

Design and selection of efficient metabolic pathways is critical for the success of metabolic engineering endeavors. Convenient pathways should not only produce the target metabolite in high yields but also are required to be thermodynamically feasible under production conditions, and to prefer efficient enzymes. To support the design and selection of such pathways, different computational approaches have been proposed for exploring the feasible pathway space under many of the above constraints. In this review, an overview of recent constraint-based optimization frameworks for metabolic pathway prediction, as well as relevant pathway engineering case studies that highlight the importance of rational metabolic designs is presented. Despite the availability and suitability of in silico design tools for metabolic pathway engineering, scarce-although increasing-application of computational outcomes is found. Finally, challenges and limitations hindering the broad adoption and successful application of these tools in metabolic engineering projects are discussed.

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.

Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

Reliability of Hand-Held Transcranial Doppler with M-mode Ultrasound in Middle Cerebral Artery Measurement.

PURPOSE: To determine the intra- and interrater agreement of mean flow velocity (MFV) and pulsatility index (PI) measurement in middle cerebral arteries, assessed by transcranial Doppler (TCD) with M-mode. METHODS: Masked experienced neurosonologists performed TCD with M-mode using handheld probe in healthy adult volunteers. The Bland-Altman method for concordance and intraclass correlation coefficient were used. RESULTS: Seventy-seven healthy volunteers and seven raters participated (3 on regular TCD shift and 4 off-shift). The intrarater absolute mean difference between measurements was 5.5 cm/s [95% confidence interval (CI), 4.7-6.3] for MVF and 0.073 (95% CI, 0.063-0.083) for PI. The difference between MFV measurements was significantly higher in off-shift raters (p = 0.015). The interrater absolute mean difference between measurements was 6.5 cm/s (95% CI, 5.5-7.5) for MVF and 0.065 (95% CI, 0.059-0.071) for PI. No influence was found for the middle cerebral artery side, volunteer's sex, or age, and there was no significant difference between raters. The intraclass correlation coefficient was 82.2% (95% CI 77.8-85.6) and 72.9% (95% CI 67.4-77.6) for MFV and PI, respectively. CONCLUSIONS: There exists good intra- and interrater agreement in MFV and PI measurements using M-mode TCD. These results support the use of this noninvasive tool and are important for clinical and investigational purposes.

Transcranial Doppler as a Predictor of Ischemic Events in Carotid Artery Dissection.

BACKGROUND: We aim to evaluate clinical features and transcranial Doppler (TCD) elements, as predictors of the development of ischemic events (IEs) in patients suffering from spontaneous carotid arterial dissection without stroke (CCADW). METHODS: Consecutive patients with CCADW, seen in Clínica Alemana de Santiago between April 2004 and January 2015, were evaluated clinically, and with TCD, microembolic signals (MES) monitoring and breath hold Index (BHI) test were performed. RESULTS: Forty-one patients with 45 CCADW were included. Mean age 41.9 years, 31 male, and 12 (29.1%) patients present with multiple CCADW. At the moment of TCD evaluation, 17 (41.4%) patients were being treated with antiplatelets and the rest under Heparin. TCD monitoring lasted in average 53.3 minutes and demonstrated at the moment of evaluation, MES in four carotid arteries (11.1%) of 3 patients and 13 (28.8%) abnormal BHI in 11 patients. Six IEs occurred in 3 patients, 3 strokes, and 3 transient ischemic attacks. In the univariate analysis correlating IE with clinical and ultrasonographic findings, the degree of carotid stenosis, the presence of multiple CAD, and the presence of MES plus abnormalities of BHI were significantly associated with the risk of an IE. Multivariable analysis showed that only the presence of MES plus abnormal BHI were significant (P < .001). MES and abnormal BHI were present in the 3 patients and in four arterial territories that had IE. CONCLUSIONS: TCD can identify a subgroup of patients with CCADW who are at high risk of IE.

Chemical vs. biotechnological synthesis of C13-apocarotenoids: current methods, applications and perspectives.

Apocarotenoids are natural compounds derived from the oxidative cleavage of carotenoids. Particularly, C13-apocarotenoids are volatile compounds that contribute to the aromas of different flowers and fruits and are highly valued by the Flavor and Fragrance industry. So far, the chemical synthesis of these terpenoids has dominated the industry. Nonetheless, the increasing consumer demand for more natural and sustainable processes raises an interesting opportunity for bio-production alternatives. In this regard, enzymatic biocatalysis and metabolically engineered microorganisms emerge as attractive biotechnological options. The present review summarizes promising bioengineering approaches with regard to chemical production methods for the synthesis of two families of C13-apocarotenoids: ionones/dihydroionones and damascones/damascenone. We discuss each method and its applicability, with a thorough comparative analysis for ionones, focusing on the production process, regulatory aspects, and sustainability.

Production of ß-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.

BACKGROUND: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid ß-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. RESULTS: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of ß-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased ß-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of ß-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase. CONCLUSIONS: An efficient ß-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest ß-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.

Improved calibration of a solid substrate fermentation model

Background: Calibration of dynamic models in biotechnology is challenging. Kinetic models are usually complex and differential equations are highly coupled involving a large number of parameters. In addition, available measurements are scarce and infrequent, and some key variables are often non-measurable. Therefore, effective optimization and statistical analysis methods are crucial to achieve meaningful results. In this research, we apply a metaheuristic scatter search algorithm to calibrate a solid substrate cultivation model. Results: Even though scatter search has shown to be effective for calibrating difficult nonlinear models, we show here that a posteriori analysis can significantly improve the accuracy and reliability of the estimation. Conclusions: Sensibility and correlation analysis helped us detect reliability problems and provided suggestions to improve the design of future experiments.

ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle.

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca(2+) homeostasis and muscle physiology.

Accuracy of transcranial Doppler compared with CT angiography in diagnosing arterial obstructions in acute ischemic strokes.

BACKGROUND AND PURPOSE: Patients with acute ischemic stroke and intracranial arterial obstructions have a poor prognosis and a high probability of deteriorating at 24 hours. We aimed to evaluate the diagnostic accuracy of power motion mode Doppler (PMD-TCD) compared with CT angiography as standard in diagnosing intracranial arterial obstructions in patients presenting with ischemic stroke of <24 hours. METHODS: Consecutive patients presenting with acute ischemic stroke to the emergency department underwent high-resolution brain CT angiography and PMD-TCD within a 6-hour difference. RESULTS: A total of 100 patients were included. PMD-TCD demonstrated 34 intracranial occlusions and CTA 33. There were 6 false-positives and 4 false-negative diagnoses with PMD-TCD. PMD-TCD had a positive likelihood ratio of 13.7, a negative likelihood ratio of 0.19, sensitivity of 81.8%, and specificity of 94% for detecting an arterial occlusion in any specific artery. Results for the middle cerebral artery were: positive likelihood ratio 24.6, negative likelihood ratio 0.045, sensitivity 95.6%, and specificity 96.2%. For the anterior circulation, the results were: positive likelihood ratio 18.5, negative likelihood ratio 0, sensitivity 100%, and specificity 94.5%. For the posterior circulation, the results were: positive likelihood ratio >1000, negative likelihood ratio 0.42, sensitivity 57.1%, and specificity 100%. The post-test probability for any occluded artery when PMD-TCD was positive increased for any admission National Institutes of Health Stroke Scale score but was especially remarkable for National Institutes of Health Stroke Scale scores between 7 and 15 points. CONCLUSIONS: PMD-TCD is valid compared with CT angiography for the diagnosis of arterial occlusions in patients with acute ischemic stroke, especially in middle cerebral artery obstructions.