WetLab-2: Wet Lab RNA SmartCycler Providing PCR Capability on ISS

The WetLab-2 system will provide sample preparation and qRT-PCR analysis on-board the ISS, a capability to enable using the ISS as a real laboratory. The system will be validated on SpX-7, and is planned for its first PI use on SpX-9

WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015

Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium

Background The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. Results The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. Conclusions The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.España, Ministerio de Economía y Competitividad, project BIO2015–69613-REspaña, Junta de Andalucía project CTS-6638 CTS-66

In Flight MiRNA Isolation and Recovery on the ISS Using the Wetlab-2 System

Due to advancements in RNA research, mi (micro) RNAs and other small nucleotide RNAs have become a major research field in biology including spaceflight research. The regulation of RNA transcription and processing by miRNAs makes miRNAs an appealing topic for genetics and molecular research. It has been estimated that over 60% of human gene transcripts are targets of miRNA regulation. In fact, this is true for all organisms, including plants and insects. Small nucleotide RNAs can also play a role in regulating gene expression, meaning that gene expression alone is not a complete picture of the potential genetic changes that occur in an organism during spaceflight. The goal of the WetLab-2 project is to isolate and recover miRNAs from various tissue sources on the International Space Station (ISS). No system currently exists that can isolate and recover small nucleotide RNA in space. However, the WetLab-2 system that was validated on the ISS in 2016 can be adapted to fit this purpose. We are currently testing the new modified protocols by running plant and mouse blood experiments in parallel, allowing us to demonstrate the effectiveness of the procedure on different sample types. We expect to be able to optimize and implement the modified miRNA protocols for use on future ISS flights

In Flight miRNA Isolation and Recovery on the ISS

Due to advancements in RNA research, mi (micro) RNAs and other small nucleotide RNAs have become a major research field in biology including spaceflight research. The regulation of RNA transcription and processing by miRNAs makes miRNAs an appealing topic for genetics and molecular research. It has been estimated that over 60% of human gene transcripts are targets of miRNA regulation. In fact, this is true for all organisms, including plants and insects. Small nucleotide RNAs can also play a role in regulating gene expression, meaning that gene expression alone is not a complete picture of the potential genetic changes that occur in an organism during spaceflight. The goal of the WetLab-2 project is to isolate and recover miRNAs from various tissue sources on the International Space Station (ISS). No system currently exists that can isolate and recover small nucleotide RNA in space. However, the WetLab-2 system that was validated on the ISS in 2016 can be adapted to fit this purpose. We are currently testing the new modified protocols by running plant and mouse blood experiments in parallel, allowing us to demonstrate the effectiveness of the procedure on different sample types. We expect to be able to optimize and implement the modified miRNA protocols for use on future ISS flights

Ocean-Atmosphere CO2 Fluxes in the North Atlantic Subtropical Gyre: Association with Biochemical and Physical Factors during Spring

We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Sea surface partial pressure of CO2 (pCO2) was measured continuously in a transect of the North Atlantic subtropical gyre between Santo Domingo, Dominican Republic (18.1° N, 68.5° W) and Vigo, Spain (41.9° N, 11.8° W) during spring 2011. Additional biogeochemical and physical variables measured to identify factors controlling the surface pCO2 were analyzed in discrete samples collected at 16 sites along the transect at the surface and to a depth of 200 m. Sea surface pCO2 varied between 309 and 662 μatm, and showed differences between the western and eastern subtropical gyre. The subtropical gyre acted as a net CO2 sink, with a mean flux of −5.5 ± 2.2 mmol m−2 day−1. The eastern part of the transect, close to the North Atlantic Iberian upwelling off the Galician coast, was a CO2 source with an average flux of 33.5 ± 9.0 mmol m−2 day−1. Our results highlight the importance of making more surface pCO2 observations in the area located east of the Azores Islands since air-sea CO2 fluxes there are poorly studied.The authors would like to thank those responsible for the two projects Proyecto Buque Escuela de Oceanografía 2011 (Research Project CTM 2009-08399-E/MAR) and the Malaspina Circumnavigation Expedition 2010 (Research Project Consolider-Ingenio, CSD 2008-00077), both funded by the Spanish Government. Their collaboration made this study possible. The authors would also like to thank J. Gómez-Enri and G. Navarro for their help with the satellite images.Peer reviewe

Synthesis and Bactericidal Properties of Hyaluronic Acid Doped with Metal Nanoparticles

A study on the nanoparticles size and the antibacterial properties of hyaluronic acid (HA) doped with nanoparticles is reported. Nanoparticles from gold, silver, copper, and silver palladium with HA support were performed. The solvated metal atom dispersion (SMAD) method with 2-propanol and HA was used. High-resolution transmission electron microscopy (HRTEM), infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA) were conducted. The average sizes of nanoclusters were as follows: HA-Au = 17.88 nm; HA-Ag = 50.41 nm; HA-Cu = 13.33 nm; and HA-AgPd = 33.22 nm. The antibacterial activity of solutions and films containing nanoparticles against American Type Culture Collection (ATCC) bacterial strains Escherichia coli (EC), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), and Pseudomonas aeruginosa (PA) was determined. Inhibition was observed for HA-Ag, HA-Cu, and HA-AgPd. Toxicological tests were performed in rats that were injected intraperitoneally with two concentrations of gold, copper, silver, and silver-palladium nanoparticles. No alterations in hepatic parameters, including ALT (alanine aminotransferase), GGT (gamma-glutamyl transpeptidase) bilirubin, and albumin, were observed after 14 days. These films could be used as promoters of skin recovery and Grades I and II cutaneous burns and as scaffolds

Evaluation of quality of life in Chilean patients with orthognathic surgery. A cohort study.

Objective: To compare the presurgical and immediate postsurgical quality of life in Chilean patients with orthognathic surgery. Material and Methods: Cohort study. The study included 30 patients (mean age 20.73, 53.33% male) who underwent orthognathic surgery primarily for severe skeletal abnormalities, (17 surgeries, 56.7%) and moderate skeletal abnormalities (12 surgeries, 40%), from three Chilean hospitals between February and June 2016. Patients were asked to answer the World Health Organization quality of life Bref version questionnaire (WHOQOL-BREF) validated in Spanish to measure quality of life (QoL) two weeks before and three months after the surgery. Scores for general QoL and for every domain of QoL were described. Variations in the scores of general QoL were analyzed according to sex and severity of orofacial malformation (mild, moderate or severe) (ttest p <0.05; STATA 10.0). Results: The average score for QoL according to the WHOQOL-BREF scale was 76.43±13.83 before surgery and 90.5±7.18 three months after surgery (p<0.001). Statistically significant differences according to sex and type of orofacial malformation were found (p<0.01). An increase in the score in all the domains of the WHOQOOL-BREF scale was observed. Conclusion: Orthognathic surgery significantly improved QoL scores in Chilean patients according to the WHOQOL-BREF scale

Carotenoid Biosynthesis in Fusarium

Many fungi of the genus Fusarium stand out for the complexity of their secondary metabolism. Individual species may differ in their metabolic capacities, but they usually share the ability to synthesize carotenoids, a family of hydrophobic terpenoid pigments widely distributed in nature. Early studies on carotenoid biosynthesis in Fusarium aquaeductuum have been recently extended in Fusarium fujikuroi and Fusarium oxysporum, well-known biotechnological and phytopathogenic models, respectively. The major Fusarium carotenoid is neurosporaxanthin, a carboxylic xanthophyll synthesized from geranylgeranyl pyrophosphate through the activity of four enzymes, encoded by the genes carRA, carB, carT and carD. These fungi produce also minor amounts of β-carotene, which may be cleaved by the CarX oxygenase to produce retinal, the rhodopsin’s chromophore. The genes needed to produce retinal are organized in a gene cluster with a rhodopsin gene, while other carotenoid genes are not linked. In the investigated Fusarium species, the synthesis of carotenoids is induced by light through the transcriptional induction of the structural genes. In some species, deep-pigmented mutants with up-regulated expression of these genes are affected in the regulatory gene carS. The molecular mechanisms underlying the control by light and by the CarS protein are currently under investigation.Junta de Andalucía project CTS-6638España, Gobierno BIO2012-39716, BIO2015-69613-R, AGL2014-53195R BIO2015-71703-RED

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS