IntroductionThe future of human space missions relies on the ability to provide adequate food resources for astronauts and also to reduce stress due to the environment (microgravity and cosmic radiation). In this context, microgreens have been proposed for the astronaut diet because of their fast-growing time and their high levels of bioactive compounds and nutrients (vitamins, antioxidants, minerals, etc.), which are even higher than mature plants, and are usually consumed as ready-to-eat vegetables.MethodsOur study aimed to identify the best light recipe for the soilless cultivation of two cultivars of radish microgreens (Raphanus sativus, green daikon, and rioja improved) harvested eight days after sowing that could be used for space farming. The effects on plant metabolism of three different light emitting diodes (LED) light recipes (L1—20% red, 20% green, 60% blue; L2—40% red, 20% green, 40% blue; L3—60% red, 20% green, 20% blue) were tested on radish microgreens hydroponically grown. A fluorimetric-based technique was used for a real-time non-destructive screening to characterize plant methabolism. The adopted sensors allowed us to quantitatively estimate the fluorescence of flavonols, anthocyanins, and chlorophyll via specific indices verified by standardized spectrophotometric methods. To assess plant growth, morphometric parameters (fresh and dry weight, cotyledon area and weight, hypocotyl length) were analyzed.ResultsWe observed a statistically significant positive effect on biomass accumulation and productivity for both cultivars grown under the same light recipe (40% blue, 20% green, 40% red). We further investigated how the addition of UV and/or far-red LED lights could have a positive effect on plant metabolite accumulation (anthocyanins and flavonols).DiscussionThese results can help design plant-based bioregenerative life-support systems for long-duration human space exploration, by integrating fluorescence-based non-destructive techniques to monitor the accumulation of metabolites with nutraceutical properties in soilless cultivated microgreens.

Space exploration beyond the Low Earth Orbit requires the establishment of Bioregenerative Life Support Systems (BLSSs), which, through bioprocesses for primary resource recycling, ensure crew survival. However, the introduction of new organisms in confined space habitats must be carefully evaluated in advance to avoid unforeseen events that could compromise the mission. In this work, we have designed and built an experimental chamber, named Growing/Rearing Module (GRM), completely isolated and equipped with micro-environmental monitoring and control systems. This unit is specially intended for the study of single bioprocesses, which can be composed to design functional BLSSs. GRM can be implemented with specific devices for the biological system under study and the control of environmental parameters such as temperature, humidity, lighting and if required, pressure of gaseous components. GRM was validated in experiments of both microgreen cultivation, as a source of fresh food for astronauts, and rearing of the decomposer insect Hermetia illucens for bioconversion of organic waste. During the study of each bioprocess, the environmental and biological data were recorded, allowing to make preliminary assessments of the system efficiency. The GRM, as a completely confined environment, represents the first self-consistent unit that allows to fine-tune the optimal parameters for the operability of different bioprocesses. Furthermore, the upgradability according to the mission needs and the functional integrability of modules differently equipped are the keys to GRM versatility, representing a valuable tool for BLSSs' design.

IntroductionFuture long-term space missions will focus to the solar system exploration, with the Moon and Mars as leading goals. Plant cultivation will provide fresh food as a healthy supplement to astronauts’ diet in confined and unhealthy outposts. Ionizing radiation (IR) are a main hazard in outer space for their capacity to generate oxidative stress and DNA damage. IR is a crucial issue not only for human survival, but also for plant development and related value-added fresh food harvest. To this end, efforts to figure out how biofortification of plants with antioxidant metabolites (such as anthocyanins) may contribute to improve their performances in space outposts are needed.MethodsMicroTom plants genetically engineered to express the Petunia hybrida PhAN4 gene, restoring the biosynthesis of anthocyanins in tomato, were used. Seeds and plants from wild type and engineered lines AN4-M and AN4-P2 were exposed to IR doses that they may experience during a long-term space mission, simulated through the administration of gamma radiation. Plant response was continuously evaluated along life cycle by a non-disturbing/non-destructive monitoring of biometric and multiparametric fluorescence-based indices at both phenotypic and phenological levels, and indirectly measuring changes occurring at the primary and secondary metabolism level.ResultsResponses to gamma radiation were influenced by the phenological stage, dose and genotype. Wild type and engineered plants did not complete a seed-to-seed cycle under the exceptional condition of 30 Gy absorbed dose, but were able to cope with 0.5 and 5 Gy producing fruits and vital seeds. In particular, the AN4-M seeds and plants showed advantages over wild type: negligible variation of fluorimetric parameters related to primary metabolism, no alteration or improvement of yield traits at maturity while maintaining smaller habitus than wild type, biosynthesis of anthocyanins and maintained levels of these compounds compared to non-irradiated controls of the same age.DiscussionThese findings may be useful in understanding phenotypic effects of IR on plant growth in space, and lead to the exploitation of new breeding efforts to optimize plant performances to develop appropriate ideotypes for future long-term space exploration extending the potential of plants to serve as high-value product source.

Background: Releasing considerable numbers of radiation-sterilized males is a promising strategy to suppress mosquito vectors. However, releases may also include small percentages of biting females which translate to large numbers when releases are large.Currently, the effects of irradiation on the host-seeking and host-biting behaviors have not been exhaustively investigated. Information is also lacking regarding the effects of sterilizing treatment on the endosymbiotic bacterium Wolbachia , which is known to affect the vector competence of infected mosquitos. Methods: To ascertain the effects of irradiation on females, the pupae of two Aedes albopictus strains, differing in their natural or artificial Wolbachia infection type, and Ae. aegypti— which is not infected by Wolbachia— were treated with various doses of X-rays and monitored for key fitness parameters and biting behavior over a period of two weeks. The effect of radiation on Wolbachia was investigated by qPCR and FISH analysis. Results: Partial Ae. albopictus female sterility was achieved at 28 Gy but the number of weekly bites more than doubled compared to that of the controls. Radiation doses of 35 and 45 Gy completely inhibited progeny production but did not significantly affect the survival or flight ability of Ae. albopictus females and caused a tripling of the number of bites per female per week (compared to untreated controls). These results were also confirmed in Ae. aegypti after treatment at 50 Gy. Wolbachia density decreased significantly in 45-Gray-irradiated females, with the greatest decreases in the early irradiation group (26±2-hour-old pupae). Wolbachia density also decreased as adults aged. This trend was confirmed in ovaries but not in extra-ovarian tissues. FISH analysis showed a strongly reduced Wolbachia -specific fluorescence in the ovaries of 13±1-day-old females. Conclusions: These results suggest that, under SIT programs, the vector capacity of a target population could increase with the frequency of the irradiated females co-released with the sterile males due to an increased biting rate. In the context of a successful suppression, the related safety issues could be generally negligible, but they should be conservatively evaluated when large scale programs relying on imperfect sexing and high overflooding release ratios are run for long time in areas endemic for arboviral diseases. Also, the effects of irradiation on the vector competence deserve further investigation.

Soilless cultivation of saffron (Crocus sativus) in a controlled environment represents an interesting alternative to field cultivation, in order to obtain a standardized high-quality product and to optimize yields. In particular, pharma-grade saffron is fundamental for therapeutic applications of this spice, whose efficacy has been demonstrated in the treatment of macular diseases, such as Age-related Macular Degeneration (AMD). In this work, a hydroponic cultivation system was developed, specifically designed to meet the needs of C. sativus plant. Various cultivation recipes, different in spectrum and intensity of lighting, temperature, photoperiod and irrigation, have been adopted to study their effect on saffron production. The experimentation involved the cultivation of corms from two subsequent farm years, to identify and validate the optimal conditions, both in terms of quantitative yield and as accumulation of bioactive metabolites, with particular reference to crocins and picrocrocin, which define the 'pharma-grade' quality of saffron. Through HPLC analysis and chromatography it was possible to identify the cultivation parameters suitable for the production of saffron with neuroprotective properties, evaluated by comparison with an ISO standard and the REPRON® procedure. Furthermore, the biochemical characterization was completed through NMR and high-resolution mass spectrometry analyses of saffron extracts. The whole experimental framework allowed to establish an optimized protocol to produce pharma-grade saffron, allowing up to 3.2 g/m2 harvest (i.e., more than three times higher than field production in optimal conditions), which meets the standards of composition for the therapy of AMD.

Releasing considerable numbers of radiation-sterilized males is a promising strategy to suppress mosquito vectors. However, under large-scale settings, small percentages of biting females can also be released in the field.Currently, the effects of the irradiation on host-seeking and host-biting behaviors have not been exhaustively investigated. Information is also lacking regarding the effects of a sterilizing treatment on the endosymbiotic bacterium Wolbachia, that is known to attenuate the vector competence of infected mosquito species.To ascertain the risks associated with the release of irradiated females, the pupae of two Aedes albopictus strains, differing by their natural or artificial Wolbachia infection type, were X-ray-treated using various doses to monitor key fitness parameters and biting behavior over two weeks. The effect of the radiation on Wolbachia was investigated by qPCR and FISH analysis. Radiation doses of 35 and 45 Gy completely inhibited progeny production but did not significantly affect Ae. albopictus female survival and flight ability, and caused a tripling of the number of bites per female per week compared to untreated controls. These results were also confirmed in Ae. aegypti after treatment at 50 Gy. A partial Ae. albopictus female sterility was achieved at 28 Gy but the number of weekly bites more than doubled compared to the controls. The Wolbachia density showed a significant downward trend in 45-Gy-irradiated females and decreased with adult aging and in younger treated pupae. This trend was confirmed in ovaries but not in extraovaric tissues. FISH analysis evidenced a strongly reduced Wolbachia-specific fluorescence in the ovaries of 13±1-day-old females. These findings suggested that irradiation could markedly increase the vectorial capacity of female mosquitoes, raising concerns about open field programs involving the release of substantial numbers of irradiated mosquitoes in the absence of perfect sexing methods.To reduce the reproductive potential of a target wild population of mosquitoes, X- or gamma-ray-based irradiation is a common procedure used to induce sterility in males prior to their release in the field. A small percentage of females escaping the sexing procedure may be also unintentionally released. Herein, we tested the effect of the radiation treatment on Aedes albopictus females and found that doses commonly used in SIT programs were not sufficient to significantly reduce survival and flight abilities, and the treated females continued to seek hosts even after engorgement, tripling the weekly number of bites compared to the controls. This phenomenon was confirmed in irradiated Ae. aegypti females. Moreover, the radiation treatment reduced the density of the endosymbiotic Wolbachia bacteria and markedly affected their distribution in the tissues of Ae. albopictus possibly reducing their ability to affect the vector competence, which is common in infected Aedes species. This was more evident when radiation had been applied on younger pupae and it increased with female aging. Results suggest that large-scale control programs based on mosquito irradiation should include rigorous methods for perfect sexing to avoid the risk of release of a myriad enhanced vectors.

Plant cultivation on spacecraft or planetary outposts is a promising and actual perspective both for food and bioactive molecules production. To this aim, plant response to ionizing radiations, as an important component of space radiation, must be assessed through on-ground experiments due to the potentially fatal effects on living systems. Hereby, we investigated the effects of X-rays and γ-rays exposure on tomato “hairy root” cultures (HRCs), which represent a solid platform for the production of pharmaceutically relevant molecules, including metabolites and recombinant proteins. In a space application perspective, we used an HRC system previously fortified through the accumulation of anthocyanins, which are known for their anti-oxidant properties. Roots were independently exposed to different photon radiations, namely X-rays (250 kV) and γ-rays (Co60, 1.25 MeV), both at the absorbed dose levels of 0.5, 5, and 10 Gy. Molecular changes induced in the proteome of HRCs were investigated by a comparative approach based on two-dimensional difference in-gel electrophoresis (2D-DIGE) technology, which allowed to highlight dynamic processes activated by these environmental stresses. Results revealed a comparable response to both photon treatments. In particular, the presence of differentially represented proteins were observed only when roots were exposed to 5 or 10 Gy of X-rays or γ-rays, while no variations were appreciated at 0.5 Gy of both radiations, when compared with unexposed control. Differentially represented proteins were identified by mass spectrometry procedures and their functional interactions were analyzed, revealing variations in the activation of stress response integrated mechanisms as well as in carbon/energy and protein metabolism. Specific results from above-mentioned procedures were validated by immunoblotting. Finally, a morphometric analysis verified the absence of significant alterations in the development of HRCs, allowing to ascribe the observed variations of protein expression to processes of acclimation to ionizing radiations. Overall results contribute to a meaningful risk evaluation for biological systems exposed to extra-terrestrial environments, in the perspective of manned interplanetary missions planned for the near future.

BACKGROUND:A better understanding of locally advanced cervical cancer (LACC) is mandatory for further improving the rates of disease control, since a significant proportion of patients still fail to respond or undergo relapse after concurrent chemoradiation treatment (CRT), and survival for these patients has generally remained poor.METHODS:To identify specific markers of CRT response, we compared pretreatment biopsies from LACC patients with pathological complete response (sensitive) with those from patients showing macroscopic residual tumor (resistant) after neoadjuvant CRT, using a proteomic approach integrated with gene expression profiling. The study of the underpinning mechanisms of chemoradiation response was carried out through in vitro models of cervical cancer.RESULTS:We identified annexin A2 (ANXA2), N-myc downstream regulated gene 1 (NDRG1) and signal transducer and activator of transcription 1 (STAT1) as biomarkers of LACC patients' responsiveness to CRT. The dataset collected through qPCR on these genes was used as training dataset to implement a Random Forest algorithm able to predict the response of new patients to this treatment. Mechanistic investigations demonstrated the key role of the identified genes in the balance between death and survival of tumor cells.CONCLUSIONS:Our results define a predictive gene signature that can help in cervical cancer patient stratification, thus providing a useful tool towards more personalized treatment modalities.

Among the strategies targeting vector control, the exploitation of the endosymbiont Wolbachia to produce sterile males and/or invasive females with reduced vector competence seems to be promising. A new Aedes albopictus transinfection (ARwP-M) was generated by introducing wMel Wolbachia in the ARwP line which had been established previously by replacing wAlbA and wAlbB Wolbachia with the wPip strain. Various infection and fitness parameters were studied by comparing ARwP-M, ARwP and wild-type (SANG population) Ae. albopictus sharing the same genetic background. Moreover, the vector competence of ARwP-M related to chikungunya, dengue and zika viruses was evaluated in comparison with ARwP. ARwP-M showed a 100% rate of maternal inheritance of wMel and wPip Wolbachia. Survival, female fecundity and egg fertility did not show to differ between the three Ae. albopictus lines. Crosses between ARwP-M males and SANG females were fully unfertile regardless of male age while egg hatch in reverse crosses increased from 0 to about 17% with SANG males aging from 3 to 17 days. When competing with SANG males for SANG females, ARwP-M males induced a level of sterility significantly higher than that expected for an equal mating competitiveness (mean Fried index of 1.71 instead of 1). The overall Wolbachia density in ARwP-M females was about 15 fold higher than in ARwP, mostly due to the wMel infection. This feature corresponded to a strongly reduced vector competence for chikungunya and dengue viruses (in both cases, 5 and 0% rates of transmission at 14 and 21 days post infection) with respect to ARwP females. Results regarding Zika virus did not highlight significant differences between ARwP-M and ARwP. However, none of the tested ARwP-M females was capable at transmitting ZIKV. These findings are expected to promote the exploitation of Wolbachia to suppress the wild-type Ae. albopictus populations.

Static magnetic fields created by superconducting magnets have been proposed as an effective solution to protect spacecrafts and planetary stations from cosmic radiations. This shield can deflect high-energy particles exerting injurious effects on living organisms, including plants. In fact, plant systems are becoming increasingly interesting for space adaptation studies, being useful not only as food source but also as sink of bioactive molecules in future bioregenerative life-support systems (BLSS). However, the application of protective magnetic shields would generate inside space habitats residual magnetic fields, of the order of few hundreds milli Tesla, whose effect on plant systems is poorly known. To simulate the exposure conditions of these residual magnetic fields in shielded environment, devices generating high-intensity static magnetic field (SMF) were comparatively evaluated in blind exposure experiments (250 mT, 500 mT and sham - no SMF-). The effects of these SMFs were assayed on tomato cultures (hairy roots) previously engineered to produce anthocyanins, known for their anti-oxidant properties and possibly useful in the setting of BLSS. Hairy roots exposed for periods ranging from 24 h to 11 days were morphometrically analyzed to measure their growth and corresponding molecular changes were assessed by a differential proteomic approach. After disclosing blind exposure protocol, a stringent statistical elaboration revealed the absence of significant differences in the soluble proteome, perfectly matching phenotypic results. These experimental evidences demonstrate that the identified plant system well tolerates the exposure to these magnetic fields. Results hereby described reinforce the notion of using this plant organ culture as a tool in ground-based experiments simulating space and planetary environments, in a perspective of using tomato 'hairy root' cultures as bioreactor of ready-to-use bioactive molecules during future long-term space missions.

The mosquito Aedes albopictusi is a competent vector of harmful human pathogens, including viruses causing dengue and chikungunya. Cytoplasmic incompatibility (CI) induced by endosymbiotic Wolbachia can be used to produce functionally sterile males that can be released in the field as a suppression tool against this mosquito. Because the available sexing methods are not efficient enough to avoid unintentional release of a few transinfected females, we assessed the CI pattern in crosses between wPip Wolbachia-transinfected (ARwP) females and wild-type males of Ae. albopictus in this study. Quantitative polymerase chain reaction was used to monitor the titer of the Wolbachia strains that naturally infect Ae. albopictus, that is, wAlbA and wAlbB, in age-controlled males and females. Data were coupled with incompatibility level detected when the above-mentioned males were crossed with ARwP females. Wolbachia infection titer was also monitored in samples of wild caught males. Incompatibility level was positively correlated only with wAlbA density. Crosses between wild-type males having very low wAlbA density (<0.001 wAlbA/actin copy numbers) and ARwP females were partially fertile (CIcorr = 68.06 ± 6.20). Individuals with low wAlbA titer were frequently found among sampled wild males (30%-50% depending on the site and period). ARwP males can be as considered as a very promising tool for suppressing Ae. albopictus. However, crosses between wild males having low wAlbA density and ARwP females may be partially fertile. In the case of local establishment of the transinfected mosquito line, this occurrence may favor the replacement of the wild-type mosquitoes with the ARwP line, thus reducing the long-term efficacy of incompatible insect technique. Various alternative strategies have been discussed to prevent this risk and to exploit Wolbachia as a tool to control Ae. albopictus.

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a similar to 144?000 customized Compositae protein database, which included about 19?000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.

The analysis of grapevine (Vitis vinifera) berries at the transcriptomic, proteomic, and metabolomic levels can provide great insight into the molecular events underlying berry development and postharvest drying (withering). However, the large and very different data sets produced by such investigations are difficult to integrate. Here, we report the identification of putative stage-specific biomarkers for berry development and withering and, to our knowledge, the first integrated systems-level study of these processes. Transcriptomic, proteomic, and metabolomic data were integrated using two different strategies, one hypothesis free and the other hypothesis driven. A multistep hypothesis-free approach was applied to data from four developmental stages and three withering intervals, with integration achieved using a hierarchical clustering strategy based on the multivariate bidirectional orthogonal projections to latent structures technique. This identified stage-specific functional networks of linked transcripts, proteins, and metabolites, providing important insights into the key molecular processes that determine the quality characteristics of wine. The hypothesis-driven approach was used to integrate data from three withering intervals, starting with subdata sets of transcripts, proteins, and metabolites. We identified transcripts and proteins that were modulated during withering as well as specific classes of metabolites that accumulated at the same time and used these to select subdata sets of variables. The multivariate bidirectional orthogonal projections to latent structures technique was then used to integrate the subdata sets, identifying variables representing selected molecular processes that take place specifically during berry withering. The impact of this holistic approach on our knowledge of grapevine berry development and withering is discussed.

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.

Pepper (Capsicum spp.) is an important vegetable crop that provides a unique pungent sensation when eaten. Through construction of a pepper variome map, we examined the main groups that emerged during domestication and breeding of C. annuum, their relationships and temporal succession, and the molecular events underlying the main transitions. The results showed that the initial differentiation in fruit shape and pungency, increase in fruit weight, and transition from erect to pendent fruits, as well as the recent appearance of large, blocky, sweet fruits (bell peppers), were accompanied by strong selection/fixation of key alleles and introgressions in two large genomic regions. Furthermore, we identified Up, which encodes a BIG GRAIN protein involved in auxin transport, as a key domestication gene that controls erect vs pendent fruit orientation. The up mutation gained increased expression especially in the fruit pedicel through a 579-bp sequence deletion in its 5′ upstream region, resulting in the phenotype of pendent fruit. The function of Up was confirmed by virus-induced gene silencing. Taken together, these findings constitute a cornerstone for understanding the domestication and differentiation of a key horticultural crop.