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Abstract Background Common bean (Phaseolus vulgaris) is one of the main nutritional resources in the world, and a low environmental impact source of protein. However, the majority of its cultivation areas are affected by drought and this scenario is only expected to worsen with climate change. Stomatal closure is one of the most important plant responses to drought and the MYB60 transcription factor is among the key elements regulating stomatal aperture. If targeting and mutating the MYB60 gene of common bean would be a valuable strategy to establish more drought-tolerant beans was therefore investigated. Results The MYB60 gene of common bean, with orthology to the Arabidopsis AtMYB60 gene, was found to have conserved regions with MYB60 typical motifs and architecture. Stomata-specific expression of PvMYB60 was further confirmed by q-RT PCR on organs containing stomata, and stomata-enriched leaf fractions. Further, function of PvMYB60 in promoting stomata aperture was confirmed by complementing the defective phenotype of a previously described Arabidopsis myb60-1 mutant. Conclusions Our study finally points PvMYB60 as a potential target for obtaining more drought-tolerant common beans in the present context of climate change which would further greatly contribute to food security particularly in drought-prone countries.

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.

Abstract Global climate change, causing large parts of the world to become drier with longer drought periods, severely affects production of common beans (Phaseolus vulgaris L.). The bean is worldwide the most produced and consumed food grain legume in the human diet. In common beans, adapted to moderate climates, exposure to drought/heat stress not only results in significant reduction of bean yield but also the nutritional value. This review explores the contribution of common beans to food and nutrient security as well as health. Also discussed is the existing knowledge of the impact of drought/heat stress, associated with a changing climate, specifically on iron (Fe) and phytic acid (PA) that are both among the most important mineral and anti‐nutritional compounds found in common beans. Further discussed is how the application of modern “omics” tools contributes in common beans to higher drought/heat tolerance as well as to higher Fe and reduced PA content. Finally, possible future actions are discussed to develop new common bean varieties with both improved drought/heat tolerance and higher mineral (Fe) content.

The capsule robot (CR) is a promising endoscopic method in gastrointestinal diagnosis because of its low discomfort to users. Most CRs are used to acquire image information only and lack the ability to collect samples. Although some biopsy capsule robots (BCRs) have been developed, it remains challenging to acquire the intestinal tissue while avoiding tearing and adhesion due to the flexibility of colonic tissue. In this study, we develop a BCR with a novel sampling strategy in which soft tissue is scratched with sharp blades rotating at high speed to avoid tissue tearing. In the BCR design, a spiral spring with prestored energy is used to release high energy within a short period of time, which is difficult for a motor or magnet to perform within a small capacity installation space. The energy of the tightened spiral spring is transmitted to drive sharp blades to rotate quickly via a designed gear mechanism. To guarantee reliable sampling, a Bowden cable is used to transmit the user’s manipulation to trigger the rotation of the blades, and the triggering force transmitted by the cable can be monitored in real time by a force sensor installed at the manipulating end. A prototype of the proposed BCR is designed and fabricated, and its performance is tested through in vitro experiments. The results show that the proposed BCR is effective and the size of its acquired samples satisfies clinical requirements.

Phytic acid (PA) represents the major storage form of seed phosphate (P). During seed maturation, it accumulates as phytate salts chelating various mineral cations, therefore reducing their bioavailability. During germination, phytase dephosphorylates PA releasing both P and cations which in turn can be used for the nutrition of the growing seedling. Animals do not possess phytase, thus monogastric animals assimilate only 10% of the phytate ingested with feed, whilst 90% is excreted and may contribute to cause P pollution of the environment. To overcome this double problem, nutritional and environmental, in the last four decades, many low phytic acid (lpa) mutants (most of which affect the PA-MRP transporters) have been isolated and characterized in all major crops, showing that the lpa trait can increase the nutritional quality of foods and feeds and improve P management in agriculture. Nevertheless, these mutations are frequently accompanied by negative pleiotropic effects leading to agronomic defects which may affect either seed viability and germination or plant development or in some cases even increase the resistance to cooking, thus limiting the interest of breeders. Therefore, although some significant results have been reached, the isolation of lpa mutants improved for their nutritional quality and with a good field performance remains a goal so far not fully achieved for many crops. Here, we will summarize the main pleiotropic effects that have been reported to date in lpa mutants affected in PA-MRP transporters in five productive agronomic species, as well as addressing some of the possible challenges to overcome these hurdles and improve the breeding efforts for lpa mutants.

Low-phosphorus stress is a challenging factor in limiting plant development. Soybean is cultivated in soils often low in phosphorus. However, on average 65% of total P is in the form of organic phosphates, which are unavailable to plants unless hydrolyzed to release inorganic phosphate. One approach for enhancing crop P acquisition from organic P sources is boosting the activity of acid phosphatases (APases). This study seeks to understand the role of an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP18) in soybean. Thus, the gene was isolated and a final vector (AtPAP18/pK7GWG2D) was built. Composite soybean plants were created using Agrobacterium rhizogenes-mediated transformation. A. rhizogenes K599 carrying the AtPAP18/pK7GWG2D vector with egfp as a reporter gene was used for soybean hairy root transformation. Analysis of Egfp expression detected fluorescence signals in transgenic roots, whereas there was no detectable fluorescence in control hairy roots. The enzyme assay showed that the APase activity increased by 2-fold in transgenic hairy roots. The transformed hairy roots displayed an increase in plant soluble P and total P contents, as compared with the control plants, leading to improved biomass production. RT-PCR analysis revealed high expression levels of AtPAP18 in transformed hairy roots. It is noteworthy that these primers amplified no PAP18 transcript in control hairy roots. Taken together, the findings demonstrated that overexpression of the AtPAP18 gene offers an operative tactic to reduce the utilization of inorganic phosphorus (Pi) fertilizer through increased acquisition of soil Pi, especially improving the crop yield on soils low in available P.

Seed size is the result of complex molecular networks controlling the development of the seed coat (of maternal origin) and the two fertilization products, the embryo and the endosperm. In this study we characterized the role of Arabidopsis thaliana MADS-domain transcription factor SEEDSTICK (STK) in seed size control. STK is known to regulate the differentiation of the seed coat as well as the structural and mechanical properties of cell walls in developing seeds. In particular, we further characterized stk mutant seeds. Genetic evidence (reciprocal crosses) of the inheritance of the small-seed phenotype, together with the provided analysis of cell division activity (flow cytometry), demonstrate that STK acts in the earlier phases of seed development as a maternal activator of growth. Moreover, we describe a molecular mechanism underlying this activity by reporting how STK positively regulates cell cycle progression via directly activating the expression of E2Fa, a key regulator of the cell cycle. Altogether, our results unveil a new genetic network active in the maternal control of seed size in Arabidopsis.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

The role of secondary metabolites in the determination of cell identity has been an area of particular interest over recent years, and studies strongly indicate a connection between cell fate and the regulation of enzymes involved in secondary metabolism. In Arabidopsis thaliana, the maternally derived seed coat plays pivotal roles in both the protection of the developing embryo and the first steps of germination. In this regard, a characteristic feature of seed coat development is the accumulation of proanthocyanidins (PAs - a class of phenylpropanoid metabolites) in the innermost layer of the seed coat. Our genome-wide transcriptomic analysis suggests that the ovule identity factor SEEDSTICK (STK) is involved in the regulation of several metabolic processes, providing a strong basis for a connection between cell fate determination, development and metabolism. Using phenotypic, genetic, biochemical and transcriptomic approaches, we have focused specifically on the role of STK in PA biosynthesis. Our results indicate that STK exerts its effect by direct regulation of the gene encoding BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This is consistent with the idea that MADS-domain proteins control the expression of their target genes through the modification of chromatin states. STK might thus recruit or regulate histone modifying factors to control their activity. In addition, we show that STK is able to regulate other BAN regulators. Our study demonstrates for the first time how a floral homeotic gene controls tissue identity through the regulation of a wide range of processes including the accumulation of secondary metabolites.

Abstract Background The restoration of complex hand functions by creating a novel bidirectional link between the nervous system and a dexterous hand prosthesis is currently pursued by several research groups. This connection must be fast, intuitive, with a high success rate and quite natural to allow an effective bidirectional flow of information between the user's nervous system and the smart artificial device. This goal can be achieved with several approaches and among them, the use of implantable interfaces connected with the peripheral nervous system, namely intrafascicular electrodes, is considered particularly interesting. Methods Thin-film longitudinal intra-fascicular electrodes were implanted in the median and ulnar nerves of an amputee's stump during a four-week trial. The possibility of decoding motor commands suitable to control a dexterous hand prosthesis was investigated for the first time in this research field by implementing a spike sorting and classification algorithm. Results The results showed that motor information (e.g., grip types and single finger movements) could be extracted with classification accuracy around 85% (for three classes plus rest) and that the user could improve his ability to govern motor commands over time as shown by the improved discrimination ability of our classification algorithm. Conclusions These results open up new and promising possibilities for the development of a neuro-controlled hand prosthesis.

Abstract Background One of the main scientific and technological challenges of rehabilitation bioengineering is the development of innovative methodologies, based on the use of appropriate technological devices, for an objective assessment of patients undergoing a rehabilitation treatment. Such tools should be as fast and cheap to use as clinical scales, which are currently the daily instruments most widely used in the routine clinical practice. Methods A human-centered approach was used in the design and development of a mechanical structure equipped with eight force/torque sensors that record quantitative data during the initiation of a predefined set of Activities of Daily Living (ADL) tasks, in isometric conditions. Results Preliminary results validated the appropriateness, acceptability and functionality of the proposed platform, that has become now a tool used for clinical research in three clinical centres. Conclusion This paper presented the design and development of an innovative platform for whole-body force and torque measurements on human subjects. The platform has been designed to perform accurate quantitative measurements in isometric conditions with the specific aim to address the needs for functional assessment tests of patients undergoing a rehabilitation treatment as a consequence of a stroke. The versatility of the system also enlightens several other interesting possible areas of application for therapy in neurorehabilitation, for research in basic neuroscience, and more.

Abstract Background In the past, several research groups have shown that when a velocity dependent force field is applied during upper limb movements subjects are able to deal with this external perturbation after some training. This adaptation is achieved by creating a new internal model which is included in the normal unperturbed motor commands to achieve good performance. The efficiency of this motor control mechanism can be compromised by pathological disorders or by muscular-skeletal modifications such as the ones due to the natural aging process. In this respect, the present study aimed at identifying the age-related modifications of upper limb motor control strategies during adaptation and de-adaptation processes in velocity dependent force fields. Methods Eight young and eight elderly healthy subjects were included in the experiment. Subjects were instructed to perform pointing movements in the horizontal plane both in a null field and in a velocity dependent force field. The evolution of smoothness and hand path were used to characterize the performance of the subjects. Furthermore, the ability of modulating the interactive torque has been used as a paradigm to explain the observed discoordinated patterns during the adaptation process. Results The evolution of the kinematics during the experiments highlights important behavioural differences between the two groups during the adaptation and de-adaptation processes. In young subjects the improvement of movement smoothness was in accordance with the expected learning trend related to the consolidation of the internal model. On the contrary, elders did not show a coherent learning process. The kinetic analysis pointed out the presence of different strategies for the compensation of the external perturbation: older people required an increased involvement of the shoulder with a different modulation of joint torque components during the evolution of the experiments. Conclusion The results obtained with the present study seem to confirm the presence of different adaptation mechanisms in young and senior subjects. The strategy adopted by young subjects was to first minimize hand path errors with a secondary process that is consistent with the optimization of the effort. Elderly subjects instead, seemed to shift the importance of the two processes involved in the control loop slowing the mechanism optimizing kinematic performance and enabling more the dynamic adaptation mechanism.

Abstract Background Motivation is an important factor in rehabilitation and frequently used as a determinant of rehabilitation outcome. Several factors can influence patient motivation and so improve exercise adherence. This paper presents the design of two robot devices for use in the rehabilitation of upper limb movements, that can motivate patients during the execution of the assigned motor tasks by enhancing the gaming aspects of rehabilitation. In addition, a regular review of the obtained performance can reinforce in patients' minds the importance of exercising and encourage them to continue, so improving their motivation and consequently adherence to the program. In view of this, we also developed an evaluation metric that could characterize the rate of improvement and quantify the changes in the obtained performance. Methods Two groups (G1, n = 8 and G2, n = 12) of patients with chronic stroke were enrolled in a 3-week rehabilitation program including standard physical therapy (45 min. daily) plus treatment by means of robot devices (40 min., twice daily) respectively for wrist (G1) and elbow-shoulder movements (G2). Both groups were evaluated by means of standard clinical assessment scales and the new robot measured evaluation metric. Patients' motivation was assessed in 9/12 G2 patients by means of the Intrinsic Motivation Inventory (IMI) questionnaire. Results Both groups reduced their motor deficit and showed a significant improvement in clinical scales and the robot measured parameters. The IMI assessed in G2 patients showed high scores for interest, usefulness and importance subscales and low values for tension and pain subscales. Conclusion Thanks to the design features of the two robot devices the therapist could easily adapt training to the individual by selecting different difficulty levels of the motor task tailored to each patient's disability. The gaming aspects incorporated in the two rehabilitation robots helped maintain patients' interest high during execution of the assigned tasks by providing feedback on performance. The evaluation metric gave a precise measure of patients' performance and thus provides a tool to help therapists promote patient motivation and hence adherence to the training program.

Studies have shown that educational robotics (ER) may impact student learning, especially in relation to STEM (science, technology, engineering, and mathematics) areas. In the STEM framework, particularly for younger children, the "E" and the "T" are considered to be missing letters, because few studies have concentrated on teaching and evaluating technology and engineering through ER activities. This study aimed to develop and test the efficacy of an ER protocol to teach robotics in a sample of 389 students, hypothesizing that girls would be as successful as boys. A Robotics Questionnaire assessing the basics of robotics was developed for this study. A Wilcoxon nonparametric test was performed in order to evaluate improvements (p < 0.05). A Mann-Whitney nonparametric test was performed in order to test the presence of gender differences (p < 0.05). Data indicated significant improvements for all the age ranges considered. No gender differences were found. In order to evaluate the efficacy of a didactic intervention utilizing ER, it is important to assess the impact on children's technological and engineering (robotics, in particular) knowledge.