Piperonylic acid alters growth, mineral content accumulation and reactive oxygen species-scavenging capacity in chia seedlings.

p-Coumaric acid synthesis in plants involves the conversion of phenylalanine to trans-cinnamic acid via phenylalanine ammonia-lyase (PAL), which is then hydroxylated at the para-position under the action of trans-cinnamic acid 4-hydroxylase. Alternatively, some PAL enzymes accept tyrosine as an alternative substrate and convert tyrosine directly to p-coumaric acid without the intermediary of trans-cinnamic acid. In recent years, the contrasting roles of p-coumaric acid in regulating the growth and development of plants have been well-documented. To understand the contribution of trans-cinnamic acid 4-hydroxylase activity in p-coumaric acid-mediated plant growth, mineral content accumulation and the regulation of reactive oxygen species (ROS), we investigated the effect of piperonylic acid (a trans-cinnamic acid 4-hydroxylase inhibitor) on plant growth, essential macroelements, osmolyte content, ROS-induced oxidative damage, antioxidant enzyme activities and phytohormone levels in chia seedlings. Piperonylic acid restricted chia seedling growth by reducing shoot length, fresh weight, leaf area measurements and p-coumaric acid content. Apart from sodium, piperonylic acid significantly reduced the accumulation of other essential macroelements (such as K, P, Ca and Mg) relative to the untreated control. Enhanced proline, superoxide, hydrogen peroxide and malondialdehyde contents were observed. The inhibition of trans-cinnamic acid 4-hydroxylase activity significantly increased the enzymatic activities of ROS-scavenging enzymes such as superoxide dismutase, ascorbate peroxidase, catalase and guaiacol peroxidase. In addition, piperonylic acid caused a reduction in indole-3-acetic acid and salicylic acid content. In conclusion, the reduction in chia seedling growth in response to piperonylic acid may be attributed to a reduction in p-coumaric acid content coupled with elevated ROS-induced oxidative damage, and restricted mineral and phytohormone (indole-3-acetic acid and salicylic) levels.

DNA damage response of haematopoietic stem and progenitor cells to high-LET neutron irradiation.

The radiosensitivity of haematopoietic stem and progenitor cells (HSPCs) to neutron radiation remains largely underexplored, notwithstanding their potential role as target cells for radiation-induced leukemogenesis. New insights are required for radiation protection purposes, particularly for aviation, space missions, nuclear accidents and even particle therapy. In this study, HSPCs (CD34+CD38+ cells) were isolated from umbilical cord blood and irradiated with 60Co γ-rays (photons) and high energy p(66)/Be(40) neutrons. At 2 h post-irradiation, a significantly higher number of 1.28 ± 0.12 γ-H2AX foci/cell was observed after 0.5 Gy neutrons compared to 0.84 ± 0.14 foci/cell for photons, but this decreased to similar levels for both radiation qualities after 18 h. However, a significant difference in late apoptosis was observed with Annexin-V+/PI+ assay between photon and neutron irradiation at 18 h, 43.17 ± 6.10% versus 55.55 ± 4.87%, respectively. A significant increase in MN frequency was observed after both 0.5 and 1 Gy neutron irradiation compared to photons illustrating higher levels of neutron-induced cytogenetic damage, while there was no difference in the nuclear division index between both radiation qualities. The results point towards a higher induction of DNA damage after neutron irradiation in HSPCs followed by error-prone DNA repair, which contributes to genomic instability and a higher risk of leukemogenesis.

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, γ-H2AX. The use of an automated microscopy platform to score the number of γ-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the γ-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive γ-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

The Impact of Dose Rate on DNA Double-Strand Break Formation and Repair in Human Lymphocytes Exposed to Fast Neutron Irradiation.

The lack of information on how biological systems respond to low-dose and low dose-rate exposures makes it difficult to accurately assess the carcinogenic risks. This is of critical importance to space radiation, which remains a serious concern for long-term manned space exploration. In this study, the γ-H2AX foci assay was used to follow DNA double-strand break (DSB) induction and repair following exposure to neutron irradiation, which is produced as secondary radiation in the space environment. Human lymphocytes were exposed to high dose-rate (HDR: 0.400 Gy/min) and low dose-rate (LDR: 0.015 Gy/min) p(66)/Be(40) neutrons. DNA DSB induction was investigated 30 min post exposure to neutron doses ranging from 0.125 to 2 Gy. Repair kinetics was studied at different time points after a 1 Gy neutron dose. Our results indicated that γ-H2AX foci formation was 40% higher at HDR exposure compared to LDR exposure. The maximum γ-H2AX foci levels decreased gradually to 1.65 ± 0.64 foci/cell (LDR) and 1.29 ± 0.45 (HDR) at 24 h postirradiation, remaining significantly higher than background levels. This illustrates a significant effect of dose rate on neutron-induced DNA damage. While no significant difference was observed in residual DNA damage after 24 h, the DSB repair half-life of LDR exposure was slower than that of HDR exposure. The results give a first indication that the dose rate should be taken into account for cancer risk estimations related to neutrons.

Physico-elemental analysis of roasted organic coffee beans from Ethiopia, Colombia, Honduras, and Mexico using X-ray micro-computed tomography and external beam particle induced X-ray emission.

The physico-elemental profiles of commercially attained and roasted organic coffee beans from Ethiopia, Colombia, Honduras, and Mexico were compared using light microscopy, X-ray micro-computed tomography, and external beam particle induced X-ray emission. External beam PIXE analysis detected P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, Br, Rb, and Sr in samples. Linear discriminant analysis showed that there was no strong association between elemental data and production region, whilst a heatmap combined with hierarchical clustering showed that soil-plant physico-chemical properties may influence regional elemental signatures. Physical trait data showed that Mexican coffee beans weighed significantly more than beans from other regions, whilst Honduras beans had the highest width. X-ray micro-computed tomography qualitative data showed heterogeneous microstructural features within and between beans representing different regions. In conclusion, such multi-dimensional analysis may present a promising tool in assessing the nutritional content and qualitative characteristics of food products such as coffee.

A Novel Application of a Cryosectioning Technique to Aid Scat Hair Microanalysis.

Scat hair presents a diverse profile of hairs for morphological assessment that may find versatile applications in wildlife forensic investigations. Successful morphological assessment of scat hair microstructure, however, depends on a robust sectioning methodology. We assessed the feasibility and efficacy of a cryosectioning technique compared to that of a gold standard hand-sectioning technique. Scat hairs were embedded in paraffin wax and hand-sectioned, while cryopreserved scat hairs were sectioned with a cryostat. The results showed that cryosectioning preserved the pristine morphology of the scat hair and provided cross sections more amenable to high-resolution imaging of hair internal microstructure than hand-sectioning. The cryosectioning technique may find novel applications as a more reliable and robust technique to aid (i) scat hair internal microstructure analysis for cross-referencing with species identification keys in wildlife forensic studies and (ii) downstream toxicological analysis in wildlife forensic studies as hair biochemistry is not altered during cryopreservation.

A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality.

Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression.

Novel in situ evaluation of the role minerals play in the development of the hard-to-cook (HTC) defect of cowpeas and its effect on the in vitro mineral bioaccessibility.

Cowpea is a nutritionally important drought-resistant legume in sub-Saharan Africa. It is, however, underutilised, in part due to the hard-to-cook (HTC) defect caused by adverse storage conditions resulting in seeds not softening during cooking. This study introduced a novel evaluation of the potential role that minerals play in the development of the HTC defect. The mineral distribution in the cotyledons of normal and HTC cowpeas were analysed by Proton Induced X-ray Emission (PIXE) spectrometry. The phytate, tannin and total phenolic contents were analysed together with in vitro mineral bioaccessibility. In HTC cowpeas, Ca and Mg were more concentrated in the cell wall-middle lamella area of the parenchyma cells. This, together with the reduction in phytate content, confirmed the 'phytase-phytate-mineral' hypothesis as a mechanism for development of the HTC defect. Despite the phytate reduction in stored cowpeas, the HTC defect decreased the bioaccessibility of Ca, Fe and Zn in cowpeas.

Biofuels as a sustainable energy source: an update of the applications of proteomics in bioenergy crops and algae.

Sustainable energy is the need of the 21st century, not because of the numerous environmental and political reasons but because it is necessary to human civilization's energy future. Sustainable energy is loosely grouped into renewable energy, energy conservation, and sustainable transport disciplines. In this review, we deal with the renewable energy aspect focusing on the biomass from bioenergy crops to microalgae to produce biofuels to the utilization of high-throughput omics technologies, in particular proteomics in advancing our understanding and increasing biofuel production. We look at biofuel production by plant- and algal-based sources, and the role proteomics has played therein. This article is part of a Special Issue entitled: Translational Plant Proteomics.

A decade of plant proteomics and mass spectrometry: translation of technical advancements to food security and safety issues.

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world's population will reach 9-12 billion people demanding a food production increase of 34-70% (FAO, 2009) from today's food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future.

Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings.

Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghum variety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.