Metabolomic analysis of tomato seed germination

Introduction

Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development.

Objective

This study aimed to assess an integrative approach to explore genetics modulating seed metabolism in different developmental stages and the link between seed metabolic- and germination traits.

Methods

We have utilized gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) metabolite profiling to characterize tomato seeds during dry and imbibed stages. We describe, for the first time in tomato, the use of a so-called generalized genetical genomics (GGG) model to study the interaction between genetics, environment and seed metabolism using 100 tomato recombinant inbred lines (RILs) derived from a cross between Solanum lycopersicum and Solanum pimpinellifolium.

Results

QTLs were found for over two-thirds of the metabolites within several QTL hotspots. The transition from dry to 6 h imbibed seeds was associated with programmed metabolic switches. Significant correlations varied among individual metabolites and the obtained clusters were significantly enriched for metabolites involved in specific biochemical pathways.

Conclusions

Extensive genetic variation in metabolite abundance was uncovered. Numerous identified genetic regions that coordinate groups of metabolites were detected and these will contain plausible candidate genes. The combined analysis of germination phenotypes and metabolite profiles provides a strong indication for the hypothesis that metabolic composition is related to germination phenotypes and thus to seed performance.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Phosphorus availability from bone char in a P-fixing soil influenced by root-mycorrhizae-biochar interactions

Aims

The objectives of this study were to evaluate (1) the fertilizer potential of bone char, (2) the effects of wood biochar on plant-available phosphorus (P), and (3) the role of root-mycorrhizae-biochar interactions in plant P acquisition from a P-fixing soil.

Methods

Incubation and pot experiments were conducted with a P-fixing soil and maize with or without root hairs and arbuscular mycorrhizae (AM) inoculation. Olsen-, resin-P and plant P accumulation were used to estimate P availability from bone char, co-pyrolyzed bone char-wood biochar, and separate bone char and wood biochar additions produced at 60, 350 and 750 °C, and Triple Superphosphate (TSP).

Results

Maize inoculated with AM showed similar P accumulation when fertilized with either 750 °C bone char or TSP. Pyrolyzing bone did not increase extractable P in soil in comparison to unpyrolyzed bone, apart from a 67 % increase in resin-extractable P after additions of bone char pyrolyzed at 350 °C. Despite greater Olsen-P extractability, co-pyrolysis of bone with wood reduced maize P uptake. Wood biochars reduced resin-P from bone char by 14–26 %, whereas oven-dried wood increased resin-P by 23 %.

Conclusions

Bone char is an effective P fertilizer, especially if root-AM interactions are simultaneously considered. Biochar influences plant access to soil P and requires careful management to improve P availability.

     

Molecular dynamics in germinating,endophyte-colonized quinoa seeds

Aims

The pseudo-cereal quinoa has an outstanding nutritional value. Seed germination is unusually fast, and plant tolerance to salt stress exceptionally high. Seemingly all seeds harbor bacterial endophytes. This work examines mitogen-activated protein kinase (MAPK) activities during early development. It evaluates possible contribution of endophytes to rapid germination and plant robustness.

Methods

MAPK activities were monitored in water- and NaCl-imbibed seeds over a 4-h-period using an immunoblot-based approach. Cellulolytic and pectinolytic abilities of bacteria were assessed biochemically, and cellular movement, biofilm, elicitor and antimicrobial compound synthesis genes sequenced. GyrA-based, cultivation-independent studies provided first insight into endophyte diversity.

Results

Quinoa seeds and seedlings exhibit remarkably complex and dynamic MAPK activity profiles. Depending on seed origin, variances exist in MAPK patterns and probably also in endophyte assemblages. Mucilage-degrading activities enable endophytes to colonize seed surfaces of a non-host species, chia, without apparent adverse effects.

Conclusions

Owing to their motility, cell wall-loosening and elicitor-generating abilities, quinoa endophytes have the potential to drive cell expansion, move across cell walls, generate damage-associated molecular patterns and activate MAPKs in their host. Bacteria may thus facilitate rapid germination and confer a primed state directly upon seed rehydration. Transfer into non-native crops appears both desirable and feasible.

     

A novel method for single-grain-based metabolic profiling of Arabidopsis seed

Introduction

In plant metabolomics, metabolite contents are often normalized by sample weight. However, accurate weighing of very small samples, such as individual Arabidopsis thaliana seeds (approximately 20 µg), is difficult, which may lead to irreproducible results.

Objectives

We aimed to establish alternative normalization methods for seed-grain-based comparative metabolomics of A. thaliana.

Methods

Arabidopsis thaliana seeds were assumed to have a prolate spheroid shape. Using a microscope image of each seed, the lengths of major and minor axes were measured by fitting a projected 2-dimensional shape of each seed as an ellipse. Metabolic profiles of individual diploid or tetraploid A. thaliana seeds were measured by our highly sensitive protocol (“widely targeted metabolomics”) that uses liquid chromatography coupled with tandem quadrupole mass spectrometry. Mass spectrometric analysis of 1 µL of solution extract identified more than 100 metabolites. The data were normalized by various seed-size measures, including seed volume (single-grain-based analysis). For comparison, metabolites were extracted from 4 mg of diploid and tetraploid A. thaliana seeds and their metabolic profiles were analyzed by normalization of weight (weight-based analysis).

Results

A small number of metabolites showed statistically significant differences in the single-grain-based analysis compared to weight-based analysis. A total of 17 metabolites showed statistically different accumulation between ploidy types with similar fold changes in both analyses.

Conclusion

Seed-size measures obtained by microscopic imaging were useful for data normalization. Single-grain-based analysis enables evaluation of metabolism of each seed and elucidates the metabolic profiles of precious bioresources by using small amounts of samples.

     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

     

Fusion expression of cecropin B-like antibacterial peptide in <Emphasis Type="Italic">Pichia</Emphasis> GS115 and its antibacterial mechanism

Objectives

To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB.

Results

The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 μg/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity.

Conclusion

The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Establishing rates of lateral expansion of cyanobacterial biological soil crusts for optimal restoration

Aims

Biocrusts that form on topsoils contribute ecosystem services to drylands, and their loss under anthropogenic pressure has negative ecological consequences. Therefore, development of biocrust inoculation technology for restoration is of interest. This requires knowledge of biocrust growth and dispersal. To contribute to this, we determined the speed at which biocrusts expand laterally based on the self-propelled motility of cyanobacteria.

Methodology

We inoculated sterile soil with natural biocrusts and incubated them over a year in a greenhouse under conditions mimicking local precipitation, monitoring the crust’s lateral expansion using time-course photography, chlorophyll a content, and microscopic inspection. Concurrent uninoculated controls served to monitor, and discount, natural inoculation by aeolian propagules.

Results

While the expansion front was highly variable in space, biocrusts expanded in the order of 2 cm month^?1, but only in seasons with moderate temperatures (Spring and Fall). Microcoleus vaginatus, Microcoleus steenstrupii, and Scytonema spp. advanced at averages of 1 cm month^?1, the crust advance front being preferentially driven by specialized propagules (hormogonia). These rates are within expectations based on instantaneous gliding motility speeds of cyanobacteria.

Conclusions

Based on the expansion capability of biocrusts during growth seasons, greenhouse inoculum units can be optimally spaced to fill 4–8 cm gaps.

     

Enhancing the production of phenolic compounds during barley germination by using chitooligosaccharides to improve the antioxidant capacity of malt

Objective

To enhance the production of phenolic compounds during barley germination using chitooligosaccharide as an elicitor to improve the antioxidant capacity of malt.

Results

When used as an elicitor for barley germination, chitooligosaccharide with a molecular weight of 3 kDa, added at 10 mg/kg barley kernels during the first steeping cycle, led to the maximum production of phenolic compounds. Compared with the control with no chitooligosaccharide added to the steeping water, the total phenolic content was increased by 54.8%. Increases in the total phenolic content of the barley malt occurred when chitooligosaccharide was applied during the first or both the first and the second steeping cycles. Thus the antioxidant capacity of barley malt was increased significantly by adding chitooligosaccharide during the steeping process.

Conclusion

Applying chitooligosaccharides during the steeping process increased the content of phenolic compounds thus improving the antioxidant capacity of the barley malt.

     

Asymmetric effects of litter removal and litter addition on the structure and function of soil microbial communities in a managed pine forest

Aims

The effect of different MeJA doses applied prior to or simultaneously with toxic Al on biochemical and physiological properties of Vaccinium corymbosum cultivars with contrasting Al resistance was studied.

Methods

Legacy (Al-resistant) and Bluegold (Al-sensitive) plants were treated with and without toxic Al under controlled conditions: a) without Al and MeJA, b) 100 μM Al, c) 100 μM Al + 5 μM MeJA, d) 100 μM Al + 10 μM MeJA and e) 100 μM Al + 50 μM MeJA. MeJA was applied to leaves 24 h prior to or simultaneously with Al in nutrient solution. After 48 h, Al-concentration, lipid peroxidation (LP), H₂O₂, antioxidant activity, total phenols, total flavonoids, phenolic compounds and superoxide dismutase activity (SOD) of plant organs were analyzed.

Results

Al-concentrations increased with Al-treatment in both cultivars, being Al, LP and H₂O₂ concentrations reduced with low simultaneous MeJA application. Higher MeJA doses induced more oxidative damage than the lowest. Legacy increased mainly non-enzymatic compounds, whereas Bluegold increased SOD activity to counteract Al³⁺.

Conclusions

Low MeJA doses applied simultaneously with Al³⁺ increased Al-resistance in Legacy by increasing phenolic compounds, while Bluegold reduced oxidative damage through increment of SOD activity, suggesting a diminution of its Al-sensitivity. Higher MeJA doses could be potentially toxic. Studies are needed to determine the molecular mechanisms involved in the protective MeJA effect against Al-toxicity.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Pig slurry acidification,separation technology and thermal conversion affect phosphorus availability in soil amended with the derived solid fractions,chars or ashes

Aims

The aim was to study effects of slurry acidification, separation technology and thermal processing on the availability of P in soil amended with the solid fraction of pig slurry.

Methods

Acidified and non-acidified slurry were separated using different technologies: screw press (SCR), decanting centrifuge (DEC) and drainage after chemical pretreatment (CHE). Solids and pyrolysed (400 °C or 600 °C, char) or combusted (625 °C, ash) solids were applied to two soils, with triple superphosphate (TSP) as a reference. Soil P availability was determined over 12 weeks using diffusive gradients in thin films (DGT).

Results

The initial availability of P from DEC and CHE solids was similar to TSP in both soils. After 6 weeks no significant difference was observed between DEC solids and DEC chars. Acidification did not significantly affect P availability in the solids-amended soils over time, but it did affect P availability with the thermally processed materials.

Conclusions

Application of separated pig slurry solids generally increased soil P availability initially, but declining with time, indicating soil P fixing reactions. Chars and ashes on the other hand showed lower initial P availability, but remained constant or increased slightly with chars yielding P availability similar to solids after 12 weeks.

     

R-wave synchronised atrial pacing in pediatric patients with postoperative junctional ectopic tachycardia: the atrioventricular interval investigated by computational analysis and clinical evaluation

Background

R-wave synchronised atrial pacing is an effective temporary pacing therapy in infants with postoperative junctional ectopic tachycardia. In the technique currently used, adverse short or long intervals between atrial pacing and ventricular sensing (AP–VS) may be observed during routine clinical practice.

Objectives

The aim of the study was to analyse outcomes of R-wave synchronised atrial pacing and the relationship between maximum tracking rates and AP–VS intervals.

Methods

Calculated AP–VS intervals were compared with those predicted by experienced pediatric cardiologist.

Results

A maximum tracking rate (MTR) set 10 bpm higher than the heart rate (HR) may result in undesirable short AP–VS intervals (minimum 83 ms). A MTR set 20 bpm above the HR is the hemodynamically better choice (minimum 96 ms). Effects of either setting on the AP–VS interval could not be predicted by experienced observers. In our newly proposed technique the AP–VS interval approaches 95 ms for HR > 210 bpm and 130 ms for HR < 130 bpm. The progression is linear and decreases strictly (? 0.4 ms/bpm) between the two extreme levels.

Conclusions

Adjusting the AP–VS interval in the currently used technique is complex and may imply unfavorable pacemaker settings. A new pacemaker design is advisable to allow direct control of the AP–VS interval.

     

Embedded rock fragments affect alpine steppe plant growth,soil carbon and nitrogen in the northern Tibetan Plateau

Background and Aims

Rock fragments within topsoil have important effects on soil properties and plant growth. This study mainly aimed to investigate the relationships between rock fragments, soil carbon (C) and nitrogen (N) densities and vegetation biomass in an alpine steppe.

Methods

Rock fragments, plant and soil samples were collected from four topographic positions (top, upper, lower, and bottom) on a hillslope.

Results

Volumetric rock fragment content within the 0–30 cm soil profile varied from 17.8 to 30.5%, the upper position value was significantly greater (P < 0.05) than those at other positions. The highest aboveground biomass was observed at the lower position (921 kg ha^?1), while the highest belowground biomass within the 0–30 cm profile was found at the upper position (4460 kg ha^?1). More fine earth and plant litter input accompanied by lower C and N losses induced by rainfall erosion resulted in higher soil organic C and total N densities (28.6 Mg C ha^?1 and 2.87 Mg N ha^?1) at the lower position.

Conclusions

Rock fragments may promote root growth but limit aboveground biomass production, and can therefore change the biomass distribution pattern. Our findings provide more evidence for scientifically assessing alpine steppe productivity.

     

A seed-recruited microbiome protects developing seedlings from disease by altering homing responses of <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> zoospores

Aims

We investigated potential mechanisms by which a seed microbiome recruited from vermicomposted dairy manure alters Pythium aphanidermatum zoospore mediated pathogenesis in cucumber.

Methods

Bioassays were conducted to measure arrival of zoospores at the seed surface via qPCR and subsequent seedling disease incidence. Seed exudates were collected at relevant time points for use in zoospore microscopy assays. Metabolomic analysis was used to characterize seed exudates.

Results

Microbes recruited by the germinating seed from a disease suppressive substrate within 8 hours of sowing prevented zoospore arrival at the seed surface, modified seed exudates and reduced disease incidence. In vitro exposure to microbially modified seed exudates altered zoospore homing responses and reduced both encystment and germination compared to control exudates. Combining modified and control exudates failed to restore zoospore attraction to levels observed with control exudates. Observed zoosporolytic activity of the modified exudates was unique to the ethyl acetate fraction and metabolomic analysis revealed several putative zoosporolytic compounds present at higher relative abundance when compared to control exudates.

Conclusions

The observed disease suppression was likely due to the production of a specific zoosporolytic compound or set of compounds in the spermosphere by one or more members of the seed-recruited vermicompost microbiome.

     

Association of 24 h–systolic blood pressure variability and cardiovascular disease in patients with obstructive sleep apnea

Background

To evaluate association of 24 h–systolic blood pressure (SBP) variability and obstructive sleep apnea (OSA) as defined by the apnea-hypopnea index ≥5/h; and association of 24 h–SBP variability and prevalent cardiovascular disease (CVD) in OSA patients.

Methods

Participants underwent polysomongraphy to evaluate the presence of OSA, and 24 h–ambulatory blood pressure monitoring was applied to evaluate 24 h–SBP variability as indexed by weighted 24 h–standard deviation (SD) of SBP. Between-group differences were evaluated in participants with and without OSA. Participants with OSA were divided into high and low 24 h–SBP variability groups and between-group differences were evaluated.

Results

Mean age of 384 participants was 50 years old and 42.2% had OSA. Mean 24 h–systolic/diastolic BP were 130/78 mmHg, with mean weighted 24 h–SD of systolic/diastolic BP were 12.9/7.3 mmHg. Compared to those without OSA, OSA participants had higher clinic-, 24 h-, daytime- and nighttime-SBP, and weighted 24 h, daytime- and nighttime-SD of SBP. Age, prevalent CVD and OSA, usage of angiotensin converting enzyme inhibitor/angiotensin receptor blocker, calcium channel blocker and diuretic were significantly associated with 24 h–SBP variability. In OSA patients, compared to those with low variability, participants with high variability had higher weighted 24 h, daytime- and nighttime-SD of SBP. After adjusted for covariates including clinic-SBP and 24 h–SBP, per 1-SD increment weighted 24 h–SD of SBP was associated with 21% increased prevalent CVD.

Conclusions

Patients with newly-diagnosed OSA have higher 24 h–SBP variability compared to those without OSA; in OSA patients, increased 24 h–SBP variability is associated with increased prevalence of CVD.