Storage-dependent Changes Research Articles

Influence of seed pre-treatment and storage on germination and physiological characteristics of seeds of common mullein (Verbascum thapsus L.)

We report the seed germination and associated metabolic responses of differentially stored seeds of Verbascum thapsus L. (Scrophulariaceae), an economically and medicinally important herb from the cold desert region of Lahaul (Himachal Pradesh, India) in trans Himalaya, to certain physico-chemical and GA₃ treatments. The freshly harvested seeds of V. thapsus exhibited high (96 %) viability but low germination (55 %). During the storage of seeds under ambient conditions, viability did not change until 1-year but declined progressively thereafter showing a 33 % decline in 4-year stored seeds. The seed germination was promoted to varying extents by acid scarification (AS), SNP (sodium nitroprusside), SHC (sodium hypochlorite) and GA₃ pre-treatments. Storage-dependent changes in seed germination performance and responsiveness to different effectors were evident. Germination performance improved significantly due to 1-year storage followed by a sharp decline subsequently. The responsiveness of seeds to various treatments tended to be higher in seeds stored for ≥2 years. The changes in germination were paralleled by those in α-amylase activity in seeds. Lipid peroxidation generally increased during storage that was paralleled by a gradual decline in superoxide dismutase (SOD) activity and phenolic contents. Catalase (CAT) activity increased transiently in 1-year stored seeds and declined thereafter. Data are expected to have implications for multiplication and cultivation of V. thapsus.

Determination of the Time since Deposition of blood-traces in a forensic context: Application of untargeted LC-HR-MS/MS metabolomics profiling.

Being able to attest when a bloodstain was deposited at a crime scene can be invaluable to a prosecution process, and methods to provide that information have long been desired. Determining the Time since Deposition (TsD) of a trace would allow placing a subject both in space and time to the crime scene-or prove that a trace left by that person was unrelated to it because it was deposited before or after the time the crime had occurred. To this day, no method for TsD determination has made its way into routine forensic casework, mainly because of the numerous challenges that await when trying to understand and account for all the influencing and confounding factors that affect the aging process (such as, e.g., temperature, UV-light exposure, or humidity). Here, we present an untargeted metabolomics-based study using liquid chromatography high-resolution mass spectrometry (LC-HR-MS) and data-dependent acquisition to analyze blood samples aged under two distinctly different storage conditions over 48 weeks. Global differences in age- and storage-dependent changes in blood metabolomes were described, and TsD-classification strategies based on qualitative and quantitative assessment of molecular features (MFs) have been proposed. Based on the selected criteria to best predict the TsD, the dipeptide Phenylalanylalanine (PheAla) can be considered as a promising candidate for TsD prediction. In essence, changes in the blood metabolome dynamics showed a strong association with increasing TsD, but significant differences depending on storage conditioning were observed, facilitating the need to study further the influence of individual influencing factors on TsD determination.

Seed longevity and storage dependent changes in seed germination aspects of Berberis aristata and B. lycium from Western Himalaya: implications for seed storage and conservation

Seed longevity and storage dependent changes in seed germination aspects of Berberis aristata and B. lycium from Western Himalaya: implications for seed storage and conservation

Determination of the Time since Deposition of Blood Traces Utilizing a Liquid Chromatography-Mass Spectrometry-Based Proteomics Approach.

Knowledge about when a bloodstain was deposited at a crime scene can be of critical value in forensic investigation. A donor of a genetically identified bloodstain could be linked to a suspected time frame and the crime scene itself. Determination of the time since deposition (TsD) has been extensively studied before but has yet to reach maturity. We therefore conducted a proof-of-principle study to study time- and storage-dependent changes of the proteomes of dried blood stains. A bottom-up proteomics approach was employed, and high-resolution liquid-chromatography-mass-spectrometry (HR-LC-MS) and data-independent acquisition (DIA) were used to analyze samples aged over a 2 month period and two different storage conditions. In multivariate analysis, samples showed distinct clustering according to their TsD in both principal component analysis (PCA) and in partial least square discriminant analysis (PLS DA). The storage condition alters sample aging and yields different separation-driving peptides in hierarchical clustering and in TsD marker peptide selection. Certain peptides and amino acid modifications were identified and further assessed for their applicability in assessing passed TsD. A prediction model based on data resampling (Jackknife) was applied, and prediction values for selected peptide ratios were created. Depending on storage conditions and actual sample age, mean prediction performances ranges in between 70 and 130% for the majority of peptides and time points. This places this study as a first in investigating LC-MS based bottom-up proteomics approaches for TsD determination.

Seed germination in some important medicinal plants for seed standards

Studies were carried out at ICAR-Directorate of Medicinal and Aromatic Plants Research (DMAPR) with an objective to evaluate seed germination and set a laboratory seed standards of some important medicinal plants viz. Ashwagandha, Senna, Isabgol (Plantago ovata L.), P. indica L., Lepidium sativum L. (Asalio) and Artemesia. In the study, effect of different temperatures and storage duration of seeds was evaluated including freshly harvested seeds on germination. Results revealed significant difference in seed germination at different temperatures and storage duration for different medicinal plants. Among the species, P. indica exhibited decreased germination after a particular storage duration at 20oC. Seed germination in senna was found to be below 65% whereas, in ashwagandha it was more than 80%. Results also revealed that data on storage-dependent changes in seed germination and their responsiveness to temperature are of potential implication for propagation of these important medicinal plants.

Effect of storage duration and temperature on seed germination ofPlantago ovataL.,P. indicaL. andLepidium sativumL (Asalio)

A study was carried out at ICAR-Directorate of Medicinal and Aromatic Plants Research (DMAPR) with an objective to evaluate seed germination of three very important medicinal species viz. Plantago ovata L, P. indica L., and Lepidium sativum L (Asalio) for standardizing eff icient germination procedure and to determine minimum seed standards. In the study, effect of different temperatures (15, 20, 25 and 30°C) and storage duration from 7 months to 3 years old seeds was evaluated including freshly harvested seeds on germination. Results revealed that seed germination was >95% at 20°C in 7 months old seeds of all the species. However, it varied with storage duration with time taken for germination i.e. first count and final count was on 2nd and 6th day, respectively at 20°C. Seed germination more than 80% could also be recorded up to a storage limit of 24 months excluding first three months after the harvest. Among the species, P. indica exhibited decreased germination after this storage duration at 20°C, while, >95% of seed germination could be recorded in 7–8 months old seeds after harvest in all the species irrespective of temperatures indicating that there was physiological dormancy initially in these species. Results also revealed that data on storage-dependent changes in seed germination and their responsiveness to temperature of Isabgol species and L. sativum are of potential implication for propagation of these plant species.

Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells.

During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity was measured in hemolysates and not in individual RBCs. We hypothesized that analysis of G6PD activity in individual RBC identifies storage-dependent changes in G6PD function. Seven units of stored leukoreduced RBCs, stored in saline-adenine-glucose-mannitol, were sampled every week up to 6 weeks of storage. G6PD activity was determined with the cytofluorometric method and expressed as mean fluorescent intensity (MFI) per RBC. During storage, G6PD activity decreased significantly. Mean MFI after 3 days of storage was 27.8 ± 8.8 and gradually decreased significantly to 18.0 ± 8.3 after 42 days. G6PD activity decreases during storage of leukoreduced RBCs. Our results may form a new target to improve storage conditions of RBCs and subsequently improve the quality of transfusion products.

Predicting storage-dependent damage to red blood cells using nitrite oxidation kinetics, peroxiredoxin-2 oxidation, and hemoglobin and free heme measurements.

Storage-dependent damage to red blood cells (RBCs) varies significantly. Identifying RBC units that will undergo higher levels of hemolysis during storage may allow for more efficient inventory management decision-making. Oxidative-stress mediates storage-dependent damage to RBCs and will depend on the oxidant:antioxidant balance. We reasoned that this balance or redox tone will serve as a determinant of how a given RBC unit stores and that its assessment in "young" RBCs will predict storage-dependent hemolysis. RBCs were sampled from bags and segments stored for 7 to 42 days. Redox tone was assessed by nitrite oxidation kinetics and peroxiredoxin-2 (Prx-2) oxidation. In parallel, hemolysis was assessed by measuring cell-free hemoglobin (Hb) and free heme (hemin). Correlation analyses were performed to determine if Day 7 measurements predicted either the level of hemolysis at Day 35 or the increase in hemolysis during storage. Higher Day 7 Prx-2 oxidation was associated with higher Day 35 Prx-2 oxidation, suggesting that early assessment of this variable may identify RBCs that will incur the most oxidative damage during storage. RBCs that oxidized nitrite faster on Day 7 were associated with the greatest levels of storage-dependent hemolysis and increases in Prx-2 oxidation. An inverse relationship between storage-dependent changes in oxyhemoglobin and free heme was observed underscoring an unappreciated reciprocity between these molecular species. Moreover, free heme was higher in the bag compared to paired segments, with opposite trends observed for free Hb. Measurement of Prx-2 oxidation and nitrite oxidation kinetics early during RBC storage may predict storage-dependent damage to RBC including hemolysis-dependent formation of free Hb and heme.

A comparative study of the physical and chemical properties of nano-sized ZnO particles from multiple batches of three commercial products

Given the broad commercial applications for ZnO nanomaterials, accurate attribution of physicochemical characteristics that induce toxic effects is particularly important. We report on the physicochemical properties of three commercial nano-ZnO products: Z-COTE and Z-COTE HP1 from BASF, and Nanosun from Micronisers, and, for reference, “bulk” ZnO from Sigma-Aldrich. Z-COTE, Nanosun and “bulk” consist of uncoated particles with different sizes, while Z-COTE HP1 consists of nanoparticles with a hydrophobic coating. Specific batches of these ZnO products were included in the OECD Sponsorship Programme to test manufactured nanomaterials. In order to identify properties potentially susceptible to variations between production runs, three additional batches of Z-COTE and Nanosun and two additional batches of Z-COTE HP1 were also investigated here. In general, all products showed little variation between batches for properties measured from powdered samples, but batch variations in the amount of surface coating were evident for the coated Z-COTE HP1. Properties measured with samples dispersed in liquids (agglomeration, photocatalytic activity, dissolution) were highly dependent on dispersion protocols, and this made it difficult to differentiate between differences due to dispersion and due to batches. However, batch-sensitive properties did appear to be present in Z-COTE and Z-COTE HP1 (photocatalytic activity), and Nanosun (dissolution). Intra-batch time and/or storage-dependent changes in the applied surface coating, noted specifically for the OECD batch of Z-COTE HP1, highlight the need for best practice when storing and accessing stocks of nano products. Awareness of inter-batch and intra-batch variability is essential for commercial applications and for nanotoxicological studies aimed at identifying links between physicochemical properties and any adverse effects in biological systems.

Red blood cells stored for increasing periods produce progressive impairments in nitric oxide–mediated vasodilation

Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow. AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled. RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging. These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.

The Effects of Pre-Analytical Processing and Storage on Bovine Blood D- and L-Lactate Concentrations

To investigate processing- and storage-dependent changes in D- and L-lactate concentration, blood samples from eleven healthy Holstein calves were spiked with 3 mM D-lactic acid and 3 mM L-lactic acid immediately following collection (time 0) or left untreated for comparison. Serum and plasma, respectively, were separated 0.5 hours following collection or left in contact with blood cells, stored at 4°C and analyzed for D- and L-lactate concentration using enzymatic assays at 1, 2, 4, 8, 12, 24, and 48 hours. Concentrations were compared to the 1 hour sample. D- and L-Lactate concentrations in all separated samples were stable for up to 48 hours. When left in contact with cells, L-lactate concentration in untreated and spiked serum and in spiked plasma, D-Lactate concentration in untreated serum, and total lactate concentration in untreated serum increased significantly by 48 hours.

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

The effects of long‐term storage of human red blood cells on the glutathione synthesis rate and steady‐state concentration

Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production. RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed. After 6 weeks, the ATP concentration declined to 50 ± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 ± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 ± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration. This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.

Storage-Dependent Changes in Dormancy and Germination of Himalayan Mayapple (Podophyllum hexandrum Royle) Seeds and their Response to Gibberellic Acid

The storage-dependent changes in viability and dormancy/germination status of seeds of a Western population of Himalayan Mayapple (Podophyllum hexandrum), an endangered plant species of high medicinal value, have been monitored with particular reference to the changes in seed responsiveness to GA3. Shortly after harvest (3 months), seeds exhibited dormancy that was marginally overcome after storage for 30 months. GA3 treatment was effective in overcoming the dormancy, particularly when concentrated H2SO4 was used. The responsiveness of the seeds to GA3, however, decreased with the storage period. A subset of seeds that failed to germinate under a GA3-H2SO4-GA3 treatment eventually deteriorated. GA3-induced enhancement of the activities of α-amylase and dehydrogenases (2,3,5 triphenyltetrazolium chloride (TTC) reduction) corresponded to germination improvement in seeds stored for a short duration, but not in seeds stored for long period of time. Lipid peroxidation in seeds did not change owing to storage or GA3 treatment, whereas catalase activity tended to decline marginally. Seed phenolic contents were not involved in the seed germination behavior. Possible mechanisms of heterogeneity in responsiveness of seeds to GA3 are discussed.

A single assay for multiple storage‐sensitive red blood cell characteristics by means of infrared spectroscopy

To maintain a high quality of red blood cells (RBCs), RBC characteristics must be followed during storage under blood bank conditions. By means of infrared (IR) spectroscopy, several characteristics can be measured simultaneously. IR spectra were acquired for samples from RBCs that were collected and stored according to Dutch blood bank procedures for a period of up to 50 days. Spectra of the soluble cell components were acquired separately after hypotonic lysis of the cells, followed by centrifugation. Characteristic vibrational bands were analyzed with respect to storage time-dependent changes in peak position and in intensity. A decrease in corresponding peak intensities indicates that RBCs lose protein and lipid during storage. Changes in protein secondary structure during storage are largely confined to integral membrane proteins and membrane-associated proteins. A concurrent decrease in lipid packing density probably reflects the gradual change in cellular shape from discoidal to globular. By integration over a narrow range, storage-dependent changes in intracellular adenosine triphosphate (ATP) and glucose levels could be estimated. ATP levels decrease during storage, but stay above the required 75% of the initial level after 35 days of storage. Glucose concentrations stay well above 5 mmol/L over the entire storage period. IR spectroscopy is a promising technique to follow structural and metabolic changes in RBCs during storage under blood bank conditions. Several variables can be determined rapidly in a single measurement.

The proteome of red cell membranes and vesicles during storage in blood bank conditions

During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods of storage in blood bank conditions. RBCs and vesicles were isolated from RBCs after various storage periods. The proteins of RBC membranes and vesicles were separated by gel electrophoresis and identified by a semiquantitative proteomic analysis. Our findings confirm previous data, such as a storage-associated increase in hemoglobin binding to the membrane and aggregation and degradation of the integral membrane protein band 3, suggesting a remodeling of the RBC membrane during storage. Our data also show storage-dependent changes in the membrane association of proteasome and chaperone proteins, metabolic enzymes, small G proteins, and signal transduction proteins. Vesicles display similar changes in their protein composition during storage. The results of this analysis indicate that the storage-related changes in the RBC membrane are the results of disturbance and/or acceleration of physiologic processes such as cellular aging, including vesicle formation. The latter may serve to remove damaged membrane patches that would otherwise lead to accelerated RBC removal. These data provide a framework for future studies toward the development of better storage conditions and a reduction of the side effects of RBC transfusion.

Long-Time Respiration of Contaminated Soil Samples during Laboratory Storage under Different Conditions

Soil samples have to be stored during transportation and investigation in the laboratory if they are not analyzed directly at the site. Existing standards and investigations give no recommendations on handling and storage of soil materials for (eco-)toxicological investigations. The objective of this investigation was to determine if microbial turnovers and losses of volatile organic compounds mainly cause storage-dependent changes in soil samples. Furthermore, recommendations are given for storage of soil reserve samples for toxicological investigations. During 18 months of storage, the microbial respiration of six highly contaminated soil samples was determined. Physicochemical characteristics, such as contaminant and nutrient content, were analyzed before and after storage. From the investigations it can be concluded that the oxygen consumption depends on the storage temperature, organic matter content, nutrient content, and total content of toxic substances. Based on the results, a flow scheme was derived that could be a useful tool for a sequential approach to determine the storage capacity of contaminated soil samples and sites for toxicological investigations.

Evidence that electrofusion yield is controlled by biologically relevant membrane factors

Rabbit + rabbit and human + human combinations of erythrocyte ghost membranes were fused under the same conditions with an electric pulse. Storage at 4°C of ghost membranes from both rabbit and human erythrocytes showed no change with time but storage of the erythrocytes for various periods before ghost preparation showed consistent storage-dependent changes in fusion yield.

Alterations in blood components during storage and their clinical significance.

Storage-dependent changes in blood and components as affecting a patient with acute haemorrhage are described. With currently used preservatives and additives blood qualifies as "fresh" for two weeks after collection. Modified red cell concentrates are the optimal form of initial haemotherapy, with the possible exception of massive blood transfusion. Availability of platelet concentrates and other haemostatic blood components as routinely issued by a modern blood bank should meet almost all requirements for an acutely bleeding patient.