Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

Download Full-text

Unconventional effects of long-term storage of microwave-modified chicken egg white lysozyme preparations

Scientific Reports ◽

10.1038/s41598-021-89849-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Grzegorz Leśnierowski ◽

Tianyu Yang ◽

Renata Cegielska-Radziejewska

Keyword(s):

Microwave Radiation ◽

Egg White ◽

Process Time ◽

High Quality ◽

Chicken Egg ◽

Egg White Lysozyme ◽

Physicochemical Changes ◽

Long Term Storage ◽

Term Storage

AbstractThermal modification is an effective method that induces significant expansion of the antimicrobial properties and other valuable properties of chicken egg white lysozyme. In our latest research, a new innovative method of enzyme modification was developed, in which microwave radiation was used as an energy source to process liquid lysozyme concentrate (LLC). After modification, high-quality preparations were obtained. However, long-term storage in a concentrated form initiated various processes that caused darkening over time and could also lead to other significant changes to their structure and, consequently, to their functional properties. This necessitated multidirectional research to explain this phenomenon. This paper presents the results of research aimed at assessing the physicochemical changes in the properties of microwave-modified lysozyme in the form of a liquid concentrate after long-term storage under refrigeration conditions. The assessment also considered the conditions under the acidity of the modifying medium and the duration of the microwave modification. The analysis showed that the values of the basic parameters determining the quality and usefulness of the modified enzyme significantly improved during long-term storage of the preparations. The greatest changes were observed in the preparations modified for the longest time and in the most acidic environment (process time 260 s, pH 2.0), the number of oligomers under these conditions increased by 18% after 12 months of holding, and the surface hydrophobicity increased by as much as 31%. In addition, microbiological tests showed that the preparations of microwave-modified lysozyme had an effect on gram-positive bacteria as well as on gram-negative, and this effect was significantly enhanced after 12 months. The results confirm that LLC modification with microwave radiation is a highly efficient method to prepare high-quality and high utility potential lysozyme. Notably, an interesting and important phenomenon was the observation of the unconventional behaviour of the preparations during their long-term storage, which increased their utility potential significantly.

Download Full-text

Summer Pruning: An Ecological Alternative to Postharvest Calcium Treatment to Improve Storability of High Quality Apple cv. ‘Reinette du Canada’

Food Science and Technology International ◽

10.1177/1082013210366977 ◽

2010 ◽

Vol 16 (4) ◽

pp. 343-350 ◽

Cited By ~ 6

Author(s):

M. Guerra ◽

P.A. Casquero

Keyword(s):

Titratable Acidity ◽

Soluble Solids ◽

High Quality ◽

Long Term Storage ◽

Postharvest Application ◽

Bitter Pit ◽

Best Parameter ◽

Term Storage ◽

Organic Orchards

Two strategies, summer pruning and postharvest Ca treatment, were studied in apple (Malus domestica Borkh) cv. ‘Reinette du Canada’ in order to analyze its effect on the fruit quality during storage. Summer pruning and Ca treatment reduced external and internal bitter-pits; so after 180 days of storage, both treatments decreased external bitter-pit by 10.0% and 16.7%, respectively. Summer pruning influenced color, firmness, total soluble solids and titratable acidity (TA) of fruit during storage, whereas Ca treatment only affected firmness and TA. Fruit from pruned trees had significant lower K and Mg than those from unpruned trees and Ca treatment increased Ca content. Orchard management, by means of summer pruning, combined with Ca postharvest application would be useful to prevent losses due to bitter-pit during storage in commercial orchards. However, in organic orchards, summer pruning would be the ecological alternative to decrease bitter-pit incidence during storage in high quality apple cv. ‘Reinette du Canada’. K/Ca ratio, on the peel at harvest, turned out to be the best parameter to correlate with external and internal bitter-pits during storage; so this ratio would be useful to predict bitter-pit on long-term storage.

Download Full-text

3′ MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage

Laboratory Investigation ◽

10.1038/s41374-020-0446-z ◽

2020 ◽

Vol 100 (10) ◽

pp. 1345-1355 ◽

Cited By ~ 2

Author(s):

Stefaniya Boneva ◽

Anja Schlecht ◽

Daniel Böhringer ◽

Hans Mittelviefhaus ◽

Thomas Reinhard ◽

...

Keyword(s):

Rna Sequencing ◽

Storage Time ◽

Transcriptome Profiling ◽

Rna Seq ◽

Fresh Tissue ◽

Long Term Storage ◽

Ffpe Samples ◽

The Impact ◽

Term Storage

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

Download Full-text

Effect of Short- and Long-Term Storage on Human Serum and Recombinant Apolipoprotein E Concentration

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2000.077 ◽

2000 ◽

Vol 38 (6) ◽

Cited By ~ 6

Author(s):

Françoise Schiele ◽

Monique Vincent-Viry ◽

Bernard Herbeth ◽

Athanase Visvikis ◽

Gérard Siest

Keyword(s):

Apolipoprotein E ◽

Human Serum ◽

Long Term Storage ◽

Short And Long Term ◽

Term Storage

Download Full-text

Understanding Actinides through the Role of 5f Electrons

MRS Bulletin ◽

10.1557/mrs2001.178 ◽

2001 ◽

Vol 26 (9) ◽

pp. 684-688 ◽

Cited By ~ 3

Author(s):

T. Gouder ◽

F. Wastin ◽

J. Rebizant ◽

G.H. Lander

Keyword(s):

Predictive Power ◽

Quality Data ◽

Nuclear Fuels ◽

High Quality ◽

High Quality Data ◽

Long Term Storage ◽

5F Electrons ◽

Term Storage

Studies of the actinide elements and compounds were (and are) motivated by the need to characterize their structural and thermodynamic properties for the development of nuclear fuels and the treatment of waste, whether it be for long-term storage or ideas involving transmutation in high-powered accelerators. For the most part, tables giving these data exist, although the data for transuranium compounds are rather sparse. A much more difficult task is to understand the data and develop theories that have predictive power in this part of the periodic table. In doing this, however, we are confronted with the extremely complicated electronic structure of the 5f shell and the great paucity of high-quality data on materials containing transuranium isotopes.

Download Full-text

The Influence of the Long-Term Storage of Hardwood on the Selected Physical and Mechanical Properties in View of Pulping

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.688.10 ◽

2016 ◽

Vol 688 ◽

pp. 10-16

Author(s):

Blažej Seman ◽

Anton Geffert ◽

Jarmila Geffertova

Keyword(s):

Mechanical Properties ◽

Impact Strength ◽

Continuous Production ◽

Beech Wood ◽

Physical And Mechanical Properties ◽

Long Term Storage ◽

The Impact ◽

Term Storage ◽

Strength In Bending

Wood is loosely stored to ensure continuous production inside paper mills where it is exposed to the effect of external factors. The impact of storage leads to some changes of mechanical and physical properties of wood, but these changes are not the same in all specimens. In this paper, it has been observed that the long term storage of wood influences the impact strength in bending and the permeability of wood for fluids. During the storage, there was a decrease of impact strength in bending of poplar heartwood by 28.3% and oak by 22.1% and mature beech wood by 37.3%. Also, there was decreased a permeability of wood, poplar sapwood 18.3 % and heartwood of 53.9%; oak sapwood by 20.0% and heartwood by 20.3%; beech sapwood 45.8% and mature wood by 48.2%. By decrease of the observed properties of the stored wood, a deterioration a quality of produced pulp can be expected (a higher Kappa number, amount reject and decrease the mechanical properties of pulp).

Download Full-text

Stability of Folate and Vitamin B12in Human Serum after Long-Term Storage: A Follow-Up after 13 Years

Journal of Nutrition and Metabolism ◽

10.1155/2018/9834181 ◽

2018 ◽

Vol 2018 ◽

pp. 1-4 ◽

Cited By ~ 5

Author(s):

Eugène H. J. M. Jansen ◽

Piet K. Beekhof

Keyword(s):

Human Serum ◽

Storage Time ◽

Vitamin B ◽

Serum Samples ◽

Long Term Storage ◽

Nutrition Research ◽

The Stability ◽

Term Storage

In epidemiological and nutrition research, it is very important to evaluate the stability of biomarkers as function of both storage time and temperature. In this study, the stability of folate and vitamin B12in human serum samples has been tested after long-term storage at −80°C up to 13 years. Serum samples of 16 individuals were used in this study. The concentration of folate and vitamin B12has been determined att=0and at 1, 8, and 13 years after storage at −80°C. The folate concentrations in serum samples remained stable at −80°C. The concentration of vitamin B12was decreasing during the time of the study to about 50%. The correlation of the folate and also of the vitamin B12concentrations in the stored samples compared with the starting values was still good. Therefore, although the concentration of vitamin B12decreased upon storage, reliable comparative analyses can still be performed.

Download Full-text

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.728273 ◽

2021 ◽

Vol 8 ◽

Author(s):

Giscard Lima ◽

Alexander Kolliari-Turner ◽

Fernanda Rossell Malinsky ◽

Fergus M. Guppy ◽

Renan Paulo Martin ◽

...

Keyword(s):

Whole Blood ◽

Differentially Expressed ◽

Rna Seq ◽

Short Term ◽

Long Term Storage ◽

Short Term Storage ◽

Short And Long Term ◽

Rna Preservation ◽

Term Storage

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation.Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes.Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments.Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Download Full-text