The proteins cleaved by endogenous tryptic proteases in normal EDTA plasma by C18 collection of peptides for liquid chromatography micro electrospray ionization and tandem mass spectrometry

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E?9 (i.e. FDR = 0.000000001) to E?227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.     

Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

Background

Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature.

Methods

Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) and correlated with X!TANDEM.

Results

Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than “no enzyme” correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours–days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra.

Conclusion

The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.

     

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.

     

Cryopreservation of conditionally dormant orthodox seeds of Betula pendula

To evaluate the feasibility of long-term cryopreservation of Polish provenances of silver birch (Betula pendula), the sensitivity of conditionally dormant seeds to extreme desiccation and/or the ultra-low temperature of liquid nitrogen (LN; ?196°C) was evaluated. The critical water content (WC) of desiccated seeds and the high-moisture freezing limit of seeds desiccated or moistened to various WCs and frozen for 24 h or for 2 years in LN was also determined. Germination tests revealed no critical WC for seeds [to 0.02 g H₂O g^?1 dry mass (g g^?1)]. Seeds tolerated freezing in LN within specific safe range of WC 0.02–0.23 g g^?1 (nuts). Seeds desiccated to the safe WC and stored in LN for 2 years had similar or higher germination as seeds stored at ?3°C for 2 years, depending on provenance. Therefore, long-term cryopreservation of B. pendula seeds in gene banks is feasible.     

Destabilization of mayonnaise induced by lipid crystallization upon freezing

The thermal and rheological history of mayonnaise during freezing and its dispersion stability after the freeze-thaw process were investigated. Mayonnaise was cooled to freeze and stored at ?20 to ?40 °C while monitoring the temperature; penetration tests were conducted on the mayonnaise, which was sampled at selected times during isothermal storage at ?20 °C. Significant increases in the temperature and stress values due to water-phase crystallization and subsequent oil-phase crystallization were observed. The water phase crystallized during the cooling step in all the tested mayonnaise samples. The oil phases of the prepared mayonnaise (with rapeseed oil) and commercial mayonnaise crystallized during isothermal storage after 6 and 4 h, respectively, at ?20 °C. The dispersion stability was evaluated from the separation ratio, which was defined as the weight ratio of separated oil after centrifuging to the total amount of oil in the commercial mayonnaise. The separation ratio rapidly increased after 4 h of freezing. This result suggests that crystallization of the oil phase is strongly related to the dispersion stability of mayonnaise.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

The plasma peptidome

Background

It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.

Methods

A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.

Results

Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~?14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥?E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~?26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~?0.001 or less. Log₁₀ peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.

Conclusions

The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log₁₀ intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

     

Towards the development of innovative multi-mycotoxin reference materials as promising metrological tool for emerging and regulated mycotoxin analyses

The interest in LC-MS/MS multi-mycotoxin methods unveiled an urgent need for multi-mycotoxin reference material. A multi-fusariotoxin, including deoxynivalenol (DON); zearalenone (ZEN); T-2 toxin (T-2); HT-2 toxin (HT-2); enniatin A, A1, B, and B1 (ENNs); and beauvericin (BEA), contaminated wheat flour was obtained by inoculation Fusarium spp. strains. The candidate material has successfully passed the homogeneity test and submitted to an international interlaboratory study achieved by 19 laboratories from 11 countries using their routine analytical method. The dispersion of the results for ZEN and BEA did not allow the derivation of reliable consensus values, while the assignment was only possible for DON, HT-2, T-2, and ENN A. No link was found between the methods used by the participants and the results. Significant changes in dry matter contents (≥±1.4 % of the initial dry matter) and significant changes in ergosterol contents (≥±10 %) did not occur. Using the mycotoxin contents in wheat flour stored at ?80 °C as reference values, statistically significant decreases were observed only for T-2 contents at +24 °C, in contrast to the storage at ?20 and +4 °C. For the other involved toxins, the candidate material was found to be stable at ?20, +4, or +24 °C. Based on the T-2 decreases, a shelf life of 6 years was derived from isochronous study when the material is kept at ?20 °C. At room temperature (e.g., +24 °C) or higher, this time validity drastically decreases down to 6 months. The development of this metrological tool is an important step towards food and feed quality control using multi-mycotoxin analyses. In vivo animal experiments using multi-mycotoxin-contaminated feeds dealing with the carryover or mitigation could further benefit from the methodology of this work.     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4?°C for up to 7 days. We also analyzed storage at ?80?°C across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24?h, followed by shipping the samples on ice over 2–3?days, and finally, long-term storage in the ?20?°C or ?80?°C freezer.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

野生鸡枞菌种长期保存方法比较

野生鸡枞菌种质资源的有效保存是对野生鸡枞加以保护和利用的前提。以自行分离的5个野生鸡枞菌株作为研究对象,采用蒸馏水保藏法和-80°C冻结保藏法对野生鸡枞菌种长期保存的方法进行了实验研究,蒸馏水法分别保存于室温和4°C,-80°C冻结保藏同时采用程控降温法和泡沫盒降温法,保存20个月后对4种不同方法保存的5个菌株的保存效果进行比较。实验结果表明:蒸馏水室温保存法菌种存活率为100%,萌发期较短,为4-10 d,是一种简便、实用、有效而成本低廉的长期保存方法;-80°C冻结保藏法的存活率为56%-76%,萌发期7-16 d,泡沫盒降温法可以很好地控制降温速度,是一种简便有效的控温方法。     

Protocols for dry DNA storage and shipment at room temperature

The globalization of DNA barcoding will require core analytical facilities to develop cost‐effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry‐state DNA stabilization systems: commercial Biomatrica^® DNAstable^® plates, home‐made trehalose and polyvinyl alcohol (PVA) plates on 96‐well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at ?20 °C. PCR and selective sequencing were performed over a 4‐year interval to test the condition of DNA extracts. Biomatrica^® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica^® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at ?20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long‐term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography–mass spectrometry (GC–MS) analysis. A one-step derivatization using 100 μL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations of all targeted compounds showed good intra- and inter-day (within 7 days) precision (<10 %), and good stability (<20 %) within 4 days at room temperature (23–25 °C), or 7 days when stored at ?20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistical analysis of the concentrations of these targeted metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.     

Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Proteomic Studies of Saliva: A Proposal for a Standardized Handling of Clinical Samples

Background

In recent years, differential analysis of proteins from human saliva, i.e., proteomic analysis, has received much attention mainly due to its unstressful sampling and its great potential for biomarker research. It is widely considered that saliva is a highly stable medium for proteins thanks to a large amount of antiprotease agents, even at ambient and physiological temperatures.

Objective

To find the best protocol for the handling of samples, we have investigated the stability of saliva proteins stored at different temperatures (from ?80 to 20°C) by one- and two-dimensional electrophoresis.

Results

At 20°C, no major changes were observed on protein one-dimensional profiles following 1 day of storage; however, between 7 days and 30 days, the native alpha-amylase band decreased slightly to give several bands with molecular weight between 35 and 25 kDa. The same phenomenon appeared after 30 days of storage at 4°C. Two-dimensional analysis of salivary maps revealed degradation from day 7 of several protein groups for samples stored at 20°C.

Conclusion

All these findings have to be carefully considered when saliva is collected for clinical proteomic analysis. We can conclude that, to maintain the optimum stability of saliva proteins, saliva samples should be collected on ice followed by the addition of protease inhibitor cocktail, centrifuged to remove insoluble material, and stored at ?20 or ?80°C.     

Oligomerization of glycine and alanine on metal(II) octacynaomolybdate(IV): role of double metal cyanides in prebiotic chemistry

Condensation reactions of amino acid (glycine and alanine) on the surface of metal(II) octacyanomolybdate(IV) (MOCMo) complexes are investigated using high-performance liquid chromatography (HPLC) and electron spray ionizations–mass spectroscopy (ESI–MS). The series of MOCMo have been synthesized and the effect of outer sphere metal ions present in the MOCMo on the oligomerization of glycine and alanine at different temperature and time found out. Formation of peptides was observed to start after 7?days at 60?°C. Maximum yield of peptides was found after 35?days at 90?°C. It has been found that zinc(II) octacyanomolybdate(IV) and cobalt(II) were the most effective metal cations present in outer sphere of the MOCMo for the production of high yield of oligomerized products. Surface area of MOCMo seems to play dominating parameter for the oligomerization of alanine and glycine. The results of the present study reveal the role of MOCMo in chemical evolution for the oligomerization of biomolecules.     

Effect of freezing and freeze-drying on the viability and storage of Lilium longiflorum L. and Zea mays L. pollen

The freeze-preservation of pollen is dependent on the interaction of several factors such as freezing rate, thawing rate, freeze-drying temperature and duration, storage temperature and environment and rehydration rates. Changes in any of these variables affects the others directly or indirectly.Rapid freezing of pollen at rates of approximately 200 °C/min maintains the highest degree of viable pollen in combination with rapid thawing rates of 218 °C/min. Rapid cooling and slow rewarming resulted in a substantial loss of pollen viability. This might indicate that intracellular ice crystals formed during rapid cooling perhaps grow into larger ice masses during slow rewarming or storage at temperatures above ?50 °C.The germinability of pollen freeze-dried at temperatures below ?50 °C was also prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 0–5 °C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at the same temperature yielded considerably higher viability percentages for both lily and corn pollen. Drying time is an important factor, perhaps indicating that residual moisture is critical. Freeze-dried pollen can be stored at higher temperatures than frozen and control pollen. Freeze-dried material stored for five months at 0–5 °C, upon slow rehydration yielded intact grains which has average germination percentages of 25 for lily and 15 for corn. The same pollen upon rapid rehydration showed rupturing of 20–40% of the cells and practically no germination.