Long-term storage of gametes and gonadal tissues at room temperatures: the end of the ice age?

Long-term preservation of viable spermatozoa, eggs, embryos, and gonadal tissues of good quality is essential in human reproductive medicine and for the population management of livestock, laboratory, and wild species. Instead of using freezing temperatures, encouraging findings indicate that structures and functions of gametes or gonadal tissues can be suspended in trehalose glass after dehydration and then preserved at supra-zero temperatures. As a new era in fertility preservation and biobanking is about to start, the advantages, needs, and implications of germplasm storage at room temperatures must be carefully examined. Although very promising, the development of alternate biobanking strategies does not necessarily mean that the end of the "ice age" (cryopreservation) is near.

Potential probiotic characterization of Lactobacillus reuteri from traditional Chinese highland barley wine and application for room-temperature-storage drinkable yogurt.

The aim of this study was to select probiotic strains that could be used in drinkable yogurt to yield viable cells following storage at room temperature (RT). The uniquely high altitude conditions in Tibet and the alcoholic environment of certain products, such as the highland barley wine homemade in Tibet, may induce unusual characteristics of microbial strains. A total of 27 lactic acid bacteria were isolated from homemade highland barley wines. One strain, Lactobacillus reuteri WHH1689, demonstrated no ability for lactose utilization, exhibited a high survival rate during storage at RT in drinkable yogurts, and produced very weak post-acidification. This strain showed great resistance to conditions simulating the gastrointestinal tract, including strong adherence to HT-29 cells and inhibitory activity against Escherichia coli, Shigella flexneri, Salmonella paratyphi ß, and Staphylococcus aureus. A dextran sulfate sodium (DSS)-induced mouse model was used to evaluate the in vivo influence of Lb. reuteri WHH1689 on the intestinal flora and showed that strain WHH1689 increased viable counts of bifidobacteria in feces of mice. The probiotic strain selected in this study-with its high survival at RT and lack of serious post-acidification problems-may provide significant improvements for dairy industry products by extending the storage time of dairy products with living cells.

Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review.

The use of live probiotic bacteria as food supplement has become popular. Capability of probiotic bacteria to be kept at room temperature becomes necessary for customer's convenience and manufacturer's cost reduction. Hence, production of dried form of probiotic bacteria is important. Two common drying methods commonly used for microencapsulation are freeze drying and spray drying. In spite of their benefits, both methods have adverse effects on cell membrane integrity and protein structures resulting in decrease in bacterial viability. Microencapsulation of probiotic bacteria has been a promising technology to ensure bacterial stability during the drying process and to preserve their viability during storage without significantly losing their functional properties such acid tolerance, bile tolerance, surface hydrophobicity, and enzyme activities. Storage at room temperatures instead of freezing or low temperature storage is preferable for minimizing costs of handling, transportation, and storage. Concepts of water activity and glass transition become important in terms of determination of bacterial survival during the storage. The effectiveness of microencapsulation is also affected by microcapsule materials. Carbohydrate- and protein-based microencapsulants and their combination are discussed in terms of their protecting effect on probiotic bacteria during dehydration, during exposure to harsh gastrointestinal transit and small intestine transit and during storage.

Long-term storage stability of PGL-I coated ELISA plates for anti-Mycobacterium leprae IgM detection.

The assessment of ELISA plates coated with phenolic glycolipid-I/PGL-I revealed excellent stability during eight years of storage at room temperature, promoting consistent IgM antibody detection in multibacillary leprosy patients. These stable, standardized plates can significantly contribute to efficient leprosy serology research and support its widespread distribution and use in endemic countries.

[Effects of Carriers on ANAMMOX Sludge Activity Recovery and Microbial Flora Characteristics].

In order to realize the rapid recovery of ANAMMOX sludge bacterial activity after long-term room temperature storage, three groups of reactors were added to ANAMMOX sludge that had been stored without substrate at room temperature (15-30℃) for 9 months. Among the three groups of reactors, comet fiber carrier and K3 carrier were added to R2 and R3 reactors, respectively, as biological carriers. The effects of different carriers on the recovery rate of ANAMMOX sludge bacterial activity were investigated. The results showed that ANAMMOX reactions in the R2 and R3 reactors began taking place on the 8th and 10th day, respectively, with respective TIN removal rates of 82.25% and 80.92%, which were significantly improved compared with that in the R1 reactor, in which no carrier was added (ANAMMOX reaction started occurring on the 15th day with a TIN removal rate of 80.26%). After 42 days with influent, ρ(NH4+-N) and ρ(NO2--N) respectively increased to 300 mg·L-1 and 396 mg·L-1, and the TIN removal rates of the three groups of reactors were respectively 78.96%, 84.92%, and 84.66%. Microbial community structure analysis showed that the relative abundances of ANAMMOX bacteria in the R2 and R3 reactor were respectively 6.85% and 6.06%, two to four times that in the R1 reactor. The predominant ANAMMOX bacteria in the sludge was Candidatus Jettenia, whose relative abundances in the three groups of reactors were respectively 1.62%, 5.74%, and 5.21%. The results show that ANAMMOX biofilm-granular sludge complex systems constructed by adding carriers can considerably shorten the time for recovering ANAMMOX sludge bacterial activity after long-term room temperature storage without substrate. The carriers effectively promoted the relative abundances of ANAMMOX bacteria in the reactors, whereas the promoting effect of comet fiber carrier was slightly more significant than that of the K3 carrier.

Decline of Listeria monocytogenes on fresh apples during long-term, low-temperature simulated international sea-freight transport.

Listeria monocytogenes has caused outbreaks of foodborne illness from apples in the USA, and is also a major issue for regulatory compliance worldwide. Due to apple's significance as an important export product from New Zealand, we aimed to determine the effect of long-term, low-temperature sea-freight from New Zealand to the USA (July) and Europe (March-April), two key New Zealand markets, on the survival and/or growth of L. monocytogenes on fresh apples. Temperature and humidity values were recorded during a shipment to each market (USA and Europe), then the observed variations around the 0.5 °C target temperature were simulated in laboratory trials using open ('Scired') and closed ('Royal Gala' for the USA and 'Cripps Pink' for Europe) calyx cultivars of apples inoculated with a cocktail of 107-108 cells of seven strains of L. monocytogenes. Samples were analysed for L. monocytogenes quantification at various intervals during the simulation and on each occasion, an extra set was analysed after a subsequent 8 days at 20 °C. When both the sea-freight simulations concluded, L. monocytogenes showed 5 log reductions on the equatorial surface of skin of apples, but only about 2.5 log reduction for USA and about 3.3 log reduction for Europe in the calyx. Cultivar type had no significant effect on the survival of L. monocytogenes for both sea-freight simulations, either in the calyx or on the skin (P > 0.05). Most of the reduction in the culturable cells on the skin occurred during the initial 2 weeks of the long-term storage simulations. There was also no significant difference in the reduction of L. monocytogenes at 0.5 or 20 °C. No correlation was observed between firmness or total soluble solids and survival of L. monocytogenes. Because the inoculated bacterial log reduction was lower in the calyx than on the skin, it is speculated that the risk of causing illness is higher if contaminated apple cores are eaten. The result suggested that the international sea-freight transportation does not result in the growth of L. monocytogenes irrespective of time and temperature. The results of this study provide useful insights into the survival of L. monocytogenes on different apple cultivars that can be used to develop effective risk mitigation strategies for fresh apples during long-term, low-temperature international sea-freight transportation.

Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows.

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

Analysis of metabolomics associated with quality differences between room-temperature- and low-temperature-stored litchi pulps.

Studies on how temperature affects the postharvest quality of litchi have focused mainly on pericarp browning but rarely on the metabolites in postharvest litchi pulp. In this study, the differences in respiration rates, total soluble solid content, and titratable acid content demonstrated that room and low temperatures have different effects on the quality of "Feizixiao" litchi pulp. UHPLC-ESI-QTOF-MS/MS analysis was performed to compare the differentially expressed metabolites (DEMs) in litchi pulp after 8 days of storage at room temperature (RT-8 d) with those in litchi pulp after 28 days of storage at low temperature (LT-28 d). Nineteen carbohydrates (phosphohexoses, sorbitol, and mannose), fifteen acids, seven amino acids, nine energy metabolites and nucleotides, and six aliphatic and secondary metabolites were identified as common DEMs in RT-8 d and LT-28 d pulps. These findings indicated active fructose and mannose metabolism and increased catabolism of nicotinate, nicotinamide, alanine, aspartate, and glutamate. Four carbohydrates (mainly phosphohexoses), five acids, ten amino acids, three aliphatic and secondary metabolites, and one hormone were identified as unique DEMs in RT-8 d pulp, the consumption of key metabolites in glycolysis and the tricarboxylic acid cycle, and accumulation of phenylalanine, tyrosine, and tryptophan. Active consumption of nucleotide metabolites and biosynthesis of aliphatics in LT-28 d pulp were indicated by unique DEMs (eleven carbohydrates, four acids, seven amino acids, seven energy metabolites and nucleotides, and six aliphatic and secondary metabolites). These results provided an unambiguous metabolic fingerprint, thereby revealing how room and low temperatures differentially influenced the quality of litchi pulp.

A novel affordable reagent for room temperature storage and transport of fecal samples for metagenomic analyses.

BACKGROUND: The number of large-scale studies on the gut microbiota in human cohorts is rapidly increasing. However, the few and expensive options for storage of fecal samples at room temperature have been an obstacle for large-scale metagenomic studies and the development of clinical/commercial personal metagenomic sequencing. RESULTS: In this study, we systematically tested a novel N-octylpyridinium bromide-based fecal sample preservation method and compared it with other currently used storage methods. We found that the N-octylpyridinium bromide-based method enabled preservation of the bacterial composition in fecal samples transported and stored at room temperature for up to at least 14 days. CONCLUSIONS: We describe a novel chemical stabilizer that allows cost-effective transportation and storage at room temperature for several days with preservation of bacterial composition. This method will facilitate sample collection even in remote area and also enable transport via normal commercial transportation routes.

Long-Term Room Temperature Storage of Dry Ribonucleic Acid for Use in RNA-Seq Analysis.

RNA is an essential biological material for research in genomics and translational medicine. As such, its storage for biobanking is an important field of study. Traditionally, long-term storage in the cold (generally freezers or liquid nitrogen) is used to maintain high-quality (in terms of quantity and integrity) RNA. Room temperature (RT) preservation provides an alternative to the cold, which is plagued by serious problems (mainly cost and safety), for RNA long-term storage. In this study, we evaluated the performance of several RT storage procedures, including the RNAshell® from Imagene, where the RNA is dried and kept protected from the atmosphere, and the vacuum drying of RNA with additives such as the Imagene stabilization solution and a home-made trehalose solution. This evaluation was performed through accelerated (equivalent to 10 years for RNAshell) aging and real-time studies (4 years). To check RNA quality and integrity, we used RNA integrity number values and RNA-seq. Our study shows that isolation from atmosphere offers a superior protective effect for RNA storage compared with vacuum drying alone, and demonstrates that RNAshell permits satisfactory RNA quality for long-term RT storage. Thus, the RNA quality could meet the demand of downstream applications such as RNA-seq.

Evaluation of DNAstable for DNA storage at ambient temperature.

Preserving DNA is important for validation of prospective and retrospective analyses, requiring many expensive types of equipment (e.g., freezers and back-up generators) and energy. While freezing is the most common method for storing extracted DNA evidence or well-characterized DNA samples for validation studies, DNAstable (Biomatrica), a commercially available medium for room temperature storage of DNA extracts was evaluated in this study. Two groups of samples consisting of different DNA quantities were investigated, one ranging from 20 to 400 ng (group 1) and the other one ranging from 1.4 to 20 ng (group 2). The DNA samples with and without DNAstable were stored at four different temperatures [∼25 °C (room temperature), -20 °C, 37 °C or 50 °C]. DNA degradation over several months was monitored by SYBR Green-based qPCR assays and by PCR amplification of the core CODIS STR markers for group 1 and 2 DNA samples, respectively. For the time points tested in this study (up to 365 days), the findings indicate that the -20 °C controls and the DNAstable protected samples at room temperature provided similar DNA recoveries that were higher compared to the unprotected controls kept at RT, 37 °C or 50 °C. These results suggest that DNAstable can protect DNA samples with effectiveness similar to that of the traditional -20 °C freezing method. In addition, extrapolations from accelerated aging experiments conducted at high temperatures support that DNAstable is an effective technology for preserving purified DNA at room temperature with a larger protective impact on DNA samples of low quantity (<20 ng).