Oldies, but goldies-preserved morphology and stability of antigenic determinants in decades-old cryosections of human m. vastus lateralis.

Fibre typing by immunohistochemistry on cryosections from human skeletal muscle biopsies is an essential tool in the diagnosis and research of muscular diseases, ageing, and responses to exercise training and disuse. Preserving a good quality in these frozen specimens can be challenging especially if they are stored for longer periods before histological processing, which is often the case in studies with a large number of test subjects and/or repeated sampling separated by multiple years. We demonstrate in this article that both, the morphology and reactivity of epitopes to myosin heavy chain isoforms and dystrophin are well preserved in up to 18-year-stored unfixed and unstained cryosections of human m. vastus lateralis (n = 241). Any variation in staining intensity between samples was unrelated to the age of the biopsy donor or the storage period of the unstained cryosections, and in all cases, the obtained images were appropriate for image analysis, such as the determination of the fibre type composition and the fibre cross-sectional area, and quantitative analysis of muscle capillarisation.

Impact of protectants and the method of preservation on the stability of potentially probiotic bacteria.

Probiotics offer health advantages when consumed in adequate quantities. As ongoing research identifies promising new strains, ensuring their viability and functionality through simple preservation methods is vital for success within the probiotic industry. This study employed a factorial design to investigate the combined effects of four cryoprotectants [C1: MRS broth + 14 % (w/v) glycerol, C2: Aqueous solution containing 4 % (w/v) trehalose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate, C3: Aqueous solution containing 10 % (w/v) skimmed milk and 4 % (w/v) sodium glutamate, C4: Aqueous solution containing 4 % (w/v) sucrose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate] and three methods of preservation (P1: -86 °C freezing, P2: -196 °C liquid nitrogen freezing, and P3: storing at 4 °C after lyophilization) on the cell viability of three potentially probiotic strains over 12 months. Pediococcus sp P15 and Weissella cibaria ml6 had the highest viability under treatments C3 and C2, after 12 months of storage, respectively. Meanwhile, Lactococcus lactis ml3 demonstrated the highest viability in both treatments C2 and C4 (P ≤ 0.05). According to the results freezing, either P1 or P2, is the most effective preservation method for P. sp P15 and W. cibaria ml6. Meanwhile, L. lactis ml3 showed the highest colony count under treatment (P1) after 12 months of storage (P ≤ 0.05). Among the tested conditions, P. sp P15 and L. lactis ml3 exhibited the highest viability and bile salt resistance when stored under P1C1. For W. cibaria ml6, the optimal storage condition was P2C2 (frozen in liquid nitrogen with cryoprotectant C2).

Long-term storage does not affect the DNA methylation profiles of vitrified-warmed human embryos.

With the widespread application of embryo cryopreservation in assisted reproductive techniques, it is necessary to assess the safety of long-term cryopreservation of human embryos and it is unclear whether storage time has an impact on the DNA methylation profiles of human embryos. Nine women who received IVF treatment were recruited for this study. The retrieved eight-cell human embryos were classified into three groups including fresh embryos, cryopreserved embryos stored for 3 years, and cryopreserved embryos stored for 8 years. Single-cell whole-genome bisulfite sequencing (scWGBS) was conducted. The genome-wide methylation pattern of the fresh and two cryopreserved groups were similar. In addition, the methylation level in different genomic regions showed comparable patterns and no significant differences were observed in the methylation level of imprinted genes among the three groups. A total of 587 differentially methylated regions (DMRs) in the 3-year group and 540 DMRs in the 8-year group were identified comparing to fresh group. However, they were not enriched in promoters and had a similar genome-wide distributions, suggesting that these DMRs may not contribute to the changes in corresponding gene expressions. Our study illustrated that long-term cryopreservation will not affect the DNA methylation profiles of human eight-cell embryos at single-cell level.

DNA nanotechnology in ionic liquids and deep eutectic solvents.

Nucleic acids have the ability to generate advanced nanostructures in a controlled manner and can interact with target sequences or molecules with high affinity and selectivity. For this reason, they have applications in a variety of nanotechnology applications, from highly specific sensors to smart nanomachines and even in other applications such as enantioselective catalysis or drug delivery systems. However, a common disadvantage is the use of water as the ubiquitous solvent. The use of nucleic acids in non-aqueous solvents offers the opportunity to create a completely new toolbox with unprecedented degrees of freedom. Ionic liquids (ILs) and deep eutectic solvents (DESs) are the most promising alternative solvents due to their unique electrolyte and solvent roles, as well as their ability to maintain the stability and functionality of nucleic acids. This review aims to be a comprehensive, critical, and accessible evaluation of how much this goal has been achieved and what are the most critical parameters for accomplishing a breakthrough.

Biotechnological interventions for improving the seed longevity in cereal crops: progress and prospects.

Seed longevity is a measure of the viability of seeds during long-term storage and is crucial for germplasm conservation and crop improvement programs. Also, longevity is an important trait for ensuring food and nutritional security. Thus, a better understanding of various factors regulating seed longevity is requisite to improve this trait and to minimize the genetic drift during the regeneration of germplasm. In particular, seed deterioration of cereal crops during storage adversely affects agricultural productivity and food security. The irreversible process of seed deterioration involves a complex interplay between different genes and regulatory pathways leading to: loss of DNA integrity, membrane damage, inactivation of storage enzymes and mitochondrial dysfunction. Identifying the genetic determinants of seed longevity and manipulating them using biotechnological tools hold the key to ensuring prolonged seed storage. Genetics and genomics approaches had identified several genomic regions regulating the longevity trait in major cereals such as: rice, wheat, maize and barley. However, very few studies are available in other Poaceae members, including millets. Deploying omics tools, including genomics, proteomics, metabolomics, and phenomics, and integrating the datasets will pinpoint the precise molecular determinants affecting the survivability of seeds. Given this, the present review enumerates the genetic factors regulating longevity and demonstrates the importance of integrated omics strategies to dissect the molecular machinery underlying seed deterioration. Further, the review provides a roadmap for deploying biotechnological approaches to manipulate the genes and genomic regions to develop improved cultivars with prolonged storage potential.

Quantification and stability assessment of urinary phenolic and acidic biomarkers of non-persistent chemicals using the SPE-GC/MS/MS method.

Nowadays, people are exposed to numerous man-made chemicals, many of which are ubiquitously present in our daily lives, and some of which can be hazardous to human health. Human biomonitoring plays an important role in exposure assessment, but complex exposure evaluation requires suitable tools. Therefore, routine analytical methods are needed to determine several biomarkers simultaneously. The aim of this study was to develop an analytical method for quantification and stability testing of 26 phenolic and acidic biomarkers of selected environmental pollutants (e.g., bisphenols, parabens, pesticide metabolites) in human urine. For this purpose, a solid-phase extraction coupled with gas chromatography and tandem mass spectrometry (SPE-GC/MS/MS) method was developed and validated. After enzymatic hydrolysis, urine samples were extracted using Bond Elut Plexa sorbent, and prior to GC, the analytes were derivatized with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA). Matrix-matched calibration curves were linear in the range of 0.1-1000 ng mL-1 with R > 0.985. Satisfactory accuracy (78-118%), precision (< 17%), and limits of quantification (0.1-0.5 ng mL-1) were obtained for 22 biomarkers. The stability of the biomarkers in urine was assayed under different temperature and time conditions that included freezing and thawing cycles. All tested biomarkers were stable at room temperature for 24 h, at 4 °C for 7 days, and at -20 °C for 18 months. The total concentration of 1-naphthol decreased by 25% after the first freeze-thaw cycle. The method was successfully used for the quantification of target biomarkers in 38 urine samples.

Use of fine capillaries for cryopreservation of Caenorhabditis elegans by vitrification.

Caenorhabditis elegans is an exceptional model organism. More than twenty thousand different strains have been developed, increasing knowledge on countless topics. However, the traditional method to cryopreserve this nematode, based on slow freezing, usually reaches recovery rates of around 35% for the L1 and L2 larval stages. Here, we propose two alternative methods to cryopreserve this nematode based on vitrification that are applicable in common laboratories and allow the selective individual cryopreservation of this organism. These new methods require ultra-high warming rates, which are achieved by employing very thin capillaries as the nematode container, and a very low final concentration of cryoprotectants, which, as compared to slow freezing, reduce toxicity damage. The recovery rate was 98.5% for larvae (L1 - L4) and 84.3% for adults. Given these results, our procedures offer an alternative to cryopreserve this nematode (larvae and adults) with higher recovery rates, avoiding expensive requirements. Indeed, it only needed a container with liquid nitrogen and a warming bath for water at 37 °C. The high performance of this approach has been revealed by preserving the long-term memory and, probably, the connectome of this nematode.

Identification of DNA Methylation Changes in European Beech Seeds during Desiccation and Storage.

Ageing and deterioration of seeds is a major problem for the maintenance of seed quality and viability during long-term storage. Prediction of early stages of seed deterioration in order to point out the plantlets' regeneration time is a major challenge of successful storage. In preserved seeds, damages accumulate within cells at the rate mainly related to their moisture content and temperature of storage. Current research reveals global alterations in DNA methylation in lipid-rich intermediate seeds during desiccation and storage at various regimes covering nonoptimal and optimal conditions. We show for the first time that monitoring of 5-methylcytosine (m5C) level in seeds can be used as a truly universal viability marker regardless of postharvest category of seeds and their composition. For seeds stored up to three years, in varied conditions, moisture content, temperature, and time of storage had significant influence on seedling emergence and DNA methylation (p < 0.05). Similarities among lipid-rich intermediate and orthodox seeds regarding different reactions of embryonic axes and cotyledons to desiccation are newly revealed. Along with previous studies on seeds dramatically different in desiccation tolerance (recalcitrant vs. orthodox), results regarding lipid-rich seeds positioned in-between (intermediate) prove that maintaining global DNA methylation status is crucial for maintaining seed viability.

Impact of ultra-low temperature long-term storage on the preanalytical variability of twenty-one common biochemical analytes.

OBJECTIVES: Retrospective studies frequently assume analytes long-term stability at ultra-low temperatures. However, these storage conditions, common among biobanks and research, may increase the preanalytical variability, adding a potential uncertainty to the measurements. This study is aimed to evaluate long-term storage stability of different analytes at <-70 °C and to assess its impact on the reference change value formula. METHODS: Twenty-one analytes commonly measured in clinical laboratories were quantified in 60 serum samples. Samples were immediately aliquoted and frozen at <-70 °C, and reanalyzed after 11 ± 3.9 years of storage. A change in concentration after storage was considered relevant if the percent deviation from the baseline measurement was significant and higher than the analytical performance specifications. RESULTS: Preanalytical variability (CVP) due to storage, determined by the percentage deviation, showed a noticeable dispersion. Changes were relevant for alanine aminotransferase, creatinine, glucose, magnesium, potassium, sodium, total bilirubin and urate. No significant differences were found in aspartate aminotransferase, calcium, carcinoembryonic antigen, cholesterol, C-reactive protein, direct bilirubin, free thryroxine, gamma-glutamyltransferase, lactate dehydrogenase, prostate-specific antigen, triglycerides, thyrotropin, and urea. As nonnegligible, CVP must remain included in reference change value formula, which was modified to consider whether one or two samples were frozen. CONCLUSIONS: After long-term storage at ultra-low temperatures, there was a significant variation in some analytes that should be considered. We propose that reference change value formula should include the CVP when analyzing samples stored in these conditions.

Long-term cryostorage does not negatively affect the recovery of Caenorhabditis elegans.

Cryostorage of Caenorhabditis elegans nematodes is important to maintain the many lines used for research. The standard method uses 15% of glycerol in M9-Buffer and a cooling rate of 1 °C/min; then worms can be stored in a -80 °C freezer or in liquid nitrogen. The recovery of C. elegans from stocks stored in liquid nitrogen is reported to be in the range of 35-45% and slightly decreases after years of storage. The storage at -80 °C is also considered safe, but the recovery is not as high as in liquid nitrogen. These observations have not been experimentally reported and therefore require verification. In this study, the standard methods were used in a set of experiments to compare the recovery of larvae and adult worms stored at -80 °C or in liquid nitrogen, after short- (a week) or long-term storage (3.5 years). No differences were observed in recovery, either for the time of storage or for the temperature of storage. Recovery of larvae was 32% at -80 °C and 36% in liquid nitrogen after 3.5 yr and that was not significantly different from the 7-d recovery rates. Adult worm recovery was below 5% for all treatments. These results suggest that both methods of storage can be used to successfully store C. elegans larvae for at least 3.5 years.

Comparison of a novel lateral-flow device to galactomannan assay at different time periods for detections of invasive aspergillosis.

PURPOSE: To compare a lateral-flow device (LFD) method to the galactomannan assay (GM) for the diagnosis of invasive aspergillosis (IA). METHODS: First, 20 GM-positive serum samples stored for two years were retested with both the GM and LFD assays. Second, 153 serum samples from 91 immunocompromised patients suspected of having IA were tested prospectively, including 56 hematologic malignancies and 35 chronic illnesses with steroid therapy. RESULTS: For the twenty GM-positive stored samples, only ten were positive for the repeated GM assay and none were positive for IA according to the LFD test. The concordance of the LDF with the GM test was 79.81% (83/104) if both tests were performed on the sample collection day, with the rate reducing to 67.65% (23/34) (p < 0.05) if the LFD test was performed 2-7 days after the GM test. Furthermore, there was a significant difference in the discrepancy between the GM and LFD tests between previous and no anti-mold exposure subgroups (33.33% vs. 12.31%, p < 0.01). The sensitivity and specificity of the GM test were 89.65% and 98.66%, 68.96%, and 78.67% for the LFD assay. CONCLUSION: Serum samples that have been stored long term are not suitable for re-testing with the GM or LFD assay. There was a strong correlation between the LFD and GM assay results if the tests were performed on the same day, however, this decreased if the samples were stored for more than 2 days. Additionally, previous exposure to antibiotics and/or antifungal therapy could influence the LFD results, leading to discrepancies with the GM test results.

Reducing Pre- and Post-Treatments in Cryopreservation Protocol and Testing Storage at -80 °C for Norway Spruce Embryogenic Cultures.

Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a storage method that is able to handle large quantities of embryogenic tissues (ETs) reliably and at a low cost is required. The aim of the present study was to compare freezing at -80 °C in a freezer to cryopreservation using liquid nitrogen (LN) as a method for storing Norway spruce ETs. The possibility of simplifying both the pre-treatment and thawing processes in cryopreservation was also studied. The addition of abscisic acid (ABA) to the pre-treatment media and using polyethylene glycol PEG4000 instead of PEG6000 in a cryoprotectant solution were tested. Both the pre-and post-treatments on semi-solid media could be simplified by reducing the number of media, without any loss of genotype or embryo production capacity of ETs. On the contrary, the storage of ETs in a freezer at -80 °C instead of using LN was not possible, and the addition of ABA to the pre-treatment media did not provide benefits but increased costs. The lower regeneration rate after using PEG4000 instead of PEG6000 in a cryoprotectant solution in cryovials was unexpected and unwanted. The simplified pre-and post-treatment protocol will remarkably reduce the workload and costs in the mass-cryopreservation of future forest regeneration materials and in thawing the samples for mass propagations, respectively.

Evaluation of losses and quality maintenance of wheat during storage in a commercial unit in Brazil.

BACKGROUND: Specific studies of Brazilian wheat storage on a commercial scale on the maintenance of wheat quality are required since the continental extent of Brazil has regions of different weather and because of the diversity of the storage network. This study aimed to evaluate the technological quality (physicochemical and rheological), sanitary quality (insects, fungi and mycotoxins) and dry matter loss of wheat stored in a metal silo in a commercial storage unit. Two dynamic samples, collected during loading and unloading of wheat in silos, and four static samples, collected using a commercial pneumatic grain sampler, were used in this study. RESULTS: Silo temperature was higher than 20 °C during the summer season. The temperature was approximately 15 °C from June to December and provided excellent conditions for grain aeration, which resulted in the maintenance of wheat quality, with no changes in hectoliter weight and rheological properties of wheat (falling number, wet gluten and stability). The effect of Alternaria spp. (~220) and Aspergillus flavus (~7) infection on wheat did not differ statistically during the storage period, although a slight increase in A. flavus infection was noted in February (summer season). The wheat mycotoxins deoxynivalenol, zearalenone, aflatoxins and ochratoxin A were not detected during the studied storage period. Finally, dry matter reduced by approximately 0.4% after the storage period - approximately 0.013% per month. CONCLUSION: The management practices and climate conditions in southern Brazil provided excellent conditions for grain aeration at ambient air temperature and led to the maintenance of wheat quality during the post-harvest period. © 2021 Society of Chemical Industry.

Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage.

BACKGROUND: Stabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and maintaining a low aw during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C. RESULTS: The addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05). CONCLUSIONS: The addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.

Coagulation function of never frozen liquid plasma stored for 40 days.

BACKGROUND: Never frozen liquid plasma (LP) has limited shelf life versus fresh frozen plasma (FFP) or plasma frozen within 24 h (PF24). Previous studies showed decreasing factor activities after Day (D)14 in thawed FFP but no differences between LP and FFP until D10. This study examined LP function through D40. STUDY DESIGN AND METHODS: FFP and PF24 were stored at -20°C until assaying. LP was assayed on D5 then stored (4°C) for testing through D40. A clinical coagulation analyzer measured Factor (F)V, FVIII, fibrinogen, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thromboelastography (TEG) and thrombogram measured functional coagulation. Ristocetin cofactor assay quantified von Willebrand factor (vWF) activity. Residual platelets were counted. RESULTS: FV/FVIII showed diminished activity over time in LP, while PT and aPTT both increased over time. LP vWF declined significantly by D7. Fibrinogen remained high through D40. Thrombin lagtime was delayed in LP but consistent to D40, while peak thrombin was significantly lower in LP but did not significantly decline over time. TEG R-time and angle remained constant. LP and PF24 (with residual platelets) had initially higher TEG maximum amplitudes (MA), but by D14 LP was similar to FFP. CONCLUSION: Despite significant declines in some factors in D40 LP, fibrinogen concentration and TEG MA were stable suggesting stored LP provides fibrinogen similarly to frozen plasmas even at D40. LP is easier to store and prepare for prehospital transfusion, important benefits when the alternative is crystalloid.

The relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs.

BACKGROUND AND OBJECTIVES: In clinical practice, it has been shown that transfusion of packed red blood cells (pRBCs) with late shelf life increases the risk of post-transfusion complications. OBJECTIVE: To study relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs. MATERIALS AND METHODS: pRBCs were processed and stored according to blood bank procedure, for 42 days, at +4°C; pRBC samples were taken on days 3, 12, 19, 21, 24, 28, 35 and 42. Cytoskeleton images and membrane stiffness were studied using atomic force microscope. RESULTS: In the course of the pRBC storage, the cytoskeleton network configuration underwent structural changes. Simultaneously, pRBC membrane stiffness was increasing, with the correlation coefficient 0·88. Until 19 days, the stiffness grew slowly, in 19-24 days there occurred a transition period, after which its growth rate was three times higher than the initial. A chain of pathological processes developed in pRBC during long storage: pH reduction (linked to increased oxidative stress), then cytoskeletal destruction and an associated increase in pRBC membrane stiffness. CONCLUSION: During prolonged storage of pRBCs and their acidification, there is a progression of pRBC cytoskeletal changes and associated increase of membrane stiffness, observed to increase in rate after days 19-24. Mutual measurements of cytoskeletal integrity and membrane stiffness may be useful quality assessment tool to study the molecular mechanisms of RBC structural degradation during storage.

Barley Seeds miRNome Stability during Long-Term Storage and Aging.

Seed aging is a complex biological process that has been attracting scientists' attention for many years. High-throughput small RNA sequencing was applied to examine microRNAs contribution in barley seeds senescence. Unique samples of seeds that, despite having the same genetic makeup, differed in viability after over 45 years of storage in a dry state were investigated. In total, 61 known and 81 novel miRNA were identified in dry seeds. The highest level of expression was found in four conserved miRNA families, i.e., miR159, miR156, miR166, and miR168. However, the most astonishing result was the lack of significant differences in the level of almost all miRNAs in seed samples with significantly different viability. This result reveals that miRNAs in dry seeds are extremely stable. This is also the first identified RNA fraction that is not deteriorating along with the loss of seed viability. Moreover, the novel miRNA hvu-new41, with higher expression in seeds with the lowest viability as detected by RT-qPCR, has the potential to become an indicator of the decreasing viability of seeds during storage in a dry state.

Effects of Long-Term Storage on Radical Scavenging Properties and Phenolic Content of Kombucha from Black Tea.

Kombucha is a fermented beverage. Its consumption has significantly increased during the last decades due to its perceived beneficial effects. For this reason, it has become a highly commercialized drink that is produced industrially. However, kombucha is still also a homemade beverage, and the parameters which, besides its organoleptic characteristics, define the duration of its potential beneficial properties over time, are poorly known. Therefore, this study aimed to determine the effect of 9-month storage at 4 °C with 30-day sampling on the pH, total phenolic, and flavonoid contents, free radical scavenging properties of kombucha fermented from black tea. Our results highlighted that, after four months, the phenolic content decreased significantly from the initial value of 234.1 ± 1.4 µg GAE mL-1 to 202.9 ± 2.1 µg GAE mL-1, as well its antioxidant capacity tested by two in vitro models, DPPH, and ABTS assays. Concomitantly, the pH value increased from 2.82 to 3.16. The novel findings of this pilot study revealed that kombucha from sugared black tea can be stored at refrigerator temperature for four months. After this period the antioxidant properties of kombucha are no longer retained.

A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization.

A non-activating semen diluent does not cause motility or acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of honey bee spermatozoa stored in ambient conditions were assessed 60 days pre-cryopreservation and 24 h post-cryopreservation. Seven variations of a Tris-based non-activating diluents (FEM1 - FEM7) were compared to samples treated with conventional activating diluent and untreated semen. Semen viability (membrane integrity) was assessed after short- and long-term storage at 14.0 ± 0.2 °C. The non-activating medium FEM7 contained more viable spermatozoa than the activating medium, 24 h after cryopreservation (67.6 ± 10.9% and ~4%, respectively). After 60 days, 22.0 ± 7.8% of spermatozoa was viable in non-activating medium versus 0.0 and 60.8 ± 12.3%, in conventional media and untreated controls, respectively. Hence FEM7 was used to cryopreserve bee semen and subsequently inseminate honey bee queens. The quality of brood produced by the queens was assessed 30-60 days after insemination. The percentage of worker-bee offspring (produced from successfully fertilized eggs) was ~75% for both the non-activating medium and the conventional extender medium. Our results indicate that a non-activating medium possesses significant advantage over the conventional activating medium if the semen requires storage after treatments such as cryopreservation. The percentage of female offspring (from fertilized eggs) produced by queens inseminated with semen diluted in either the activating or non-activating medium did not differ from one another.

A Combination of Cryopreservation and Kneading Maintains the Usability of Mohs Paste.

Mohs paste is useful for controlling exudates from wounds and infections and is used to treat patients with inoperable skin tumors. Unfortunately, Mohs paste is difficult to preserve because its viscosity and stickiness increase dramatically immediately after preparation, resulting in decreased usability. In this study, the combined use of cryopreservation and kneading was shown to improve long-term storage of Mohs paste. At 25°C, Mohs pastes solidified rapidly, and viscosity reached approximately 700 Pa·s 5 h after preparation. In contrast, cryopreservation at -20°C attenuated hardening of Mohs pastes, and kneading also decreased viscosity. The viscosity of Mohs pastes cotreated with cryopreservation and kneading after 7 months of storage was <70 Pa·s. In addition, tissue invasion with these stored pastes was similar to freshly prepared Mohs paste. Results suggest that the combination of cryopreservation and kneading permits Mohs paste to be stored over extended periods, which may increase the utility of the paste for clinical use.