Repetitive Freeze-thaw Cycles Research Articles

Enhancing protein stability under stress: osmolyte-based deep eutectic solvents as a biocompatible and robust stabilizing medium for lysozyme under heat and cold shock.

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml-1. Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.

Improving Mechanical Properties of Tendon Allograft through Rehydration Strategies: An In Vitro Study.

Allogenic tendons grafts sourced from intrasynovial tendons are often used for tendon reconstruction. Processing is achieved through repetitive freeze-thaw cycles followed by lyophilization. Soaking the lyophilized tendon in saline (0.9%) for 24 h is the standard practice for rehydration. However, data supporting saline rehydration over the use of other hydrating solutions are scant. The purpose of the current study was to compare the effects of different rehydration solutions on biomechanical properties of lyophilized tendon allograft. A total of 36 canine flexor digitorum profundus tendons were collected, five freeze-thaw cycles followed by lyophilization were performed for processing, and then divided into three groups rehydrated with either saline solution (0.9%), phosphate-buffered saline (PBS), or minimum essential medium (MEM). Flexural stiffness, tensile stiffness, and gliding friction were evaluated before and after allograft processing. The flexural moduli in both fibrous and fibrocartilaginous regions of the tendons were measured. After lyophilization and reconstitution, the flexural moduli of both the fibrocartilaginous and non-fibrocartilaginous regions of the tendons increase significantly in the saline and MEM groups (p < 0.05). Compared to the saline and MEM groups, the flexural moduli of the fibrocartilaginous and non-fibrocartilaginous regions of tendons rehydrated with PBS are significantly lower (p < 0.05). Tensile moduli of rehydrated tendons are significantly lower than those of fresh tendons for all groups (p < 0.05). The gliding friction of rehydrated tendons is significantly higher than that of fresh tendons in all groups (p < 0.05). There is no significant difference in either tensile moduli or gliding friction between tendons treated with different rehydration solutions. These results demonstrate that allograft reconstitution can be optimized through careful selection of hydrating solution and that PBS could be a better choice as the impact on flexural properties is lower.

Low-temperature cracking resistance of asphalt concretes for railway substructure exposed to repeated freeze-thaw cycles

The waterproofing substructure under high-speed railway track is of importance to avoid the infiltration of surface water and maintain the bearing capacity of railway subgrade. In this study, the low-temperature cracking resistance of composite polymerized asphalt concretes for high-speed railway waterproofing layer (shortened to railway asphalt concretes) was investigated when exposed to freeze-thaw cycles. Although the measured maximum load, fracture energy and fracture toughness indicated a certain degree of low-temperature performance deterioration after repetitive freeze-thaw cycles, railway asphalt concretes showed excellent moisture susceptibility in this regard. In addition, the railway asphalt concretes prepared with binder B and gradation AC-10 were generally superior to their counterparts in this respect. On the other hand, the image processing of specimens' fracture surfaces indicated that the cracking of railway asphalt concretes at low temperatures could be mainly attributed to the binder cohesion failure. Besides, the contribution rate of aggregate-binder adhesion failure to the overall cracking gradually exceeded that of aggregate fracture with the increased freeze-thaw cycles. Finally, a statistically significant correlation between the fracture energy and interfacial adhesion failure was found. This implied that the deterioration of low-temperature cracking resistance could be primarily contributed to the reduced aggregate-binder adhesion strength during freeze-thaw cycles.

Assessing the biocompatibility of bovine tendon scaffold, a step forward in tendon tissue engineering.

Tendon is a collagen-enriched, tough, and intricately arranged connective tissue that connects muscle to the bone and transmits forces, resulting in joint movement. High mechanical demands can affect normal tissues and may lead to severe disorders, which usually require replacement of the damaged tendon. In recent decades, various decellularization methods have been studied for tissue engineering applications. One of the major challenges in tendon decellularization is preservation of the tendon extracellular matrix (ECM) architecture to maintain natural tissue characteristics. The aim of the present study was to create a decellularized bovine Achilles tendon scaffold to investigate its cytocompatibility with seeded hAd-MSCs (human adipose derived-mesenchymal stem cells) and blastema tissue in vitro. Here, we describe a reliable procedure to decellularize bovine Achilles tendon using a combination of physical and chemical treatments including repetitive freeze-thaw cycles and the ionic detergent SDS, respectively. The decellularization effectiveness and cytocompatibility of the tendon scaffolds were verified by histological studies and scanning electron microscopy for up to 30days after culture. Histological studies revealed hAd-MSC attachment and penetration into the scaffolds at 5, 10, 15 and 20days of culture. However, a decrease in cell number was observed on days 25 and 30 after culture in vitro. Moreover, migration of the blastema tissue cells into the scaffold were shown at 10 to 25days post culture, however, destruction of the scaffolds and reduction in cell number were observed on 30th day after culture. Our results suggest that this decellularization protocol is an effective and biocompatible procedure which supports the maintenance and growth of both hAd-MSCs and blastema cells, and thus might be promising for tendon tissue engineering.

Effects of Repetitive Freeze-Thaw Thermal Cycles on the In-Plane Effective Thermal Conductivity of a Gas Diffusion Layer in Polymer Electrolyte Membrane Fuel Cells

As the fundamental components of polymer electrolyte membrane fuel cells, the thermal properties of gas diffusion layers (GDLs) play a significant role in influencing their overall heat conduction and management. We have experimentally characterized the temperature dependence of the in-plane thermal conductivity of a commercial carbon paper GDL, including the progressively in-plane thermal degradation of GDLs through a series of repetitive freeze-thaw thermal aging cycles. Even though the thermal properties of carbon fibers are crucial for the thermal performance determination of GDLs, the temperature-dependent in-plane thermal conductivity of pristine GDLs depends mainly on the thermal characteristics of the binder that connects carbon fibers and the polytetrafluoroethylene (PTFE) coated on the microporous layer. The microstructural failure of aged GDLs (breakage of carbon fibers, delamination, adhesive failure of binder, and PTFE cracks) caused by the freeze-thaw thermal aging treatment dominantly affects the in-plane effective thermal degradation at different thermal aging phases. Overall, the breakage of carbon fibers positively contributes, and delamination, adhesive failure of binder, and PTFE cacks negatively contribute to the in-plane effective thermal conductivity of GDLs. In particular, the in-plane thermal conductivity of aged GDLs through 300 cycles reaches a peak.

Stability of interleukin-1β, -4, -6, -8, -10, -13, interferon-γ and tumor necrosis factor-α in human sera after repetitive freeze-thaw cycles and long storage

IntroductionCytokines (CKs) are one of the key components of the molecular network modulating multiple immunological interactions. Within such biological systems, CKs functions are associated with several processes, thus quantification of these analytes in serum samples, as well as a faithful determination of its concentration, are crucial for the translational aspect of many studies. AimThis study is focused on the evaluation of the effects of storage duration and multiple freeze-thaw cycles on CKs stability. Materials and methodsSerum samples were obtained from 24 healthy participants. Samples were prospectively stored at 4 °C for 1–7 and 30 days, and also underwent multiple freeze-thaw cycles. Afterwards, CK levels were determined by enzyme-linked immunosorbent assay. ResultsAmong the 8 examined CKs all of them showed significant degradation (determined with the two-way ANOVA and post-hoc test) after 4 days of sample storage at 4 °C. Serum were affected by freezing at −20 °C and thawing, and 2 of CKs (IL-1β and IL-8) showed significant concentration decrease after following 2 freeze-thaw cycles. It has been also determineded that CKs in serum samples after multiple freeze-thaw cycles had better stability, when samples were stored at −80 °C (compared to storage at −20 °C). ConclusionsThis study demonstrates that long storage at 4 °C, as well as multiple freeze-thaw cycles of serum samples, must be avoided and CK concentrations should be measured immediately after sample collection.

Durability of Portland Concrete Containing Polymeric Fillers and Fly Ash

Portland concrete suffers in service brittle failure, extensive crack propagation, and wear rates increasing with time. In spite of all the effort expended, these problems persisted when we had started our project. We used several polymeric fillers and fly ash. Higher compressive moduli than the starting concrete are seen for some compositions, the highest for 5 % of one of the polymers + 5 % fly ash. The same composition has the lowest Taber abrasive wear loss. All composites show lower wear loss values than Portland concrete. After 25 days of acidic degradation in 4.0 molar aq. HCl, the starting Portland concrete suffers stronger degradation that our composites. Polymer swelling mitigates acidic degradation. Repetitive freeze-thaw cycles between 15oF and 85oF show disappearance of the deep voids present before the first cycle in our composites—but not in the Portland cement. While the use of fly ash mitigates contamination of the environment, it is the combination of fly ash with polymers which provides significantly improved properties - tribological, chemical and mechanical ones – of the Portland concrete.

Degradations in porous components of a proton exchange membrane fuel cell under freeze-thaw cycles: Morphology and microstructure effects

In this study, porous components of a proton exchange membrane (PEM) fuel cell, i.e., single-layer gas diffusion layer (GDL, carbon paper), double-layer GDL (microporous layer (MPL) deposited carbon papers), and catalyzed electrodes, are subjected to 60 repetitive freeze-thaw cycles between −40 °C and 30 °C under water-submerged conditions; and their morphological and microstructural characteristics are investigated at each 15 cycles and compared with those of virgin materials. The results indicate that consecutive cycling of temperature causes different degradation patterns in different components. The single-layer GDL shows a unique degradation mechanism, in which macro-scale pores volumetrically expand, and relatively small-scale hollows and cracks form on the polymeric binder and carbon fiber interfaces, respectively. For the double-layer GDL, large-scale surface cracks form on the MPL surface after 15 cycles, and the morphology and microstructure degradation gains momentum with the formation of these cracks, and upon completion of 30 cycles, large-scale carbon/hydrophobic agent flakes start to detach from the surface. For the catalyzed electrodes, due to their inherently cracked surface, the catalyst layers (CLs) degrade first through expansion of the cracks in the in- and through-plane directions, and then through swelling and agglomeration of the ionomer; and combination of these two patterns triggers detachment of large CL flakes from the surface, negatively affecting the microstructure.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Stability of important antibodies for kidney disease: pre-analytic methodological considerations.

BackgroundThe importance of circulating antibodies as biomarkers of kidney disease has recently been recognized. However, no study has systematically described the methodology of sample preparation and storage regarding antibodies as biomarkers of kidney disease. It remains unknown whether repetitive freeze-thaw cycles, physical disturbances, storage at different temperatures or for different periods of time, or haemolytic or turbid serum samples affect antibody measurements. The aim of this study was to investigate the stabilities of antibodies associated with kidney disease in serum samples under various relevant clinical and research conditions.MethodsWe stored serum samples in the following different conditions: repetitive freeze-thaw cycles (1, 6 or 12 times), long-term storage (7 or 12 months at −80 °C), physical disturbance (1 or 8 h), and storage at 4 °C (1, 3 or 6 weeks) and room temperature (1 or 7 days). The stabilities of the anti-phospholipase A2 receptor (anti-PLA2R), anti-glomerular basement membrane, anti-myeloperoxidase and anti-proteinase 3 antibodies were evaluated with enzyme-linked immunosorbent assays (ELISA).ResultsWe found that repetitive freeze-thaw cycles did not have a significant effect on the stabilities of the abovementioned antibodies in clear serum samples. The ELISA readings of haemolytic and turbid serum samples tended to increase and decrease, respectively. Neither long-term storage at −80 °C nor physical disturbance had a significant effect on anti-PLA2R antibody stability in sealed serum samples. The concentrations of most of these antibodies increased in unsealed serum samples that were stored at 4 °C for more than 6 weeks or at room temperature for more than 7 days.DiscussionOur findings revealed that the abovementioned circulating antibodies that are used as biomarkers for kidney disease had stable physicochemical properties, structures and immunoreactivities such that they were not influenced by repetitive freeze-thaw cycles, physical disturbances or long-term storage at −80 °C. However, the ELISA readings tended to change for haemolytic, turbid and unsealed serum samples.

A novel serological assay for influenza based on DiD fluorescence dequenching that is free from observer bias and potentially automatable – A proof of concept study

BackgroundSerum hemagglutination inhibition (HAI) and microneutralization (MN) antibodies are often used as a correlate of protection for influenza. However, these manual assays are labor-intensive and difficult to standardize due to variability in biologic reagents used and subjective interpretation of the results. MethodsSera with known HAI and MN titers were used to assess a novel test based on the inhibition of fluorescence ‘dequenching’. Whole influenza virions (A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2) and B/Brisbane/60/2008) labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) were exposed to serial dilutions of serum and mixed with turkey red blood cells followed by acidification of the media (pH 5.0–5.5). The H1N1 and B/Brisbane strains were high hemagglutinating while the H3N2 strain had low hemagglutinating activity. In some experiments, labelled virions were subjected to repetitive freeze-thaw cycles prior to use in the assay. ResultsIn the absence of detectable HAI/MN antibodies, there were consistent and substantial increases from baseline DiD fluorescence upon acidification. Sera with known high titer HAI/MN antibodies reduced or completely prevented DiD dequenching at low dilutions with progressive increases in fluorescence at higher dilutions, which permitted a reproducible assignment of an antibody ‘titer’ based on baseline and acidified DiD fluorescence values. The ‘titers’ measured by the DiD dequenching assay were highly correlated with HAI/MN results for the H1N1 and B strains (Spearman’s correlation coefficients (rs) 0.874 to 0.946, p < 10−7 to 10−35). Correlations with HAI/MN titres for the low-hemagglutinating H3N2 strain tested were lower but remained statistically significant (rs 0.547–0.551, p < 0.004). Freeze-thawing of the DiD pre-stained virus stocks had no significant impact on the results of the assay. ConclusionsThe DiD dequenching assay may be a labour-saving and more objective alternative to the classic serologies. This novel assay could theoretically be standardized across laboratories using pre-stained virions and has the potential to be fully automated.

The effect of repeated freeze-thaw cycles on human muscle tissue visualized by postmortem computed tomography (PMCT).

The aim of this study was to determine whether effects of repetitive freeze-thaw cycles, with various thawing temperatures, on human muscle tissue can be quantified using postmortem computed tomography (PMCT) technology. An additional objective was to determine the preferred thawing temperature for muscle tissue in this study. Human cadaver upper extremities were divided into two different thawing temperature groups and underwent a series of four freeze-thaw cycles in total. Axial CT scans were performed after each cycle. CT attenuation (in Hounsfield units, HU) was measured in four muscles of the upper extremities. HU values changed significantly with the introduction of each subsequent freeze-thaw cycle. Moreover, the changes in HU values were different for each thawing group. There was a significant increase of HU values in both groups between t0 and t1 . Unfrozen tissue showed large variation of HU values in all samples. It was possible to distinguish between samples thawed at different thawing temperatures based on their respective HU values. It is advisable to keep the number of freeze-thaw cycles to just one, if the human cadaveric tissue is to be used for educational purposes. The preferred thawing temperature in this study is 2°C. Clin. Anat. 30:799-804, 2017. © 2017Wiley Periodicals, Inc.

Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.

Background and aimThe potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles.MethodsBlood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times).ResultsDifferent collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples.ConclusionThis study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

A simple and fast method for extraction and quantification of cryptophyte phycoerythrin

The microalgal pigment phycoerythrin (PE) is of commercial interest as natural colorant in food and cosmetics, as well as fluoroprobes for laboratory analysis. Several methods for extraction and quantification of PE are available but they comprise typically various extraction buffers, repetitive freeze-thaw cycles and liquid nitrogen, making extraction procedures more complicated. A simple method for extraction of PE from cryptophytes is described using standard laboratory materials and equipment. The cryptophyte cells on the filters were disrupted at −80°C and added phosphate buffer for extraction at 4°C followed by absorbance measurement. The cryptophyte Rhodomonas salina was used as a model organism.•Simple method for extraction and quantification of phycoerythrin from cryptophytes.•Minimal usage of equipment and chemicals, and low labor costs.•Applicable for industrial and biological purposes.

Decellularization of Large Tendon Specimens: Combination of Manually Performed Freeze-Thaw Cycles and Detergent Treatment.

Reliable decellularization techniques applicable to tendon tissue play a critical role in the field of current tissue engineering. Particularly, an application as three-dimensional culture model for in vitro research and translational approaches to establish graft-based tendon repair as a routine clinical tool represent two main application fields for decellularized tendon scaffolds. Considering methodological issues of tendon decellularization, one of the major challenges lies in the preservation of the tendon-specific extracellular matrix (ECM) architecture to reflect natural tissue characteristic as best as possible. Concurrently, further requirements for high-quality decellularized biological tendon scaffolds include not only the reduction of resident cells, but also an ensured cytocompatibility.To date, a large number and a wide variety of decellularization protocols for natural tendon tissue have already been investigated and usually, physical as well as chemical and/or enzyme-based treatments are used for the purpose of decellularization. However, to the best of our knowledge, there is a lack of evidence-based protocols for the processing of full-thickness large tendon samples, such as the equine flexor tendons.Therefore, the here presented protocol describes a reliable procedure to decellularize equine superficial digital flexor tendons by using a combined treatment of physical decellularization in the form of repetitive freeze-thaw cycles, and of chemical decellularization with the non-ionic detergent Triton X-100. The decellularization effectiveness evaluated by reduction of cell and DNA content, the influence of decellularization on the morphology of the tendon extracellular matrix (ECM) as well as the cytocompatibility of the decellularized tendon scaffolds obtained have been investigated previously. Based on this previous study, the here present protocol is an effective procedure, particularly applicable for large tendon specimens.

Mass spectrometric based approaches in urine metabolomics and biomarker discovery.

Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:115-134, 2017.

The Impact of Freeze-Thaw Cycles on Epinephrine

Epinephrine is the first-line medical treatment for anaphylaxis, a life-threatening allergic syndrome that may be triggered by an insect sting, substances, medications, and certain foods. Administering epinephrine can rapidly reverse bronchospasm and other life-threatening changes from anaphylaxis. Backcountry recreationalists and guides carry epinephrine in the form of an autoinjector. Epinephrine may be exposed to cold temperatures during these expeditions. No studies have examined the stability of epinephrine after being exposed to freezing temperatures. The purpose of this study was to determine whether freeze-thaw cycles alter the concentration of epinephrine and to compare these results with standards of therapeutic activity. The United States Pharmacopeia (USP) designates an epinephrine solution must contain between 90% and 115% of the labeled epinephrine amount to meet USP standards. Epinephrine from 6 autoinjectors (Epi-Pen, Mylan Speciality, Basking Ridge, NJ) was extracted and divided into experimental and control samples. The experimental samples underwent 7 12-hour freeze cycles at an average temperature of −25.4°C followed by 7 12-hour thaw cycles at an average temperature 22.7°C. The control samples remained at an average temperature of 23.1°C for the duration of the study. Immediately after the seventh thaw cycle, epinephrine concentrations were measured using a high-performance liquid chromatography assay with mass spectrometry detection. The mean concentration of the freeze-thaw samples increased compared to the control samples: 1.07 mg/mL (SD 8.78, 95% CI: 1.04–1.11) vs 0.96 mg/mL (SD 6.8, 95% CI: 0.94–0.99), respectively (P < .01). Epinephrine concentrations remain within the therapeutically active range (90% to 115%) after repetitive freeze-thaw cycles. This knowledge benefits those who must emergently administer epinephrine after the medication has accidentally frozen and then thawed.

Exfoliation of Graphite Oxide in Water without Sonication: Bridging Length Scales from Nanosheets to Macroscopic Materials

Dispersed graphene oxide (GO) nanosheets in water were synthesized via a new approach involving sonication-free exfoliation, as promoted by repetitive simple freeze–thaw cycles. These cycles consist of rapid freezing of an aqueous solution containing graphite oxide and subsequent thawing of the resultant solid. This approach is effective for exfoliation of graphite oxide and yields approximately 80% GO after six repetitive freeze–thaw cycles. The GO synthesized by the new method experiences minimal fragmentation during the exfoliation process and has a lateral size at least 3-fold larger than that of GO prepared by using sonication, as evidenced by atomic force microscopy and dynamic light scattering. We also demonstrate use of the resulting exfoliated GO that is synthesized using this approach as a building block for the synthesis of a low-density (≈0.02 g cm–3) macroporous monolithic material, using directional freezing, which forms ice rods within the solution that served as the template to direct the assembly of the solution-dispersed GO nanosheets into a honeycomb-like morphology with a 10 μm macropore opening and sheet-like walls. Such a unique morphology of the synthesized monolith has broad applicability for advanced functional materials that allow extremely high throughput with minimal pressure drop as well as electronic and energy storage materials.

Freeze-Thaw Cycles Enhance Decellularization of Large Tendons

The use of decellularized tendon tissue as a scaffold for tendon tissue engineering provides great opportunities for future clinical and current research applications. The aim of this study was to assess the effect of repetitive freeze-thaw cycles and two different detergents, t-octyl-phenoxypolyethoxyethanol (Triton X-100) and sodium dodecyl sulfate (SDS), on decellularization effectiveness and cytocompatibility in large tendons. Freshly collected equine superficial and deep digital flexor tendons were subjected to decellularization according to four different protocols (1 and 2: freeze-thaw cycles combined with either Triton X-100 or SDS; 3 and 4: Triton X-100 or SDS). Decellularization effectiveness was assessed based on the reduction of vital cell counts, histologically visible nuclei, and DNA content. Transmission electron microscopy was performed to evaluate cellular and extracellular matrix integrity. Further, cytocompatibility of scaffolds that had been decellularized according to the protocols including freeze-thaw cycles (protocols 1 and 2) was assessed by seeding the scaffolds with superparamagnetic iron oxide labeled mesenchymal stromal cells and monitoring the cells histologically and by magnetic resonance imaging for two weeks. Decellularization was significantly more effective when using the protocols including freeze-thaw cycles, leaving only roughly 1% residual nuclei and 20% residual DNA, whereas samples that had not undergone additional freeze-thaw cycles contained roughly 20% residual nuclei and 40% residual DNA. No morphological extracellular matrix alterations due to decellularization could be observed. Scaffolds prepared by both protocols including freeze-thaw cycles were cytocompatible, but the cell distribution into the scaffold tended to be better in scaffolds that had been decellularized using freeze-thaw cycles combined with Triton X-100 instead of SDS.

AB0058 A simple set of validation steps identifies and removes false positive measurements in a sandwich elisa caused by anti-animal protein antibodies in arthritis plasma

BackgroundIn spite of the widespred use of sandwich ELISAs in arthritis research recommendations on how to eliminate unspecific interference by rheumatoid factor and anti-animal protein antibodies frequently found in arthritis...