Freeze-thawed Samples Research Articles

Comparison of Extraction Techniques for Determining Bioactive Compounds and Antioxidant Activity of Spirulina platensis

Spirulina platensis (S. platensis) is a high-nutrient blue-green algae that has been used as a food supplement for a long time. It contains carbohydrates, lipids, proteins, vitamins, minerals, and bioactive compounds essential for basic human nutrition. It is known to have anti-cancer, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, and hypocholesterolemic properties due to the bioactive compounds it contains. In this study, the effects of freeze-thawing, a rapid freezing (-20°C) and thawing (4°C) process, and ultrasonically assisted extraction techniques on the color, antioxidant capacity, total phenolic content, and phenolic composition of Spirulina platensis extracts were investigated. The antioxidant capacity of the extracts obtained was determined by two different methods, DPPH (2,2-diphenyl-1-picryl hydrazyl) and ABTS (2,2-azinobis (3-ethylbenzothiazollin-6-sulfonic acid)). The sugar profile was determined by HPLC-RID and phenolic composition was determined by HPLC-ESI-DAD-MS/MS. The antioxidant activity and total phenolic content of samples prepared by the freeze-thawing were higher than those prepared by ultrasonic-assisted conventional extraction technique. In addition to ferulic acid 4-O-glucuronide and brevifolin carboxylate, an isocoumarin derivative, as the dominant phenolic compound in S. platensis extracts, a total of 10 phenolic compounds including catechin isomer, resveratrol C-hexoside, myricetin, ferulic acid, gallic acid, phloroglucinol, and lutein were detected. Glucose was the predominant sugar in both samples. The total sugar content was higher in the freeze-thawed samples (217.92 mg/100 g DW) than in the ultrasonic-assisted conventional extraction technique (182.91 mg/100 g DW). S. platensis has a significant amount of antioxidants, valuable secondary metabolites, and potential commercial applications and medicinal properties, but releasing these compounds is difficult due to the cell wall. This study was carried out to determine how different extraction techniques alter the release of bioactive compounds.

An assessment of a biosensor system for the quantification of microcystins in freshwater cyanobacterial blooms

Microcystin-producing cyanobacterial blooms are a global issue threatening drinking water supplies and recreation on lakes and beaches. Direct measurement of microcystins is the only way to ensure waters have concentrations below guideline concentrations; however, analyzing water for microcystins takes several hours to days to obtain data. We tested LightDeck Diagnostics’ bead beater cell lysis and two versions of the quantification system designed to give microcystin concentrations within 20 min and compared it to the standard freeze-thaw cycle lysis method and ELISA quantification. The bead beater lyser was only 30 % effective at extracting microcystins compared to freeze-thaw. When considering freeze-thaw samples analyzed in 2021, there was good agreement between ELISA and LightDeck version 2 (n = 152; R2 = 0.868), but the LightDeck slightly underestimated microcystins (slope of 0.862). However, we found poor relationships between LightDeck version 2 and ELISA in 2022 (n = 49, slopes 0.60 to 1.6; R2 < 0.6) and LightDeck version 1 (slope = 1.77 but also a high number of less than quantifiable concentrations). After the quantification issues are resolved, combining the LightDeck system with an already-proven rapid lysis method (such as microwaving) will allow beach managers and water treatment operators to make quicker, well-informed decisions.

Investigation on the high-temperature stability and fatigue behavior of cold mixed epoxy asphalt mixture with different gradations

Cold mixed epoxy asphalt mixture (CMEAM) is widely applied on steel deck bridge pavements due to its excellent high temperature and fatigue resistance. However, previous studies investigated above two properties through destructive test or without considering water-damage factor, respectively, which is far away from real servicing condition. To distinguish above two properties of EA-05 and EA-10 (mixture with nominal maximum particle size of 4.75 mm and 9.5 mm), uniaxial static creep tests, dynamic modulus test and four-point bending beam fatigue test were applied in this paper. At the same time, to comprehensively acquire elastic, viscous-elastic and plastic performance of CMEAM, four kinds of constitutive models were also put forward. Results showed that seven-element viscous-elastic-plastic creep model has the highest correlation coefficients which proves there exists plastic deformation in CMEAM. Meanwhile, EA-10 is likely to present viscous-elastic deformation while EA-05 is more likely to show plastic deformation. Dynamic modulus among different gradation type mixture at 40 °C is not so apparent. But with the increase of frequency and testing temperature, this difference would be clear. The middle gradation of EA-10 has the most excellent high temperature stability under dynamic load. In terms of the resistance to fatigue damage of control or freeze-thaw treated samples, EA-05-U(the upper limit of EA-05 gradation type) always takes the first place and has the lowest sensitivity to the change of stress level and water damage.

Experimental Study on the Effect of Freeze-Thaw Cycles on the Mechanical and Permeability Characteristics of Coal

The safe and efficient mining of coal seams with low porosity, low permeability, and high heterogeneity under complex geological conditions is a major challenge, with the permeability of coal seams playing a crucial role in coal mine gas extraction. The development of coal seam permeability enhancement technology can help coal mines produce safely and efficiently, while the extracted coal bed methane can be utilized as green energy. To study the effect of freezing and thawing on the evolution of the mechanical and permeability properties of coal, triaxial permeability tests were conducted on low-permeability coal under two different confining pressures. Simultaneously, dry, saturated, and freeze-thaw coal samples were set up for comparison, and the effects of water and freeze-thaw were isolated from each other. The triaxial mechanics and percolation laws of dry, saturated, and freeze-thaw coal rocks were obtained; the results show that saturated coal has the lowest initial permeability, while freeze-thawed coal has the highest initial permeability. Through analyzing the effects produced by water, freezing and thawing on coal specimens, the mechanism of the influence of freeze-thaw on the permeability evolution of coal was revealed. The research results can provide theoretical guidance for the development of gas extraction technology for low-permeability coal seams.

Mechanical properties and heavy metal leaching behaviors of municipal solid waste incineration bottom ash as road embankment fillings

With the development of economy and urbanization, the natural gravels and sands for new road construction materials encountered severe shortage issues in recent decades. In the meantime, the municipal Solid Waste Incineration bottom ash (MSWIBA) is regarded as a feasible alternative of mined nature aggregates due to its similar mineralogical, physical and chemical characteristics. However, the uncertainties of long-term service performance and potential environmental pollution risk of MSWIBA prevent its widespread applications in road constructions. For these reasons, this paper investigates the resilient modulus and permanent deformation properties by the repeated load triaxial test at different moisture contents, and examines the long-term environment influence using leaching tests on the samples after subjecting to 5 freeze-thaw cycles and samples after one year weathering. Besides, the existing resilient modulus and permanent deformation prediction models of unbounded granular materials are evaluated for their applicability on MSWIBA. The test results indicate that the MSWIBA shows obvious stress-dependence and moisture-dependence in resilient modulus and permanent deformation behaviors. Moreover, the existing resilient modulus and permanent deformation prediction models with stress and moisture dependence are feasible for MSWIBA as well. Additionally, heavy metal leaching concentrations obtained from column test can meet the standard of surface water quality class V in China. This work is expected to benefit the design work and provide the insight of performance of MSWIBA as road embankment material, and promote its further widespread application in road construction.

Freeze-thaw failure characteristics and strength loss of non-penetrating fractured rock mass with different fracture densities

Rock mass usually contains cracks, joints and other defects with different density and spatial combination characteristics in the diagenetic process. Under the freeze–thaw effect, they continue to expand and connect, which continuously weakens the strength property and poses a serious threat to the stability of rock mass. Previous studies paid more attentions to the strength reduction of rock, failing to directly reflecting the freeze–thaw damage characteristics of rock mass. In this paper, the freeze–thaw failure characteristics and strength loss of non-penetrating fractured rock mass with different fracture densities were studied by means of theoretical derivation and laboratory tests. Firstly, non-penetrating fractured rock mass samples with different fracture conditions were prepared. Then the freeze–thaw samples were subjected to laboratory compression tests and the loss of uniaxial compressive strength was measured. On this basis, the theoretical values of geological strength index GSI was calculated for the samples, and the rough prediction empirical model of GSI for non-penetrating fractured rock mass treated with different freeze–thaw cycles was obtained through data fitting. Finally, by combining the apparent observation method with acoustic emission monitoring technique, the effects of freeze–thaw cycles and fracture density on the failure characteristics of non-penetrating fractured rock mass samples were analyzed comprehensively from both the macro and micro perspectives.

Deterioration laws of Hoek-Brown parameters in freeze–thaw multi-fractured rock mass

The Hoek-Brown criterion can better depict the nonlinear failure characteristics of rock mass, which has been widely used in the fields of large-scaled slopes, deep-buried tunnels, foundations in complex geological conditions, hydropower dams, and energy mining. In view of the previous studies mostly focused on the property reduction of freeze–thaw rock, the deterioration study of Hoek-Brown parameters for freeze–thaw multi-fractured rock mass was investigated in this study. Firstly, Monte-Carlo method was used to generate two-dimensional discrete fracture network, according to which the multi-fractured rock-like samples were prepared. Freeze-thaw cycle experiments were subsequently designed and executed to observe the apparent damage of freeze–thaw effect on the samples. Then, laboratory direct shear tests on freeze–thaw samples under multistage normal loading were carried out. The shear performances of freeze–thaw rock mass were analyzed based on the shear stress-shear displacement curves of samples with different freeze-thawed cycles. Besides, considering the deficiencies of the existing Hoek-Brown parameter estimation methods such as complicated sample preparation and strong subjectivity, a new estimation method based on shear test was proposed by deducing the shear expression of Hoek-Brown criterion, which was programmed in MATLAB and verified by direct shear test results. On this basis, the values of Hoek-Brown parameter of those samples under different freeze–thaw cycles were determined, and the variation law with freeze–thaw cycles was summarized. It turned out that the values of GSI and mi both decrease with the increase of freeze–thaw cycles, and the decreasing rate is also lowering, according with the negative exponential function model.

Demonstration of Escherichia coli Inactivation in Sterile Physiological Saline under High Pressure (HP) Phase Transition Conditions and Analysis of Probable Contribution of HP Metastable Positions Using Model Solutions and Apple Juice.

It was demonstrated that the inactivation of high pressure (HP) treatment on Escherichia coli survival in sterile physiological saline (SPS) was influenced by the treatment conditions: unfrozen, frozen-thawed and fully frozen (phase transition). In order to probe the enhanced phase transition microbial destruction, vibration effects of phase transition position were created and discussed. Test samples were placed in HP chamber for treatment (150/240/330 MPa, no holding time) at room temperature and a special cooling device was used to maintain the phase transition conditions. Results showed that the phase transition from ice I to ice III of frozen SPS could be realized based on the cooling of a 20% sodium chloride solution. HP treatment under fully frozen conditions produced the best lethal effect compared to unfrozen and freeze-thaw samples. Vibration tests were carried out by using model solutions and apple juice to explore the behavior of phase transition. A synchronous and advance phase transition of internal apple juice was realized, respectively, by using pure water and 5% sodium chloride solution as external vibration sources, and the advance phase transitions of external pure water were realized by using 5% sodium chloride solution and 5% glucose solution as internal vibration sources.

Experimental Study on Mechanical Properties of Freeze-Thaw Damaged Red Sandstone under Combined Dynamic and Static Loading

Using the freeze-thaw cycle test chamber, the red sandstone samples are subjected to cyclic freeze-thaw tests. The physical properties, static mechanical properties of freeze-thaw damage rocks, and the compressional wave velocity at specific axial pressure are measured using conventional physical tests and uniaxial compression tests. The mechanical properties of freeze-thaw damage rocks under dynamic and static loading were studied using Hopkinson pressure bar which can exert axial pressure. The studies show that, with the increase of freeze-thaw cycles, the surface layer of the rock sample undergoes spalling phenomenon, the weight gradually decreases, the sample compactness becomes worse, there are microcracks between the cemented particles, and the compressive strength and elastic modulus decrease. Under the static loading, the longitudinal wave velocity of freeze-thaw damaged samples change significantly compared with that of samples without freeze-thaw. The freeze-thaw damage degree, axial pressure, and strain rate are coupled with each other, which together affect the dynamic mechanical properties of samples, and make the variation of mechanical parameters, such as dynamic peak strength and dynamic elastic modulus of rock. The combined action of freeze-thaw damage and axial pressure weakens the strain rate effect of samples, but when the incident wave of SHPB test is same, the dynamic strength and elastic modulus of freeze-thaw damaged samples are reduced compared with those without freeze-thaw. Combining with strain equivalence principle, the constitutive relation of freeze-thaw damage of red sandstone under dynamic and static combined loading can reflect the influence of coupling damage of axial pressure and freeze-thaw, dynamic impact parameters, and other factors, which are in good agreement with the test results.

Dual cryoprotective and antioxidant effects of silver carp (Hypophthalmichthys molitrix) protein hydrolysates on unwashed surimi stored at conventional and ultra-low frozen temperatures

The dual cryoprotective and antioxidant effects of silver carp hydrolysates on unwashed surimi, stored at conventional (−18 °C) or ultra-low (−60 °C) temperatures and subject to three and six freeze-thaw treatments, were examined. The addition of hydrolysates could alleviate the formation of carbonyls of myofibrillar protein, inhibit the decrease of free sulfhydryl content and keep the protein bands stability compared to the control and SuSo group. The lipid peroxidation product malondialdehyde, and the target volatiles hexanal, nonanal and 1-octen-3-ol of the unwashed surimi as detected by HS-SPME-GC-MS, increased slowly under the protection of hydrolysates. The oxidation for proteins and lipids in freeze-thawed samples stored at −18 °C are lower than those of samples stored at −60 °C (p < 0.05), indicating that ultra-low frozen temperature storage accompanied by freeze-thaw cycles accelerates structural deterioration of unwashed surimi. LF-NMR and SEM also revealed that the hydrolysates facilitated to form gel network with preferable structural integrity and stability during freeze-thaw treatments. This study provides theoretical supports for the application of silver carp protein hydrolysates as a cryoprotectant in transportation and storage of frozen surimi.

The retrogradation characteristics of starch in green wheat product Nianzhuan: effects of storage temperature and time

Abstract The objective of this study was to reveal the process of starch retrogradation and quality changes of Nianzhuan stored at 4, −18 °C, and freeze-thaw cycles treatment for different lengths of time. XRD revealed that Nianzhuan starch displayed an increasing trend of crystallinity with prolonged storage time and numbers of freezing-thawing cycles, which was likely due to a more orderly crystalline matrix in starch. The Raman full width at half-maximum (FWHM) of the bands at 2913 cm−1 of the three storage methods all decreased. According to DSC analysis, an increase in ∆H was detected, and a significant (P &lt; 0.05) increase in T o and T p were found at −18 °C, and freeze-thaw treated samples, indicating more thermal energy were needed to disrupt re-crystallization. Good correlations between crystallinity, FWHM, ∆H, and hardness, springiness, chewiness were tested. The results of this study would provide useful information for the process of starch-based product Nianzhuan.

Lateral flow assays for the detection of African swine fever virus antigen are not fit for field diagnosis of wild boar carcasses.

African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, 'point-of-care' (POC) tests, especially immunochromatographic assays, have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. The later topic was the starting point for our present study. In detail, we evaluated the performance of the commercially available INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) with selected high-quality reference blood samples, and with blood samples from wild boar carcasses collected under field conditions in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct testing of blood samples from carcasses without any modification, resulted in a drastically reduced sensitivity of only 12.5% with the given sample set. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of wild boar carcass samples. A diagnostic specificity of 100% was observed. In summary, the antigen LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen that could be overcome, to a certain extent, with assay modifications.

Synergistic effect of rice husk, glass and marble sludges on the engineering characteristics of eco-friendly bricks

Due to rapid population growth and industrialization, the generation of different industrial and agricultural wastes has surged in the past few decades. In this study, burnt clay bricks are prepared to investigate the synergistic and individual effect of glass sludge (GS), marble sludge (MS), and rice husk (RH) on the physical, mechanical, durability, and thermal properties of brick samples. Results show that the addition of waste materials (GS, MS, and RH) reduces the shrinkage, weight per unit area, and thermal conductivity of brick samples. All the brick samples incorporating different waste materials show efflorescence much lower than 10%. Furthermore, no brick sample faced any cracking even after 50 freeze-thaw cycles. The mass loss of brick samples incorporating 25% of GS is observed lower than the specified limit of ASTM C67 and are classified as freeze-thaw resistant brick samples. All the brick samples incorporating different waste materials satisfy the minimum compressive strength and modulus of rupture requirements for building bricks and can be used in a moderate weather-resistant environment, leading to sustainable masonry construction. Furthermore, all the brick samples show leaching toxicity values much lower than the specified limits of the Environmental Protection Agency. The scanning electron microscopic images also support the results of porosity and water absorption of brick samples observed in this study. Based on the results, all the combinations of waste materials considered in this study for making brick samples can be used for masonry construction leading towards landfill reduction and the production of eco-friendly bricks.

Laboratory study of cryogenic treatment induced pore-scale structural alterations of Illinois coal and their implications on gas sorption and diffusion behaviors

Cryogenic fracturing using liquid nitrogen is a choice for CBM reservoir stimulation. However, the development of cryogenic fracturing is still at its infancy. This work focused on induced pore structural alterations due to cryogenic treatment and their effects on gas sorption and diffusion behaviors. The changes in the pore structure of coal induced by cyclic nitrogen injections were studied by physical adsorption at low temperatures. A micromechanical model is proposed to simulate the microscopic process and predict the degree of deterioration due to low temperature treatments. As a common characteristic of modeled results and experimental results, the total volume of mesopore and macropore increased with cryogenic treatment, but the growth rate of pore volume became much smaller as freezing-thawing were repeated. Pores of different sizes experienced different degrees of deterioration. In the range of micropores, no significant alteration of pore volume occurred with the repetition of freezing and thawing. In the range of mesopores, pore volume increased with the repetition of freezing and thawing. In the range of macropores, pore volume increased after the first cycle of freezing and thawing but decreased after three cycles of freezing and thawing. Because of pore structural alteration, cryogenic treatment enhanced gas transport process as the diffusion coefficients of the freeze-thawed coal samples were increased by 18.76% and 30.18% in the adsorption and desorption process. The effect of cyclic cryogenic treatment on the pore structure of tested coal varied depending on coal properties. For the studied Illinois coal sample, repetitive applications of cryogenic treatment reduced macropore volume and increase mesopore volume. For the tested sample, the diffusion coefficient of the coal sample that underwent three cycles of freezing-thawing was lower than that of the coal sample that underwent a single cycle of freezing and thawing. The outcome of this study provides justification for a post-cryogenic fracturing effect on diffusion improvement and gas production enhancement, especially for high “sorption time” CBM reservoirs.

Damage estimation of concrete canal due to freeze and thawed effects by acoustic emission and X-ray CT methods

As a detailed inspection of a concrete structure in service, core samples are usually drilled out and then mechanical properties are measured. In this study, damage estimation of freeze-thawed concrete from concrete-core samples is developed, applying acoustic emission (AE) method. By the authors, the quantitative damage evaluation of concrete has been proposed in RILEM TC −212ACD, by applying AE and damage mechanics in the compression test. In this study, characteristics evaluation of cracking damage effects in concrete mechanical properties by detected AE energy in core test. Prior to the compression test, distribution of micro-cracks in a concrete-core sample is inspected by helical X-ray computer tomography (CT). Then, freeze-thawed damaged samples are tested by the compression test with AE monitoring. Concrete-core samples were taken out of a channel structures in Hokkaido, Japan which is extremely developed cracking damage. Thus, it is demonstrated that the concentration of material damage could be evaluated by comparing geometrical characteristics of cracks with the “energy rate” of AE generation, which is analyzed by AE parameter analysis. A relation between AE energy rate, physical properties and geometrical properties of void is correlated, and thus the damage of concrete is qualitatively estimated by AE parameters.

Experimental Research on Physical Indices and Mechanical Properties of Sandstone under Combined Action of Chemical-Freeze-Thaw Cycles

In order to study the effects of chemical solution erosion and freeze-thaw cycles on the physical parameters and mechanical properties of rocks, sandstone samples from central Shandong Province were taken as the research objects. The freeze-thaw cycle test and uniaxial compression test were carried out for rock samples eroded by 0.01mol/L H2SO4 solution, 0.01mol/L NaCl solution and 0.01mol/L NaOH solution, respectively. The results show that the mass loss and porosity of rock samples eroded by different chemical solutions increase with the increase of freeze-thaw cycles, and the peak strength and elastic modulus decrease exponentially with the increase of freeze-thaw cycles. The damage degree of acidic solution to freeze-thaw cycling sandstone samples is the greatest, that of alkaline solution is the next, and that of neutral solution is the smallest. The conclusions can be used for reference in support and design of underground engineering.

Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics.

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

Ex vivo comparison of the effect of storage temperature on canine intestinal leakage pressures.

To determine the effect of storage temperature on cadaveric small intestinal leakage pressures after enterotomy. Experimental ex vivo study. Grossly normal jejunal segments from four canine cadavers. Thirty-six jejunal segments (n = 12 segments/group) were harvested immediately after euthanasia and assigned to a fresh group (tested within 4 hours), chilled group (stored for 24 hours at 4°C before testing), or freeze-thaw group (frozen at -20°C for 7 days and thawed at 21°C for 6 hours before testing). A 2-cm antimesenteric enterotomy was performed and repaired with 4-0 monofilament suture in a simple-continuous pattern. Initial leakage pressure (ILP), maximal intraluminal pressure (MIP), and leakage location were recorded, with testing performed at room temperature. Mean ± SD ILP for fresh, chilled, and frozen-thawed specimens was 52.9 ± 8.4, 51.8 ± 11.9 and 29.8 ± 4.4 mm Hg, respectively. There was a difference in ILP among groups (P < .003), with freeze-thaw samples demonstrating lower ILP compared with other groups. There was no difference in MIP between groups (P = .186) There was a difference in leakage location among groups (P = .004), with the majority of chilled and freeze-thaw samples leaking at the suture holes compared with the incisional line in fresh samples. Freezing and subsequent thawing prior to specimen testing reduced ILP compared with use of fresh and chilled specimens but did not affect MIP among experimental groups. Cadaveric canine intestinal specimens tested immediately after collection or after chilling for 24 hours should be recommended for ex vivo burst pressure assessment in dogs. Additional studies to evaluate loss in testing viability of chilled intestinal specimens are warranted to help govern experimental methodologies.

RTHP-43 (LTBK-02). MULTI-TIME POINT EVALUATION OF PERIPHERAL BLOOD MYELOID-DERIVED SUPPRESSOR CELL AND LYMPHOCYTE POPULATIONS IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA RECEIVING ADJUVANT THERAPY

Abstract Alterations in peripheral blood cell populations, including myeloid cell subsets and lymphocytes, have been associated with poor prognostic outcomes in patients with glioblastoma (GBM). To date, evaluation of these populations has largely been on freeze-thawed samples at a limited number of time points. The purpose of our study is to identify the frequency and activation status of tumor-supportive myeloid and lymphocyte subsets during the course of standard adjuvant therapy in patients with GBM in fresh peripheral blood samples. We hypothesize that dysfunctional myeloid and lymphocyte populations expand during standard care temozolomide (TMZ)/radiation therapy (RT). We prospectively enrolled 16 patients with a new diagnosis of GBM that underwent maximal safe surgical resection. Twelve received standard adjuvant therapy consisting of six weeks of TMZ/RT. Four received alternative therapies: one experimental chemotherapeutic/three alternative radiation schemes. Peripheral blood was obtained seven times over the course of adjuvant therapy: prior to starting adjuvant therapy, five during treatment, and once one month after completion of therapy. Eleven patients completed all seven time points. Peripheral blood samples were analyzed by flow cytometry, focusing on immune checkpoints and critical markers of activation of systemic immunity. Available preliminary data demonstrate a median increase of 2.1% (-21.2–27.4%) in the frequency (%fx) of myeloid-derived suppressor cells (MDSC) after commencement of adjuvant therapy at time point 2 (TP2), compared to baseline. To date, 7/16 patients are alive and without evidence of recurrent disease on imaging. The majority of these patients had a decrease in %fx of MDSC at TP2, after starting adjuvant therapy, compared to baseline. The %fx of CD8+ T cells was greatest at TP7 (range: 21.7%-67.0%), one month after TMZ/RT, for most patients. PD-1 and FOXP3 expression by lymphocytes was variable across time points, suggesting the importance of additional factors which will be explored through future analyses. These early findings suggest that the immune response changes throughout the course of TMZ/RT. An understanding of these changes in the peripheral blood, termed immunodynamics in the context of our studies, may be valuable future tool to aid in optimizing immunity in patients with GBM.

Effects of prestressing and saw-cutting on the freeze-thaw durability

There are many factors that affect the freeze-thaw durability of concrete elements, including proper air entrainment system, freeze-thaw durable aggregates, low permeability, and proper curing. While ASTM C666 allows the excising of small freeze-thaw samples from larger sections it doesn't address the issues that can arise when excising samples from prestressed concrete and subsequent testing in freezing and thawing. In order to investigate the effects of saw-cutting, the existence of reinforcement, and use of prestressing, concrete samples of different sizes, with and without saw-cutting, with and without reinforcement, and with and without prestressing were tested in freeze-thaw. The freeze-thaw mechanism was adjusted, from that of ASTM C666, for the larger sample in terms of freezing and thawing rates and the testing method. The results indicate that saw-cutting through plain concrete had minimal effect on the freeze-thaw durability, whereas sawcuts through reinforcement were shown to be very detrimental to the measured durability in the tests.