scholarly journals Effects of Production Method and Repeated Freeze Thaw Cycles on Cytokine Concentrations and Microbial Contamination in Equine Autologous Conditioned Serum

2021 ◽  
Vol 8 ◽  
Author(s):  
Josephine Hale ◽  
Kristopher Hughes ◽  
Sarah Hall ◽  
Raphael Labens
Keyword(s):  
Repeated Measures ◽  
Production Method ◽  
Mixed Effect ◽  
Production Methods ◽  
Freeze Thaw ◽  
Standard Clinical Practice ◽  
Thaw Cycle ◽  
Autologous Conditioned Serum ◽  
Tnf Α ◽  
Two Samples

Autologous conditioned serum (ACS) is a common intra-articular treatment for osteoarthritis in horses. The objective of this study was to investigate the influence of ACS preparation method on product contamination and concentrations of relevant cytokines and the influence of multiple freeze/thaw cycles. Blood was obtained from 10 healthy Thoroughbred horses and processed in parallel using a commercial and a non-commercial method to obtain ACS. Fluorescent microsphere immunoassay (FMIA) analysis was performed to quantify Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations in ACS obtained by both production methods. Effect of 3, 4 and 5 freeze/thaw cycles on concentrations of IL-1Ra, IL-10, IL-1β and TNF-α were assessed against baseline samples (2 cycles) in commercial ACS products. Standard aerobic and anaerobic culture methods were applied to both ACS products. Mixed effect one-way analyses of variance (ANOVA) were used to compare the two ACS production method for each cytokine. Repeated measures, mixed effect ANOVA were used to assess the effect of freeze/thaw on cytokine concentrations. Significance was set at P < 0.05. There was no difference in cytokine concentration between production methods (IL-1Ra P = 0.067, IL-1β P = 0.752, IL-10 P = 0.211 and TNF-α P = 0.25). Microbial growth was only observed in two samples obtained using the commercial production method. When compared to baseline, IL-1Ra concentration was decreased following the 5th freeze/thaw cycle (P < 0.001). These results suggest that the concentration of important cytokines are not influenced by ACS production method. When storing ACS samples for future use, freeze/thaw cycles associated with standard clinical practice are unlikely to influence cytokine concentrations. However, the lack of outcome measures associated with 1 or 2 freeze/thaw cycles represents a limitation of this study.

Cryoinjury Enhancement of Breast Cancer Cells by Use of a Molecular Adjuvant (TNF-alpha)

2004 ◽  
Author(s):  
Bumsoo Han ◽  
Matthew D. Egberg ◽  
Pung-Pung Haung ◽  
David J. Swanlund ◽  
John C. Bischof
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Mcf7 Cells ◽  
Freeze Thaw ◽  
Molecular Adjuvant ◽  
Tissue Equivalents ◽  
Thaw Cycle ◽  
Tnf Α ◽  
Pre Treatment

Cryoinjury of human breast cancer cells (MCF7) in engineered tissue equivalents and the enhancement of the cryoinjury by use of a molecular adjuvant (tumor necrosis factor alpha, TNF-α) was studied. Tissue equivalents (TEs) were constructed by seeding MCF7 cells in collagen solutions at the concentration of 100,000 cells/ml. After cultured in vitro for 2 days, the TEs were exposed with 100ng/ml TNF-α and cultured for 24 hours, and then underwent a single freeze-thaw cycle by a cryosurgery simulator. With the concentration and duration of TNF-α treatment studied, no apoptotic or necrotic cell death was observed by the administration of TNF-α only. After a freeze/thaw, MCF7 cells within the frozen region of the TEs were significantly injured immediately (i.e. ≤ 20% survival), but gradually repopulated and reached approximately 80% survival in Day3 without TNF-α pre-treatment. MCF7 with TNF-α pre-treatment showed the slight enhancement of immediate injury in the frozen region (i.e. ≤ 10% survival), and the repopulation was significantly inhibited so the viability remained below 40% even in Day 3. These results imply that TNF-α can be a potent adjuvant for cryosurgery.

scholarly journals Alternatives to Viral Transport Medium for use in SARS-CoV-2 Sample Preparation

2020 ◽  
Author(s):  
Richard R Chapleau ◽  
Anthony C Fries ◽  
Mark W Lisanby ◽  
Michael G Rhode ◽  
Richard Salisbury ◽  
...  
Keyword(s):  
Repeated Measures ◽  
Limit Of Detection ◽  
Viral Rna ◽  
Published Data ◽  
Clinical Effects ◽  
Freeze Thaw ◽  
Transport Medium ◽  
Thaw Cycle ◽  
Viral Transport Medium ◽  
Freeze Thaw Cycle

Introduction: As the world struggles to manage and move forward from the clinical effects of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the ability to test for viral genomic RNA in patient samples is critical. Currently, the development and performance of SARS-CoV-2 clinical tests is impaired by a diminished supply chain of reagents needed for the tests, compelling labs to seek alternative, readily-available reagents as substitutes. Aim: To evaluate the suitability of Phosphate-Buffered Saline (PBS) and RNAlater™ as substitutes for sample transport media, to preserve the fidelity of viral RNA for use in a SARS-CoV-2 RT-PCR assay. Materials and Methods: This molecular study was conducted in Dayton, Ohio (USA) using synthetic materials and de-identified remnant patient specimens. Simulated standard clinical laboratory storage conditions were used, including prolonged storage up to 72 hours at 2-8°C and a freeze/thaw cycle. PCR amplification performance was measured for PBS and RNAlater™ against transport medium as a reference using purified viral RNA. Performance differences were determined using repeated-measures two-way ANOVA with a 5% false discovery rate. Results: Results indicate that both solutions were suitable for testing viral RNA in the short term, but the viral RNA stored in PBS began to degrade after just 24 hours at 2-8°C. In contrast, RNAlater™ preserved the viral RNA out to 72 hours when stored at 2-8°C, with no statistically significant decrease in the detection limits compared to freshly-prepared viral RNA dilutions. A single freeze/thaw cycle raised the lower limit of detection for RNAlater™-preserved viral RNA slightly. Conclusion: The current (as of April 2020) CDC sample guidelines permit the use of PBS, but have not published data to support this claim. These results offer an alternative to the transport options outlined in many Emergency Use Authorisations (EUA) currently authorised for use in diagnostic testing and may be used for possible long-term storage solutions for studies investigating SARS-CoV-2.

scholarly journals Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Katharina N. Schwaiger ◽  
Monika Cserjan-Puschmann ◽  
Gerald Striedner ◽  
Bernd Nidetzky
Keyword(s):  
Specific Activity ◽  
Dry Mass ◽  
High Yield ◽  
Sucrose Phosphorylase ◽  
Bifidobacterium Adolescentis ◽  
Enzymatic Production ◽  
Intracellular Enzyme ◽  
Whole Cell ◽  
Freeze Thaw ◽  
Thaw Cycle

Abstract Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

scholarly journals Effect of freeze–thaw cycle on physical and mechanical properties and damage characteristics of sandstone

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Longxiao Chen ◽  
Kesheng Li ◽  
Guilei Song ◽  
Deng Zhang ◽  
Chuanxiao Liu
Keyword(s):  
Mechanical Properties ◽  
Shear Failure ◽  
Triaxial Compression ◽  
Physical And Mechanical Properties ◽  
Freeze Thaw ◽  
Damage Characteristics ◽  
Large Pore ◽  
Natural Rock ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle

AbstractRock deterioration under freeze–thaw cycles is a concern for in-service tunnel in cold regions. Previous studies focused on the change of rock mechanical properties under unidirectional stress, but the natural rock mass is under three dimensional stresses. This paper investigates influences of the number of freeze–thaw cycle on sandstone under low confining pressure. Twelve sandstone samples were tested subjected to triaxial compression. Additionally, the damage characteristics of sandstone internal microstructure were obtained by using acoustic emission (AE) and mercury intrusion porosimetry. Results indicated that the mechanical properties of sandstone were significantly reduced by freeze–thaw effect. Sandstone’ peak strength and elastic modulus were 7.28–37.96% and 6.38–40.87% less than for the control, respectively. The proportion of super-large pore and large pore in sandstone increased by 19.53–81.19%. We attributed the reduced sandstone’ mechanical properties to the degenerated sandstone microstructure, which, in turn, was associated with increased sandstone macropores. The macroscopic failure pattern of sandstone changed from splitting failure to shear failure with an increasing of freeze–thaw cycles. Moreover, the activity of AE signal increased at each stage, and the cumulative ringing count also showed upward trend with the increase of freeze–thaw number.

Non-linear creep damage model of sandstone under freeze-thaw cycle

2021 ◽  
Vol 28 (3) ◽  
pp. 954-967
Author(s):  
Jie-lin Li ◽  
Long-yin Zhu ◽  
Ke-ping Zhou ◽  
Hui Chen ◽  
Le Gao ◽  
...  
Keyword(s):  
Damage Model ◽  
Creep Damage ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Linear Creep ◽  
Non Linear ◽  
Freeze Thaw Cycle ◽  
Creep Damage Model

scholarly journals Strength and Microscopic Damage Mechanism of Yellow Sandstone with Holes under Freezing and Thawing

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Huren Rong ◽  
Jingyu Gu ◽  
Miren Rong ◽  
Hong Liu ◽  
Jiayao Zhang ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Pore Size ◽  
Power Function ◽  
Damage Mechanism ◽  
Uniaxial Compression Test ◽  
Freezing And Thawing ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Microscopic Damage

In order to study the damage characteristics of the yellow sandstone containing pores under the freeze-thaw cycle, the uniaxial compression test of saturated water-stained yellow sandstones with different freeze-thaw cycles was carried out by rock servo press, the microstructure was qualitatively analyzed by Zeiss 508 stereo microscope, and the microdamage mechanism was quantitatively studied by using specific surface area and pore size analyzer. The mechanism of weakening mechanical properties of single-hole yellow sandstone was expounded from the perspective of microstructure. The results show the following. (1) The number of freeze-thaw cycles and single-pore diameter have significant effects on the strength and elastic modulus of the yellow sandstone; the more the freeze-thaw cycles and the larger the pore size, the lower the strength of the yellow sandstone. (2) The damage modes of the yellow sandstone containing pores under the freeze-thaw cycle are divided into five types, and the yellow sandstone with pores is divided into two areas: the periphery of the hole and the distance from the hole; as the number of freeze-thaw cycles increases, different regions show different microscopic damage patterns. (3) The damage degree of yellow sandstone is different with freeze-thaw cycle and pore size. Freeze-thaw not only affects the mechanical properties of yellow sandstone but also accelerates the damage process of pores. (4) The damage of the yellow sandstone by freeze-thaw is logarithmic function, and the damage of the yellow sandstone is a power function. The damage equation of the yellow sandstone with pores under the freezing and thawing is a log-power function nonlinear change law and presents a good correlation.

Studies the Properties of Polyaspartic Polyurea Coated Concrete under Coaction of Salt Fog and Freeze-Thaw

2012 ◽  
Vol 455-456 ◽  
pp. 781-785
Author(s):  
Ping Lu ◽  
Xin Mao Li ◽  
Xue Qiang Ma ◽  
Wei Bo Huang
Keyword(s):  
Elastic Modulus ◽  
Frost Resistance ◽  
Dynamic Elastic Modulus ◽  
Freeze Thaw ◽  
Decrease Rate ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Salt Fog

. This paper mainly studied the properties of PAE polyurea coated concrete under coactions of salt fog and freeze-thaw. After exposed salt fog conditions for 200d, T3, B2, F2 and TM four coated concrete relative dynamic elastic modulus have small changes, but different coated concrete variation amplitude is different. T3 coated concrete after 100 times of freeze-thaw cycle the relative dynamic elastic modulus began to drop, 200 times freeze-thaw cycle ends, relative dynamic elastic modulus variation is the largest, decrease rate is 95%, TM concrete during 200 times freeze-thaw cycle, relative dynamic elastic modulus almost no change, B2 concrete and F2 concrete the extent of change between coating T3 and TM. After 300 times the freeze-thaw cycle coated concrete didn't appear freeze-thaw damage phenomenon. Four kinds of coating concrete relative dynamic elastic modulus variation by large to small order: T3 coated concrete > B2 coated concrete >F2 coated concrete > TM coated concrete, concrete with the same 200d rule. Frost resistance order, by contrast, TM coated concrete > B2 coated concrete > F2 coated concrete > T3 coated concrete.

Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress by green tea polyphenol

2005 ◽  
Vol 27 (9) ◽  
pp. 655-660 ◽  
Author(s):  
Dong-Wook Han ◽  
Hak Hee Kim ◽  
Mi Hee Lee ◽  
Hyun Sook Baek ◽  
Kwon-Yong Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Green Tea ◽  
Osteoblastic Cells ◽  
Tea Polyphenol ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Green Tea Polyphenol ◽  
Freeze Thaw Cycle
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