Optimizing Renal Transporter Immunodetection: Consequences of Freeze-Thaw during Sample Preparation.

Renal transporters (co-transporters, channels, claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. While journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Froz) versus freshly-processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of fifteen renal transporters, and the impact of protease inhibitors during homogenization. In female Sprague Dawley rat kidneys homogenized with: aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ~50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmpleteTM Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague Dawley rats, immunoblot signal density was lower in Froz+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Froz vs Fresh by ~25-50% and was greater in Froz+. In contrast, female mice exhibited selective transporter signal degradation in Froz not improved with additional protease inhibitors. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility.

Cyanobacterial Biocrust on Biomineralized Soil Mitigates Freeze-Thaw Effects and Preserves Structure and Ecological Functions.

Biocrust inoculation and microbially induced carbonate precipitation (MICP) are tools used in restoring degraded arid lands. It remains unclear whether the ecological functions of the two tools persist when these methods are combined and subjected to freeze-thaw (FT) cycles. We hypothesized a synergetic interaction between MICP treatment and biocrust under FT cycles, which would allow both components to retain their ecological functions. We grew cyanobacterial (Nostoc commune) biocrusts on bare soil and on MICP (Sporosarcina pasteurii)-treated soil, subjecting them to repeated FT cycles simulating the Mongolian climate. Generalized linear modeling revealed that FT cycling did not affect physical structure or related functions but could increase the productivity and reduce the nutrient condition of the crust. The results confirm the high tolerance of MICP-treated soil and biocrust to FT cycling. MICP treatment + biocrust maintained higher total carbohydrate content under FT stress. Our study indicates that biocrust on biomineralized soil has a robust enough structure to endure FT cycling during spring and autumn and to promote restoration of degraded lands.

Effects of freeze-thaw cycles on soil greenhouse gas emissions: A systematic review.

In the context of global warming, increasingly widespread and frequent freezing and thawing cycles (FTCs) will have profound effects on the biogeochemical cycling of soil carbon and nitrogen. FTCs can increase soil greenhouse gas (GHG) emissions by reducing the stability of soil aggregates, promoting the release of dissolved organic carbon, decreasing the number of microorganisms, inducing cell rupture, and releasing carbon and nitrogen nutrients for use by surviving microorganisms. However, the similarity and disparity of the mechanisms potentially contributing to changes in GHGs have not been systematically evaluated. The present study consolidates the most recent findings on the dynamics of soil carbon and nitrogen, as well as GHGs, in relation to FTCs. Additionally, it analyzes the impact of FTCs on soil GHGs in a systematic manner. In this study, particular emphasis is given to the following: (i) the reaction mechanism involved; (ii) variations in soil composition in different types of land (e.g., forest, peatland, farmland, and grassland); (iii) changes in soil structure in response to cycles of freezing temperatures; (iv) alterations in microbial biomass and community structure that may provide further insight into the fluctuations in GHGs after FTCs. The challenges identified included the extension of laboratory-scale research to ecosystem scales, the performance of in-depth investigation of the coupled effects of carbon, nitrogen, and water in the freeze-thaw process, and analysis of the effects of FTCs through the use of integrated research tools. The results of this study can provide a valuable point of reference for future experimental designs and scientific investigations and can also assist in the analysis of the attributes of GHG emissions from soil and the ecological consequences of the factors that influence these emissions in the context of global permafrost warming.

Feasibility of sludge deep dewaterability improvement for incineration disposal by combined conditioning of freeze-thaw and sawdust.

Sludge incineration is the main strategy for sludge reduction in China. The combined conditioning of lime and chemical agents has been proven to achieve sludge dewatering by disrupting the extracellular polymeric substances (EPS) of sludge and reducing its compressibility. However, when incineration is the intended disposal purpose, this method poses challenges such as incomplete combustion, equipment corrosion, secondary pollution, and decreased calorific value of sludge cake. In contrast, freeze-thaw conditioning, coupled with sawdust as a high-calorific-value bio-waste, emerges as an efficient and clean alternative. The research investigates the synergistic effects of freeze-thaw and sawdust co-conditioning on various sludge properties, including dewaterability, compressibility, consolidation, permeability, microscopicity, and calorific value. The study reveals that the combined conditioning significantly reduces water content and compressibility while increasing void ratio, consolidation, permeability, and enhancing the calorific value of the sludge cake. Specifically, sludge cake conditioned with 60% dried solids (DS) sawdust and freeze-thaw achieved a water content (Wc) of 49.07% and a calorific value of 1422.3 kcal/kg, meeting standards for self-sustained incineration. With heat recovery, the combined conditioning generates an economic revenue of 25.1 $/t DS after deducting costs, thereby reducing the overall cost of sludge reduction treatment. This research offers a clean and practical solution for sludge incineration and reduction, demonstrating great economic value and application potential.

Archaeal communities change responding to anthropogenic and natural treatments of freeze-thawed soils.

The coverage of accumulated snow plays a significant role in inducing changes in both microbial activity and environmental factors within freeze-thaw soil systems. This study aimed to analyze the impact of snow cover on the dynamics of archeal communities in freeze-thaw soil. Furthermore, it seeks to investigate the role of fertilization in freeze-thaw soil. Four treatments were established based on snow cover and fertilization:No snow and no fertilizer (CK-N), snow cover without fertilizer (X-N), fertilizer without snow cover (T-N), and both fertilizer and snow cover (T-X). The research findings indicated that after snow cover treatment, the carbon, nitrogen, and phosphorus content in freeze-thaw soil exhibit periodic fluctuations. Snow covered effectively altered the community composition of bacteria and archaea in the soil, with a greater impact on archaeal communities than on bacterial communities. Snow covered improves the stability of archaeal communities in freeze-thaw soil. Additionally, the arrival of snow also enhanced the correlation between archaea and environmental factors, with the key archaeal phyla involved being Nanoarchaeota and Crenarchaeota. Further research showed that the application of organic fertilizers also had some impact on freeze-thaw soil, but this impact was smaller compared to snow cover. In summary, the arrival of snow could alter the archaeal community and protect nutrient elements in freeze-thaw soil, reducing their loss, and its effect is more pronounced compared to the application of organic fertilizers.

Optimizing Phycocyanin Extraction from Cyanobacterial Biomass: A Comparative Study of Freeze-Thaw Cycling with Various Solvents.

Cyanobacterial phycocyanin pigment is widely utilized for its properties in various industries, including food, cosmetics, and pharmaceuticals. Despite its potential, challenges exist, such as extraction methods impacting yield, stability, and purity. This study investigates the impact of the number of freeze-thaw (FT) cycles on the extraction of phycocyanin from the wet biomass of four cyanobacteria species (Arthrospira platensis, Chlorogloeopsis fritschii, Phormidium sp., and Synechocystis sp.), along with the impact of five extraction solutions (Tris-HCl buffer, phosphate buffer, CaCl2, deionized water, and tap water) at various pH values. Synechocystis sp. exhibited the highest phycocyanin content among the studied species. For A. platensis, Tris-HCl buffer yielded maximum phycocyanin concentration from the first FT cycle, while phosphate buffer provided satisfactory results from the second cycle. Similarly, Tris-HCl buffer showed promising results for C. fritschii (68.5% of the maximum from the first cycle), with the highest concentration (~12% w/w) achieved during the seventh cycle, using phosphate buffer. Phormidium sp. yielded the maximum pigment concentration from the first cycle using tap water. Among species-specific optimal extraction solutions, Tris-HCl buffer demonstrated sufficient extraction efficacy for all species, from the first cycle. This study represents an initial step toward establishing a universal extraction method for phycocyanin from diverse cyanobacteria species.

Effects of freeze-thaw leaching on physicochemical properties and cadmium transformation in cadmium contaminated soil.

This study aims to investigate the effect of the combined method of freeze-thaw and leaching on the removal of cadmium (Cd) in soil and to provide a theoretical basis for the remediation of farmland soil polluted by heavy metals. The removal process and mechanism of Cd were deduced through oscillatory leaching experiments and freeze-thaw leaching simulation experiments, and the influence of the freeze-thaw leaching technology on the soil environment was evaluated. The results of oscillatory leaching showed that a mixture consisting of 0.80 mol/L citric acid and 0.80 mol/L ferric chloride in a 1:19 vol ratio effectively remove 47.75 % of Cd, indicating that the composite leaching agent could effectively remove Cd from the soil. The results of the freeze-thaw leaching simulation experiment showed that although the freeze-thaw leaching treatment increased the total Cd content in the 0-5 cm soil layer, the total Cd content in the 5-10 cm, 10-15 cm, and 15-20 cm soil layers decreased by 5.08 %, 2.39 %, and 5.68 %, respectively. The freeze-thaw leaching increased the content of exchangeable Cd (p<0.05), carbonate bound Cd, but decreased organic bound Cd and residual Cd (p<0.05), thereby increasing the bioavailability of Cd. Freeze-thaw leaching not only increased the competitive adsorption of Cd2+ by decreasing soil pH, cation exchange capacity, and increasing the content of exchangeable calcium and exchangeable magnesium, thus reducing the adsorption of Cd in soil. And the results of XPS and FTIR similarly showed that the freeze-thaw leaching could promote the chelation between Cd2+ and hydroxyl, carboxyl and carbonyl functional groups. Although the freeze-thaw leaching destroyed the large particle structure (0.05-2 mm) and large pores in the soil, and increased the clay content (<0.002 mm) and the proportion of small pores in the soil, the XRD results showed that freeze-thaw leaching had no significant effect on the minerals in the soil. In summary, this study shows that freeze-thaw leaching has a significant effect on the removal of soil heavy metals, suggesting that the synergistic effect of freeze-thaw and leaching should be considered in the process of removing soil pollutants in seasonal freeze-thaw zones, and that this method provides a new insight into the remediation of contaminated soils.

Nitrogen removal performance of bioretention cells under freeze-thaw cycles: Effects of filler structure and microbial community.

Cold climates have an adverse effect on the nitrogen-removal capacity of bioretention cells, especially during freeze-thaw cycles (FTCs). To explore the effects of FTCs on the nitrogen removal performance of bioretention cells, this research compared the effects of FTCs on the pore structure and microbial community composition of the filler, and analyzed the nitrogen removal performance of the bioretention cell before (RT), during (FTC) and after (RRT) FTCs. The results demonstrated that RRT filler had a much greater number of pores with equivalent diameter <500 µm than RT filler, and that RRT had a higher pore volume and pore density than RT. Microbial community analysis revealed that the diversity and richness of the microbial community in FTC were lower than in RT, and the relative abundance of Lacunisphaera, Pseudomonas, and Dokdonella decreased significantly. There was no significant difference in microbial community richness between RRT and RT, however RRT diversity was lower. RRT has a higher relative abundance of nitrifying bacteria (Subgroup_10, Bryobacter, etc.) than RT, but a lower relative abundance of denitrifying bacteria (Pseudomonas, Dokdonella, Arenimonas, etc.). The nitrogen removal efficiency of FTC was inhibited, resulting in a decrease of 13.0 ± 4.86%, 19.7 ± 9.17%, and 26.6 ± 1.74% in the removal rates of ammonia nitrogen(NH4+-N), nitrate nitrogen(NO3--N), and total nitrogen(TN) when compared to RT, respectively. RRT improved nitrification and increased NH4+-N removal rate by 10.3 ± 2.69% compared to RT. However, because of denitrification inhibition, the nitrogen removal performance of RRT was not able to reach RT levels, and its NO3--N and TN removal rates decreased by 100 ± 4.70% and 58.3 ± 3.71%, respectively. This study has demonstrated that FTCs can permanently harm the bioretention cell's filler structure and microbial community, resulting in a significant decrease in the nitrogen removal performance of the bioretention cell designed according to warm climate conditions after experiencing FTCs.

Effect of eco-friendly pervious concrete pavement with travertine waste and sand on the heavy metal removal and runoff reduction performance.

To address the negative environmental and economic impact of the large amounts of solid waste generated during travertine mining and to reduce the dependence on natural aggregates and cement for pervious concrete pavement applications, travertine waste, as aggregate and powder, was used for the travertine powder pervious concrete (TPPC) to improve the utilization of solid waste and decrease CO2 emissions. The experimental results showed that using 25% travertine aggregate and 5% powder results in a compressive strength reduction of only 9.8% to 25.92 MPa but a significant improvement in water permeability of 57.1% from 3.89 to 6.11 mm/s. To improve the performance of TPPC, further research was done on the effect of sand addition rate (SAR) on TPPC's density, compressive strength, porosity, water permeability, freeze-thaw resistance and heavy metal removal capacity to obtain an optimal incorporation ratio. As SAR rises, the compressive strength of TPPC with sand (STPC) initially increases and then decreases, while permeability behaves inversely. At 3% SAR, the compressive strength reached a maximum of 26.51 MPa, primarily due to the sand added to fill in some of the pores and stabilize the gradation. After 25 cycles, the strength loss rate of STPC varies from 11.39 to 17.93% and the freeze-thaw resistance is most excellent when SAR is 3%. The removal rate of heavy metals using the immersion method was found to be significantly higher (83.4-100%) compared to the rapid method (11.7-28.1%). Therefore, the 3% SAR was recommended for the mixture design of STPC. A laboratory-scale version of the pavement was constructed to assess the efficacy of STPC pavement (STPCP) in reducing runoff and removing heavy metals. The results showed that STPCP could remove more than 94% of runoff with varying intensities after 1 h. The STPCP exhibited removal rates ranging from 42.0 to 99.4% for Cd2+ and 79.5-95.4% for Cu2+. STPCP also attained a removal rate above 98% for Pb2+ after 30 min, demonstrating its environmental friendliness.

Varieties of P fractions in biochar-amended reconstructed soils as impacted by freeze-thaw interference.

With low cost and stable chemical properties, biochar has great potential in environmental pollution control and improving soil quality. Reusing tailings slag to reconstruct soil ecosystems and applying amendments such as biochar to enhance soil quality are significant for restoring waste mine lands. Phosphorus (P) as the restrictive nutrition element for plant growth is easily affected by freeze-thaw cycles (FTCs). However, effective information about FTCs on P dynamics in biochar-amended reconstructed soil is scanty. To further understand the effect of FTCs on P in reclaimed mine soils, three reconstructed soils composed of equal brown soil and tailings slag with the respective application of no amendment, 5% biochar and 5% powder both derived from Gleditsia japonica shells (GS), were prepared to evaluate P fraction changes after FTCs. The results indicated that GS biochar increased soil pH, total organic matter (TOM), and moisture content (MC). GS biomass had a similar impact on TOM and MC but decreased soil pH. The two agricultural amendments increased active P and microbial biomass P (MBP) by 46.13%-101.63% and 162.8%-185.7%, which might be largely contributed by soil organic matter and moisture. FTC numbers (0, 3, 6, 10, 15) significantly decreased MBP contents and slightly converted non-labile P into labile fractions while FTC temperature (-20∼5 °C and -10∼5 °C) hardly influenced soil P behavior. In addition, GS conditioners simultaneously enhanced available P content and P fixation potential by soil under FTCs.

Spring applied phosphorus loss with cover crops in no-till terraced field.

Cover crops (CC) can improve phosphorus (P) cycling by reducing water related P losses and contributing to P nutrition of a rotational crop. This is particularly important in claypan soils with freeze-thaw cycles in early spring in the Midwest U.S. This 4-year study (2019-2022) examined the impact of CC monoculture and mix of CC species on P losses from a fertilizer application, and determined the P balance in soil compared to no cover crop (noCC). The CC mix consisted of wheat (Triticum aestivum L.), radish (Raphanus raphanistrum subsp. Sativus), and turnip (Brassica rapa subsp. Rapa) (3xCCmix) in 2019 and 2021 before corn, and cereal rye (Secale cereale L.) was planted as monoculture before soybean in 2020 and 2022. The 3xCCmix had no effect on total phosphorus (TP) and dissolved reactive phosphorus (PO4-P) concentration or load in 2019 and 2021. Cereal rye reduced TP and PO4-P load 70% and 73%, respectively, compared to noCC. The variation in soil moisture, temperature, and net precipitation from fertilizer application until CC termination affected available soil P pools due to variability in CC species P uptake, residue decomposition, and P loss in surface water runoff. Overall, the P budget calculations showed cereal rye had 2.4 kg ha-1 greater P uptake compared to the 3xCCmix species which also reduced P loss in water and had greater differences in soil P status compared to noCC. This study highlights the benefit of CCs in reducing P loss in surface runoff and immobilizing P through plant uptake. However, these effects were minimal with 3xCCmix species and variability in crop residue decomposition from different CC species could affect overall P-soil balance.

Impact of freeze-thaw cycles on greenhouse gas emissions in marginally productive agricultural land under different perennial bioenergy crops.

Knowledge of freeze-thaw-induced carbon (C) and nitrogen (N) cycling and concomitant nitrous oxide (N2O) and carbon dioxide (CO2) emissions in perennial bioenergy crops is crucial to understanding the contribution of these crops in mitigating climate change through reduced greenhouse gas (GHG) emissions. In this study, a 49-day laboratory incubation experiment was conducted to compare the impact of freeze-thaw cycles on N2O and CO2 emissions in different perennial bioenergy crops [miscanthus (Miscanthus giganteus L.), switchgrass (Panicum virgatum L.), and willow (Salix miyabeana L.)] to a successional site and to understand the processes controlling the N2O and CO2 emissions in these crops. The results showed that freeze-thaw cycles caused a decline in dissolved organic C (DOC) and dissolved inorganic N (DIN) concentrations but enhanced the dissolved organic N (DON) and nitrate (NO3-). Although, freeze-thaw decreased water stable soil aggregates in all the bioenergy crops and successional site, this did not have any significant impact on N2O and CO2 emissions, suggesting that the N2O and CO2 emitted during the freeze-thaw cycles may have originated mostly from cellular materials released from lysis and death of microbial biomass rather than from soil aggregate disruption. Cumulative N2O emissions measured over the 49-day incubation period ranged from 148 mg N2O-N m-2 to 17 mg N2O-N m-2 and were highest in miscanthus followed by willow, switchgrass, and successional site. Cumulative CO2 on the other hand was highest in the successional site than any of the bioenergy crops and ranged from 25,262 mg CO2-C m-2 to 15,403 mg CO2-C m-2 after the 49 days. Higher N2O emissions in the miscanthus and willow than switchgrass and successional site were attributed to accelerated N losses as N2O. Results from our study indicate that managing perennial bioenergy crops on low productive agricultural lands to reduce freeze-thaw related GHG emissions and climate change mitigation is dependent on the crop species grown.

Freeze-thaw stability of high-internal-phase emulsion stabilized by chickpea protein microgel particles and its application in surimi.

BACKGROUND: Future applications of high-internal-phase emulsions (HIPEs) are highly regarded, but poor freeze-thaw stability limits their utilization in frozen products. This study aimed to characterize the structure of chickpea protein microgel particles (HCPI) induced by NaCl and to assess its impact on the freeze-thaw stability of HIPEs. RESULTS: The results showed that NaCl induction (0-400 mmol L-1) increased the surface hydrophobicity (175.9-278.9) and interfacial adsorbed protein content (84.9%-91.3%) of HCPI. HIPEs prepared with HCPI induced by high concentration of NaCl exhibited superior flocculation index and centrifugal stability, and their freeze-thaw stability was better than that of natural chickpea protein. The increase in NaCl concentration reduced the droplet aggregation and coalescence index of the freeze-thaw emulsions, diminishing the precipitation of oil from the emulsion. Linear and nonlinear rheology showed that the strengthened gel structure (higher G' values) restricted water flow and counteracted the damage to the interfacial film by ice crystals at 100-400 mmol L-1 NaCl, thus improving the viscoelasticity of the freeze-thaw emulsions. Finally, the thawing loss of surimi gel with HCPI-200 HIPE was reduced by 2.04% compared to directly adding oil. CONCLUSION: This study provided a promising strategy to improve the freeze-thaw stability of HIPEs and reduce the thawing loss of frozen products. © 2024 Society of Chemical Industry.

Enhanced gelling property and freeze-thaw stability of potato, tapioca and corn starches modified by mild heating in aqueous ethanol solution.

BACKGROUND: Physically modified starches can be classified as natural ingredients on food labels and clean label products. Thus, the market demand for physically modified starch is increasing. Potato, tapioca and corn starches were physically modified by mild heat treatment in an alcoholic solution to enhance their gelling property and freeze-thaw stability. RESULTS: During mild heating of starch suspension (40% w/w) in 10% ethanol solution at the onset gelatinization temperature, granular swelling of starch occurred, followed by amylose leaching with medication of the surface structure of the starch granules. All treated starches exhibited increased gelatinization and pasting temperatures and decreased breakdown for pasting as a result of improved stability against shear and heat. The treated starches had higher hardness, cohesiveness and springiness of gel than the respective native starches, and these gel properties were more pronounced in potato starch than in tapioca and corn starches. The treated starches showed substantially reduced gel syneresis during freeze-thawing. CONCLUSION: Physical modification of starch by mild heat treatment in an alcoholic solution substantially improved its gelation ability and freeze-thaw stability. © 2024 Society of Chemical Industry.

Non-destructive prediction of TVB-N using color-texture features of UV-induced fluorescence image for freeze-thaw treated frozen-whole-round tilapia.

BACKGROUND: The investigation of UV-induced fluorescence imaging coupled with machine learning was conducted to non-destructively detect the total volatile basic nitrogen (TVB-N) of frozen-whole-round tilapia (FWRT) during freezing and thawing. The UV-induced fluorescence images of FWRT at the wavelength of 365 nm were acquired by self-developed fluorescence image acquisition system. In total, 169 color and texture features based on RGB, hue-saturation-intensity and L*a*b* color spaces and gray level co-occurrence matrix were extracted, respectively. Successive projections algorithm (SPA) was employed to select the optimal 16 features to achieve feature dimension reduction modeling. With full and extracted features as input, the models of partial least squares regression (PLSR), least-squares support vector machine (LSSVM) and convolutional neural network (CNN) were established for TVB-N prediction. RESULTS: Results indicated that the full features-based CNN performed better than SPA based prediction models (SPA-PLSR and SPA-LSSVM). The CNN model was determined to be the optimal with an RP2 value of 0.9779, RMSEP value of 1.1502 × 10-2 g N kg-1 and RPD value of 6.721 for TVB-N content predictiin. CONCLUSION: The CNN method based on UV fluorescence imaging technology has potential for quality and safety detection of FWRT. © 2023 Society of Chemical Industry.

Effects of pH and salt concentration on freeze-thaw fractionation of soymilk protein.

BACKGROUND: The two major storage proteins of soymilk are the globulins 7S and 11S. Freeze-thaw fractionation is a simple method for separating these proteins in raw soymilk. In this study, we assessed the freeze-thaw fractionation ability of raw soymilk under various pH (4.3-11.6) conditions and added salt (sodium chloride) concentrations (0.00-0.67 mol L-1). RESULTS: We successfully achieved fractionation within a pH range of 5.8-6.7 and when the salt concentration was 0.22 mol L-1 or lower. Analysis of particle size distribution and microscopic examination of soymilk revealed no direct correlation between particle size and freeze-thaw fractionation ability. Interestingly, it was confirmed that the ranges of zeta potential values associated with successful freeze-thaw fractionation in raw soymilk remained consistent across different pH and salt concentration conditions. These ranges were between -23 and -28 mV at pH levels ranging from 5.8 to 6.7 and between -18 and -29 mV at added salt concentrations ranging from 0 to 0.22 mol L-1. CONCLUSION: The pH and salt concentration in raw soymilk markedly influence the freeze-thaw fractionation process. We confirmed that the range of zeta potential values where fractionation was possible remained consistent under various pH and salt concentration conditions. These findings suggest that the zeta potential value might serve as an indicator for evaluating the freeze-thaw fractionation ability of raw soymilk. © 2024 Society of Chemical Industry.

Human plasma derived exosomes: Impact of active and passive drug loading approaches on drug delivery.

The aim of the current study was to explore the potential of human plasma-derived exosomes as versatile carriers for drug delivery by employing various active and passive loading methods. Exosomes were isolated from human plasma using differential centrifugation and ultrafiltration method. Drug loading was achieved by employing sonication and freeze thaw methods, facilitating effective drug encapsulation within exosomes for delivery. Each approach was examined for its effectiveness, loading efficiency and ability to preserve membrane stability. Methotrexate (MTX), a weak acid model drug was loaded at a concentration of 2.2 µM to exosomes underwent characterization using various techniques such as particle size analysis, transmission electron microscopy and drug loading capacity. Human plasma derived exosomes showed a mean size of 162.15 ± 28.21 nm and zeta potential of -30.6 ± 0.71 mV. These exosomes were successfully loaded with MTX demonstrated a better drug encapsulation of 64.538 ± 1.54 % by freeze thaw method in comparison 55.515 ± 1.907 % by sonication. In-vitro drug release displayed 60 % loaded drug released within 72 h by freeze thaw method that was significantly different from that by sonication method i.e., 99 % within 72 h (p value 0.0045). Moreover, cell viability of exosomes loaded by freeze thaw method was significantly higher than that by sonication method (p value 0.0091) suggested that there was membrane disruption by sonication method. In conclusion, this study offers valuable insights into the potential of human plasma-derived exosomes loaded by freeze thaw method suggest as a promising carrier for improved drug loading and maintenance of exosomal membrane integrity.

Transcriptome analysis of Populus × canadensis 'Zhongliao1' in response to low temperature stress.

BACKGROUND: Low temperatures are known to limit the growth and geographical distribution of poplars. Although some transcriptomic studies have been conducted to explore the response of poplar leaves to cold stress, only a few have comprehensively analyzed the effects of low temperature on the transcriptome of poplars and identified genes related to cold stress response and repair of freeze-thaw injury. RESULTS: We exposed the Euramerican poplar Zhongliao1 to low temperatures; after stems were exposed to - 40℃, 4℃, and 20℃, the mixture of phloem and cambium was collected for transcriptome sequencing and bioinformatics analysis. A total of 29,060 genes were detected, including 28,739 known genes and 321 novel genes. Several differentially expressed genes (n = 36) were found to be involved in the Ca2+ signaling pathway, starch-sucrose metabolism pathway, abscisic acid signaling pathway, and DNA repair. They were functionally annotated; glucan endo-1,3-beta-glucosidase and UDP-glucuronosyltransferase genes, for instance, showed a close relationship with cold resistance. The expression of 11 differentially expressed genes was verified by qRT-PCR; RNA-Seq and qRT-PCR data were found to be consistent, which validated the robustness of our RNA-Seq findings. Finally, multiple sequence alignment and evolutionary analysis were performed, the results of which suggested a close association between several novel genes and cold resistance in Zhongliao1. CONCLUSION: We believe that the cold resistance and freeze-thaw injury repair genes identified in this study are of great significance for cold tolerance breeding.

Repeated freeze-thaw cycles in freeze-tolerant treefrogs: novel interindividual variation of integrative biochemical, cellular, and organismal responses.

The freeze-tolerant anuran Dryophytes chrysoscelis, Cope's gray treefrog, mobilizes a complex cryoprotectant system that includes glycerol, glucose, and urea to minimize damage induced by freezing and thawing of up to 65% of body water. In this species' eastern Northern American temperate habitat, oscillations of temperature above and below freezing are common; however, the effects of repeated freezing and thawing in this species are unstudied. The biochemical and physiological effects of repeated freeze-thaw cycles were therefore evaluated and compared with cold acclimation and single freeze-thaw episodes. Glycerol was elevated in plasma, liver, and skeletal muscle of both singly and repeatedly frozen and thawed animals compared with cold-acclimated frogs. In contrast, urea was unchanged by freezing and thawing, whereas glucose was elevated in singly frozen and thawed animals but was reduced toward cold acclimation levels after repeated bouts of freezing. Overall, the cryoprotectant system was maintained, but not further elevated, in all tissues assayed in repeatedly frozen and thawed animals. For repeated freeze-thaw only, hepatic glycogen was depleted and plasma hemoglobin, indicative of erythrocyte hemolysis, increased. Postfreeze recovery of locomotor function, including limb and whole body movement, was delayed with repeated freeze-thaw and was associated with glycerol accumulation and glycogen depletion. Individuals that resumed locomotor function more quickly also accumulated greater cryoinjury. Integrated analyses of cryoprotectant and cryoinjury accumulation suggest that winter survival of D. chrysoscelis may be vulnerable to climate change, limited by carbohydrate stores, cellular repair mechanisms, and plasticity of the cryoprotectant system.

Snow, fire and drought: How alpine and treeline soil seed banks are affected by simulated climate change.

BACKGROUND AND AIMS: Seed persistence in soil depends on environmental factors that affect seed dormancy and germination, such as temperature and water availability. In high-elevation ecosystems, rapid changes in these environmental factors due to climate change can impact future plant recruitment. To date, our knowledge on how soil seed banks from high-altitude environments will respond to climate change and extreme climate-related events is limited. Here, using the seedling emergence method, we investigated the effects of reduced snow cover, fire and drought on the density and diversity of germinants from soil seed banks of two high-altitude plant communities: a tall alpine herbfield and a treeline ecotone. METHODS: In Autumn 2020, we collected soil samples and characterized the standing vegetation of both communities at Kosciuszko National Park, Australia. Then, we employed a factorial experiment and subjected the soil samples to a series of manipulative treatments using greenhouse studies. KEY RESULTS: The treeline had a larger and more diverse soil seed bank than the herbfield. A reduction in snow had a negative effect on the number of germinants in the herbfield and increased the dissimilarity with the standing vegetation, while the treeline responses were mainly neutral. Fire did not significantly affect the number of germinants but decreased the evenness values in both communities. The drought treatment reduced the number and richness of germinants and increased the dissimilarity with the standing vegetation in both communities. Plant functional forms explained some of the detected effects but seed functional traits did not. CONCLUSIONS: Our study suggests that simulated climate change will affect plant recruitment from soil seed banks in a variety of ways. Changes in snow cover, incidences of fire and drought may be key drivers of germination from the soil seed bank and therefore the future composition of alpine plant communities.