High throughput method for simultaneous screening of membrane permeability and toxicity for discovery of new cryoprotective agents.

Vitrification is the most promising method for cryopreservation of complex structures such as organs and tissue constructs. However, this method requires multimolar concentrations of cell-permeant cryoprotective agents (CPAs), which can be toxic at such elevated levels. The selection of CPAs for organ vitrification has been limited to a few chemicals; however, there are numerous chemicals with properties similar to commonly used CPAs. In this study, we developed a high-throughput method that significantly increases the speed of cell membrane permeability measurement, enabling ~100 times faster permeability measurement than previous methods. The method also allows assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 °C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation.

Impact of cryoprotectant-free sperm vitrification in pulled-glass capillary on sperm parameters and DNA integrity: A lab trial study.

Background: Vitrification is a recently introduced yet widely applied assisted reproduction technique. So far, the effects of the chemicals and devices in vitrification on sperm motility and DNA integrity are still unclear. Objective: This study aimed to examine sperm quality, as determined by semen analysis and sperm DNA integrity when vitrified with or without cryoprotectant agents (CPAs) using pulled-glass capillaries. Materials and Methods: Between February and June 2020, 50 infertile men from the Hue Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy, Vietnam, were enrolled. Sperm samples, prepared using the swim-up technique, were divided into 2 groups: vitrification with CPAs (group 1) and without CPAs (group 2). Vitrified sperm samples were preserved in 10 µL pulled-glass capillaries. Motility, sperm membrane integrity, and the DNA fragmentation index were tested. Results: Sperm motility in vitrified media with CPAs (54.4 ± 11%) was statistically higher than in media without CPAs (51.14 ± 10.6%, p < 0.05). CPAs did not affect sperm membrane integrity or large halo ratio (71.34 ± 8.47 vs. 70.38 ± 8.11 and 50.84 ± 18.92 vs. 51.98 ± 19.44, respectively). Group 2 exhibited a lower DNA fragmentation index than group 1 after vitrification (14.2 ± 8.47 vs. 12.60 ± 9.03, p = 0.021). Conclusion: Using a pulled-glass capillary for sperm vitrification, the presence of CPAs in the vitrification medium resulted in higher progressive motility and lower DNA fragmentation index than the medium without CPAs.

Basic study on cryopreservation of rat calvarial osteoblasts with different cryoprotectants.

Cryopreservation is a method adopted for storage of autologous skulls. Herein, this current research sought to explore the effects of different cryoprotectants on the biological characteristics of rat calvarial osteoblasts after cryopreservation. Neonatal Sprague-Dawley rats were selected and their skull tissues were isolated. The skull tissues were allocated into the refrigerating-3M, refrigerating-6M, M199-3M, M199-6M, povidone iodine-3M, and povidone iodine-6M groups according to the usage of cryoprotectants and treatment time (month) and the fresh group. Osteoblasts were isolated from skull tissues in each group through digestion. The histomorphology of the skull was evaluated by H&E staining and cell morphology was observed by microscopy. The viability, proliferation, apoptosis, and osteogenic activity of osteoblasts were assessed by trypan blue staining, MTT, flow cytometry, and alkaline phosphatase (ALP) staining. The skull histomorphology and osteoblast morphology were similar between the fresh and refrigerating groups. Osteoblast viability was weakened after cryopreservation. The longer the refrigeration time, the lower the number of living cells and the higher the apoptosis rate. However, cryopreservation using different cryoprotectants did not evidently affect osteoblast proliferation and ALP activity. Different cryoprotectants show no apparent effect on the osteogenic activity of rat calvarial osteoblasts after cryopreservation.

Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study.

BACKGROUND AND AIM: An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. METHODS: A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5-10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. RESULTS: MSCs had an average viability of 94.3% (95% CI: 87.2-100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03-9.0%; P: 0.049) and 11.4% (95% CI: 6.9-15.8%; P< 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7-100.0%), and it was lower by 5.6% (95% CI: 1.3-9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. CONCLUSION: MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.

L-Proline as a Cryoprotective Agent for the Preservation of Galea Spixii Skin Fibroblasts.

Somatic cell biobanking is a promising strategy for developing reproductive techniques. Although cryopreservation, a technique used for creating biobanks, has been performed on Galea spixii, structural and physiological damage to its cells highlight the need to optimize the cryoprotective solution being used. Therefore, the osmoprotective activity of 5 mM L-proline was evaluated as an alternative cryoprotectant for G. spixii fibroblast conservation. The concentration was defined based on previous studies conducted on mammalian cells. Cells derived from the skin of six individuals were cultured until the fifth passage were cryopreserved under the following treatments: (i) control (non-cryopreserved); (ii) a solution with 10% dimethyl sulfoxide (Me2SO), 10% fetal bovine serum (FBS), and 0.2 M sucrose; (iii) a solution with 10% Me2SO, 10% FBS, and 5 mM L-proline; and (iv) a solution with 10% Me2SO, 10% FBS, 0.2 M sucrose, and 5 mM L-proline. Tests were conducted to analyze cell morphology, viability, metabolism, proliferation, and apoptosis; reactive oxygen species (ROS) levels; and mitochondrial membrane activity (ΔΨm). A reduction in the number of viable cells (72.3% ± 1.2%) was observed in the sucrose-containing group compared to the control (86.7% ± 2.0%) and L-proline (88.4% ± 1.8% and 87.8% ± 2.1%) groups. After apoptotic analysis, a reduction in the number of viable cells was observed in the group with sucrose alone (74.6% ± 4.1%) compared to the control group (88.2% ± 1.1%). The ROS levels (1.03 ± 0.5 and 1.07 ± 0.5, respectively) and ΔΨm values (0.99 ± 0.42 and 1.22 ± 0.73, respectively) observed in the groups with L-proline were similar to that observed in the control group (1.00 ± 0.5 and 1.00 ± 0.4, respectively). Moreover, no difference was observed between groups for cell morphology, metabolism, or proliferation. Thus, L-proline is a cryoprotectant agent that can be used during G. spixii fibroblast cryopreservation, alone or with sucrose. In addition, we developed an adequate biobank for G. spixii, whereby stored cells could be used for reproductive techniques.

Enzymatic production of xylooligosaccharide from lignocellulosic and marine biomass: A review of current progress, challenges, and its applications in food sectors.

Over the last decade, xylooligosaccharides (XOS) have attracted great attentions because of their unique chemical properties and excellent prebiotic effects. Among the current strategies for XOS production, enzymatic hydrolysis is preferred due to its green and safe process, simplicity in equipment, and high control of the degrees of polymerization. This paper comprehensively summarizes various lignocellulosic biomass and marine biomass employed in enzymatic production of XOS. The importance and advantages of enzyme immobilization in XOS production are also discussed. Many novel immobilization techniques for xylanase are presented. In addition, bioinformatics techniques for the mining and designing of new xylanase are also described. Moreover, XOS has exhibited great potential applications in the food industry as diverse roles, such as a sugar replacer, a fat replacer, and cryoprotectant. This review systematically summarizes the current research progress on the applications of XOS in food sectors, including beverages, bakery products, dairy products, meat products, aquatic products, food packaging film, wall materials, and others. It is anticipated that this paper will act as a reference for the further development and application of XOS in food sectors and other fields.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

BACKGROUND: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. RESULTS: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. CONCLUSIONS: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

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

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

Primordial germ cells of Astyanax altiparanae, isolated and recovered intact after vitrification: A preliminary study for potential cryopreservation of Neotropical fish germplasm.

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

24 Factorial Design Formulation Optimization and In vitro Characterization of Desloratadine Nanosuspension Prepared Using Antisolvent Precipitation.

INTRODUCTION: Desloratadine, a second-generation antihistaminic drug, is poorly watersoluble and requires amelioration of the dissolution rate to improve its pharmacokinetics properties. METHOD: This study investigated the impact of polymer, surfactant types, and concentration on the particle size, zeta potential, and dissolution efficiency of nanosuspensions formulated through the solvent antisolvent precipitation method. To optimize the delivery of Desloratadine nanosuspension, we used Minitab software and a 4-factor, 2-level full factorial design. Physicochemical properties and drug release studies were conducted to evaluate the suggested nanosuspension formulations. The optimization goals included minimizing particle size and zeta potential while maximizing dissolution efficiencies. RESULT: The selected optimal nanosuspension demonstrated favourable values, including a particle size of 478.63 ± 15.67 nm, a zeta potential of -36.24 ± 3.21 mV, and dissolution efficiencies in double distilled water and buffer of 90.29 ± 3.75 % and 93.70 ± 3.67 %, respectively. The optimized formulation was subjected to additional analysis using X-ray powder diffraction (XPRD), scanning and transmission electron microscopy (SEM and TEM), and Fourier-transform infrared spectroscopy (FTIR). CONCLUSION: The optimized nanosuspension formulation also underwent further studies under optimal lyophilization conditions, revealing the effectiveness of mannitol as a cryoprotectant at a concentration of 8%.

Thawed drip and its membrane-separated components: Role in retarding myofibrillar protein gel deterioration during freezing-thawing cycles.

Myofibrillar proteins are crucial for gel formation in processed meat products such as sausages and meat patties. Freeze-thaw cycles can alter protein properties, impacting gel stability and product quality. This study aims to investigate the potential of thawed drip and its membrane-separated components as potential antifreeze agents to retard denaturation, oxidation and gel deterioration of myofibrillar proteins during freezing-thawing cycles of pork patties. The thawed drip and its membrane-separated components of > 10 kDa and < 10 kDa, along with deionized water, were added to minced pork at 10 % mass fraction and subjected to increasing freeze-thaw cycles. Results showed that the addition of thawed drip and its membrane separation components inhibited denaturation and structural changes of myofibrillar proteins, evidenced by reduced surface hydrophobicity and carbonyl content, increased free sulfhydryl groups, protein solubility and α-helix, as compared to the deionized water group. Correspondingly, improved gel properties including water-holding capacity, textural parameters and denser network structure were observed with the addition of thawed drip and its membrane separation components. Denaturation and oxidation of myofibrillar proteins were positively correlated with gel deterioration during freezing-thawing cycles. We here propose a role of thawed drip and its membrane separation components as cryoprotectants against myofibrillar protein gel deterioration during freeze-thawing cycles.

Effects of chilling and cryoprotectants on glycans in shrimp embryos.

Glycans are carbohydrates present in every organism that bind to specific molecules such as lectins, a diverse group of proteins. Glycans are vital to cell proliferation and protein trafficking. In addition, embryogenesis is a critical phase in the development of marine organisms. This study investigated the effects of chilling and cryoprotective agents (CPAs) on glycans in the embryos of Stenopus hispidus. The glycan profiles of embryos of S. hispidus at the heartbeat stage were analyzed using lectin arrays. The results of analyses revealed that mannose was the most abundant glycan in the S. hispidus embryos; mannose is crucial to cell proliferation, providing the energy required for embryonic growth. Additionally, the results reveled that chilling altered the content of several glycans, including fucose and Gla-GlcNAc. Chilling may promote monosaccharide accumulation, facilitating osmotic regulation of cells and signal molecules to aid S. hispidus embryos in adapting to cold conditions. Changes were also observed in the lectins NPA, orysata, PALa, ASA, discoidin II, discoidin I, UDA, PA-IIL, and PHA-P after the samples were treated with different CPAs. DMSO may minimize cell damage during exposure to chilling by preserving cell structures, membrane properties, and functions. The present study is the first to investigate the profiles and functions of glycans in shrimp embryos subjected to low-temperature injuries. This study enhances the understanding of cell reproduction during embryogenesis and provides valuable information for the study of glycans in embryos.

Influence of different penetrating cryoprotectants and egg yolk level on the post-thaw quality of sperm.

Objective: The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. Methods: In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), wobble movement coefficient (WOB), average motion degree (MAD), functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. Results: When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p < 0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p < 0.05) decreased the ROS level of sperm. In this study, the TM, PM and membrane integrity of the 6% EG were significantly (p < 0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p < 0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. Conclusion: In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

Cryoprotective role of vacuum infused inulin on the quality of artichoke: Interactive effects of freezing, thawing and storage period.

Freezing of artichoke is a promising alternative to storing it in brine and canning. The perishable vegetable was vacuum infused with inulin to improve freezing tolerance. Artichokes with and without inulin were frozen by static, air blast and individual quick freezing (IQF) methods and thawed by microwave, 25 °C and 4 °C temperature levels at each month of 6-months storage. Process conditions were evaluated by multivariate analysis of variance (MANOVA) and were found significant on the quality parameters. Inulin infusion better conserved the aw, color, texture, ascorbic acid and overall integrity of artichokes during frozen storage. Inulin incorporation and IQF showed mutual positive effect on drip loss. Polyphenol oxidase (PPO) activity values fitted to 2nd order kinetic and the highest residuals were determined in static freezing. PPO showed alleviating effect on total phenolic content. Vacuum impregnation caused a color difference prior to freezing, but was found effective for maintaining color during storage. As a result, the use of quick freezing techniques together with the addition of cryoprotectant was effective in the preservation of artichoke quality attributes during frozen storage.

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

Cereus jamacaru DC. (mandacaru) fruit as a source of lactic acid bacteria with in vitro probiotic-related characteristics and its protective effects on Pediococcus pentosaceus during lyophilization and refrigeration storage.

This study isolated and identified autochthonous lactic acid bacteria (LAB) from mandacaru fruit and evaluated their potential probiotic and technological aptitudes in vitro, as well as the protective effects of freeze-dried mandacaru fruit on the most promising LAB isolate during lyophilization and refrigeration storage. Initially, 212 colonies were isolated from mandacaru fruit, and 34 were preliminarily identified as LAB. Thirteen isolates identified by 16S-rRNA sequencing as Pediococcus pentosaceus were negative for DNase, gelatinase, hemolytic, and biogenic amine production. The selected isolates showed proteolytic activity, diacetyl and exopolysaccharide production, and good tolerance to different NaCl concentrations while having low cellular hydrophobicity and antagonistic activity against pathogens. The survival of isolates sharply decreased after 3 h of exposure to pH 2 and had a good tolerance to 1 % bile salt. A principal component analysis selected P. pentosaceus 57 as the most promising isolate based on the examined technological and probiotic-related physiological properties. This isolate was lyophilized with mandacaru fruit and stored under refrigeration for 90 days. P. pentosaceus 57 lyophilized with mandacaru fruit had high viable cell counts (9.69 ± 0.03 log CFU/mL) and >50 % of physiologically active cells at 90 days of refrigeration storage. The results indicate that mandacaru fruit is a source of P. pentosaceus with aptitudes to be explored as potential probiotic and technological characteristics of interest for the food industry, besides being a good candidate for use in lyophilization processes and refrigeration storage of LAB due to its cryoprotective effects.

Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue.

Recently, there has been increasing emphasis on the gonadotoxic effects of cancer therapy in prepubertal boys. As advances in oncology treatments continue to enhance survival rates for prepubertal boys, the need for preserving their functional testicular tissue for future reproduction becomes increasingly vital. Therefore, we explore cutting-edge strategies in fertility preservation, focusing on the cryopreservation and transplantation of immature testicular tissue as a promising avenue. The evolution of cryopreservation techniques, from controlled slow freezing to more recent advancements in vitrification, with an assessment of their strengths and limitations was exhibited. Detailed analysis of cryoprotectants, exposure times, and protocols underscores their impact on immature testicular tissue viability. In transplantation strategy, studies have revealed that the scrotal site may be the preferred location for immature testicular tissue grafting in both autotransplantation and xenotransplantation scenarios. Moreover, the use of biomaterial scaffolds during graft transplantation has shown promise in enhancing graft survival and stimulating spermatogenesis in immature testicular tissue over time. This comprehensive review provides a holistic approach to optimize the preservation strategy of human immature testicular tissue in the future.

Effects of exogenous glycine betaine on growth and development of tomato seedlings under cold stress.

Low temperature is a type of abiotic stress affecting the tomato (Solanum lycopersicum) growth. Understanding the mechanisms and utilization of exogenous substances underlying plant tolerance to cold stress would lay the foundation for improving temperature resilience in this important crop. Our study is aiming to investigate the effect of exogenous glycine betaine (GB) on tomato seedlings to increase tolerance to low temperatures. By treating tomato seedlings with exogenous GB under low temperature stress, we found that 30 mmol/L exogenous GB can significantly improve the cold tolerance of tomato seedlings. Exogenous GB can influence the enzyme activity of antioxidant defense system and ROS levels in tomato leaves. The seedlings with GB treatment presented higher Fv/Fm value and photochemical activity under cold stress compared with the control. Moreover, analysis of high-throughput plant phenotyping of tomato seedlings also supported that exogenous GB can protect the photosynthetic system of tomato seedlings under cold stress. In addition, we proved that exogenous GB significantly increased the content of endogenous abscisic acid (ABA) and decreased endogenous gibberellin (GA) levels, which protected tomatoes from low temperatures. Meanwhile, transcriptional analysis showed that GB regulated the expression of genes involved in antioxidant capacity, calcium signaling, photosynthesis activity, energy metabolism-related and low temperature pathway-related genes in tomato plants. In conclusion, our findings indicated that exogenous GB, as a cryoprotectant, can enhance plant tolerance to low temperature by improving the antioxidant system, photosynthetic system, hormone signaling, and cold response pathway and so on.

Development of sperm cryopreservation protocol for patin buah, Pangasius nasutus.

The development of sperm cryopreservation for Pangasius nasutus is necessary in order to serve the growing demand of this species through artificial fertilization and the preservation of valuable strains of male broodstocks. In the present study, the basic protocol of sperm cryopreservation for P. nasutus was established by identifying the optimal conditions for optimum cryoprotectant, toxicity of cryoprotectants, extenders, freezing condition and dilution ratio. Methanol (MeOH) at 10% concentration had the best post-thaw motility (26.3 ± 0.9%) and curvilinear velocity (VCL) compared to dimethyl acetamide and dimethyl sulfoxide. MeOH was the least toxic cryoprotectant; sperm suspended in 5 and 10% MeOH maintained motility up to 50 min. No significant differences were detected between the three types of extenders tested (0.9% sodium chloride, Calcium-free Hanks' Balance salt solution and ringer solution). P. nasutus sperm had a narrow range of optimal cooling rate. Significantly higher post-thaw motility was identified when cooling at 9.23 °C min-1, obtained by freezing at height of 14 cm above liquid nitrogen vapor for 7 min, showing lower cooling rate is suitable for this species. However, when cooling below and above the optimal cooling rate, post-thaw motility dropped drastically. There were no significant differences among the dilution ratios investigated, indicating the volume of cryodiluent at all tested ratios (1:9, 1:19 and 1:49) was sufficient for the protection of cells during the cryopreservation process. The development of the protocol for cryopreserved P. nasutus sperm will assist artificial seed production and provide an important tool for genetic and breeding research.