Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

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.

OBJECTIVE: 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.
CONCLUSIONS: 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.

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.

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.

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.

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.

BACKGROUND: Desloratadine, a second-generation antihistaminic drug, is poorly watersoluble and requires amelioration of the dissolution rate to improve its pharmacokinetics properties.
METHODS: 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.
RESULTS: 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).
CONCLUSIONS: 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%.