Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Biochar addition regulates soil phosphorus fractions and improves release of available phosphorus under freezing-thawing cycles.

Currently, the shortage of phosphorus resources is becoming more and more serious. In general, phosphorus fertilizer is poorly utilized in soil and tends to gradually accumulate. Freezing-thawing cycles (FT) are seasonal phenomenon occurring in high latitudes and altitudes regions, which have obvious influence on the form of phosphorus in soil. This study investigates the effect of biochar on soil physicochemical properties, phosphorus form and availability under FT and thermostatic incubation (TH) condition. Compared with treatment without biochar, 4 % biochar addition increased the soil pH value, electrical conductivity, organic matter and Olsen-P of soil by a maximum of 0.76, 285.55 µS/cm, 28.60 g/kg and 139.27 mg/kg, respectively. Moreover, according to Hedley-P classification results, under FT condition, the content of labile phosphorus pool is always higher than those under TH. FT may promote the conversion of phosphorus from other fractions to labile phosphorus pool. Redundancy analysis results show that biochar addition and FT can not only directly change the soil phosphorus pool, but also alter the soil physicochemical properties and microbial community, which further affect the adsorption and mineralization of phosphorus in soil. The results of this study will be devoted to understanding the changes in soil phosphorus fractions under the effects of biochar addition and FT, providing references for agricultural production in areas where FT occur.

Release and transformation of phosphorus in sediment following seasonal freezing-thawing cycles.

River ice in the upper Yarlung Zangbo River is characterized by seasonal freezing-thawing cycles (SFTC). It is important to explore the effects of SFTC on phosphorus release and transformation from upstream surface sediments to protect the ecosystem of the Yarlung Zangbo River. The process and mechanism of phosphorus release and transformation in sediments following SFTC were investigated in a laboratory simulation experiment. The results showed that after freezing, sediment particles were broken, the specific surface area was increased by 14%, and the particle size was decreased by 43%, which resulted in weakened adsorption of phosphorus by sediments. Moreover, the destruction of organic matter (OM) on the sediment surface will release more ion adsorption sites and promote the release of phosphorus. The bioavailabilities of exchangeable phosphorus (Ex-P), aluminum phosphorus (Al-P) and iron phosphorus (Fe-P) increased by 60.09%, 86.86% and 31.86%, respectively, after freezing. Organic phosphorus (O-P) is used indirectly by organisms, and O-P content showed a significant correlation with OM content. Water affected the oxygen content in sediments during the freezing period, and continuous hypoxia promoted the release and transformation of Fe-P and Al-P.

Effect of pre-swelling and freezing/thawing cycles on the structure of molecular, morphological, and functional properties of potato starch.

This study aimed to investigate the effect of pre-swelling at 55°C for 1 hr followed by freezing-thawing cycles (PFTCs), and freezing-thawing cycles (FTCs) in the starch granules to improve the freeze-thaw stability and evaluate its impact on the molecular, morphological, and functional properties of potato starch (PS). FTCs at 1 cycle and 7 cycles were applied for both treated PS. Microscopical structure, thermal, molecular, and functional properties (i.e., swelling power, solubility, shear viscosity, and gel strength) were comprehensively analyzed. In terms of granule structures, treated PS by FTC showed a slightly affected on the surface of starch granules, while treating PS by PFTC showed an affected in the form of small cracks and holes in the outer surface of starch granules. The gelatinization enthalpy (∆Hgel ) values decreased in the treated PS compared with the native. Thus, decreasing was systemically increased with the number of applied cycles from 1- to 7-cycle. The viscosity of treated PS decreased systematically with molecular degradation or the physical modification, with remarkable reduction, particularly at a higher shear rate (150°C). Treated PS by FTC showed a clear difference (p ≤ .05) in gel values compared with the native at disintegration temperature 115°C. Finally, the degradation of the molecular properties showed significant differences between the native and treated PS either by the FTC or PFTC in molecular weight of starch and amylose without debranching and after debranching by pullulanase enzyme. PRACTICAL APPLICATIONS: Freezing is one of the standard preservation methods used for ready-to-eat products. When this type of food's exposed to more freeze-thaw cycles, the phase separation will be increased due to the increase in retrogradation of amylopectin. To avoid such changes during frozen storage, native potato starch (PS) was modified using both pre-swelling followed by freezing-thawing cycles (PFTCs) and freezing-thawing cycles (FTCs) at 1- and 7-cycle to enhance starch properties, such as swelling power, solubility, shear viscosity, and gel strength. The findings of this study might add to the theoretical understanding of modified PS and act as a guideline for modified starch manufacturing.

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa.

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.

Protective effects of Tribulus terrestris and Cinnamomum zeylanicum extracts and trehalose added to diluents on goat epididymal sperm freezability.

This study investigates the effect of adding Tribulus terrestris ethanol extract (TEE) and Cinnamomum zeylanicum ethanol extract (CEE) and trehalose on freezability of goat epididymal spermatozoa. In Experiment 1, the treatments consist of basic extender containing 25, 50 or 100 µg/ml of TEE or CEE. The control contained no additives. Experiment 2 was carried out to compare the effect of best concentrations resulted in the first experiment with 150 mM trehalose added to basic extender. The results of experiment 1 showed that supplementation of 50 µg/ml TEE and 50 µg/ml CEE increased significantly the percentages of motility, progressive motility and viability of cryopreserved spermatozoa, while the level of malondialdehyde concentration was decreased. Moreover, the 50 µg/ml TEE treatment indicate significantly) P < 0.05) the lowest DNA fragmentation among the other treatments. The data obtained from experiment 2 show that all treatments increased significantly) P < 0.05) the percentages of total motility, viability and membrane integrity, and concurrently decreased the rate of MDA compared to control. In addition, the rates of viability and progressive motility were significantly (P < 0.05) higher in diluents contained herb extracts and trehalose. Regarding DNA fragmentation, the results demonstrate that using the extracts and trehalose in diluents decreased the DNA damages and thereby improved the rate of intact sperm heads. In conclusion, the results of this study indicate that 50 µg/ml of Tribulus terrestris and Cinnamomum zeylanicum ethanolic extracts alone and plus trehalose improved the spermatozoa quality and could be used for cryopreservation.

Effects of supplementation of Tris-egg yolk extender with royal jelly on chilled and frozen-thawed ram semen characteristics.

This study was conducted to examine the effect of supplementation of Tris-egg yolk extender with lyophilized royal jelly (RJ) on chilled and frozen-thawed ram semen parameters. Ejaculates were collected by artificial vagina from 4 mature rams, twice a week for 4 weeks. Only samples with motility of ≥70% were included, pooled and divided into four equal parts and then diluted in extenders with various concentrations of RJ (0, 1, 3 and 5%, vol/vol) to a final concentration of 200 × 106 sperm/mL and was incubated at 37 °C for 30 min and were subsequently evaluated. After equilibration of extended semen for 2 h at 4 °C, some semen samples were packed in 0.25 mL plastic straws. Then, the straws were frozen in the liquid nitrogen vapor phase for 15 min and stored at -196 °C in liquid nitrogen. The frozen straws were thawed in warm water (37 °C) for 30 s and evaluated; whereas, other semen samples were stored in the refrigerator (4 °C) up to 7 days. The chilled samples were kept in water bath (37 °C) for 5 min and then were evaluated. After dilution, the lowest and highest sperm total abnormality was recorded in 3 and 5% RJ supplemented groups, respectively (P < 0.05). The chilled sperm total motility and membrane integrity were significantly (P < 0.05) higher in 3% than those in 0% and 5% RJ supplemented groups. The chilled sperm progressive motility and viability was significantly (P < 0.05) higher in 1 and 3% than those in 0 and 5% RJ supplemented groups. The frozen-thawed sperm total motility, progressive motility, membrane integrity and viability were significantly higher in 3% RJ supplemented group (P < 0.05). In conclusion, supplementation of Tris-egg yolk extender with 3% lyophilized RJ had a protective effect on chilled and cryopreserved ram spermatozoa.

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes' zona pellucida.

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 µM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.