Supplemented phase diagrams for vitrification CPA cocktails: DP6, VS55 and M22.

DP6, VS55 and M22 are the most commonly used cryoprotective agent (CPA) cocktails for vitrification experiments in tissues and organs. However, complete phase diagrams for the three CPAs are often unavailable or incomplete (only available for full strength CPAs) thereby hampering optimization of vitrification and rewarming procedures. In this paper, we used differential scanning calorimetry (DSC) to measure the transition temperatures including heterogeneous nucleation temperatures (Thet), glass transition temperatures (Tg), rewarming phase crystallization (devitrification and/or recrystallization) temperatures (Td) and melting temperatures (Tm) while cooling or warming the CPA sample at 5 °C/min and plotted the obtained transition temperatures for different concentrations of CPAs into the phase diagrams. We also used cryomicroscopy cooling or warming the sample at the same rate to record the ice crystallization during the whole process, and we presented the cryomicroscopic images at the transition temperatures, which agreed with the DSC presented phenomena.

A guide to successful mL to L scale vitrification and rewarming.

Cryopreservation by vitrification to achieve an "ice free" glassy state is an effective technique for preserving biomaterials including cells, tissues, and potentially even whole organs. The major challenges in cooling to and rewarming from a vitrified state remain ice crystallization and cracking/fracture. Ice crystallization can be inhibited by the use of cryoprotective agents (CPAs), though the inhibition further depends upon the rates achieved during cooling and rewarming. The minimal rate required to prevent any ice crystallization or recrystallization/devitrification in a given CPA is called the critical cooling rate (CCR) or critical warming rate (CWR), respectively. On the other hand, physical cracking is mainly related to thermomechanical stresses, which can be avoided by maintaining temperature differences below a critical threshold. In this simplified analysis, we calculate deltaT as the largest temperature difference occurring in a system during cooling or rewarming in the brittle/glassy phase. This deltaT is then used in a simple "thermal shock equation" to estimate thermal stress within the material to decide if the material is above the yield strength and to evaluate the potential for fracture failure. In this review we aimed to understand the limits of success and failure at different length scales for cryopreservation by vitrification, due to both ice crystallization and cracking. Here we use thermal modeling to help us understand the magnitude and trajectory of these challenges as we scale the biomaterial volume for a given CPA from the milliliter to liter scale. First, we solved the governing heat transfer equations in a cylindrical geometry for three common vitrification cocktails (i.e., VS55, DP6, and M22) to estimate the cooling and warming rates during convective cooling and warming and nanowarming (volumetric heating). Second, we estimated the temperature difference deltaT and compared it to a tolerable threshold (deltaTmax) based on a simplified "thermal shock" equation for the same cooling and rewarming conditions. We found, not surprisingly, that M22 achieves vitrification more easily during convective cooling and rewarming for all volumes compared to VS55 or DP6 due to its considerably lower CCR and CWR. Further, convective rewarming (boundary rewarming) leads to larger temperature differences and smaller rates compared to nanowarming (volumetric rewarming) for all CPAs with increasing failure at larger volumes. We conclude that as more and larger systems are vitrified and rewarmed with standard CPA cocktails, this work can serve as a practical guide to successful implementation based on the characteristic length (volume/surface area) of the system and the specific conditions of cooling and warming. doi.org/10.54680/fr22610110112.

De novo transcriptome assembly, annotation and SSR mining data of Hellula undalis (Fabr.) (Lepidoptera: Pyralidae), the cabbage webworm.

BACKGROUND: The cabbage webworm, Hellula undalis (Fabricius) (Lepidoptera: Pyralidae), is a significant pest of brassicas and other cruciferous plants in warm regions worldwide. Transcriptome analysis is valuable for investigation of molecular mechanisms underlying the insect development and reproduction. De novo assembly is particularly useful for acquiring complete transcriptome information of insect species when there is no reference genome available. In case of Hellula undalis, only 17 nucleotide records are currently available throughout NCBI nucleotide database. Genes associated with metabolic processes, general development, reproduction, defense and functional genomics were not previously predicted in the Hellula undalis at the genomic level. METHODS & RESULTS: To address this issue, we constructed Hellula undalis transcriptome using Illumina NovaSeq6000 technology. Approximately 48 million 150 bp paired-end reads were obtained from sequencing. A total of 30,451 contigs were generated by de novo assembly of sample and were compared with the sequences in the NCBI non-redundant protein database (Nr). In total, 71 % of contigs were matched to known proteins in public databases including Nr, Gene Ontology (GO), and Cluster Orthologous Gene Database (COG), and then, contigs were mapped to 123 via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, we compared the ortholog gene family of the Hullula undalis, transcriptome to Spodoptera frugiperda, spodotera litura and spodoptera littoralis and found that 391 orthologous gene families are specific to Hullula undalis. A total of 1,913 potential SSRs was discovered in Hullula undalis contigs. CONCLUSIONS: This study is the first transcriptome data for Hullula undalis. Additionally, it serves as a valuable resource for identifying target genes and developing effective and environmentally friendly strategies for pest control.