Targeted development and optimization of small-molecule ice recrystallization inhibitors (IRIs) for the cryopreservation of biological systems.

Despite the routine use of cryopreservation for the storage of biological materials, its outcomes are often sub-optimal (including reduced post-thaw viability, recovery, and functionality) due to the damage caused by uncontrolled ice growth. Traditional cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), fail to prevent damage caused by ice growth and concerns over CPA cytotoxicity have fostered an increased interest in developing improved CPAs and cryoprotection strategies. The inhibition of ice recrystallization by natural antifreeze (glyco)proteins [AF(G)Ps] to improve cryopreservation outcomes has been examined; however, the ice binding properties of these substances and their challenging large-scale production make them poor CPA candidates. Therefore, the development and deployment of biocompatible, small-molecule ice recrystallization inhibitors (IRIs) for use as CPAs is a worthwhile objective. Extensive structure-activity relationship studies on AF(G)Ps revealed that simple carbohydrate derivatives could inhibit ice recrystallization. It was later discovered that this activity could be fine-tuned by delicately balancing the molecule's hydrophobicity and hydrophilicity. Current generation small-molecule IRIs have been meticulously designed to avoid binding to the surface of ice and subsequent biological testing (for both cytotoxicity and cryopreservation efficacy) has demonstrated significant improvements to the cryopreservation outcomes of several cell types. However, an individualized cell-specific approach for the simultaneous assessment of multiple cryopreservation outcomes is necessary to realize the full potential of IRIs as CPAs. This article provides a detailed overview of the development of small-molecule carbohydrate-based IRIs and highlights the crucial cell-specific biological considerations that must be taken into account when assessing cryopreservation outcomes. https://doi.org/10.54680/fr24210110112.

Biosynthesis of 1α-hydroxycorticosterone in the winter skate Leucoraja ocellata: evidence to suggest a novel steroidogenic route.

The present study explores the ability of intracellular bacteria within the renal-inter-renal tissue of the winter skate Leucoraja ocellata to metabolize steroids and contribute to the synthesis of the novel elasmobranch corticosteroid, 1α-hydroxycorticosterone (1α-OH-B). Despite the rarity of C1 hydroxylation noted in the original identification of 1α-OH-B, literature provides evidence for steroid C1 hydroxylation by micro-organisms. Eight ureolytic bacterial isolates were identified in the renal-inter-renal tissue of L. ocellata, the latter being the site of 1α-OH-B synthesis. From incubations of bacterial isolates with known amounts of potential 1α-OH-B precursors, one isolate UM008 of the genus Rhodococcus was seen to metabolize corticosteroids and produce novel products via HPLC analysis. Cations Zn2+ and Fe3+ altered metabolism of certain steroid precursors, suggesting inhibition of Rhodococcus steroid catabolism. Genome sequencing of UM008 identified strong sequence and structural homology to that of Rhodococcus erythropolis PR4. A complete enzymatic pathway for steroid-ring oxidation as documented within other Actinobacteria was identified within the UM008 genome. This study highlights the potential role of Rhodococcus bacteria in steroid metabolism and proposes a novel alternative pathway for 1α-OH-B synthesis, suggesting a unique form of mutualism between intracellular bacteria and their elasmobranch host.

Fully convergent solid phase synthesis of antifreeze glycoprotein analogues.

The convergent solid phase synthesis of C-linked analogues of antifreeze glycoprotein (AFGP) has been achieved. In this approach, three to six carbohydrate residues are simultaneously coupled to a resin-bound polypeptide. Glycopeptides ranging from 1.6 to 3.0 kDa are easily prepared in 26-44% yield demonstrating the utility of this approach.

A general synthesis of structurally diverse building blocks for preparing analogues of C-linked antifreeze glycoproteins.

A synthetic methodology to afford unusual glycoconjugate building blocks useful for the solid-phase synthesis of C-linked antifreeze glycoprotein (AFGP) analogues is described. Such compounds are urgently required in order to elucidate the molecular mechanism by which AFGPs function. All reactions are general in nature and accommodate structural variation in the carbohydrate moiety, polypeptide backbone, and amino acid side chain.

Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.

Little is known of the post-absorptive, metabolic fate of gamma-tocopherol, the major form of vitamin E in North American diets. The objective of this study was to determine the extent of urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a recently identified metabolite of gamma-tocopherol. A method for measurement of urinary gamma-CEHC was developed, using gas chromatography-mass spectrometry (GC-MS) with a deuterated internal standard, 2,7,8-trimethyl-2-(beta-carboxyethyl)-(3, 4-2H2)-6-hydroxychroman (d2-gamma-CEHC). This standard was synthesized by dehydrogenation of 6-acetyl-gamma-CEHC followed by deuteration of the resulting 3,4-double bond. The use of d2-gamma-CEHC resulted in accurate determinations of the concentration of d0-gamma-CEHC in human urine. Urine samples containing added d2-gamma-CEHC were treated with beta-glucuronidase, extracted with an organic solvent, and analyzed by GC-MS. Analysis of 24-h urine pools from healthy subjects revealed gamma-CEHC concentrations, normalized against creatinine, ranging from 2.5 to 31.5 micromol/g creatinine, or a total of 4.6 to 29.8 micromol per day. These results correspond to 2-12 mg gamma-tocopherol excreted daily as gamma-CEHC in the urine. Given an estimated mean intake of gamma-tocopherol of 20 mg/day, catabolism of gamma-tocopherol to gamma-CEHC, followed by glucuronide conjugation and urinary excretion, is a major pathway for elimination of gamma-tocopherol in humans.