It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Probing the Mechanism of Action of Small-Molecule Ice Recrystallization Inhibitors Using Proton Nuclear Magnetic Resonance Relaxation.

N-2-Fluorophenyl-d-gluconamide (2FA) improves the recovery and function of cryopreserved biological materials by inhibiting ice recrystallization. However, as for many small-molecule ice recrystallization inhibitors, the mechanism of action of 2FA is not well-understood. In this study, the IC50 of 2FA for ice recrystallization was determined to be 3.5 mM (95% CI [3.41-3.52]). 1H transverse and longitudinal relaxations were then characterized by NMR at 2FA concentrations from 0 to 10 mM and at temperatures between -15 °C and +30 °C. Corresponding activation energy of water molecule motion (EAH2O) was calculated, showing that at each concentration 2FA did not affect EAH2O in the solid state, whereas in the liquid state EAH2O was significantly higher with 2FA than for pure water. Therefore, 2FA is excluded from the ice lattice upon freezing and concentrated in the interstitial liquid phase. This restricts the migration of water molecules between ice crystals via the liquid phase, inhibiting ice recrystallization.

Synthesis of natural/13C-enriched d-tagatose from natural/13C-enriched d-fructose.

A concise, easily scalable synthesis of a rare ketohexose, d-tagatose, was developed, that is compatible with the preparation of d-[UL-13C6]tagatose. Epimerization of the widely available and inexpensive ketohexose d-fructose at the C-4 position via an oxidation/reduction (Dess-Martin periodinane/NaBH4) was a key step in the synthesis. Overall, fully protected natural d-tagatose (3.21 g) was prepared from d-fructose (9 g) on a 50 mmol scale in 23% overall yield, after five steps and two chromatographic purifications. d-[UL-13C6]Tagatose (92 mg) was prepared from d-[UL-13C6]fructose (465 mg, 2.5 mmol) in 16% overall yield after six steps and four chromatographic purifications.

Use of Ice Recrystallization Inhibition Assays to Screen for Compounds That Inhibit Ice Recrystallization.

Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay to measure IRI activity.

Inhibition of ice recrystallization during cryopreservation of cord blood grafts improves platelet engraftment.

BACKGROUND: Platelet engraftment following cord blood (CB) transplantation remains a significant hurdle to this day. The uncontrolled growth of ice, a process referred to as ice recrystallization, is one of several mechanisms that lead to cell loss and decreased potency during freezing and thawing. We hypothesized that reducing cell damage induced by ice recrystallization in CB units (CBUs) would reduce losses of stem and progenitor cells and therefore improve engraftment. We previously demonstrated that the ice recrystallization inhibitor (IRI) N-(2-fluorophenyl)-D-gluconamide (IRI 2) increases the postthaw recovery of CB progenitors. Herein, we set out to ascertain whether IRI 2 can enhance platelet and bone marrow engraftment activity of hematopoietic stem cells (HSCs) in cryopreserved CBUs using a serial transplantation model. STUDY DESIGN AND METHODS: CBUs were processed following standard volume/red blood cell reduction procedure and portions frozen with dimethyl sulfoxide (DMSO) supplemented or not with IRI 2. Thawed CB samples were serially transplanted into immunodeficient mice. RESULTS: Our results show that supplementation of DMSO with IRI 2 had several beneficial effects. Specifically, higher levels of human platelets were observed in the peripheral blood (p < 0.05; n = 4) upon transplant of CBUs preserved with the IRIs. In addition, human BM chimerism and the number of human CFU progenitors in the bone marrow were superior in IRI 2 recipients compared to DMSO recipients. Moreover, IRI 2 had no negative impact on the multilineage differentiation and self-renewal activities of HSCs. DISCUSSION: Taken together, these results demonstrate that supplementation of a hematopoietic graft with IRI can improve the postthaw engraftment activities of HSCs.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ±â€¯0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 µM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

Modulating Intracellular Ice Growth with Cell-Permeating Small-Molecule Ice Recrystallization Inhibitors.

Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-ß-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.

1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics.

The promise of organ and tissue preservation to transform medicine.

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

Photoswitchable carbohydrate-based fluorosurfactants as tuneable ice recrystallization inhibitors.

Cryopreservation is an important technique employed for the storage and preservation of biological tissues and cells. The limited effectiveness and significant toxicity of conventionally-used cryoprotectants, such as DMSO, have prompted efforts toward the rational design of less toxic alternatives, including carbohydrate-based surfactants. In this paper, we report the modular synthesis and ice recrystallization inhibition (IRI) activity of a library of variably substituted, carbohydrate-based fluorosurfactants. Carbohydrate-based fluorosurfactants possessed a variable mono- or disaccharide head group appended to a hydrophobic fluoroalkyl-substituted azobenzene tail group. Light-addressable fluorosurfactants displayed weak-to-moderate IRI activity that could be tuned through selection of carbohydrate head group, position of the trifluoroalkyl group on the azobenzene ring, and isomeric state of the azobenzene tail fragment.

QSAR Accelerated Discovery of Potent Ice Recrystallization Inhibitors.

Ice recrystallization is the main contributor to cell damage and death during the cryopreservation of cells and tissues. Over the past five years, many small carbohydrate-based molecules were identified as ice recrystallization inhibitors and several were shown to reduce cryoinjury during the cryopreservation of red blood cells (RBCs) and hematopoietic stems cells (HSCs). Unfortunately, clear structure-activity relationships have not been identified impeding the rational design of future compounds possessing ice recrystallization inhibition (IRI) activity. A set of 124 previously synthesized compounds with known IRI activities were used to calibrate 3D-QSAR classification models using GRid INdependent Descriptors (GRIND) derived from DFT level quantum mechanical calculations. Partial least squares (PLS) model was calibrated with 70% of the data set which successfully identified 80% of the IRI active compounds with a precision of 0.8. This model exhibited good performance in screening the remaining 30% of the data set with 70% of active additives successfully recovered with a precision of ~0.7 and specificity of 0.8. The model was further applied to screen a new library of aryl-alditol molecules which were then experimentally synthesized and tested with a success rate of 82%. Presented is the first computer-aided high-throughput experimental screening for novel IRI active compounds.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing.

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

O-Aryl-Glycoside Ice Recrystallization Inhibitors as Novel Cryoprotectants: A Structure-Function Study.

Low-molecular-weight ice recrystallization inhibitors (IRIs) are ideal cryoprotectants that control the growth of ice and mitigate cell damage during freezing. Herein, we describe a detailed study correlating the ice recrystallization inhibition activity and the cryopreservation ability with the structure of O-aryl-glycosides. Many effective IRIs are efficient cryoadditives for the freezing of red blood cells (RBCs). One effective cryoadditive did not inhibit ice recrystallization but instead inhibited ice nucleation, demonstrating the significance of inhibiting both processes and illustrating the importance of this emerging class of cryoprotectants.

Small-Molecule Ice Recrystallization Inhibitors Improve the Post-Thaw Function of Hematopoietic Stem and Progenitor Cells.

The success of hematopoietic stem cell transplantation depends in part on the number and the quality of cells transplanted. Cryoinjuries during freezing and thawing reduce the ability of hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate after thawing. Up to 20% of the patients undergoing umbilical cord blood (UCB) transplant experience delayed or failed engraftment, likely because of the inadequate hematopoietic potency of the unit. Therefore, the optimization of cryopreservation protocols, with an emphasis on the preservation of HSPCs, is an important issue. Current protocols typically utilize a 10% dimethyl sulfoxide cryoprotectant solution. This solution ensures 70-80% post-thaw cell viability by diluting intracellular solutes and maintaining the cell volume during cryopreservation. However, this solution fails to fully protect HSPCs, resulting in the loss of potency. Therefore, a new class of cryoprotectants (N-aryl-d-aldonamides) was designed and assessed for the ability to inhibit ice recrystallization and to protect HSPCs against cryoinjury. Several highly active ice recrystallization inhibitors were discovered. When used as additives to the conventional cryoprotectant solution, these nontoxic small molecules improved the preservation of functionally divergent hematopoietic progenitors in the colony-forming unit and long-term culture-initiating cell assays. By contrast, structurally similar compounds that did not inhibit ice recrystallization failed to improve the post-thaw recovery of myeloid progenitors. Together, these results demonstrate that the supplementation of cryopreservation solution with compounds capable of controlling ice recrystallization increases the post-thaw function and potency of HSPCs in UCB. This increase may translate into reduced risk of engraftment failure and allow for greater use of cryopreserved cord blood units.

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as ß-PMP-Glc and ß-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM ß-PMP-Glc or 30 mM ß-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.