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.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

In vivo methods for imaging blood-brain barrier function and dysfunction.

The blood-brain barrier (BBB) is the interface between the central nervous system and systemic circulation. It tightly regulates what enters and is removed from the brain parenchyma and is fundamental in maintaining brain homeostasis. Increasingly, the BBB is recognised as having a significant role in numerous neurological disorders, ranging from acute disorders (traumatic brain injury, stroke, seizures) to chronic neurodegeneration (Alzheimer's disease, vascular dementia, small vessel disease). Numerous approaches have been developed to study the BBB in vitro, in vivo, and ex vivo. The complex multicellular structure and effects of disease are difficult to recreate accurately in vitro, and functional aspects of the BBB cannot be easily studied ex vivo. As such, the value of in vivo methods to study the intact BBB cannot be overstated. This review discusses the structure and function of the BBB and how these are affected in diseases. It then discusses in depth several established and novel methods for imaging the BBB in vivo, with a focus on MRI, nuclear imaging, and high-resolution intravital fluorescence microscopy.

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.

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation.

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5-6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at -80 °C. Alternatively, platelets were frozen with 5-6% Me2SO at -30 °C or with 3% Me2SO at -80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at -30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at -30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at -30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.

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.

Synthesis of Highly Biocompatible and Temperature-Responsive Physical Gels for Cryopreservation and 3D Cell Culture.

There is considerable interest in the cryopreservation in 3D cell culture, as structurally preserving intact cells and tissues is critical in utilizing these systems to promote cell differentiation and tissue organization. Temperature-responsive physical gels and zwitterionic polymers are useful materials as 3D scaffolds for cell culture which may also provide cryoprotection to the composite cells. Nevertheless, there has been a lack of relevant data for polymer systems that have both of these properties. In this study, highly biocompatible triblock copolymers were examined for their effectiveness both as gelators and as cryo-protectants. The triblock copolymers were synthesized with 2-methacryloyloxyethyl phosphorylcholine (MPC) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via atom transfer radical polymerization (PDEGMA113-b-PMPC243-b-PDEGMA113). ABA triblock copolymers composed of hydrophilic "B" block and temperature responsive "A" block could form physical gels above their lower critical solution temperatures (LCST). PDEGMA113-b-PMPC243-b-PDEGMA113 triblock copolymer exhibited the LCST derived from DEGMA and assembled in micellar structures forming physical gels above the LCST. The mechanical properties of the physical gels were evaluated by rheological tests, and the low toxicity of PDEGMA113-b-PMPC243-b-PDEGMA113 was confirmed by MTT assay. Interestingly, the triblock copolymer showed ice recrystallization inhibition (IRI) activity which was determined to be suitable for the cryopreservation of several cell lines. In vitro studies were conducted to demonstrate the cryo-protectant properties and the formation of two and three-dimensional (2D/3D) cell culture scaffolds with high biocompatibility. This stimuli-responsive gelator polymers can therefore be useful for cryopreservation of different cells models, and a promising material for 3D cell culture.

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.

Percutane Image-Guided Cryoablation of Painful Osseous Metastases: A Retrospective Single-Center Review.

PURPOSE: Painful osseous metastases are a common problem in patients with malignancy, and they can be associated with significant morbidity owing to immobility, pain, pathologic fracture, or neurovascular compromise or all of these. We retrospectively evaluated pain levels and tumor enhancement in patients who underwent palliative percutaneous cryoablation for painful bone metastasis. METHODS: In this institutional review board-approved, health insurance portability and accountability act-compliant study, we retrospectively searched our department׳s picture archiving system for patients who underwent computed tomography (CT)-guided percutaneous cryoablation for treatment of painful metastatic osseous disease over a 6-year period (1/1/2005-12/31/2011). The preprocedure and postprocedure images and imaging reports, primary tumor type, CT-guided cryoablation procedure details, treated tumor response, immediate and 3-month postprocedure complications, reported pain response to cryoablation, postprocedural tumor imaging characteristics, and imaging response of noncryoablated systemically treated metastatic lesions were reviewed in patients with metastatic osseous disease who underwent cryoablation. RESULTS: All 16 patients reported improvement in pain within 1 week after the procedure and at 3-month clinical follow-up. A total of 6.2% had tumor growth and 93.8% had tumor arrest or shrinkage on follow-up CT, although all study patients had progression of noncryoablated metastases at other sites despite systemic therapy. A total of 62.5% of patients with posttreatment contrasted CT demonstrated marginal enhancement at the ablation site, although only single patient had interval growth. CONCLUSION: Most of our patients had tumor arrest or shrinkage on follow-up imaging, despite progression of noncryoablated metastases treated with preprocedure and postprocedure systemic therapy. Radiation therapy, chemotherapy, and analgesics have a moderate failure rate and require repeat treatments where quality of life is the foremost objective. CT-guided cryoablation is a safe palliative treatment to reduce pain in patients with painful osseous metastatic disease, achieve effective local tumor control, and in some cases, provide a curative option for a target lesion.

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.