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1. The role of antifreeze glycoprotein (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) as ice modulators during partial freezing of rat livers

   https://doi.org/10.3389/fphy.2022.1033613  

   Tessier, Shannon N.; Haque, Omar; Pendexter, Casie A.; Cronin, Stephanie E.; Hafiz, Ehab O.; Weng, Lindong; Yeh, Heidi; Markmann, James F.; Taylor, Michael J.; Fahy, Gregory M.; et al (December 2022, Frontiers in Physics)

   Introduction:The current liver organ shortage has pushed the field of transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at high subzero storage temperatures (−10°C to −15°C), while simultaneously maintaining a sufficient unfrozen fraction to limit ice-mediated injury. Methods and results:Using glycerol as the main permeating cryoprotectant agent, this research first demonstrated that partially frozen rat livers showed similar outcomes after thawing from either −10°C or −15°C with respect to subnormothermic machine perfusion metrics. Next, we assessed the effect of adding ice modulators, including antifreeze glycoprotein (AFGP) or a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Discussion:Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, including testing additional ice modulators, can help improve the viability of these partially frozen organs. 

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2. Impact of Oxygen and Sulfur Heteroatom Core Substitution on Catalyst Properties of Phenoxazines and Their Performance in Organocatalyzed Atom Transfer Radical Polymerization (O‐ATRP)

   https://doi.org/10.1002/chem.202501179  

   Lathrop, Jessica_L; Portela, Brandon_S; Paton, Robert_S; Miyake, Garret_M (July 2025, Chemistry – A European Journal)

   Abstract Phenoxazines are a successful class of organic photoredox catalysts (PCs) with tunable redox and photophysical properties. Originally, we aimed to realize more reducing phenoxazine PCs through heteroatom core substituted (HetCS) derivatives, while maintaining an efficiently oxidizing PC^·+. However, core modification with thioether or ether functionality to a PC that exhibits photoinduced intramolecular charge transfer (CT) negligibly alters the singlet excited state reduction potential (E_S1°*), while yielding a less oxidizing PC^·+(E_1/2) (E_1/2 = 0.50–0.64 V vs. SCE) compared to the noncore modified PC1(0.68 V vs. SCE). Photophysical characterization of HetCS PCs revealed that increasing electron density on the core of a CT exhibiting PC stabilizes the emissive state and PC^·+, resulting in a relatively unchangedE_S1°* compared to PC1. In contrast, modifying the core of a PC that does not exhibit CT yields a highly reducingE_S1°* (PC3= −2.48 V vs. SCE) compared to its CT equivalent (PC1d= −1.68 V vs. SCE). The impact of PC property on photocatalytic ability was evaluated through organocatalyzed atom transfer radical polymerization (O‐ATRP). HetCS PCs were able to yield poly(methyl methacrylate) with low dispersity and moderate targeted molecular weight as evaluated by initiator efficiency (I*) in DMAc (Ð= 1.20–1.26;I*= 47–57%). Ultimately, this work provides insight into how phenoxazine PC properties are altered through structural modification, which can inform future PC design. 

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3. Partial freezing of rat livers extends preservation time by 5-fold

   https://doi.org/10.1038/s41467-022-31490-2  

   Tessier, Shannon N.; de Vries, Reinier J.; Pendexter, Casie A.; Cronin, Stephanie E. J.; Ozer, Sinan; Hafiz, Ehab O. A.; Raigani, Siavash; Oliveira-Costa, Joao Paulo; Wilks, Benjamin T.; Lopera Higuita, Manuela; et al (July 2022, Nature Communications)

   Abstract The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between −4 and −6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (−10 to −15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes. 

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4. Sub-Zero Non-Freezing of Vascularized Composite Allografts Preservation in Rodents

   https://doi.org/10.21203/rs.3.rs-3750450/v1  

   Filz von Reiterdank, Irina; Tawa, Pierre; Berkane, Yanis; de Clermont-Tonnerre, Eloi; Dinicu, Antonia; Pendexter, Casie; Goutard, Marion; Lellouch, Alexandre G; Mink van der Molen, Aebele B; Coert, JH; et al (December 2023, Research Square)

   Abstract Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4-6 hours of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by cryoprotective agent (CPA) cocktail developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation.*Irina Filz von Reiterdank & Pierre Tawa Contributed equally. 

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5. Evaluation of RNA later as a Field-Compatible Preservation Method for Metaproteomic Analyses of Bacterium-Animal Symbioses

   https://doi.org/10.1128/Spectrum.01429-21  

   Jensen, Marlene; Wippler, Juliane; Kleiner, Manuel (October 2021, Microbiology Spectrum)

   Gralnick, Jeffrey A. (Ed.)

   ABSTRACT Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities “ in situ ,” which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNA later using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNA later storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNA later and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNA later for protein preservation of field-collected samples targeted for metaproteomic analyses. IMPORTANCE Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNA later can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNA later preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature. 

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