RESUMO
A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.
Assuntos
Armazenamento de Medicamentos , Hidrogéis , Desnaturação Proteica , Estabilidade Proteica , Proteínas , Seringas , Humanos , Excipientes , Liofilização , Hidrogéis/química , Proteínas/administração & dosagem , Proteínas/química , Proteínas/economia , Trealose , Congelamento , Refrigeração , Vacinas contra Papillomavirus/química , Armazenamento de Medicamentos/economia , Armazenamento de Medicamentos/métodosRESUMO
The Greenland Ice Sheet has a central role in the global climate system owing to its size, radiative effects and freshwater storage, and as a potential tipping point1. Weather stations show that the coastal regions are warming2, but the imprint of global warming in the central part of the ice sheet is unclear, owing to missing long-term observations. Current ice-core-based temperature reconstructions3-5 are ambiguous with respect to isolating global warming signatures from natural variability, because they are too noisy and do not include the most recent decades. By systematically redrilling ice cores, we created a high-quality reconstruction of central and north Greenland temperatures from AD 1000 until 2011. Here we show that the warming in the recent reconstructed decade exceeds the range of the pre-industrial temperature variability in the past millennium with virtual certainty (P < 0.001) and is on average 1.5 ± 0.4 degrees Celsius (1 standard error) warmer than the twentieth century. Our findings suggest that these exceptional temperatures arise from the superposition of natural variability with a long-term warming trend, apparent since AD 1800. The disproportionate warming is accompanied by enhanced Greenland meltwater run-off, implying that anthropogenic influence has also arrived in central and north Greenland, which might further accelerate the overall Greenland mass loss.
Assuntos
Clima , Aquecimento Global , Temperatura , Aquecimento Global/estatística & dados numéricos , Groenlândia , Camada de Gelo , Atividades Humanas/tendências , Movimentos da Água , CongelamentoRESUMO
The abyssal ocean circulation is a key component of the global meridional overturning circulation, cycling heat, carbon, oxygen and nutrients throughout the world ocean1,2. The strongest historical trend observed in the abyssal ocean is warming at high southern latitudes2-4, yet it is unclear what processes have driven this warming, and whether this warming is linked to a slowdown in the ocean's overturning circulation. Furthermore, attributing change to specific drivers is difficult owing to limited measurements, and because coupled climate models exhibit biases in the region5-7. In addition, future change remains uncertain, with the latest coordinated climate model projections not accounting for dynamic ice-sheet melt. Here we use a transient forced high-resolution coupled ocean-sea-ice model to show that under a high-emissions scenario, abyssal warming is set to accelerate over the next 30 years. We find that meltwater input around Antarctica drives a contraction of Antarctic Bottom Water (AABW), opening a pathway that allows warm Circumpolar Deep Water greater access to the continental shelf. The reduction in AABW formation results in warming and ageing of the abyssal ocean, consistent with recent measurements. In contrast, projected wind and thermal forcing has little impact on the properties, age and volume of AABW. These results highlight the critical importance of Antarctic meltwater in setting the abyssal ocean overturning, with implications for global ocean biogeochemistry and climate that could last for centuries.
Assuntos
Congelamento , Temperatura Alta , Oceanos e Mares , Água do Mar , Movimentos da Água , Regiões Antárticas , Água do Mar/análise , Água do Mar/química , Aceleração , Incerteza , Mudança ClimáticaRESUMO
Melting of the Greenland ice sheet (GrIS) in response to anthropogenic global warming poses a severe threat in terms of global sea-level rise (SLR)1. Modelling and palaeoclimate evidence suggest that rapidly increasing temperatures in the Arctic can trigger positive feedback mechanisms for the GrIS, leading to self-sustained melting2-4, and the GrIS has been shown to permit several stable states5. Critical transitions are expected when the global mean temperature (GMT) crosses specific thresholds, with substantial hysteresis between the stable states6. Here we use two independent ice-sheet models to investigate the impact of different overshoot scenarios with varying peak and convergence temperatures for a broad range of warming and subsequent cooling rates. Our results show that the maximum GMT and the time span of overshooting given GMT targets are critical in determining GrIS stability. We find a threshold GMT between 1.7 °C and 2.3 °C above preindustrial levels for an abrupt ice-sheet loss. GrIS loss can be substantially mitigated, even for maximum GMTs of 6 °C or more above preindustrial levels, if the GMT is subsequently reduced to less than 1.5 °C above preindustrial levels within a few centuries. However, our results also show that even temporarily overshooting the temperature threshold, without a transition to a new ice-sheet state, still leads to a peak in SLR of up to several metres.
Assuntos
Modelos Climáticos , Congelamento , Aquecimento Global , Camada de Gelo , Elevação do Nível do Mar , Temperatura , Aquecimento Global/estatística & dados numéricos , Groenlândia , Camada de Gelo/química , Fatores de TempoRESUMO
Water resources sustainability in High Mountain Asia (HMA) surrounding the Tibetan Plateau (TP)-known as Asia's water tower-has triggered widespread concerns because HMA protects millions of people against water stress1,2. However, the mechanisms behind the heterogeneous trends observed in terrestrial water storage (TWS) over the TP remain poorly understood. Here we use a Lagrangian particle dispersion model and satellite observations to attribute about 1 Gt of monthly TWS decline in the southern TP during 2003-2016 to westerlies-carried deficit in precipitation minus evaporation (PME) from the southeast North Atlantic. We further show that HMA blocks the propagation of PME deficit into the central TP, causing a monthly TWS increase by about 0.5 Gt. Furthermore, warming-induced snow and glacial melt as well as drying-induced TWS depletion in HMA weaken the blocking of HMA's mountains, causing persistent northward expansion of the TP's TWS deficit since 2009. Future projections under two emissions scenarios verified by satellite observations during 2020-2021 indicate that, by the end of the twenty-first century, up to 84% (for scenario SSP245) and 97% (for scenario SSP585) of the TP could be afflicted by TWS deficits. Our findings indicate a trajectory towards unsustainable water systems in HMA that could exacerbate downstream water stress.
Assuntos
Altitude , Mudança Climática , Dessecação , Previsões , Abastecimento de Água , Humanos , Ásia , Mudança Climática/estatística & dados numéricos , Abastecimento de Água/estatística & dados numéricos , Tibet , Congelamento , Neve , Imagens de Satélites , Chuva , Oceano Atlântico , Camada de Gelo , Conservação dos Recursos HídricosRESUMO
Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1 that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops.
Assuntos
Brassinosteroides , Congelamento , Regulação da Expressão Gênica de Plantas , Metiltransferases , Proteínas de Plantas , Ácido Salicílico , Transdução de Sinais , Triticum , Triticum/genética , Triticum/fisiologia , Triticum/metabolismo , Brassinosteroides/metabolismo , Brassinosteroides/farmacologia , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genéticaRESUMO
Particular frost patterns on natural leaves had prompted Yao et al. [Y. Yao et al., Proc. Natl. Acad. Sci. U.S.A. 117, 6323-6329 (2020)] to investigate the underlying physics. Their work revealed why on corrugated surfaces ice forms on crests and dries out adjacent grooves. In the absence of frost, in contrast, grooves tend to constitute niches on a leaf where microorganisms are less limited by moisture than in other locations. Here, we show that microorganisms able to nucleate ice before it forms on crests can modify the frosting pattern to their advantage. This ability might drive in cold arid environments the association between certain microorganisms and plants.
Assuntos
Congelamento , Folhas de Planta , GeloRESUMO
Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.
Assuntos
Gelo , Água , Congelamento , Temperatura , Proteínas da Membrana Bacteriana Externa/metabolismoRESUMO
Cryogenic electron microscopy (cryo-EM) can reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by focused-ion-beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage it causes to a thinned cell section has not yet been determined. We recently described an approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here, we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This layer of damage limits the recovery of information for in situ structural biology. We find that the mechanism of FIB milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB milling damage, we estimate that FIB milling damage with current protocols will negate the potential improvements from lamella thinning beyond 90 nm.
Assuntos
Gálio , Microscopia Eletrônica , Congelamento , Elétrons , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.
Assuntos
Proteínas Anticongelantes , alfa-Fetoproteínas , Cristalização , Proteínas Anticongelantes/química , Congelamento , CrioprotetoresRESUMO
The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach integrating both methods with near-native ultrastructural preservation is currently lacking. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.
Assuntos
Dictyostelium , Mycobacterium marinum , Mycobacterium , Dictyostelium/metabolismo , Dictyostelium/microbiologia , Congelamento , Microscopia EletrônicaRESUMO
The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Resposta ao Choque Frio/fisiologia , Membrana Celular/metabolismo , Temperatura Baixa , Congelamento , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Thawing of viably frozen human tissue T cells, ILCs, and NK cells and subsequent single-cell RNA sequencing reveals that recovery of cellular subclusters is variably impacted. While freeze-thawing does not alter the transcriptional profiles of cells, it upregulates genes and gene pathways associated with stress and activation.
Assuntos
Células Matadoras Naturais , Linfócitos T , Humanos , Congelamento , Análise de Sequência de RNARESUMO
In recent decades, meltwater runoff has accelerated to become the dominant mechanism for mass loss in the Greenland ice sheet1-3. In Greenland's high-elevation interior, porous snow and firn accumulate; these can absorb surface meltwater and inhibit runoff4, but this buffering effect is limited if enough water refreezes near the surface to restrict percolation5,6. However, the influence of refreezing on runoff from Greenland remains largely unquantified. Here we use firn cores, radar observations and regional climate models to show that recent increases in meltwater have resulted in the formation of metres-thick, low-permeability 'ice slabs' that have expanded the Greenland ice sheet's total runoff area by 26 ± 3 per cent since 2001. Although runoff from the top of ice slabs has added less than one millimetre to global sea-level rise so far, this contribution will grow substantially as ice slabs expand inland in a warming climate. Runoff over ice slabs is set to contribute 7 to 33 millimetres and 17 to 74 millimetres to global sea-level rise by 2100 under moderate- and high-emissions scenarios, respectively-approximately double the estimated runoff from Greenland's high-elevation interior, as predicted by surface mass balance models without ice slabs. Ice slabs will have an important role in enhancing surface meltwater feedback processes, fundamentally altering the ice sheet's present and future hydrology.
Assuntos
Camada de Gelo , Modelos Teóricos , Congelamento , Aquecimento Global , GroenlândiaRESUMO
About 800 million people depend in part on meltwater from the thousands of glaciers in the high mountains of Asia. Water stress makes this region vulnerable to drought, but glaciers are a uniquely drought-resilient source of water. Here I show that seasonal glacier meltwater is equivalent to the basic needs of 221 ± 59 million people, or most of the annual municipal and industrial needs of Pakistan, Afghanistan, Tajikistan, Turkmenistan, Uzbekistan and Kyrgyzstan. During drought summers, meltwater dominates water inputs to the upper Indus, Aral and Chu/Issyk-Kul river basins. This reduces the risk of social instability, conflict and sudden migrations triggered by water scarcity, which is already associated with the large, rapidly growing populations and hydro-economies of these basins. Regional meltwater production is, however, unsustainably high-at 1.6 times the balance rate-and is expected to increase in future decades before ultimately declining. These results update and reinforce a previous publication in Nature on this topic, which was retracted after an inadvertent error was discovered.
Assuntos
Secas , Congelamento , Camada de Gelo/química , Abastecimento de Água/estatística & dados numéricos , Aclimatação , Ásia , Desidratação/prevenção & controle , Ecossistema , Humanos , Política , Chuva , Rios , Estações do Ano , TemperaturaAssuntos
Criopreservação , Congelamento , Refrigeração , Gerenciamento do Tempo , Criopreservação/economia , Criopreservação/instrumentação , Criopreservação/métodos , Refrigeração/economia , Refrigeração/instrumentação , Refrigeração/métodos , Gerenciamento do Tempo/economia , Gerenciamento do Tempo/métodosRESUMO
Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1's efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.
Assuntos
Criopreservação , Crioprotetores , Rim , Organoides , Criopreservação/métodos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Humanos , Rim/citologia , Crioprotetores/farmacologia , Vitrificação , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Congelamento , Sobrevivência Celular/efeitos dos fármacosRESUMO
Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this "delta-melt" approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.
Assuntos
DNA , Conformação de Ácido Nucleico , RNA , Sequência de Bases , DNA/química , Congelamento , RNA/química , Termodinâmica , Raios UltravioletaRESUMO
Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.