Calpain-3 is an intracellular Ca2+-dependent cysteine protease abundant in skeletal muscle. Its physiological role in the sarcomere is thought to include removing damaged muscle proteins after exercise. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These mutations, of which there are over 500 in humans, are spread all along this 94-kDa multi-domain protein that includes three 40+-residue sequences (NS, IS1, and IS2). The latter sequences are unique to this calpain isoform and are hypersensitive to proteolysis. To investigate the whole enzyme structure and how mutations might affect its activity, we produce the proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in E. coli. During size-exclusion chromatography, this calpain-3 was consistently eluted as a much larger 0.5-MDa complex rather than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS, Blue Native PAGE, and AUC, made the complex amenable to single-particle cryo-EM analysis. From two data sets, we obtained a 3.85-Å reconstruction map that shows the complex is a trimer of calpain-3 dimers with six penta-EF-hand domains at its core. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When this 37-kDa region of titin was co-expressed with calpain-3 the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to several copies of the titin fragment. We suggest that newly synthesized calpain-3 is kept as an inactive hexamer until it binds the N2A region of titin in the sarcomere, whereupon it dissociates into functional dimers.

Vibrio cholerae, the causative agent of cholera, uses surface proteins such as the repeats-in-toxin (RTX) adhesin FrhA to colonize hosts and initiate infection. Blocking bacterial adhesion represents a promising therapeutic strategy to treat infections without promoting drug resistance. FrhA contains a peptide-binding domain (PBD) that is key for hemagglutination, human epithelial cell binding, and V. cholerae biofilm formation. Previous studies identified a lead pentapeptide ligand with the sequence Ala-Gly-Tyr-Thr-Asp (AGYTD) that blocks V. cholerae colonization of the mouse small intestine at high micromolar concentrations. In this study, a structure-guided approach identified a minimal D-amino acid-containing tripeptide motif with higher affinity for the FrhA-PBD and predicted metabolic stability. Our results contribute to the development of anti-adhesion strategies to combat infections. Impact statement Our study elucidates the molecular basis of peptide recognition by the Vibrio cholerae adhesin FrhA and develops minimal D-amino-acid peptides that block adhesion with nanomolar affinity. These findings advance understanding of RTX adhesins and provide a structural blueprint for next-generation anti-adhesion therapeutics against cholera and related infections.

Magnetotactic bacteria (MTB), inherently motile and self‐powered, are promising biorobotic candidates for targeted anti‐cancer drug delivery since they can actively deliver the therapeutic agent to the tumor, decreasing adverse side effects. However, the directed navigation of these bacteria through intricate microenvironments mimicking the natural microvasculature has not been investigated. Here, the directed navigation of MTB is demonstrated within a vasculature‐on‐a‐chip platform. A perfusable vascular network is developed to investigate MTB at the single‐microorganism level. MTB is demonstrated to successfully align and navigate along the magnetic field inside the microvessels. Surface interaction with the microvessel walls, hydrodynamic forces, and counterdirectional flows in the order of 10 µm∙s −1 are examined as potential factors that may interfere with the MTB alignment and magnetotaxis. The average swimming speed of the studied bacteria within the vasculature‐on‐a‐chip device is 13.9 µm s −1 . Finite Element Analysis reveals that under these conditions, MTB experience shear stresses of up to 30 Pa, and drag forces between 10 and 40 pN, depending on their relative orientation to the flow field. Altogether, this work provides a first demonstration of effective directed navigation of MTB in a vasculature‐on‐a‐chip platform, and the influence of external factors on their field alignment and magnetotactic behavior.

Spiders are among the most important natural enemies of pests in orchards. Clubiona spp. are active even during winter, when other pest predators are dormant. Such activity is possible because these spiders produce antifreeze proteins (AFPs) that bind to the surface of ice crystals to prevent their growth at subzero temperatures. To investigate their AFPs, we collected Clubiona spiders from a pome fruit orchard in the Czech Republic on subzero days in December and February. The AFPs were isolated through five successive rounds of ice‐affinity purification as a family of ~30‐kDa isoforms. Tryptic fragments from these AFPs were sequenced using tandem mass spectrometry and corresponding transcripts were obtained from an Illumina metatranscriptome. Homologs were absent from the GenBank protein database. Protein modelling with AlphaFold2 shows that these spider AFPs form a previously unseen β‐solenoid with a flat surface populated by arrays of threonine (Thr) residues. Although these features are like those of the β‐solenoid AFPs from beetles and moths, the proteins are not homologous. This is a remarkable example of convergent evolution of proteins at both the structural and sequence levels for the task of binding ice.

ABSTRACT Bacteria often make initial contact with their hosts through the ligand-binding domains of large adhesin proteins. Recent analyses of repeats-in-toxin (RTX) adhesins in Gram-negative bacteria suggest that ligand-binding domains can be identified by the way they emerge from “split” domains within the adhesin. Here, using this criterion and an AlphaFold3 model of a 5047-residue RTX adhesin from Aeromonas hydrophila, we identified three different ligand-binding domains in this fibrillar protein. The crystal structures of the two novel domains were solved to 1.4 and 1.95 Å resolution, respectively, and demonstrate excellent agreement with their modeled structures. The other domain was recognized as a carbohydrate-binding module based on its beta-strand topology and confirmed by its micromolar affinity for fucosylated glycans, including the Lewis B and Y antigens. This lectin-like module, which was recombinantly produced with its companion split domain and nearby extender domain, bound to a wide variety of cells including yeasts, diatoms, erythrocytes, and human endothelial cells. In each case, 50 mM free fucose prevented this binding and may offer some protection from infection. The carbohydrate-binding module with its neighboring domains also caused aggregation of yeast and erythrocytes, which was again blocked by the addition of free fucose. The second putative ligand-binding domain has a beta-roll structure supported by a parallel alpha-helix, and the third is a homolog of a von Willebrand Factor A domain. These two domains bind to a more limited range of cell types, and their ligands have yet to be identified.IMPORTANCECharacterizing the ligand-binding domains of fibrillar adhesins is important for understanding how bacteria can colonize host surfaces and how this colonization might be blocked. Here, we show that the opportunistic pathogen, Aeromonas hydrophila, uses a carbohydrate-binding module (CBM) to attach to several different cell types. The CBM is one of three ligand-binding domains at the distal tip of the adhesin. Identifying the glycans bound by the CBM as Lewis B and Y antigens has helped explain the range of cell types that the bacterium will bind and colonize, and it has suggested sugars that might interfere with these processes. Indeed, fucose, which is a constituent of the Lewis B and Y antigens, is effective at 50 mM concentrations in blocking the attachment of the CBM to host cells. This will lead to the design of more effective inhibitors against bacterial infections.

Calpain-1 and calpain-2 are heterodimeric proteases consisting of a common small regulatory subunit CAPNS1 and a large catalytic subunit, CAPN1 or CAPN2, respectively. These calpains have emerged as potential therapeutic targets in cancer and other diseases through their roles in cell signaling pathways affecting sensitivity to chemotherapeutic and targeted drugs and in promoting metastasis. While inhibition of calpains has the potential to provide therapeutic benefit to cancer patients, there are currently no clinically approved active site-directed drugs that specifically and effectively inhibit them. However, the structures of calpain-1 and calpain-2 make them susceptible to allosteric inhibition aimed at interfering with heterodimerization of the catalytic and regulatory subunits, which is necessary for stability and proteolytic activity. Split-Nanoluciferase biosensors were generated to quantify the protein-protein interactions between the calcium-binding penta-EF-hand domains of CAPN1 or CAPN2 and CAPNS1. These biosensors were used to quantify the heterodimer dissociation constants (KD) of calpain-1 and calpain-2, estimated at 185 nM and 509 nM, respectively, in the presence of 5 mM Ca2+; and 362 nM and 1651 nM, respectively, in the presence of Mg2+. The half-maximal Ca2+ concentrations supporting these protein-protein interactions for calpain-1 and calpain-2 were 59.9 μM and 940.8 μM, respectively. Molecular modeling, based on the crystal structure of calpain-2, was used to predict 20 residues of the penta-EF-hand domains that contribute to heterodimerization. Individual point mutation of CAPNS1 at Q263 reduced the catalytic activity of calpain-2 to 51.0 ± 6.4% in live cells.

Dysregulated calpain-1 and calpain-2 protease activity linked to several diseases has encouraged efforts to explore inhibiting calpain to provide therapeutic benefits. However, there are currently no clinically approved drugs that specifically target calpain functionality. To address this unmet need, we carried out in silico drug discovery efforts to identify small molecules capable of modulating calpain activity. Our approach is based on the observation that heterodimer formation of the calpain-2 catalytic (CAPN2) and regulatory (CAPNS1) subunits is needed for both proteolytic activity and CAPN2 stability. In recognition of this obligate protein-protein interaction (PPI), the CAPN2-CAPNS1 interface was targeted with nearly 3.6 million small molecules to find candidates that bind at the interface with high affinity and introduce steric clashes capable of altering heterodimerization or the conformation of CAPN2, thereby modulating proteolytic activity. Twenty small molecules predicted to disrupt the most hydrogen bonds at the CAPN2-CAPNS1 interface were validated experimentally. Five small molecules inhibited calpain activity by 53.6 ± 4.1, 36.8 ± 38.3, 31.1 ± 17.5, 69.8 ± 27.3, and 47.1 ± 18.5%, while two enhanced protease activity by 163.0 ± 41.9 and 129.2 ± 11.9%. Unexpectedly, the effects of these seven molecules on the CAPN2-CAPNS1 PPI assay did not correlate with their effects on protease activity. Molecular simulation showed that small molecules that modulate calpain activity without abolishing heterodimerization do so by altering the conformational changes needed for the activity. This apparent allosteric mechanism paves the way for developing novel therapeutic solutions for modulating the calpain activity in various diseases associated with calpain dysregulation.

Gram-negative bacteria attach to host surfaces using ligand-binding domains (LBDs) at the distal tips of fibrillar RTX adhesins. Blocking the initial binding interaction(s) can potentially prevent colonization and subsequent biofilm formation and infection. To this end, adhesins must be identified, and it is also essential to determine the dominant adhesin type for those species that have more than one. RTX adhesins are frequently the largest proteins within each species (ranging from 1500 to 15,000 aa) and are often misannotated as incomplete/pseudogene products because their highly repetitive nature confounds genome assemblies from short-read technologies. Our bioinformatic process collates predicted proteins from long-read assemblies, which are then clustered based on the similarity of their C-terminal regions where the LBDs are typically located. RTX adhesins are identified by their length and domain structure and are modelled using AlphaFold3. An exhaustive search of multiple strains from seven species revealed a total of 35 different RTX adhesins that map to 16 different loci, with differing arrangements of LBDs that include putative carbohydrate-binding modules and von Willebrand Factor A-like domains. Notably, similar adhesins are sometimes found in multiple species, either by descent or through DNA uptake, and three species have an RTX adhesin of uncertain function because it lacks an obvious LBD. Many bacteria initiate infection by reaching out with large, complex proteins called adhesins to attach themselves to a host cell. The DNA sequences of adhesins are difficult to read, due to their length and repetitiveness. This study leverages recent technological advances like “long-read sequencing” and structural modelling to identify and characterize the adhesins in seven species of harmful bacteria: Acinetobacter baumannii , Aeromonas hydrophila , Aeromonas salmonicida , Bordetella parapertussis , Legionella pneumophila , Vibrio parahaemolyticus , and Vibrio vulnificus . We identified thirty-five unique versions of adhesins and demonstrated their mix-and-match architecture. This research provides a foundation for strategies to block bacteria from binding surfaces, offering a vital alternative treatment as antibiotic resistance continues to rise.

In this method, a bacterial lectin is allowed to bind to its target polysaccharide in the form of a matrix suspension that can be recovered by centrifugation or filtration. After an initial test to show that the lectin is fully bound to the matrix, a measured amount of competing free sugar is added to the matrix resuspended in buffer. After mixing, the suspension is centrifuged, and the protein content of the supernatant fraction is measured before being returned to the matrix with the addition of another aliquot of sugar. The cycle of mixing, centrifugation, and quantification of protein (lectin) in the supernatant fraction is continued until the released protein reaches plateau levels. The procedure is repeated with a variety of different sugars to compare their efficiencies at binding and displacing the lectin from its polysaccharide ligand. This procedure is illustrated here using a PA14-type bacterial lectin that has affinity for fucosylated glycans and will, as a result, bind to dextran-based resins like Superdex 200 (S200). A second example features the release of Escherichia coli maltose-binding protein from amylose resin by mono- and disaccharides. The purpose of this assay is to determine the relative binding affinity of different sugars for a bacterial lectin.

Fishing gears are designed to catch marine species, but when lost at sea, they can continue to trap and kill aquatic life, a phenomenon known as ghost fishing. This study evaluated the use of a biodegradable twine made from poly(butylene succinate)/poly(butylene adipate terephthalate) (PBS/PBAT) in trawl fishing. The assessment included mechanical testing, trawl modelling, and seawater ageing simulations to explore potential loss scenarios. Mechanical tests indicated that the PBS/PBAT braid was about half as strong as high-density polyethylene (HDPE) but was suitable for braiding and netting. After 3 years in seawater, PBS/PBAT monofilaments showed biotic degradation, with strength losses of 20 % at 15 °C and 80 % at 25 °C. This suggests that gear made from this polymer would degrade faster than conventional gear if lost. Trawl modelling further demonstrated that trawl performance was only slightly affected by material change and ageing, highlighting the potential for reducing ghost fishing with less persistent twine.

Alanine-rich, alpha-helical type I antifreeze proteins (AFPs) in fishes are thought to have arisen independently in the last 30 Ma on at least four occasions. This hypothesis has recently been proven for flounder and sculpin AFPs, which both originated by gene duplication and divergence followed by substantial gene copy number expansion. Here, we examined the origins of the cunner (wrasse) and snailfish (liparid) AFPs. The cunner AFP has arisen by a similar route from the duplication and divergence of a GIMAP gene. The coding region for this AFP stems from an alanine-rich region flanking the GTPase domain of GIMAPa. The AFP gene has remained in the GIMAP gene locus and has undergone amplification there along with the GIMAPa gene. The AFP gene originated after the cunner diverged from its common ancestor with the closely related spotty and ballan wrasses, which exhibit similar gene synteny but lack AFP genes. Snailfish AFPs have also recently evolved because they are confined to a single genus of this family. In these AFP-producing species, the AFP locus does not share any similarity to functional genes. Instead, it is replete with repetitive DNAs and transposons, several stretches of which could encode alanine tracts with a dominant codon (GCC) that matches the bias observed in the AFP genes. All four known instances of type I AFPs occurring in fishes are independent evolutionary events that occurred soon after the onset of Northern Hemisphere Cenozoic glaciation events. Collectively, these results provide a remarkable example of convergent evolution to one AFP type.

Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins.

Antifreeze proteins (AFPs) are found in a variety of marine cold‐water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine‐rich α‐helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate‐ and glutamine‐rich segment, was converted to an alanine‐rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine‐rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly β-sandwich extender modules, but other domains like a β-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

The phenomenon of marine plastic pollution is now well-established, with documented impacts on marine biodiversity and biogeochemical cycles. In order to mitigate this environmental impact, a significant amount of research has been conducted in recent years with the objective of developing biodegradable alternatives to conventional polymers and their composites in marine environments. The findings of this research significantly enhanced our understanding of biodegradation mechanisms and identified promising candidates. However, the majority of these studies have been conducted in coastal marine environments, which represent a minor component of the marine ecosystem. Recent models on the transport of plastic debris in the oceans indicate that deep-sea environments are likely to be the ultimate sink for a significant proportion of plastics entering the oceans. The aim of this review is to provide an overview of the processes of biodegradation of polymers in these deep-sea environments. The diversity and specific characteristics of these environments with respect to degradation mechanisms are discussed. While the majority of deep-sea conditions are not conducive to biodegradation, studies on organic falls (wood and whale carcasses) and a few investigations into materials previously shown to be biodegradable in coastal marine environments demonstrate mechanisms that are similar to those observed in shallow waters. Nevertheless, further research is necessary to reach definitive conclusions. It is essential to extend these studies to a broader range of deep-sea environments. Additionally, new methodologies that integrate microbiology and polymer science are required to accurately assess the process of assimilation of these materials in these environments.

In order to reduce the contamination of marine ecosystems by plastic materials, the scientific community is engaged in the development of biodegradable substitutes for conventional plastics. While certain candidates have been successfully tested in coastal marine environments, the degradation process in deep-sea environments remains poorly understood. This study examined the degradation of two industrial biopolyesters, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a polybutylene-succinate (PBS), in two deep marine environments of the Middle and Eastern Atlantic, at depths of 780 and 1740 m, as well as under laboratory conditions under hydrostatic pressure and without micro-organisms. The findings reveal a considerable biodeterioration of PHBV and a pronounced influence of flax fibre reinforcement on the degradation mechanisms. Conversely, PBS exhibits minimal to no indications of degradation. Additionally, the results confirm that biotic factors are the primary determinants of the degradation processes, with no degradation observed under abiotic conditions.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows.

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temper-atures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 & DEG;C) and at-6 & DEG;C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 & DEG;C, those stored at-6 & DEG;C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.