Pab1 has recently been shown to be a key element in this response in fission yeast, where it regulates cell cycle progression and sexual differentiation. Importantly, a recent study from our group has shown that PP2A-B55Pab1 acts as a mediator between the activities of the two TOR signaling modules, enabling a crosstalk that is required to engage in the differentiation program. In this review, we recapitulate the studies that have led to our current understanding of the interplay between TOR complexes. Moreover, we discuss several aspects of the response to nitrogen availability that still require further attention, and which will be important in the future to fully realize the implications of phosphatase activity in the context of TOR signaling.The control of cell fate, growth and proliferation in response to nitrogen availability is a tightly controlled process, with the two TOR complexes (TORC1 and TORC2) and their effectors playing a central role. PP2A-B55 Saccharomyces cerevisiae  leads to different outcomes. Thus, while deletion of Raptor impairs adipogenic differentiation and enhances osteogenic differentiation, deletion of Rictor has the opposite effect  to enhance TORC1 activity in detriment of TORC2. Failure to favor the balance towards TORC1 brings about epidermal defects that can be rescued by specific inhibition of TORC2 or enhancement of TORC1 . In the case of TORC1, stimulation of PI3K leads to the phosphorylation of AKT on its activation loop (Thr308) by PDK1  displayed phenotypes reminiscent of rapamycin treatment   regulate entry into quiescence through PP2A-B55Cdc55 , phosphorylation of Gad8 Ser546 gradually increased in parallel to Igo1 phosphorylation. This was largely prevented in igo1-deleted cells : Ser527 in the turn motif and Thr387 in the activation loop  we could detect basal Gad8 Ser546 phosphorylation. This can be understood as the result of a lower catalytic efficiency in the dephosphorylation reaction compared to the phosphorylation by TORC2. Yet, it could also indicate that the entire population of Gad8 is not available for dephosphorylation by PP2A-B55Pab1. PP2A-B55Pab1 localizes throughout the cell, but it is enriched in the nucleus. Gad8 localization has been rather contentious in the field. Previous studies had shown it to be primarily cytoplasmic  protects it from degradation Jiang . WhetherPab1 in the regulation of sexual differentiation, but is it the only relevant one? Most likely there are other substrates of PP2A-B55Pab1 that also contribute to the phenotype of enhanced differentiation that we observed in a pab1Δ mutant. Mutation of Gad8 Ser546 to Ala completely blocked the mating response, both in the presence or absence of PP2A-B55Pab1. However, it reduced but did not completely block expression of mei2, suggesting that other proteins also contribute to this effect. In budding yeast, the Greatwall-Endosulfin pathway has been shown to regulate the expression of stress and stationary phase genes during a diauxic shift through the inhibition of PP2A-B55Cdc55  has also been identified in a screen for mutants resistant to TORC1 inhibition (Rallis et al. Finally, what other protein phosphatases participate in the differentiation response of fission yeast? Clearly, the Tap42 and Ppe1 roles are worth revisiting. Moreover, a second PP2A complex (PP2A-B56The two TOR complexes are key mediators of nutritional signaling with seminal implications in cell differentiation, proliferation and metabolism. In this review, we have summarized their multiple functions and interplay, with special focus on the role of phosphatases in these events. Great challenges still lie ahead, but the latest findings bring us one step closer to fully comprehending the significance of protein phosphatases in the TOR-mediated cellular responses.


However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant “desensitization” of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a “sensitized intermediate” state that is in effect primed to trigger PKAc activity.cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG)  XS) that is phosphorylated by PKAc, RI subunits have a “pseudosubstrate” IS (RRX(A/G)) that is unable to be phosphorylated by PKAc; therefore, substrate competition is required to trigger full PKA holoenzyme dissociation  (–101 residue (P + 2 to the pseudosubstrate IS) was shown to be an in vitro substrate of cGMP-dependent protein kinase (PKG) 1–5–9–ociation . Additiod Ser101 A 18–20)–20XS) thse (PKG) , 22. Giv101 might lead to changes in type I PKA activity. The protein structure of the RIα–PKAc heterodimer was solved by X-ray crystallography techniques by Kim et al. (2ATP) presents a kinetic barrier for activation, whereas this is not critical for RII subunits capable of phosphorylation by PKAc . Upon analysis of polar interactions in this region, one can see that the hydroxyl moiety of Ser101 makes hydrogen bonds with residues from the αC-helix of PKAc (particularly residues Gln84 and His87). It is inferred that introduction of a phosphate group at this position will introduce steric hindrance that will likely alter the interaction of these residues. Furthermore, the interaction of Ser101 with Gln84 and His87 facilitates a hydrogen-bond network near the activation-loop phosphorylation site Thr197 in PKAc. Thus, we can further postulate that phosphorylation of Ser101 will also bring about a negative charge–charge repulsion effect caused by the juxtaposition of two phosphate groups within this binding interface. These effects in combination would lend to opening of the active site cleft of PKAc. Taking these observations under consideration, we hypothesized that phosphorylation of Ser101 by PKG would promote enhanced PKAc activity by removing the additional requirement of substrate competition in RIα holoenzyme activation. Finding evidence of direct PKG–PKA cross-talk in the context of cellular signaling would aid our understanding of how these two related pathways could regulate physiologically relevant systems where both proteins are expressed (i.e. smooth muscle and cardiac tissues)  , 26.in vitro phosphorylation reactions using purified recombinant bovine RIα (bRIα)–PKAc holoenzyme and PKGIα. The reactions contained [γ-32P]ATP, and phosphate incorporation was determined by autoradiography. We observed robust phosphorylation of bRIα only in the presence of PKGIα . RIα was phosphorylated by PKGIα to a similar extent in the absence and presence of cyclic nucleotides (lanes 5–8). These results are a bit unexpected, because cyclic nucleotide free and cAMP-bound PKGIα should not be as active as the cGMP-bound kinase. However, under the conditions used, it is likely that PKGIα activity was driven by PKGIα autophosphorylation and was cyclic nucleotide–independent. We originally reasoned that cAMP would disassociate bRIα from PKAc and thus provide easier access for PKGIα to phosphorylate Ser101; however, although there was a trend toward increased RIα phosphorylation in the presence of cAMP, the difference did not reach significance (compare lane 5 with 7 and 6 with 8). Thus, it appears that holoenzyme association/dissociation has no effect on PKGIα's access to the phosphorylation site.To extend previous reports demonstrating that PKA RIα could be phosphorylated by PKGIα, we performed 32P]orthophosphate, and the differences between WT and S101A-mutant RIα phosphorylation were compared in the presence or absence of PKGIα, as well as with and without 2-h stimulus with 8-CPT-cGMP . In cells transfected with WT RIα alone, we observed a small amount of basal RIα phosphorylation, which was slightly increased by treatment with 8-CPT-cGMP . In cells co-transfected with PKGIα, treatment with 8-CPT-cGMP induced a much higher level of RIα phosphorylation, which was dramatically reduced in cells transfected with PKGIα and S101A-mutant RIα . The lower panel of Fig. 2C is an immunoblot using anti-FLAG antibodies, showing an equal expression of transfected FLAG-tagged RIα in all samples tested. Our results indicate that RIα Ser101 is a specific phosphorylation target of PKGIα in cells.Next, we examined whether PKGIα could phosphorylate RIα in intact cells. HEK293T cells were transfected with expression vectors for PKGIα and FLAG-tagged WT or S101A-mutant human RΙα. The cells were labeled with  and hRIa[S101E]) were introduced by site-directed mutagenesis via QuikChange® PCR as per the manufacturer's instructions . The same genetic material was also incorporated into pRSET-Xa3 vector for bacterial recombinant protein expression. Point mutations (bRIa[S101A] and bRIa[S101E]) were introduced into both constructs by site-directed mutagenesis via QuikChange® PCR as per the manufacturer's instructions (Stratagene). FLAG-tagged RIα was constructed by PCR using an untagged RIα vector as a template. The PCR product was digested and inserted into the pFLAG-D expression vector. The S101A and S101E mutant RIα constructs were generated using overlapping extension PCR. All constructs that went through a PCR step were sequenced to ensure that the expected coding sequence was present.E. coli and purified as described (m MES (pH 6.5), 100 mm NaCl, 2 mm EGTA, 2 mm EDTA, and 5 mm DTT. RIα proteins were further purified on a gel filtration column using Superdex 200 and concentrated for purposes of biochemical assays using Amicon® centrifugal filters .The C-subunit was expressed in escribed . Full-lem HEPES (pH 7.4), 10 mm MgCl2, 10 mm β-glycerol phosphate, 100 mm ATP, 0.05 mCi of [γ-32P]ATP, and 1 μm of the indicated cyclic nucleotides. The reactions were incubated at 30 °C for 2 h. The reactions were terminated by adding 20 μl of 2× SDS-PAGE loading buffer containing 100 mm EDTA followed by boiling. Phosphate incorporation was determined by SDS-PAGE/autoradiography. Densiometric analysis was performed using ImageJ.To examine RIα–PKAc holoenzyme phosphorylation by PKGIα, purified PKGIα  was addeHEK293T and mouse embryonic fibroblast (MEF) cells were used throughout this study. Propagating cultures were grown in 10-cm dishes using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. MEF cultures contained 1% penicillin/streptomycin, unless otherwise stated.2 atmosphere. The day before transfection, the cells were spit into a 6-well cluster dish such that the cells were 90–95% confluent at the time of transfection. The cells were transfected with the indicated expression constructs with Lipofectamine 2000 using conditions recommended by the manufacturer (Thermo Fisher). The next day, the medium was removed, and 1 ml of phosphate-free media and 100 μCi of 32PO4 was added to each well. The cells were incubated at 37 °C for 2 h, and then 250 μm 8-CPT-cGMP was added to the appropriate wells. The cells were incubated for an additional 2 h at 37 °C, at which point the medium was removed, the cells were washed once with ice-cold PBS, and the cells were lysed on the plate in PBS, 0.1% Nonidet P-40, 1× protease inhibitor mixture , and 1× HALT phosphatase inhibitor . Cleared lysates were added to 20 μl of anti-FLAG affinity gel and incubated for 1 h at 4 °C with constant mixing. The beads were washed and boiled in 30 μl of 1× SDS sample buffer, and phosphorylation was analyzed by SDS-PAGE/autoradiography. The relative amount of immunoprecipitated FLAG-tagged RIα was determined by immunoblotting.HEK293T cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a 37 °C incubator with a 5% COi.e. 20 μm forskolin and 100 μm IBMX). 10 μl of cell lysate  was added to 5 μl each of “5× peptide,” “5× buffer,” and distilled water for all reactions. The samples were mixed and incubated at room temperature for 30 min and then heat-inactivated at 95 °C for 10 min. For recombinant protein experiments: RIα and PKAc proteins were incubated in situ at a 1.2:1 molar stoichiometry within a 10-μl volume (for RIα(1–379) dimer protein, 5 μl of 1.2 μm R-subunit was added to 5 μl of 2 μm PKAc). Then 5 μl each of 5× peptide, 5× buffer, and either 5 mm cAMP or distilled water was added to start the reactions. Samples were mixed and incubated at room temperature for 30 min and then heat-inactivated at 95 °C for 10 min. For all experiments, phosphopeptides were separated by 0.8% agarose gel as per the manufacturer's directions. Densiometric analysis of gel images was performed using ImageJ. Activity is expressed as the percentage of phosphorylated peptide, calculated by the ratio of phosphorylated peptide signal divided by total signal.Assays using both cell lysates and recombinant proteins were performed in a modified procedure from the manufacturer's directions. For cell-based experiments, MEF cell samples were plated on 6-well dishes at passage 3. Transfections were conducted using Lipofectamine 2000® (Thermo Fisher) as per the manufacturer's directions. For activity experiments, MEF cells were serum-starved for 2 h before conducting 10 min of Fsk/IBMX stress treatment . Protein concentrations were normalized between samples, and then SDS-PAGE was performed using NuPAGE 4–12% gradient gels  in MES running buffer . The gels were resolved at 150 V for 70 min and then subsequently transferred to 0.45-μm nitrocellulose membranes (200 mA/blot for 60 min). The membranes were blocked using 5% milk protein (or with 5% BSA for phosphoblots) in 1× PBS, 0.05% Tween for 60 min at room temperature, and then primary antibodies were incubated overnight at 4 °C in 1× PBS, 0.05% Tween 20 or 1× TBS, 0.1% Tween 20 at the concentrations specified by the manufacturer. Densiometric analysis was performed using ImageJ.SDS-PAGE samples were prepared using 5× Laemmli buffer  and RIα tagged with F2 fragment (RIα-F2) were generated previously . S101E am MOPS, pH 7.0, 35 mm NaCl, 10 mm MgCl2, 1 mm ATP, 2 mm DTT, and 0.005% Triton X-100). End-point fluorescence polarization measurements were performed using a GENios Pro® plate-reader spectrometer , configured with 485-nm excitation and 535-nm emission filters and using optimal gain settings. Concentrations of proteins, [5/6-FAM]-IP20, and cAMP for each experimental method are listed below: for cAMP response experiments, 12.0 nm PKA catalytic subunit, 14.4 nm PKA regulatory subunit bRIα (91–379) or bRIα (1-379) (based on monomer concentration), 2.0 nm [5/6-FAM]-IP20, 1 nm-1 μm cAMP (dissolved in 1× buffer without ATP or DTT); and for R-subunit inhibition response experiments, 10.0 nm PKA catalytic subunit, 1 nm-1 μm PKA regulatory subunit bRIα(91–379) or bRIα(1–379) (based on monomer concentration), 1.67 nm [5/6-FAM]-IP20.Use of the LiReC assay has been described previously . All reat test of planned comparisons.All activity, immunoblot, and PCA data are presented as means ± S.D., and all fluorescence polarization assay data are presented as means ± S.E. GraphPad Prism 4 software  was used for all statistical analysis. Statistical analyses were performed by unpaired Student's K. J. H. designed and conducted most of the experiments, built expression constructs, generated recombinant proteins, analyzed the results, and wrote most of the paper. D. E. C. built expression constructs, conducted radioactive phosphorylation and co-immunoprecipitation experiments, and analyzed results. A. R. conducted protein complementation assay experiments and analyzed results. E. S. conducted protein complementation assay experiments, analyzed results, and provided materials for the study. H. H. P. provided material and facilities for the study and wrote the paper with K. J. H. S. S. T. conceived the idea for the project, provided material and facilities for the study, and wrote the paper with K. J. H.


In most industrialized countries, allergies have increased in frequency quite dramatically during the past 50 years. Estimates show that 20–30% of the populations are affected. Allergies have thereby become one of the major medical challenges of the twenty-first century. Despite several theories including the hygiene hypothesis, there are still very few solid clues concerning the causes of this increase. To trace the origins of allergies, we have studied cells and molecules of importance for the development of IgE-mediated allergies, including the repertoire of immunoglobulin genes. These studies have shown that IgE and IgG most likely appeared by a gene duplication of IgY in an early mammal, possibly 220–300 million years ago. Receptors specific for IgE and IgG subsequently appeared in parallel with the increase in Ig isotypes from a subfamily of the recently identified Fc receptor-like molecules. Circulating IgE levels are generally very low in humans and laboratory rodents. However, when dogs and Scandinavian wolfs were analyzed, IgE levels were found to be 100–200 times higher compared to humans, indicating a generally much more active IgE synthesis in free-living animals, most likely connected to intestinal parasite infections. One of the major effector molecules released upon IgE-mediated activation by mast cells are serine proteases. These proteases, which belong to the large family of hematopoietic serine proteases, are extremely abundant and can account for up to 35% of the total cellular protein. Recent studies show that several of these enzymes, including the chymases and tryptases, are old. Ancestors for these enzymes were most likely present in an early mammal more than 200 million years ago before the separation of the three extant mammalian lineages; monotremes, marsupials, and placental mammals. The aim is now to continue these studies of mast cell biology and IgE to obtain additional clues to their evolutionary conserved functions. A focus concerns why the humoral immune response involving IgE and mast cells have become so dysregulated in humans as well as several of our domestic companion animals. During the past 50 years, allergies has increased in prevalence quite dramatically and in most industrialized countries 20–30% of the population are affected. In some school classes, the percentage of affected children can reach as high as 50. Allergies have thereby become one of the major medical challenges of the twenty-first century. Although relatively few die from an anaphylactic shock, the severest form of allergic reaction, there are extensive burdens for sufferers, which in turn can also cause major economic loss. Asthma in children has in the USA been estimated to cost 56 billion $ per year and asthma in adults to cost 19 billion € per year in the EU so allergies involves large costs for the society . These sThe absolute majority of allergies in humans belong to the immunoglobulin (Ig) E-mediated allergies, which also are named atopic allergies. The major focus of this review will be on this type of allergy, where dendritic cells, IgE, and IgE-binding cells, primarily mast cells, and basophils are central players .via uptake of the protein antigen, the allergen, by local dendritic cells. Generally, allergens are proteins of a relatively low molecular weight and are often also relatively stable proteins, indicating they can more easily enter the mucosa in a native conformation. Dendritic cells are abundant at sites where allergens generally enter the body, for example, the skin, the lung, or the intestinal mucosa. These cells then process some of the protein into peptides of variable length in endosomal compartments using lysosomal proteases. The peptides, generally between 12 and 18 amino acids, are then presented onto major histocompatibility complex (MHC) class II molecules on the cell surface of the dendritic cell  with all four polypeptide subunits  FiguresA–C. The 10 . Monomer10 . These a10 C. The phMast cells or mast cell-like cells have been described in most vertebrate lineages including mammals, birds, reptiles, amphibians, and bony fishes –32. MastXenopus laevis or tropicalis have five Ig classes, also here with only one isotype each, i.e., IgM, IgD, IgY, IgX, and IgF (Anolis carolinensis) having only three Ig classes also here with one isotype for each class, IgM, IgD, and IgY whereas the American and Chinese alligators have 4 Ig classes and 10 isotypes . Re. ReXenops Figure –41. The s Figure .In general, mammals have five Ig classes IgM, IgD, IgG, IgE, and IgA, and a varying number of isotypes. Quite big differences have been observed between different mammalian species. For example, humans have nine isotypes, due to four different IgG isotypes and two IgA isotypes. Mice have 8 due to that they have only 1 IgA isotype, and rabbits have as many as 17 because of having 13 different copies of the IgA gene , IgG, and IgE (IgY) appeared with frogs and other amphibians. The most recent addition to the complexity came with early mammals from a gene duplication of IgY. One of the copies lost the second, CH2 encoding domain, and became a four domain Ig class with V, CH1, CH2, and CH3 domains. This gene was named IgG whereas the second duplicate, which became IgE, maintained all domains of IgY and thereby containing V, CH1, CH2, CH3, and CH4 domains Figure –46. Simivia IgE, and the major plasma antibody, IgG, with functions in complement activation, immune complex clearance, and antibody-dependent cellular cytotoxicity (ADCC). Interestingly the marsupials, exemplified by the American opossum only have four Ig classes, IgM, IgG, IgE, and IgA, and only one isotype for each Ig class , 53via IIg class . The selOne noteworthy finding concerning IgE is in the pattern of glycosylation. Almost all of the Igs are glycosylated. Most often on asparagines, so called N-linked glycosylation. The glycosylation pattern does often differ between different species and the amount of carbohydrate also correlates with the amount of positive charge . TherefoThe IgE receptor α chain is one member of a complex set of proteins interacting with the constant domains of the Igs –59. ThesAll of the FcRs, including the PIGRs and the FcRL molecules, contain one or several Ig type domains. These domains have a similar fold to the Ig constant or variable domains and belong to the large family of Ig-like domains. The Ig domains have been classified into four basic types depending on the number of antiparallel beta-sheets and the positions of cysteine and other conserved amino acids. These are described as the V, C1, C2, and I type of domains , 65. V dThe important signaling molecule for the classical FcRs, the common γ chain, is a member of a small family of non Ig-domain-containing molecules including the TCR zeta chain, DAP10, and DAP12 , 66–70. Xenopus tropicalis) and the Chinese alligator, indicating that the processes forming the subfamily of receptors that later became the classical IgG and IgE receptors may have started already during early tetrapod evolution. However, this subfamily probably did not appear as a distinct subfamily until the appearance of the mammals . Al. Al60). via its binding and activation of anti-thrombin , 90via gD Figure . The fouD Figure  95–97).–97.via gIn order to obtain additional functional information concerning these proteases, we have used an unbiased technique with a very large number of potential target sequences. This consists of a phage library with 50 million different 9 amino acid long random sequences to study the extended cleavage specificity of a number of these hematopoietic serine proteases , 98.in vivo function based on available model systems  proposed a theory that IgE together with mast cells may actually function as a door or gate keeper, stopping the antigen at the site of entry . Much ofNon-allergic persons have generally very low levels of IgE in their circulation, ranging from 20 to 400 ng/ml in blood . In compAn interesting question here is if these very low levels of IgE are reflected in the general situation in both domestic and wild animal populations, and how this affects our view of the function of IgE. Most inbred mouse and rat strains have similar low IgE levels to non-allergic humans, below 200 ng/ml, with many strains having less than 50 ng/ml , 126. HoHowever, other factors are also very significant. Both humans and rodents living under laboratory conditions are generally free from worm infections, which are known to be potent inducers of IgE production . By contA few years ago we developed a reliable assay testing for dog IgE based on monoclonals raised against recombinant dog IgE. This made it possible to study IgE levels in both dogs and wolves, with high accuracy, which had not been possible with previously existing reagents. Analysis of a panel of 76 adult dogs showed that adult dogs have IgE levels that are between 10 and 40 μg/ml, which is almost 100 times higher than non-allergic humans, but that young dogs started with low with levels often below 1 μg/ml of IgE Figure  128). A. A128). A very interesting study of Ethiopian Jews has also been performed, which provides indications further in line with these animal findings. When IgE levels were analyzed on people that had moved from Ethiopia to Israel at an adult age their IgE levels were high and stayed high during the entire study, whereas their children who were born and raised in Israel had similar low levels of IgE as other children born in Israel . The sitA number of factors have been indicated to be of importance for the high incidence of allergies in industrialized countries. One factor that seems to have a major impact is general domestication, as it is primarily among ourselves and among domestic animals that we find allergic individuals. To our knowledge, allergies have not been described in wild animals. One potential factor could be a genetic drift due to strong selection for phenotypic characteristics like coat color, long or short noses, running fast, or wanted social behaviors. Such strong selections are seen in the breeding programs for dogs, horses, and cats, but a questionable cause for human allergies. However, it is possible that we constantly need to be selecting against hypersensitivities, which may occur due to minor shift in immune functions caused by spontaneous point mutations. A strong such selection process most likely exists in wild animals under tough environmental conditions but not in domestic animals and in humans. Another factor could be parasite infections. However, the presence or absence of parasites cannot be the only explanation as we see allergies both in ourselves, who are generally free from intestinal parasites, at least in the industrialized world, whereas dogs and horses are often parasite infected.There could also be numerous other contributing factors, which are only partly dependent on domestication such as hygiene including a reduced complexity of intestinal microbiome. For humans, one factor could be the reduced use of fermented food. The list has become relatively long for potential causes of this increase and there are studies, which favor and disfavor almost all of them, making the situation very complex. It is clear that not only one single factor is involved but a combination of many and often also diverse factors, which together provides the imbalance of the immune system to overreact against often harmless substances such as pollen, nuts, animal fur, cow milk proteins, eggs, fish, and shellfish.By studying only a few species, which live under very similar conditions we most likely get a fairly limited view of the regulation of the immune system. For example, many of the knockout strains of mice have been shown to display a very mild phenotype, indicating that many cell types and molecules are dispensable and not essential. However, this may be the situation under low pathogen loads and under conditions of good access to nutrients and clean water. A more pronounced phenotype may be seen under stress, under conditions of limited water and food supply, when individuals have to fight for territory and mating partners and under heavy parasite loads. Similarly, we may not see the factors regulating a normal immune response by only looking at a few species, which are essentially living under very similar and limited environmental conditions, including absence of parasites, a lack of fighting for territory, and an almost unlimited supply of food and water. One interesting finding relating to this idea is that persons lacking IgE seem to live a relatively normal life in spite of the fact that the connection between IgE and mast cells has been evolutionary conserved for what it seems at least 200 million years . By studIn addition to giving us a more detailed view of the regulation and the evolution of our immune system, wild animals may also be a rich source of therapeutic proteins and other potential therapeutic molecules. As a separate line of research we have also been trying to develop new treatment strategies against atopic allergies. One such line of research has been the development of therapeutic vaccines targeting IgE and several of the early TH2 inducing cytokines including IL-33, IL-18, and TSLP Figure , 144. InWild animals can teach us a lot about our own immune system, including how it is regulated, how it has evolved, and which functions are essential for a potent immune defense. These reasons are but a few to consider, where non-domestic animals may facilitate new solutions to difficult therapeutic challenges.The review was co-written by LH, SA, MT, and ZF.The patents for the VLR-B sequence used to enhance immunogenicity to therapeutic vaccines targeting self-molecules is owned by a company owned by LH. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We investigated the involvement of glycogen synthase kinase-3β (GSK3β) and the voltage-dependent anion channel (VDAC) in livers subjected to cold ischemia–reperfusion injury (I/R) associated with orthotopic liver transplantation (OLT). Rat livers were preserved in University of Wisconsin (UW) and Institute Georges Lopez (IGL-1) solution, the latter enriched or not with trimetazidine, and then subjected to OLT. Transaminase (ALT) and HMGB1 protein levels, glutamate dehydrogenase (GLDH), and oxidative stress (MDA) were measured. The AKT protein kinase and its direct substrates, GSK3β and VDAC, as well as caspases 3, 9, and cytochrome C and reticulum endoplasmic stress-related proteins , were determined by Western blot. IGL-1+TMZ significantly reduced liver injury. We also observed a significant phosphorylation of AKT, which in turn induced the phosphorylation and inhibition of GSK3β. In addition, TMZ protected the mitochondria since, in comparison with IGL-1 alone, we found reductions in VDAC phosphorylation, apoptosis, and GLDH release. All these results were correlated with decreased ER stress. Addition of TMZ to IGL-1 solution increased the tolerance of the liver graft to I/R injury through inhibition of GSK3β and VDAC, contributing to ER stress reduction and cell death prevention. Cold ischemia–reperfusion (I/R) injury is a major cause of primary graft non-function after liver transplantation (LT) . As a prIn addition, in previous publications, we have shown that the use of additives such as trimetazidine (TMZ) to UW ,6 and IGGlycogen synthase kinase-3β (GSK3β) is a serine/threonine kinase that participates in the regulation of many cell functions. Its activity is controlled by multiple mechanisms, including its phosphorylation and intracellular translocation. GSK3β is active in its dephosphorylated form, showing a pleiotropic function in the regulation of cell activation, differentiation, and apoptosis ,12. In aGSK3β has been implicated in liver I/R pathophysiology. Its inhibition improves liver microcirculation and hepatocellular function after hemorrhagic shock . In addiIn this report we assessed the effect of GSK3β modulation by trimetazidine, as an additive in IGL-1 solution, for enhancing liver graft preservation followed by OLT. We also evaluated the relevance of GSK3β inhibition during cold storage for the modulation of ER stress and mitochondrial damage.In this study we evaluated whether the addition of trimetazidine to IGL-1 solution protected liver grafts against cold I/R associated with liver transplantation, and whether this protection might be mediated by changes in the protein levels of AKT and its direct substrate GSK3β after 8 h of ischemic cold storage followed by 24 h orthotopic liver transplantation. As indicated in In view of the potential relationship between GSK3β and VDAC, one of the major resident proteins of the outer membrane mitochondria (the active non-phosphorylated GSK3β activates VDAC by increasing its phosphorylation) , we nextThese findings were consistent with the reductions in mitochondrial damage, oxidative stress and liver injury evidenced respectively by reductions in GLDH activity, MDA, and ALT .Since the mitochondrial alteration was generally correlated with apoptotic cell death after liver transplantation, we evaluated the changes in protein levels of cytochrome C and cleaved caspase 3 and 9 as well as the percentage of TUNEL-positive cells. Our results indicated that the activation of GSK3β and VDAC in UW group was in accordance with the increased protein levels of cytochrome C and cleaved caspase 3 and 9 compared with the sham group A–C. AlthThere is growing evidence that perturbations in the endoplasmic reticulum (ER) are novel subcellular effectors, possibly involved in the promotion of cell death during cold organ preservation . For thiThe benefits of ER stress reduction achieved by using TMZ as an additive to IGL-1 were correlated with an increased expression of other cytoprotective factors such as HO-1 and HSP70. As shown in Finally, because of the association of inflammatory response and hepatocellular damage in liver transplantation with ER stress response, we explored the expression of HMGB-1, a well-known early mediator in inflammation and liver damage after liver transplantation , in a hiIt is well established today that cold I/R injury is a major cause of primary graft non-function after liver transplantation . The comIn view of the recent data published by the European Liver Transplant Registry  and our In addition, it is well established that phosphorylation of mitochondrial VDAC is a key element of the complex network of regulatory and signaling pathways involved in mechanism for numerous cellular processes, such as those occurring in the mitochondrial dysfunction associated with liver transplantation ,30. For Given the close relationship between mPTP opening and apoptotic cell death, we next evaluated the key mediators of mitochondrial-induced apoptosis such as cytochrome c, cleaved caspase 3 and 9 levels and the percentage of TUNEL-positive cells. Our results indicated that IGL-1 prevented cytochrome C release and consequently reduced caspase 3 and 9 activation when compared with UW solution. IGL-1 enrichment with TMZ enhanced mitochondrial protection even more and inhibited apoptosis. These results are in agreement with other data suggesting that TMZ exerts its cytoprotective effect during early reperfusion essentially by reducing the mitochondrial dysfunction after renal I/R .More recently, ER stress alteration has been proposed as a new contributor to the poor tolerance of livers to I/R injury ,34. It hFinally, in order to assess the impact of AKT/GSK3β pathway modulation on inflammation and liver damage after orthotopic liver transplantation we examined changes in the protein levels of HMGB-1, an early inflammatory mediator after acute I/R injury ,37, as wIn summary, IGL-1 solution, especially when supplemented with TMZ, increases the tolerance of the liver graft to I/R associated with OLT through the inhibition of GSK3β and VDAC, thus preventing cell apoptosis. GSK3β inhibition contributes to reinforcing mitochondrial protection and leads to reduced ER stress. The inhibition of GSK3β is an issue of growing interest in the search for new therapeutic strategies for the prevention of I/R injury.Liver transplantation protocol was applied to male Sprague–Dawley rats (250–300 g bodyweight) under general anesthesia with isofluorane respecting the European Union rules for animal experiments (EC-guideline 86/609/CEE) and approved by the Ethics Committees for Animal Experimentation (CEEA)of the University of Barcelona (number 483/16).The following experimental groups were defined as follows.n = 12): Sprague–Dawley rats were immediately subjected to laparotomy and the subsequent ligation of suprarenal and diaphragmatic vein and hepatic artery, respectively. Group 2 : donor livers were flushed and stored in UW preservation solution for 8 h at 4 °C, and then underwent OLT according to Kamada’s cuff technique without hepatic artery construction [n = 12, six transplantation): same as 2, but using IGL-1 preservation solution instead of UW. Group 4 : same as 3, but IGL-1 was supplemented with TMZ at 10−6 mol/L [Group 1  levels using commercial kits from RAL . ALT was determined at 365 nm with an Ultraviolet spectrophotometer  and calculated following the supplier’s instructions .Activity of glutamate dehydrogenase (GLDH), a mitochondrial enzyme, was used as an indirect determination of mitochondrial damage, as previously reported ,42.Liver lipoperoxides were determined by measuring the formation of malondialdehyde (MDA) with the thiobarbiturate reaction, as previously described .The evaluation of DNA fragmentation was carried out by using TUNEL assay, as previously indicated in our studies ,8.The severity of hepatic injury was assessed in the haematoxylin and eosin-stained sections by a point-counting method using optical microscopy. Liver injury was scored on an ordinal scale as previously reported .Liver tissue was homogenized as previously described ,34: 50 μFrozen liver sections (16 μm thick) were fixed in pre-cooled acetone for 10 min at room temperature. After washing and permeabilization, HO-1 was detected by using a primary HO-1 antibody . Following, rinsing in Phosphate-buffered saline solution (PBS) permeabilization was performed again three times with 0.5% Triton X-100 for 10 min. The secondary antibody  was applied at the dilution of 1:400 in the blocking solution for 50 min at room temperature. Negative controls were prepared as previously described [Frozen liver sections (16 μm thick) were fixed in 1% p-formaldehyde for 10 min at room temperature. After washing with PBS, post-fixation with ethanol:acetic acid 2:1 for 5 min and permeabilization twice with 0.5% Triton X-100 for 15 min were performed. Antibody especific binding was blocked for 1 h in PBS with 10% normal goat serum, 3% bovine serum albumin, 1.5% NaCl and 0.5% Triton X-100 for 1 h. Immunohistochemistry analyses were performed using HSP70  and Alexa 594 Fluor labelled , as previously described . Negativp < 0.05 was considered significant.Data are expressed as means ± standard error, and were compared statistically by variance analysis, followed by the Student–Newman–Keuls test .


Antifreeze proteins from polar fish species are remarkable biomacromolecules which prevent the growth of ice crystals. Ice crystal growth is a major problem in cell/tissue cryopreservation for transplantation, transfusion and basic biomedical research, as well as technological applications such as icing of aircraft wings. This review will introduce the rapidly emerging field of synthetic macromolecular (polymer) mimics of antifreeze proteins. Particular focus is placed on designing polymers which have no structural similarities to antifreeze proteins but reproduce the same macroscopic properties, potentially by different molecular-level mechanisms. The application of these polymers to the cryopreservation of donor cells is also introduced. Ice crystal growth is a major problem in cell and tissue cryopreservation for transplantation, transfusion, icing of aircraft wings and many other applications. Here the authors review the emerging field of synthetic macromolecular mimics of antifreeze proteins that can be used overcome such problems. Lithobates sylvaticus) which can freeze solid over winter, tardigrades which can be desiccated and rehydrated and cold tolerant plants2. The mechanisms of these cryoprotectants are varied, from enabling freeze-tolerance (being able to be frozen and then thawed) to freeze avoidance (preventing ice forming) and even freeze promotion (as a predatory mechanism)6.Water is fundamental to all life on our planet, and despite it having a freezing point of 0 ˚C, Nature has evolved a series of unique adaptations to enable life to flourish in sub-zero climates, at high altitudes and at the Earth’s poles. Such extremophiles include the wood frog  which modulate ice formation and growth, and are found in freeze avoidant organisms. These can be broadly split into the antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). AFGPs are highly conserved, with a relatively simple repeat tripeptide structure and a disaccharide on every third amino acid, but are produced in a range of chain lengths. Conversely, AFPs are far more diverse, with several subdivisions, and can assume different structures; from beta barrels to alpha helices and vary in size Fig. . They al11. A key potential field where AF(G)Ps could be applied is in cell and tissue cryopreservation where ice recrystallization is a major problem14. The availability of high quality cells and tissues underpins all modern biomedical science and drug screening, and is crucial for emerging regenerative medicine applications. Progenitor (stem) cells remain challenging to cryopreserve, requiring large amounts of organic solvents which give less than 100% cell recovery and can affect their future differentiation pathways15. AF(G)Ps are not always suitable for cryopreservation due to potential immunogenicity and toxicity concerns16, expense of production and the formation of needle like ice crystals (dynamic ice shaping), which is undesirable for cell cryopreservation, leading to mixed successes and failures17. There is a clear rationale to develop synthetic mimics with tunable and tailored function.Some AFPs have already found application in improving the texture of ice cream products by preventing ice crystal growth, and other food uses are under investigation18 and even some progress towards introducing monomer sequence has been made19. This has enabled the emergence of synthetic materials which have protein-like function but with the scalability and tunability associated with polymers. For example, nanoscale drug delivery devices from block copolymers20, biochemically responsive polymers21, glycopolymer mimics of natural glycoproteins and polysaccharides22 and nucleobase containing polymers23.In the past 20 years, huge advances have been made in synthetic polymer chemistry, which now enables the design and precise synthesis of a plethora of complex architectures incorporating nearly any functional group24—polymers designed to mimic the complex function of antifreeze (glyco)proteins, without necessarily mimicking their chemical structure. This focus on function, not structure, is enabling the identification of new materials with unexpected properties. These synthetic entities are providing new insights into the mechanisms of action, enabling translation towards real world applications and a particular focus will be placed on their application in cellular cryopreservation. The physical properties of AF(G)Ps and how they are measured will be summarized, along with small molecule mimetics, followed by a comprehensive summary of the state of the art within macromolecular mimics.The aim of this perspective is to introduce the rapidly emerging field of macromolecular cryoprotectants10; dynamic ice shaping (DIS); the modification of the morphology of a growing ice crystal; ice recrystallization inhibition (IRI); crystal growth  is kinetically suppressed such that growth is inhibited; thermal hysteresis (TH); a non-equilibrium depression of the freezing point, resulting in a lower freezing than melting point, which is greater than what is expected on a colligative basis Ps to aqueous solutions significantly affects both the ice formation and growth processes, with 3 key macroscopic effects associated with growth sis Fig. 24–26. Wh27. Without AF(G)Ps the curvature of the crystal is unaffected and a round flat crystal is produced  the observed growth rate is slowed and progresses from the basal plane and hence shaping occurs, giving rise to the unique morphologies P function in relation to TH and DIS activity, which remain the most studied macroscopic properties. TH and DIS appear to be interlinked with all proteins which show TH also demonstrating DIS, although the exact magnitude of each property varies between proteins. This is most often explained by an adsorption-inhibition mechanism Fig. 26, 27. Wced Fig. . When AFies Fig. 29. Early11. For example, AFPs such as TmAFP, winter flounder type I-Hyp and M. primoryenisis are considered hyperactive, with high TH activity, but show relatively low IRI activity. Conversely AFGPs and winter flounder type I are moderately TH active but with high IRI34. These observations indicate that multiple molecular-level mechanisms may be present, and exploited differentially by the different proteins. The gold standard method to test for TH and DIS activity is nanoliter osmometry35. Samples are frozen and then slowly melted until a single crystal remains. To determine the TH of a sample the crystal is cooled further until there is a sudden burst in growth. TH is defined as the difference between the melting point and non-equilibrium freezing point33. There are limitations in determining TH in this way, mainly the technical challenges of avoiding burst growth and precise temperature control required, alongside the relatively high concentrations of material (>20 mg mL−1) needed to induce this effect. Sonocrystallization is a method proposed by Voets et al. to overcome these issues and allow the measurement of freezing and melting points in one experiment. The method involves supercooling a solution before the application of a short ultrasound pulse, which induces nucleation and freezing. The sample is then melted for melting point determination. Data obtained from sonocrystallization links to TH though does not scale with values obtained from nanoliter osmometry, which may provide new insights or indicate it is probing different molecular-level processes25. Low temperature solid-state NMR is emerging as a new tool in the field as it is possible to monitor the liquid water phase, the ice and the protein component all in the same experimental set-up.1H-1H cross-saturation and cross studies of frozen AFP:ice solutions have recently provided detailed structural information regarding the AFP:ice interface. These studies present solid-state NMR as a technique which is likely to lead to breakthroughs in the near future36.While thermal hysteresis and dynamic ice shaping appear correlated, the third property (IRI) appears to operate independently, with little or no correlation to the magnitude of TH activity37. In short, this method involves the formation of a polynucleated ice wafer by dropping a small volume of a buffered solution containing the AFP/inhibitor onto a pre-cooled surface. The ice crystals are then annealed at a sub-zero temperature, above the eutectic phase transition, and the growth of the ice crystals monitored. Due to the large number (100’s) of ice crystals obtained, various image analysis methods have been developed to facilitate this, but due to the non-regular size and shape of the ice crystals this remains non-trivial38. The length of time of the annealing will also affect the outcome, as this is only a kinetic slowing with slow growth still occurring.The ‘splat cooling’ IRI assay is widely used to probe recrystallization39. Microcapillary methods for screening for IRI have been shown by Davies and co-workers, with the advantage of allowing the sample to be archived40. It is very important to note, that when interpreting IRI data, either crystal area (mean grain size) or crystal length (MLGS) are used as measures. As area has a squared term (e.g. nm2) these values tend to be smaller at equal activity. It is important to consider this when comparing inhibitors. As a guide, 40% MLGS would be equivalent to 16% MGS.A related assay using high concentrations of sucrose (40 wt%) is also used to study growth in complex media with the advantage of simpler image analysis due to a lower density of ice crystals being obtained and does not require apparatus to ‘splat’ the dropletThe following sections will discuss the synthesis and application of various AF(G)P mimetic strategies, focusing on the macromolecular mimics, and their application to cellular cryopreservation, while referring to their activity in the above-named assays.39. AFGPs, which show greater IRI activity than AFPs, are harder to access as bacterial protein expression does not routinely enable the installation of the post-translation modification (in this case the disaccharide) necessitating chemical synthesis41. Therefore, efforts have been made to use chemical synthesis as a route to accessing these materials.AFPs are routinely produced by recombinant expression methods or, for shorter sequences, by solid phase synthesis−1), Table 42. Nishimura and co-workers reported the most comprehensive study of AFGPs to date43. In this work 10 different carbohydrates and different chain lengths were synthesized via a polymerization/fractionation approach. Interestingly, only TH and DIS were tested as an indicator of activity. It was concluded that an N-acetyl sugar, the γ-methyl on threonine and an α-glycosidic linkage were all essential components for TH and DIS to be retained. Solution phase NMR analysis revealed a largely unstructured secondary structure similar to that of a polyproline II helix (as seen in native AFGP), but interestingly all the glycans were presented on a single face creating an amphipathic structure. This is remarkably similar to crystal structures for AFPs which have clearly segregated hydrophobic (ice binding) and hydrophilic domains, suggesting that certain generic macro-scale features are crucial for function, rather than just the primary sequence; this is a key observation for the design of polymer mimics, later in this review. While the above observations were conclusive, in a sense, the study omitted any IRI activity testing  backbone, rather than alanine, were highly potent IRI’s, functioning below 0.05 µM, which is more active than AFP Type III45. Interestingly, no thermal hysteresis or dynamic ice shaping activity was observed, providing the first evidence that these properties were resolvable from IRI, which is uniquely tolerant to structural deviations from the core AFGP motif. Further studies from Ben and co-workers have explored the tolerance of glycans and backbone hydrophilicity effects on activity in detail, with galactose being preferred in general, and hydrophilic modifications to the backbone reducing activity  was also synthesized as a peptide mimetic and tested for activity; surprisingly it displayed greater activity without the glycan (as hydroxyproline) than with, highlighting the challenge associated with rational IRI synthesis, and indicating the ambiguity of the role of the sugar in AFGP; functional, or structural (to direct folding)48.Ben and co-workers reported that glycopeptides bearing a ty Table 16, 47. I49. Ben and co-workers were the first to report the synthesis of low molecular weight carbohydrate derived small molecules possessing IRI activity50. Inspired by the obvious presentation of hydrophobic faces in AF(G)Ps, , which is a remarkable level of activity for a small molecule. Detailed studies revealed it was actually forming long facially amphipathic fibres similar to a rigid AFP  55. ZrAc is a well-known co-ordination polymer, and it is hypothesized to have a solution structure56 related to poly (PVA), a highly potent IRI (see later) which may explain activity57. Nisin A, an antimicrobial peptide with pH switchable amphiphilicity was used to further exemplify the role of segregating domains, and the first example of a ‘stimuli-responsive’ IRI  Fig. . ZrAc waIRI Fig. 58. The aThe previous sections have served to highlight the attempts to reproduce the function of natural macromolecular antifreezes, especially in inhibiting ice recrystallization, and small molecule mimetics. There are clearly still no design rules and the de novo design of new non-peptide IRI-active compounds remains elusive. Compared to small molecules, synthetic polymers are often low cost, and can be assembled from a vast range of monomers and into diverse topologies and architectures offering vast scope for modulating function.37. This was one of the first instances of a synthetic polymer displaying such a property, and can be considered the birth of this field. Inada et al. evaluated the AF(G)P-like properties using commercial PVA samples60. They found that PVA had a molecular weight dependence on IRI activity and that on a molar basis had activity comparable to the shortest AFGPs. A key point to note here is that due to the high molar mass (Mw) of the polymers they were actually 10–100 fold less active than AFGP on a mass basis, but still remain the most active synthetic polymers reported today. Due to the activity of PVA, a simple assumption would be that any poly(hydroxylated) polymer could show IRI, as AFGPs are also poly-ols to a first approximation. Gibson et al. have tested various polysaccharides and glycopolymers which were found to be weak IRIs; in short, a poly-ol alone is not the minimum required feature of a synthetic IRI62. Koop et al. have postulated that the spacing between hydroxyls on PVA are a good match for the basal plane of a growing ice crystal and hence may explain its activity63. However, this does not take into account the observations with small-molecule IRIs about the role of amphiphilicity, and that an AFP’s ice-binding face is actually the most hydrophobic, with the hydrophilic face directed into the unfrozen water layer; in short, this is still not clear, but multiple molecular-level mechanisms could give rise to the same macroscopic affects and it therefore warrants more study.However, given the precise folding of AF(G)Ps and the largely unstructured nature of synthetic polymers this would seem to be an ambitious goal. In 1995, however, Knight observed that PVA, was a potent IRI but did not display significant thermal hysteresis57. By using hydrazine (rather than NaOH) to remove the acetate groups it was possible to obtain homogenous PVA. Selective re-acetylation, or co-polymerization with isopropenylacetate, revealed that incorporation of more than 20 mol% of any additional functionality lead to substantial reduction in the IRI activity. Crucially, this explains the inconsistent results from commercial PVAs due to their residual acetates, but also shows how sensitive the PVA structure is to structural modifications. Interestingly, similar effects are demonstrated by AFGP’s which lose activity if >35% of the hydroxyl groups are removed, but whether this is due to the loss of hydroxyl ‘function’ or a change in the folding of the protein has not been resolved64. Conversely, block co-polymerization of PVA appeared to be tolerated, with no change in activity. Fusion proteins of AFPs with N-terminus maltose binding protein have been found to have identical activity to free proteins also supporting that large chain-end modifications are tolerated65.The above studies were all conducted using commercial PVA samples; these are characterized by broad molecular weight distributions (Mw/Mn>2), low molecular weight contaminants  and the presence of 0–20 mol% acetate groups remaining from its synthesis from poly(vinyl acetate). This inhomogeneity meant that drawing structure-activity relationships was challenging, if not impossible. To enable a more systematic approach, Congdon et al. employed controlled radical polymerization (RAFT) to obtain well-defined PVA of predictable chain length and narrow molecular weight dispersity Fig. 57. By us60. Gibson and co-workers confirmed that even at relatively low molecular weights  there was significant IRI. An increase in chain length generally increases IRI activity but the most startling increase was seen to be between DP 10 (~450 g mol−1) and DP 20 (~900 g mol−1)  Fig.  where thIRI Fig. 66. Such 18. 3-armed, star branched PVA was found to have essentially identical activity to a 2-armed equivalent suggesting the third arm is redundant, and that total hydrodynamic volume rather than valency (number of hydroxyls) is a crucial parameter. Voets and co-workers synthesized bottlebrush PVAs and observed that the increased molecular weight did not correspond to an increase in activity, unlike with linear polymers, and hence determined that steric confinement limits activity  via post-polymerization modification of poly(aminoethyl methacrylate) from RAFT polymerization. This revealed that when an exact 50:50 ratio of carboxylic acids to amines were present, the polymer showed clear IRI but excess of either charge removed activity69. PVA10, a relatively inactive chain length, is still around 6× more active than the most active ampholyte (10% MLGS vs. ~70% for ampholytes at the same concentration). This was, however, still a remarkable observation, in that there were no obvious ice-binding sites, and that charge balance appears to be crucial. A further feature is that the basic requirement of an ampholyte is very synthetically accessible; an equal balance of cations and anions. Matsumura and co-workers have reported copolymers of 2-(dimethylamino)ethyl methacrylate and methacrylic acid70 and also carboxylated polysaccharides (dextran) as IRI-active polymers71. To date, there is no clear mechanistic explanation for why these work. Due to the synthetic routes used, only on average, were these materials actually 1:1 cationic to anionic, and the sequence distribution within the polymers was random. To overcome both these challenges, maleic anhydride copolymers have been exploited; maleic anhydride has a low propensity to self-propagate, and hence when a second monomer is added, near-perfect alternative (ABABx) structures are formed. Furthermore, the anhydride ring can be opened with a nucleophile to install the desired functionality. Mitchell et al72. and Stubbs et al. have exploited this to generate libraries of sequentially modified poly(ampholytes) to probe their function73. Increasing the backbone hydrophobicity lead to increased IRI activity, but at a cost of significantly reduced solubility—the role of hydrophobicity is again crucial to highlight here. It was found that dimethyl amino side chains lead to the most active polymers, despite being less hydrophobic than ethyl and propyl analogues; this subtle balance between hydrophobicity and hydrophilicity underlies the challenges in the rational design of AFP mimics. These polymers, while more active than the ampholytes previously mentioned, are still much less active than PVA of a similar molecular weight. Interestingly, these sequence-regulated polymers appeared to have higher activity than random copolymers but again the fundamental mechanisms governing activity remains unknown73. Other synthetic materials have also been investigated as cryoprotectants, with suggested IRI activity, but are only just emerging. Graphene oxide has also been shown to have IRI activity, with its ability to inhibit ice growth demonstrated as being as effective as PVA at similar concentrations74.While PVA has been the most studied polymeric-IRI, the findings from small molecule IRIs suggest that hydroxyl groups are not essential components and that a wide chemical space should be studied. Also, PVA itself is a relatively challenging polymer to access, as vinyl acetate is not easy to polymerize by controlled RAFT methods, only a limited range of additional functionality can be included by co-polymerization and the removal of acetate groups requires harsh conditions. Inspired by this, Matsumura et al., reported that poly(ampholytes), polymers with mixed cationic and anionic groups, were remarkable cryopreservatives and seemed to influence ice crystal morphologies at high concentrations Table Table 2PoPseudomonas syringae which promotes frost formation on plant leaves, to release nutrients for feeding76. As with AF(G)Ps the mechanism of action is also not clear but water ordering is thought to be involved77. The underlying mechanism of ice nucleation (even in the absence of promoters) is still not understood and is a major challenge in modelling and theory78, made harder by the lack of sequentially modifiable materials which can be used to carry out systematic structure-activity studies and the fact that, due to the stochastic nature of ice nucleation, time-consuming multi-point assays must be used Ps, which inhibit the growth of ice crystals, there are also ice nucleating (formation) proteins. It is important to include a brief discussion of these, as the promotion of nucleation by biological macromolecules is also an interesting, and still a misunderstood process with many potential applications and scope for synthetic materials chemists to mimic. The most widely used ice nucleating protein is from sed Fig. . Currentres Fig. 80. Covalnge Fig. 81. The e83. Cryopreservation is the most successful method for storage of these biological materials but is a complex process in which the sample volume, cooling rates, and cryoprotectants, such as dimethyl sulfoxide (DMSO), are all extremely important. Unfortunately, DMSO exhibits cytotoxicity at room temperature85, damages mitochondrial integrity86, has been shown to impact the epigenetic profile of cells87, disrupts intracellular cell signalling88, and if transfused into patients leads to a vast range of clinically undesirable symptoms. Due to this, there is a real need for new cryoprotectants with innovative modes of action, and AF(G)P inspired materials are presenting an exciting new avenue.Tissue engineering, gene therapy, and cell transfusion all rely on the ability to store and transport cells and tissues in order to be clinically successful83. Formation of extracellular ice creates an increased osmotic pressure across the cell membrane and this “osmotic flux” intensifies as ice growth continues after the nucleation event; cells will rupture if they cannot dehydrate fast enough. However, it has been shown that dehydration along with exposure to severely high concentrations of solutes is also lethal to the cell and will facilitate irreparable damage to the cell membrane89. Penetrating cryoprotectants, such as DMSO, readily cross the cell membrane and decrease the concentration of intracellular electrolytes while maintaining greater cell volumes but still carry with them the cytotoxic effects mentioned previously. Non-penetrating cryoprotectants do not cross the cell membrane, which results in an increase of osmolarity in the extracellular solution, facilitates dehydration prior to freezing, and prevents the formation of intracellular ice. Therefore, controlling and limiting extracellular ice growth, should be able to modulate and improve cell survival.The formation of ice under typical cryopreservation conditions is inevitable and in most cases ice will form outside the cell90. This was found to be due to the secondary effect of dynamic ice shaping, promoting bipyramidal ice crystals which could pierce the cell membranes. This was further demonstrated by Capicciotti et al. who showed various AF(G)Ps in the presence of 5% DMSO resulted in good cell post-freeze viabilities but cells frozen with a LpAFP mutant, which had a much smaller thermal hysteresis gap (and hence less dynamic ice shaping activity), resulted in the least amount of cellular damage91. In addition to ice-shaping, a challenge of using native AF(G)Ps is the potential for immunogenicty and toxicity. AFGPs have been found to have some cytotoxicity against human cell lines16 and conflicting reports showing either benefits or problems93 of AFGPs in cryopreservation, and hence polymer mimics with specific IRI (and not TH/DIS) could make a major impact.In 1992, Carpenter and Hansen demonstrated that addition of AFPs to blood cryopreservation solutions increased the post-thaw recovery levels. However, the effect was limited, as the AFP concentration was increased, the cell recovery actually decreased95. PVA also improved the protection of A549 and BeWo cells during DMSO-mediated cryopreservation, with PVA alone not enabling recovery of nucleated cells  this method is not as widely used for cells, with the slow-thawing method being preferred, but has been used to cryopreserve a rabbit kidney98. In addition to PVA, polyampholytes have been found to be extremely potent cryopreservation enhancers, despite their significantly lower IRI activity, relative to PVA or AF(G)Ps69. L929 and RMSCs cells were cryopreserved using polyampholytes as the sole CPA, which appear to directly interact with cell membranes, in addition to slowing ice growth68 which is similar to the proposed mechanisms of AF(G)P100. Ampholyte cryopreservation is optimal with an exact balance of cationic to anionic groups101, which also maps to IRI activity P mimetics can clearly improve cryopreservation outcomes to increase cell yield post-thawing. However, for most cases, there is still the need for low molecular weight cryoprotectants/osmolytes to ensure efficient cryopreservation. Yet another challenge in this field is the question of how much IRI (or TH) activity is needed to enhance cryopreservation and how little cryoprotectant can be used while still achieving a clinically/biotechnologically useful recovery rate. If these questions can be answered, AF(G)P mimetics could have a huge impact, especially in emerging regenerative applications and the storage of primary stem cells, or those emerging from stem-cell factories.Do several molecular-level mechanisms give rise to the observed macroscopic effects and if so can these be separated?Do polymeric mimics function by the same mechanisms as AF(G)Ps?What is the exact role of amphipathicity in IRI?Is there a minimum/maximum IRI activity required to increase cell cryopreservation?Can AF(G)P mimics be used to remove the need for organic solvents in cryopreservation?This focused review has summarized the current state-of-the-art in the rapidly emerging field of synthetic macromolecules which can mimic the function of antifreeze (glyco) proteins. In particular, recent developments in polymer chemistry have enabled a wide range of polymers to be accessed and although identification of any new material which is active remains rare, an increasingly large number are being discovered. It has become clear that the precise primary sequence and 3-D structure of natural antifreezes is essential for thermal hysteresis and ice shaping to occur, supporting a molecular-recognition type mechanism on the ice crystal surface. However, ice recrystallization inhibition appears to be an easier to access property, with a diverse range of polymers, supramolecular assemblies and even small molecules being reported, despite no structural similarities between these. The application of IRIs in cellular cryopreservation is highlighted, where slowing the growth of ice during thawing has been found to lead to remarkable enhancements in cell recovery post-storage proving innovative solutions to the logistical challenges associated with emerging regenerative medicine treatments. These advances have triggered renewed interest in this area, and we anticipate that as more research groups enter this exciting, highly interdisciplinary, field advances will continue to the point where synthetic materials may be able to outperform natural proteins, but with both increased selectivity (in terms of antifreeze properties) and also additional, currently unforeseen, functionality. We would suggest that (non-exhaustively) the following key challenges need to be addressed for the field to continue to advance:


Many cardiomyopathy-related mutations affect Ca2+ regulation and/or disrupt the relationship between Ca2+ binding and phosphorylation. Unlike the mechanism of heart activation, the modulation of Ca2+-sensitivity by phosphorylation of the cardiac specific N-terminal segment of TnI (1–30) is structurally subtle and has proven hard to investigate. The crystal structure of cardiac troponin describes only the relatively stable core of the molecule and the crucial mobile parts of the molecule are missing including TnI C-terminal region, TnI (1–30), TnI (134–149) (“inhibitory” peptide) and the C-terminal 28 amino acids of TnT that are intrinsically disordered. Recent studies have been performed to answer this matter by building structural models of cardiac troponin in phosphorylated and dephosphorylated states based on peptide NMR studies. Now these have been updated by more recent concepts derived from molecular dynamic simulations treating troponin as a dynamic structure. The emerging model confirms the stable core structure of troponin and the mobile structure of the intrinsically disordered segments. We will discuss how we can describe these segments in terms of dynamic transitions between a small number of states, with the probability distributions being altered by phosphorylation and by HCM or DCM-related mutations that can explain how Ca2+-sensitivity is modulated by phosphorylation and the effects of mutations.Troponin plays a central role in regulation of muscle contraction. It is the Ca The troponin molecule complex is shown in Figure The structure of the actin filament has been solved to high resolution  and switch (148–154) regions of TnI as well as the C-terminal mobile domain (164–210) and the C-terminal 18 amino acids of TnT. Thus, for understanding troponin function we need to define the structure of the whole molecule and take into account its dynamic nature. Recent concepts describe that order-disorder transitions in the dynamic regions of troponin are important for muscle contraction regulation  affinity. Phosphorylation disrupts the interactions and the loss of positioning leads to changes in Ca2+-sensitivity. Disorder in proteins is currently studied using molecular dynamics (MD) simulations in combination with structural studies, such as NMR.Baryshnikova demonstrated that phosphorylation of TnI Ser22 and 23 modulates the overall affinity of the N-terminal lobe of TnC to TnI (147–163) rather than the CaMolecular dynamics (MD) is a computational technique whereby proteins can be viewed as moving objects rather than stable peptides. Thus, intrinsically disordered peptides can be analyzed in terms of their dynamic, as well as their static properties. The idea behind MD is to calculate the energy of a system as a function of the position of its atoms. MD simulations can help us understand biological functions of proteins, such as conformational changes caused by interactions with other proteins or ligands. Additional properties can be measured, for example flexibility of proteins traditionally measured in nanosecond (ns) timescales, although biological events happen within microseconds (μs).2+ binding to the TnC N-terminal domain over a μs timescale, much longer that commonly used  simulations of the cardiac troponin were performed in a study by Cheng et al. . In thatRecent studies have come to the conclusion that cardiac troponin contains intrinsically disordered regions, and one of them is the TnI (1–30), which contains the phosphorylatable Ser22 and 23. In fact, being disordered is significant for the phosphorylation itself and it has been found that the majority of proteins that get phosphorylated in nature have intrinsically disordered domains close to the phosphorylation site  and Ca2+ would give information about the Ca2+-sensitivity change. Ser69 GO was found in two different conformations: at a high distance and a low distance. Cheng et al observed the low distance conformation only 10% of the time, while Zamora et al found the opposite; the Ser69 GO is located within the coordination sphere (~3Å) most of the time but flips to a longer distance (~6Å) outside the Ca2+ coordination sphere occasionally, where it can stay for ~5% of the time. Phosphorylation doubles the time spent at the long distance, which may explain the Ca2+-sensitivity change upon phosphorylation. The difference is ascribed by Zamora et al to the shorter simulation times in the Cheng et al study that have only sampled the longer distance state. In addition, it is thought that the small box size used may result in periodic artifacts mostly due to the L shaped configuration of the starting molecule and the hinge motion that elongates the molecule, increasing the probability of self-association. The study of Zamora et al used real phosphorylation in Ser22 and 23 whereas the study of Cheng et al used phosphomimetic mutations S23/S24D, although there are no structural or physiological differences between the effects of phosphorylation and phosphomimetic mutations in the myofilament . The N-terminal TnC-TnI (1–30) interaction is also crucial for the phosphorylation signal to modulate Ca2+-sensitivity. Finally, the disordered C-terminus of TnT (212–298), which has not previously been simulated, maybe involved in these interactions.The contacts between the intrinsically disordered segments and the troponin core are interesting since they are involved in the mechanism of regulation by troponin , TnI K36Q, and TnC G159D (both DCM) is that they trap Ser69 GO in the short distance configuration  and related compounds could restore the Ca2+ at a resolution of 25Å and have located troponin on the previously determined actin-tropomyosin, showing details never observed before .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DDIs) perpetrated by cimetidine, trimethoprim and pyrimethamine, resulting in decreased active renal clearance due to inhibition of organic cation transport proteins and increased plasma exposure of metformin. To understand whether area under the plasma concentration–time curve (AUC) increases relate to absorption, in vitro inhibitory potencies of these drugs against metformin transport by human organic cation transporter (OCT) 1, and the apical to basolateral absorptive permeability of metformin across Caco‐2 cells in the presence of therapeutic intestinal concentrations of cimetidine, trimethoprim or pyrimethamine, were determined. Whilst all inhibited OCT1, none enhanced metformin's absorptive permeability (~0.5 × 10−6 cm/sec) suggesting that DDI AUC changes are not related to absorption. Subsequently, to understand whether inhibition of renal transporters are responsible for AUC increases, in vitro inhibitory potencies against metformin transport by human OCT2, multidrug and toxin extrusion (MATE) 1 and MATE2‐K were determined. Ensuing IC50 values were incorporated into mechanistic static equations, alongside unbound maximal plasma concentration and transporter fraction excreted values, in order to calculate theoretical increases in metformin AUC due to inhibition by cimetidine, trimethoprim or pyrimethamine. Calculated theoretical fold‐increases in metformin exposure confirmed solitary inhibition of renal MATE1 to be the likely mechanism underlying the observed exposure changes in clinical DDIs. Interestingly, clinically observed increases in metformin AUC were predicted more closely when the renal transporter fraction excreted value derived from oral metformin administration, rather than intravenous, was utilized in theoretical calculations, likely reflecting the “flip‐flop” pharmacokinetic profile of the drug.Metformin is a common co‐medication for many diseases and the victim of clinical drug‐drug interactions ( AUCarea under the plasma concentration–time curveBCAbicinchoninic acidCmaxmaximum plasma concentrationDDIdrug‐drug interactioneƒfraction excreted value50ICinhibitory concentration that produces 50% inhibitionKiabsolute inhibition constantMATEmultidrug and toxin extrusionOCTorganic cation transportere, of 0.66) and 25% due to passive filtration   with metformin are of clinical concern as elevated plasma concentrations of metformin are associated with an increased risk of lactic acidosis  values that could be used in mechanistic static predictions of theoretical exposure change due to DDI. Furthermore, we assessed whether these same perpetrator compounds could enhance the absorptive permeability of metformin in Caco‐2 cell monolayers, using substrate and inhibitor concentrations relevant to expected clinical intestinal concentrations, in order to investigate whether an intestinal interaction may contribute towards observed DDIs.In this study, we sought to investigate the in vitro inhibitory potential of cimetidine, trimethoprim and pyrimethamine against metformin transport mediated by the organic cation transporters OCT1, OCT2, MATE1 and MATE2‐K in order to provide IC14C]‐Metformin was purchased from American Radiolabeled Chemicals  and Optiphase Supermix liquid scintillation cocktail, 24‐well liquid scintillation counting visiplates and 96‐well isoplates were purchased from PerkinElmer Life and Analytical Sciences . Hanks balanced salt solution , Dulbecco's modified eagle medium , fetal bovine serum  and mammalian protein extraction reagent (M‐PER) were purchased from Fisher Scientific . All other chemicals, solvents, and reagents were purchased from Fisher Scientific.Metformin, cimetidine, trimethoprim, pyrimethamine, ammonium chloride, sodium butyrate, non‐essential amino acids, MES, human serum albumin (HSA), lucifer yellow and HEPES were purchased from Sigma‐Aldrich . ‐metformin  was incubated at 37°C for 1, 2, 3, 5, 10 and 15 min with OCT1, MATE1 or MATE2‐K cells, and 2, 5, 10, 15, 30, and 45 min with OCT2 cells. At the end of the incubation, active transport processes were terminated by removing (via aspiration) the incubation solutions, immediately washing the cells twice with ice cold uptake buffer and then placing plates on ice. Following the wash steps, M‐PER (400 μL) was added to each well and cells were lysed for at least 5 min at 250 rpm on an orbital shaker. An aliquot (300 μL) of cell lysate was added to a white walled, clear bottomed 24‐well visiplate, liquid scintillation cocktail (2 mL) was added, and samples were counted on a Microbeta2 scintillation counter (PerkinElmer) in order to determine the total radioactivity  taken up in cells. Separately, the protein content of cell lysates (25 μL) was determined using a Bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instructions.OCT1, OCT2, MATE1, MATE2‐K and control cell lines were seeded in cell culture medium (consisting of DMEM supplemented with 10% (w/v) fetal bovine serum and 1% (v/v) nonessential amino acids) at 3–4 × 1014C]‐metformin into transporter‐expressing cells and vector control cells was determined at 37°C over a range of concentrations  in order to determine an apparent Km.Using the optimum linear incubation time determined above , the uptake of [m determinations and a metformin probe substrate concentration that was at least 10× lower than the apparent Km determined for each respective transporter.Incubations were performed as described above using the optimum linear incubation times stated for the K14C]‐metformin (100 μmol/L) was determined  at 37°C in OCT1‐expressing cells and vector control cells, in the absence and presence of cimetidine (1‐3000 μmol/L), trimethoprim (1‐300 μmol/L) or pyrimethamine (0.3‐100 μmol/L). For OCT2 assessment, uptake of the probe substrate [14C]‐metformin (100 μmol/L) was determined  at 37°C in OCT2‐expressing cells and vector control cells, in the absence and presence of cimetidine (3‐3000 μmol/L), trimethoprim (1‐1000 μmol/L) or pyrimethamine (0.3‐100 μmol/L). For MATE1 assessment, uptake of the probe substrate [14C]‐metformin (100 μmol/L) was determined  at 37°C in MATE1‐expressing cells and vector control cells, in the absence and presence of cimetidine (0.1‐100 μmol/L), trimethoprim (0.1‐100 μmol/L) or pyrimethamine (0.003‐1 μmol/L). For MATE2‐K assessment, uptake of the probe substrate [14C]‐metformin (100 μmol/L) was determined  at 37°C in MATE2‐K‐expressing cells and vector control cells, in the absence and presence of cimetidine (0.1‐100 μmol/L) or trimethoprim (0.03‐30 μmol/L).For OCT1 assessment, uptake of the probe substrate [14C]‐metformin into cells (pmol) was normalised to the protein (mg) content of each well to calculate the uptake activity (pmol/mg). For Km determination experiments the calculated uptake activity (pmol/mg) was also normalized for incubation time (min) to give uptake rate (pmol/min/mg). Uptake rate of probe substrate into transporter‐expressing cells was corrected for that determined into vector control cells to calculate the transporter‐mediated (corrected) uptake rate. For Km determinations, corrected uptake rate (pmol/min/mg) was plotted against nominal substrate concentration and fitted using SigmaPlot 12.5 (Michaelis–Menten equation). For IC50 determinations, corrected uptake activity (pmol/mg) was converted to percentage (vehicle) control activity, which was subsequently plotted against nominal inhibitor concentration. Curves were fitted using SigmaPlot 12.5 (four‐or five parameter logistic equation) to determine the concentration that produces half‐maximal inhibition of probe substrate transport (IC50).The determined total uptake of probe substrate ‐metformin with or without test inhibitor were added to basolateral  or apical compartments , respectively. Donor compartments also contained the cell monolayer integrity marker lucifer yellow (100 μmol/L). Transport buffer or MES‐transport buffer containing DMSO or test inhibitor was added to the corresponding receiver compartments. Donor and receiver solutions contained a final DMSO concentration of ≤1 % (v/v). Following a 90‐min incubation at 37°C, receiver compartments were sampled (50 μL) into 96‐well isoplates (followed by the addition of 200 μL liquid scintillation cocktail) and the amount of [14C]‐metformin was quantified by liquid scintillation counting on a Microbeta2 counter, and used to calculate apparent permeability (Papp), as described previously .Following this preincubation period, donor solutions of transport buffer, or MES‐transport buffer, containing [14C]‐metformin across Caco‐2 cell monolayers  was assessed in triplicate wells per incubation condition at metformin concentrations of 10, 100, 1000 and 10,000 μmol/L. Further assessment in buffer (pH 6.5/7.4) was conducted at a single [14C]‐metformin concentration of 10,000 μmol/L in the absence and presence of cimetidine (1000 μmol/L), trimethoprim (500 μmol/L) or pyrimethamine (200 μmol/L). Subsequently, this latter experiment was repeated in buffer containing FaSSIF (pH 6.5) and buffer containing 1 % (w/v) HSA (pH 7.4), present in the apical and basolateral compartments, respectively.The bidirectional [apical‐to‐basolateral (A–B) and basolateral‐to‐apical (B–A)] apparent permeability of e value (0.39) is derived from the renal clearance determined in human mass balance studies following oral administration of metformin, whereas the higher ƒe value (0.66) is derived from the more usual determination of renal clearance contribution following intravenous administration.Additionally, the predicted change in metformin AUC based upon inhibition of OCT2/MATE1 with a lower fraction excreted value of 0.39 was also determined. This lower ƒ14C]‐metformin (100 μmol/L) into transporter‐expressing cells was linear from 5 to 15 min for OCT1, up to 5 min for OCT2, up to 2 min for MATE1 and up to 2 min for MATE2‐K  giving the mean IC50 curves shown in Figure 50 values for each inhibitor from three separate experiments are shown in Table 50) of cimetidine towards MATE1 was approximately 225‐fold and 170‐fold more potent than towards OCT1 and OCT2, respectively. A similar trend was observed for trimethoprim which produced a MATE1 IC50 that was approximately 10‐fold and 52‐fold more potent compared to OCT1 and OCT2. Of all the inhibitors tested, pyrimethamine gave the lowest IC50 versus MATE1 (0.131 μmol/L), which again was more potent (35‐fold) compared with OCT1 and OCT2 transporters.Cimetidine, trimethoprim, and pyrimethamine demonstrated concentration‐dependent inhibition of OCT1‐, OCT2‐, MATE1‐ and MATE2‐K‐mediated transport of [2]/Ki ratios of 23, 99, and 180 for cimetidine , trimethoprim  and pyrimethamine , respectively. Determined Cmax free/Ki ratios for the inhibition potential of cimetidine, trimethoprim, and pyrimethamine toward OCT2 and MATE1 are shown in Tables Assessment of DDI potential versus intestinal OCT1 inhibition using FDA static equation approaches gave theoretical intestinal luminal concentration [Iapp (cm/sec × 10−6) of a range of concentrations of metformin in buffer (pH6.5/7.4) is shown in Figure −6) in both the A‐B and B‐A directions across all metformin concentrations . Furthermore, there was no change to the determined A‐B or B‐A Papp values of metformin  in the presence of cimetidine, trimethoprim or pyrimethamine when incubations were conducted either in buffer alone (pH6.5/7.4), or in FaSSIF containing buffer (pH6.5) apically with 1 % HSA (w/v) containing buffer (pH 7.4) basolaterally  gave AUC increases that were below bioequivalence  indicating no DDI potential via this mechanism.Using the drug inhibitory affinities determined above for cimetidine, trimethoprim, and pyrimethamine versus OCT2 and their pharmacokinetic parameters given in Table e value of 0.66, or 1.51/1.43, 1.41/1.32, and 1.37 using an ƒe value of 0.39, respectively. Predicted AUC increases were within 39‐86 % or 2‐11 % of clinically observed AUC increases when using an ƒe value of 0.66, or an ƒe value of 0.39, respectively.In contrast, the calculated theoretical fold increases in AUC due to inhibition of renal MATE1 by cimetidine, trimethoprim, and pyrimethamine were 2.32/2.03, 1.98/1.69, and 1.85 using an ƒ® label, DeFronzo et al. Metformin is widely prescribed to patients due to the prevalence of diabetes as a comorbidity in many diseases  values , confirming that DDI via this mechanism is unlikely in vivo , using IC50 values from this study (Table −6) of 10, 100, 1000 and 10,000 μmol/L metformin in Caco‐2 cells  of 250 mg and 500 mg doses of metformin. Additionally, inhibitor concentrations chosen ensured (1) that they were greater than theoretical enterocyte concentrations , trimethoprim (230 μmol/L) or pyrimethamine (67 μmol/L) and, (2) that they were >3 times determined OCT1 IC50 values to give reasonable or complete inhibition of OCT1. Inhibition of OCT1 would act to reduce the apical uptake of metformin making more available for paracellular absorption  in Mate1(−/−) null mice compared with wildtype mice  observed in Oct1/2(−/−) null mice  for MATE1. For conventional oral drugs (whose elimination rate <<<absorption rate) ƒe values are derived from total plasma clearance obtained in human intravenous balance studies  the clinically observed increases in metformin AUC based on inhibition of MATE1 by cimetidine, trimethoprim, and pyrimethamine. For future studies, it would be interesting to see if the model of Burt et al. mice, deriving values of 0.66 and 0.76, following oral and intravenous administration, respectively. If such a differential did not exist then the fold increase in metformin AUC in null mice (due to the absence of Oct1/2 protein) compared to wild type mice would be identical regardless of metformin route of administration. Interestingly, oral AUC profiles of metformin in mice showed the same protracted absorption profile (Higgins et al. Such a differential for renal transporter ƒIn conclusion, in vitro DDI studies, coupled with mechanistic static approaches for predicting maximal theoretical increases in exposure, indicate solitary inhibition of renal MATE1 as the likely mechanism underlying the metformin AUC increases in observed clinical interactions perpetrated by cimetidine, trimethoprim, and pyrimethamine.Participated in research design: Elsby; Conducted experiments: Chidlaw, Outteridge, Pickering, Radcliffe, and Sullivan; Performed data analysis: Chidlaw, Elsby, Outteridge, Pickering, Radcliffe, and Sullivan; Wrote or contributed to the writing of the manuscript: Butler, Chidlaw, Elsby, Jones, Outteridge, Pickering, and Radcliffe.None declared.