Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions. IK values in the low nanomolar range  and two hairpin loops (loops L1 and L2). Essentially, the N terminus binds to the nonprimed side, whereas the two adjacent hairpin loops occupy the primed substrate–binding sites.Cysteine proteases are key regulators in many physiological processes. Consequently, dysregulation of protease activity can have severe effects resulting in a variety of pathologies, including cancer and Alzheimer's disease , 2. Therar range , 7. The 39 residue, localized on a reactive center loop different from the papain-inhibitory site to specifically bind to the legumain active site  harbor, in addition to their papain-binding site, a legumain binding site –10. Huma–12–ive site . Furtherive site –20. Wherve site –, 10, 21.ve site –.Cystatins not only encode a high intrinsic variability because of their function as dual protease inhibitors but also because of their ability to transform to distinct oligomerization states upon conformational destabilization. Factors trigging this oligomerization include N-terminal truncation by proteolytic enzymes, acidic pH, heating, and point mutations. These cause dimer formation via a domain-swapping mechanism –25. Esse–30–Both legumain and cystatins became attractive drug targets due to their relevance in different types of cancer and dementia. Among the cystatins, the family 2 cystatins became especially interesting, because of their function as dual protease inhibitors and because they are secreted to the extracellular space, where legumain and cathepsins are similarly observed under pathophysiologic conditions. Cystatin E is the most potent physiologic legumain inhibitor, binding 100-fold more tightly as compared with cystatin C . Thereby33–However, cystatin E's physico-chemical properties, including its propensity to form dimers and higher oligomers, have hardly been studied so far. Therefore, we set out to challenge the conformation of cystatin E by different triggering factors, including N-terminal truncation, pH, and heating. Furthermore, we analyzed the effect of these destabilizing factors on its three-dimensional structure and inhibitory function.4–Leu13)hCE; ΔhCE in Fig. S1). Size-exclusion experiments (SEC) revealed that whereas full-length WT hCE migrated at the expected elution volume of the monomeric 15-kDa protein, the N-terminally truncated ΔhCE variant showed an additional peak eluting at the size of a dimer (A). This observation was already the first indication for a similar tendency of hCE to oligomerize as described for other family members. As this oligomerization tendency correlated with conformational destabilizations, we next investigated the effect of heating on full-length hCE. Interestingly, we found that cystatin E monomer efficiently converted to a dimeric form upon incubation at ≥70 °C (A and B). Using SEC, we could determine the transition temperature where 50% conversion was reached to be 65 °C (B). This was further cross-confirmed by ThermoFluor experiments . In this particular case, thermally induced increase in fluorescence at 65 °C corresponded to the transition to dimeric hCE rather than to protein unfolding. To highlight the relevance of these experiments, we used cystatin C as a standard to compare the thermal energy barriers for dimer formation between the two family members. Significantly, cystatin C showed qualitatively and quantitatively the same behavior as hCE with 50% conversion to dimeric human cystatin C (hCC) at 65 °C and 100% conversion at 70 °C . Similarly, we tested the effect of pH on hCE dimerization. Indeed, incubation of monomeric hCE at pH 3.0 could significantly reduce the transition temperature. SEC experiments revealed efficient conversion to dimeric hCE already at 60 °C . Additionally, using the ThermoFluor method, we observed a reduction of the transition temperature by 8 °C upon incubation at pH 3.5 .To mimic the effect of N-terminal proteolytic processing, we recombinantly produced an N-terminally truncated human cystatin E (ΔhCE) variant, lacking the first 10 amino acids following the mature N terminus . Dimeric hCE was generated by incubation of the monomer at 80 °C. Interestingly, the dimer showed an increased melting temperature, indicating that once the energy burden of dimerization has been overcome, the dimer is the structurally more stable conformation. Additionally, we found the monomer–dimer transition to be irreversible (i.e. dimeric hCE could not be converted back to monomeric hCE). Monomeric hCE thus represents a metastable state that is kinetically preferred in the folding process over the more stable dimeric hCE. Together, these observations were consistent with a cystatin C–like mode of dimerization, which is mediated by domain swapping  and Table S1). The N-terminal region β1-α1-β2-L1 of one cystatin E molecule (hCE) swapped out and integrated at the equivalent position of a second cystatin E molecule (hCE′), and vice versa. To allow this subdomain movement, loop L1, which connects β-strands 2 and 3 in the monomer, adopted an extended conformation. Two elongated β-strands βII and βIII composed of the monomeric β2-L1-β3 of monomers A and B, respectively, aligned in an antiparallel fashion and thereby bridged the two monomers (B and C) and A and B)Fig. S2 . The monomeric loops L1 and L1′ of either hCE are integrated within the resulting extended β-sheet, with the local symmetry dyad passing through them (C). Consequently, dimeric hCE has integrated four additional L1, L1′-derived hydrogen bonds, contributing to its higher thermal stability and energetically more stable conformation as compared with the monomeric structures (C). Additionally, the monomer–dimer transition is accompanied by a conformational relaxation of the strictly conserved Val57, relaxing the unfavorable monomer conformation, (ϕ ψ) = (−121°|−144°), to a favorable dimer conformation, (ϕ ψ) = (−140°|137°)   . Togethe2 and an interface area of ∼3354 Å2. The high content in nonbonded contacts is also reflected in a very low-solvation free energy gain ΔiG upon complex formation of −43.5 kcal/m. Furthermore, the free energy of complex dissociation, ΔGdiss, of 55.4 kcal/m indicates that the dimer is thermodynamically stable.Analysis with PDBePISA  and PDBsα positions (0.33 Å). The deviations between the two subdomains may relate to different packing environments within the crystal lattice. Interestingly, superposition of dimeric hCE with monomeric hCE revealed an average RMSD of 0.66 Å, indicating significant structural differences in hCE monomer as compared with the corresponding dimeric subdomain (A). Structural differences accumulated from a number of small rearrangements that accompanied the integration of the N-terminal β1-α1-β2 region from one molecule into the second. Therefore, the cystatin E monomer and dimer are two structurally distinct subspecies; the dimer is not the sole combination of two monomers.From a structural perspective, the hCE dimer was assembled from two hCE monomer conformers that are connected via their L1 loops. When we compared the structural similarity of the two half-domains hCEE′ and hCE′E within the dimer, we observed a relatively low root mean square deviation (RMSD) of the Cubdomain A. Structα positions of 1.3 Å . Interestingly, the difference between the hCE and hCC dimer structures is increased by 2-fold . Most of this high difference can be accounted for by the flexibility of the connecting hinge region (L1–L1′). Small structural changes within this region translate into large motions in the peripheral regions (e.g. the legumain-binding site). Given the flexibility of the connecting hinge, differences in crystal packing may also contribute to differences in the relative spatial orientation of the two subdomains, both in the hCE and hCC dimer structures . A similar assembly was described previously for stefin B (PDB code 2OCT) and cystatin C (PDB code 1G96). The stefin B tetramer forms via a handshaking mechanism of the L2 loops that is triggered by a proline switch . Pro105 is conserved throughout the whole cystatin family. However, the L2 loop is shortened by 3 amino acids in the family 2 cystatins . Hence, a direct handshake between two L2 loops is not possible. Nevertheless, the overall assembly  is similar between stefin B, cystatin C, and cystatin E and therefore suggests a conserved mechanism of oligomerization. The dimers interact via hydrophobic contacts involving residues of the former L1 loops. The hCE tetramer is visible in the crystal structure but not in SEC experiments, indicating a low affinity of this assembly.When we superposed hCE and hCC monomer structures, we found an RMSD of the Cructures . Therefoe.g. legumain-binding site 1 is built up from the RCL′ (reactive center loop) of one chain, hCE′, and the LEL of the second chain, hCE, and vice versa (B and 3A). Despite this large structural rearrangement, the local conformations of the legumain RCL and LEL were virtually identical in monomeric and dimeric hCE structures (B). Thus, we hypothesized that dimeric hCE is still functional as a legumain inhibitor. Indeed, when we incubated legumain with dimeric hCE, we observed a complete loss of its Ala-Ala-Asn-AMC substrate turnover (C). The crystal structure of dimeric hCE uncovered two symmetric and functional legumain-binding sites, either of which is accessible for legumain binding. To test whether a simultaneous binding of two legumain molecules by one hCE dimer (i.e. a tetrameric assembly) is sterically feasible, we prepared a model of such a complex based on the crystal structures of the hCE dimer and the legumain–hCE complex (PDB entry 4N6O (D). We found virtually identical intermolecular contacts for either of the two possible docking modes . However, from these docking models, it became immediately clear that simultaneous binding to both sites is sterically impossible. When legumain binds to the hCEE′ site, the free hCE′E site is in close proximity to the legumain insertion loop on legumain and vice versa. Hence, a trimer composed of one hCE dimer and one legumain (AEP) seemed to be the most likely assembly (D). To test this hypothesis, we performed size-exclusion experiments of a preformed legumain–hCE dimer complex. Indeed, we observed a peak at the size of a trimer, but not at the expected size of a tetramer (E). Additionally, SDS-PAGE revealed a 2-fold higher hCE concentration in the fractions of the legumain–hCE dimer complex (dAEP–hCE) as compared with legumain–hCE monomer complex . The legumain concentration was identical in both experiments. The difference in hCE concentration within the fractions containing the hCE–legumain complex indicates a 2:1 stoichiometry rather than 2:2. Thus, cystatin E virtually loses 50% of its legumain binding sites via domain swapping.The hCE dimer is a unique folding state that encodes two legumain-binding sites on one molecule. In contrast to monomeric hCE, the legumain-binding sites of the dimer are built up by contributions of both hCE chains . First,try 4N6O D. We fouassembly D. To testetramer E. AdditiIK of 10.7 ± 5.6 pm for monomeric hCE and 13.5 ± 6.7 pm for dimeric hCE. Molar concentrations of monomeric and dimeric hCE were calculated assuming molecular masses of 15 and 30 kDa, respectively. Although docking models of the hCE dimer–legumain complex showed some minor clashes between the unbound legumain binding site on the hCE dimer and legumain (D), these did not translate into weakened inhibition constants. (Mostly) unhindered complex formation is possible due to the flexible hinge region (L1–L1′).The observation of symmetric legumain binding sites within the cystatin E dimer suggested that binding to both sites is possible at the same affinity. Furthermore, structurally identical binding sites in the monomer and dimer implied similar affinity constants toward legumain. Indeed, we could determine a legumain D, these IK constants of dimeric versus monomeric cystatin E implied that the mode of binding of dimeric hCE to legumain is qualitatively similar to binding of monomeric hCE. To further test this conclusion, we investigated features that are characteristic for the hCE–legumain complex. First, we could previously show that interaction of the LEL on cystatin E with the legumain-primed substrate-binding site has a positive effect on its thermal stability at near neutral pH, where the isolated enzyme by itself is unstable (G). Second, we observed pH-dependent cleavage and religation of monomeric hCE at the conserved Asn39 residue located on the legumain reactive center loop of hCE by legumain (H). Together, these observations are in nice agreement with the crystal structure of dimeric hCE that uncovered structurally and functionally uncompromised legumain-binding sites.Similar unstable , 41. Theunstable G. Secondlegumain . Cleavaglegumain H. TogethA and B). Upon dimerization, the L1 loop, which is essential for binding to the papain S1′ site, was incorporated into the newly formed βII-βIII sheet. Thus, loop L1 is not available for binding anymore, and the papain-inhibitory site is destroyed by the dimer interface. In agreement with this observation, dimeric hCE did not show inhibition of papain and human cathepsin S in a peptide substrate hydrolysis assay (C). Consequently, dimerization of cystatin E led to 100% loss of its cathepsin-inhibitory function.In contrast to the legumain-inhibitory site, the papain-inhibitory site structurally differs in monomeric and dimeric hCE , A and Bis assay C. ConseqN-glycosylation site on its L2 loop (Asn112). Because both glycosylated and nonglycosylated hCE were previously reported in vivo, we were interested in the relevance of glycosylation for hCE dimerization (A). To test whether this is the case, we incubated glycosylated hCE produced in the Leishmania tarentolae expression system (LEXSY) at 85 °C and subjected it to SEC. Indeed, we observed a shift corresponding to dimeric, glycosylated hCE (B). In agreement with the domain-swapping mechanism, this dimer similarly inhibited legumain but not papain in a fluorescent peptide substrate assays (C). Furthermore, ThermoFluor experiments of glycosylated hCE revealed a similar thermal unfolding behavior as observed for unglycosylated hCE .Native cystatin E harbors an rization , 42. Therization A. To tesated hCE B. In agre assays C. FurtheFig. S4). This observation can probably be understood by the low sequence identity of only 30% between cystatin E and C. Sequence variations on the α1 helix, which is a critical connecting element, may result in steric clashes, thereby preventing heterodimerization. Inspection of the hCE and hCC monomer structures revealed, for example, a hCEAla29PhehCC variation that may be in steric conflict with Ser114-Gln-Leu116 (β5) on hCE. Thus, the affinity for homodimer formation is supposedly much higher than for heterodimerization. It might be possible to outcompete this affinity difference by having a large excess of hCC relative to hCE.Because the overall structures of stefin B and cystatin C and E dimers are superficially similar, we questioned whether dimerization might not be restricted to homodimer formation but rather open to the possibility of heterodimerization as well. To test this, we performed pulldown assays using a cystatin E construct carrying a C-terminal Strep-tag and WT cystatin C. Both proteins were co-incubated at 70 °C to ensure efficient domain swapping. Subsequently, we immobilized hCE on a Strep-Tactin® resin via its Strep-tag. If heterodimerization had occurred, we would have expected co-purification of the untagged hCC. However, using SDS-PAGE, we could not detect co-migration of hCC; solely hCE homodimer had bound (C). Additionally, we observed a second transition between 80 and 90 °C for both monomeric and dimeric hCE, pointing toward the presence of another conformational state like fibrils, analogous to cystatin C. To test this hypothesis, we incubated cystatin E at a temperature above the second transition point (90 °C). Thereby, we observed the formation of an insoluble protein pellet. To test whether this pellet contains misfolded, aggregated protein or rather structured amyloid fibrils, we performed X-ray diffraction experiments. Interestingly, we observed two diffraction maxima at 10 and 4.7 Å resolution, which are indicative of cross-β structures, which typically build up amyloid fibrils (A). Furthermore, we could confirm the presence of fibrils using thioflavin T, a dye specifically binding to amyloid structures (B). Even more, negative staining TEM experiments also revealed the presence of fibril structures . In agreement with thermal denaturation curves, fibril formation is more efficient at 90 °C as compared with 80 °C because the energy barrier is only partially overcome at 80 °C (C and 5B).Cystatin E domain swapping results in the formation of an energetically more favorable conformation. In the simplest situation, two domain-swapped monomers assemble to form a dimer. However, domain swapping is not restricted to dimerization, but in principle also allows the formation of multimers via concerted swapping reactions (chain reaction). The existence of such multimers was previously reported for stefin B and cystatin C and resulted in the assembly of highly ordered fibrils , 27, 30.Fig. 1 fibrils A. Furtheructures B. Even mat 80 °C C and 5B.A). This observation was the first strong indicator of the presence of folded protein within hCE fibrils. However, inhibition might have also been caused by nonspecific precipitation mediated by insoluble hCE fibrils. To test whether the inhibition we observed is via specific interaction of hCE with the legumain active site, we performed a pulldown assay. Specifically, we co-incubated the insoluble hCE fibrils with active site–free and active site–blocked legumain variants. To block the active site, we used a covalent acetyl-Tyr-Val-Ala-Asp-chloromethylketone (acetyl-YVAD-cmk) inhibitor. hCE fibrils are insoluble and for that reason served as stationary phase to extract potentially bound legumain. If the interaction of legumain with hCE fibrils was specific, via its active site, we would have expected to see binding of active site–free legumain but not of the acetyl-YVAD-cmk–blocked variant. Indeed, we observed co-migration of WT legumain with hCE fibrils, whereas active site–blocked legumain remained in the soluble fraction (B). Furthermore, we observed specific processing of hCE fibrils at the Asn39 cleavage site on the legumain reactive center loop. Both results supported the presence of functional, folded protein within hCE fibrils. To test the structural integrity of the legumain exosite loop, we collected thermal denaturation curves of legumain pre-incubated with hCE fibrils. Analogous to monomeric and dimeric hCE, we observed a stabilizing effect of hCE fibrils on legumain at pH 6.5 . The effect is not as pronounced as for the legumain–hCE (dimer) complex. However, this can be understood by the complexity of the fibrils.Based on the assumption that fibril formation is a result of concerted domain-swapping reactions, it seemed possible that hCE fibrils contained structured, functional protein. Because domain swapping did not compromise the functionality of the legumain-binding site, we tested whether hCE fibrils can, like dimeric hCE, inhibit legumain activity. Indeed, upon co-incubation of legumain with hCE fibrils, we observed complete inhibition of the Ala-Ala-Asn-AMC substrate turnover A. This ofraction B. FurtheA). Thus, hCE fibrils must contain, to some extent, monomeric hCE protein, which has not undergone domain swapping. Presumably, hCE fibrils are built up by domain-swapped monomers but are at the same time heterogeneously decorated with monomeric WT protein  (C). Indeed, we found fibril formation of dimeric hCE upon incubation at 90 °C, as evidenced in a thioflavin T (ThT) test (B). Therefore, the dimer is not a dead end, but most likely a folding intermediate on the route to multimers. Because both monomeric and dimeric hCE are capable of fibril formation, both might potentially serve as a building block for fibrils. However, based on ThermoFluor experiments, we would rather suggest that the dimer is the critical intermediate. To study this hypothesis, we set up a fibril nucleation assay where we tested the ability of monomeric and dimeric hCE to extend preformed fibrils. Specifically, we co-incubated monomeric and dimeric hCE with purified fibrils at 90 °C for 10 min. Interestingly, we observed an increase in ThT signal for dimeric hCE spiked with fibrils as compared with the control reaction without preaddition of fibrils  C. IndeedhT) test B. Theref fibrils . Thus, mA). Furthermore, ThermoFluor experiments revealed a reduction of the fibril transition temperature by ∼8 °C at pH 3.5 (C). Consequently, changing the pH environment will have an effect on the hCE oligomerization state. Along that line, we were also curious about the effect of glycosylation on hCE multimerization. Although dimerization is possible for the glycosylated variant, multimerization might be negatively affected because of potential steric conflicts. Indeed, we did not observe fibril formation of glycosylated cystatin E (glyco-hCE) upon incubation at elevated temperatures (B and C). This suggested to us a regulatory function of glycosylation for hCE function.Domain swapping is triggered by conformational destabilization of monomeric hCE. Besides heating, we could identify low pH as a trigger factor for hCE dimerization. Similarly, we tested the effect of acidic pH on fibril formation and thereby found an about 4-fold increase in ThT signal following incubation at pH 3.0 A. Furthet pH 3.5 C. Conseqeratures , B and Cin vivo, a similar event seems likely because of the flexible loop structure of the N-terminal region, which makes it prone to proteolytic processing. In vitro, we could previously demonstrate processing of hCC and hCE on their legumain RCL by legumain  can only bind and retain the cystatin E monomer.Although the conversion of monomeric to dimeric hCE is kinetically trapped, dimeric hCE can further convert to fibrils. Thus, domain swapping seems to be key for multimer formation. Whereas the addition of preformed fibrils to dimeric hCE led to an increase in ThT fluorescence, monomeric hCE was unaffected. This further confirms the relevance of the hCE dimer as a folding intermediate on the route to higher cystatin multimers. Additionally, it implies some prion-like behavior encoded in hCE. Because the hCE dimer has already been prone to domain swapping, it seems plausible that it is a better acceptor for a folding template than the monomer. However, it remains unclear whether monomeric hCE directly converts to fibrils if enough energy is supplied (1-step mechanism) or whether the dimer is formed first but immediately converted to fibrils , suggesting different  functions for both variants . Glycosylation at this site will probably have an effect on dimerization, because this part of the cystatin molecule is directly involved in domain swapping. Indeed, there is indirect evidence from the literature that N-terminal glycosylation is incompatible with or at least restricting hCC dimerization  vector to allow expression to the Escherichia coli periplasmic space. During secretion, the signal peptide was removed, thereby liberating the new Arg4 N terminus of full-length WT hCE (hCC numbering), Met14 of the truncated ΔhCE variant, and Ser1 of hCC. Additionally, the expression constructs carried a C-terminal His6 tag for purification. Furthermore, another WT cystatin E construct carrying a C-terminal His6 tag followed by a Strep-tag was prepared. For that purpose, a WT cystatin E construct was used as a template, and the Strep-tag was introduced following a protocol based on the inverse-PCR method. As primers, we used TCAGTTCGAAAAGTGAGATCCGGCTGCTAACAAAGCCCGAAAGG (forward) and GGGTGTGACCAGTGGTGGTGGTGGTGGTGCTCGAGCATCTGC (reverse). Correctness of expression constructs was confirmed via DNA sequencing by Eurofins MWG Operon .WT hCE and hCC constructs lacking the N-terminal signal sequence were cloned into the pET22b(+) vector (Novagen) as described earlier . A truncE. coli Bl21(DE3) cells. Briefly, the expression plasmid was transformed into Bl21(DE3) cells. For large-scale expression, cells were grown in 2-liter flasks filled with 600 ml of lysogeny broth medium  supplemented with 100 μg/ml ampicillin at 37 °C with agitation at 220 rpm until an A600 of 0.8–1.0 was reached. Expression was induced at 25 °C by the addition of 1 mm isopropyl β-d-1-thiogalactopyranoside. After overnight expression, cells were harvested by centrifugation  and frozen. The periplasmic fraction containing recombinant proteins was extracted by cold osmotic shock. Briefly, frozen cell pellets of 1.8 liters of expression culture were gently resuspended in 150 ml of lysis buffer composed of 30 mm Tris, pH 7.5, and 20% sucrose and stirred for 20 min at ambient temperature. The lysate was centrifuged at 17,500 × g for 20 min at 4 °C. The supernatant containing soluble periplasmic proteins was batch-incubated with nickel-nitrilotriacetic acid Superflow resin  for 30 min at 4 °C. Following a washing step using a buffer composed of 20 mm Tris, pH 7.5, 300 mm NaCl, and 10 mm imidazole, bound protein was eluted with washing buffer containing 250 mm imidazole. Elutions were concentrated using Amicon Ultra centrifugal filter units  and further purified by SEC. The chromatography was run on an Äkta FPLC system equipped with a Superdex 75 10/300 GL column  which was equilibrated in a buffer composed of 20 mm citric acid, pH 5.5, and 100 mm NaCl. Glyco-hCE was produced using LEXSY following a protocol described previously  with dimeric hCE at a molar ratio of 1:1 (1 AEP and 1 hCE dimer) at pH 5.5 for 10 min on ice before injection of the sample on a S200 10/300 GL column. The hCE dimer was prepared by incubation of monomeric hCE at 80 °C for 10 min. Similarly, legumain was incubated with monomeric hCE at a molar ratio of 1:2 (1 AEP and 2 hCE monomers) for 10 min at pH 5.5. To calculate molar concentrations of monomeric and dimeric hCE, a molecular mass of 15 and 30 kDa, respectively, was assumed. Both samples contained equal amounts of cystatin E either in monomeric or dimeric state. For all samples investigated, fractions were collected and analyzed on SDS-PAGE.m citric acid pH 5.5/3.0 and 100 mm NaCl. Thermal denaturation curves were collected in a 7500 real-time PCR system (Applied Biosystems) from 20 to 95 °C. The fluorescence data were analyzed as described previously  before the addition of AEP premixed with SYPRO Orange. Fibril preparation is described under “Co-precipitation assay.”Thermal denaturation curves of different protein variants after different treatments were determined using the ThermoFluor method . Brieflyeviously . Proteinm citric acid, pH 5.5, 100 mm NaCl) containing 100 μm benzyloxycarbonyl-Ala-Ala-Asn-7-amino-4-methylcoumarin substrate . Assays were carried out in an Infinite M200 plate reader (Tecan). Briefly, the assay buffer was preincubated with 8 nm cystatin, followed by the addition of 4 nm legumain. Increase in fluorescence was measured at 460 nm upon excitation at 380 nm at 37 °C. Inhibition of papain  and recombinant human cathepsin S was assayed in the same assay buffer containing 100 μm Z-FR-AMC (Bachem). The assay buffer was preincubated with 100 nm cystatin, followed by the addition of 50 nm papain or cathepsin S, and fluorescence was similarly recorded at 460 nm. Dimeric cystatin E was prepared by incubation of monomeric cystatin E at 80 °C for 10 min. The sample was filtered to remove higher oligomers. Similarly, glycosylated cystatin E was also incubated at 80 °C for 10 min to generate the dimeric form. All experiments were carried out in triplicate.Inhibition of WT legumain was tested in legumain assay buffer . To calculate molar concentrations of dimeric hCE, a molecular mass of 30 kDa was assumed. All measurements were performed in triplicates.Inhibition constants of monomeric and dimeric hCE toward legumain were determined in assay buffer composed of 50 mhibitors . First, m citric acid, pH 5.5, and 50 mm NaCl. Fractions containing dimeric protein were concentrated to ∼30 mg/ml final concentration using Amicon Ultra centrifugal filter units . Initial crystallization screening was performed in a sitting-drop vapor diffusion setup. 0.4 μl of concentrated dimeric hCE were mixed with 0.4 μl of screen solution (Hampton Index HT or JBScreen Classic) and equilibrated with 60 μl of reservoir solution in 96-well Intelli Plates (Art Robbins Instruments) at 20 °C. After 1–2 weeks, crystals were observed in a condition composed of 20% PEG 8000 and 0.1 m Ches, pH 9.5. Crystals were harvested after stepwise addition of a cryo-protectant solution containing 22% PEG 8000, 0.1 m Ches, pH 9.5, and 20% glycerol and flash-frozen in liquid nitrogen. A native data set was collected at 100 K on beamline ID23-2  equipped with a Pilatus3_2 M detector to a resolution of 2.8 Å. 720 images were collected at a wavelength of 0.8726 Å at 0.5° oscillation range and 0.1-s exposure time.Dimeric hCE was prepared by incubation of 1 mg/ml WT hCE at 80 °C for 10 min. Subsequently, the protein sample was filtered to remove higher oligomers and subjected to SEC using a S75 10/300 GL column equilibrated in a buffer composed of 20 m4N6L) by removing the N-terminal region up to loop L1. This model was used as an initial search model for molecular replacement using PHASER (6FK0. PyMOL (Data processing was performed utilizing iMOSFLM  and SCALg PHASER . Repeateg PHASER  and refig PHASER  were car0. PyMOL  was used4N6O) served as a template to align the structure of dimeric hCE.A model of dimeric hCE in complex with legumain was created using Topmatch . Specifi39 residue on cystatin E, monomeric and dimeric cystatin E were incubated with legumain in a 1:2 molar ratio  at pH 4.0 at 37 °C until 80% turnover was observed, as judged by SDS-PAGE. Subsequently, the pH was shifted to 7.5, and the samples were incubated for 1 h at 37 °C. As control samples, we used legumain only and monomeric/dimeric hCE only. Progress of proteolysis and ligation was investigated after different time points via SDS-PAGE. Dimeric hCE was prepared from N-terminally truncated hCE.To test proteolysis at the P1-Asng, 10 min, 4 °C). To wash off residual monomeric protein, the pellet was resuspended in double-distilled H2O and again harvested by centrifugation. The supernatant was discarded and washed another two times. The pellet was then mounted at the edge of a quartz glass capillary, and diffraction was assayed in house using a Bruker Microstar rotating anode generator mounted with a Mar345dtb detector.Monomeric hCE was incubated at 90 °C for 10 min at 20 mg/ml protein concentration. Subsequently, the insoluble fraction potentially containing amyloid fibrils was harvested by centrifugation  or 50 mm Tris and 100 mm NaCl (pH 7.0). Afterward, samples were put on ice for at least 10 min before testing fibril formation using a ThT test. Briefly, a ThT stock solution was prepared by dissolving 8 mg of ThT (Sigma-Aldrich) in 10 ml of PBS buffer. A working solution was prepared freshly each day by diluting the ThT stock solution 1:50 in PBS buffer. 24 μl of working solution were mixed with 1 μl of sample in a 386-well black polystyrene plate (Corning), and fluorescence was measured for 2 min at 25 °C with excitation at 440 nm and emission at 482 nm. Mean and S.D. values from three measurements were calculated.To test for formation of amyloid fibrils, cystatin E was incubated at 80 °C for 10 min at a protein concentration of 10 mg/ml in a buffer composed of 50 mm citric acid, pH 5.5, and 100 mm NaCl to induce oligomerization. Subsequently, the sample was incubated on ice for at least 10 min before analysis in a ThT assay.To test the effect of L2 loop glycosylation of cystatin E on the formation of higher oligomers, we incubated cystatin E produced in LEXSY at 90 °C for 10 min at a concentration of 20 mg/ml in a buffer composed of 50 mg, 10 min); resuspended in washing buffer composed of 50 mm citric acid, pH 5.5, and 100 mm NaCl; and centrifuged again. This washing step was repeated three times. Afterward, the fibrils were resuspended in 30 μl of wash buffer. To test inhibition of legumain and papain, the substrate solution described before was preincubated with 1 μl of fibrils, and the reaction was started by the addition of the enzyme. In each case, the final reaction volume was 50 μl. Control reactions contained only the enzyme in substrate solution. All reactions were measured in triplicate.Cystatin E fibrils were prepared by incubation of a 10 mg/ml sample at pH 3.0 and 80 °C for 10 min. The insoluble fraction was harvested by centrifugation . To remove any residual monomeric or dimeric protein contaminants, the pellet was washed three times with a buffer composed of 50 mm citric acid, pH 5.5, 100 mm NaCl. The final pellet was resuspended in 20 μl of wash buffer. 5 μl of fibrils were mixed with 5 μl of legumain  and incubated for 10 min at 20 °C. Afterward, the insoluble fraction was harvested by centrifugation and washed two times with washing buffer. Control reactions contained fibrils only, fibrils + AEP precomplexed with the covalent acetyl-YVAD-cmk, and legumain only.Cystatin E fibrils were prepared by incubation of monomeric hCE at a 20 mg/ml concentration at 90 °C for 10 min. The insoluble fraction was harvested by centrifugation  were supplemented with 1 μl of cystatin E fibrils and incubated at 90 °C for 10 min. Control samples contained fibrils only or cystatin E monomer/dimer only. Afterward, samples were incubated on ice for at least 10 min before setting up a ThT assay. The reaction was set up in a buffer composed of 50 mm Tris, pH 8.0, 150 mm NaCl). The flow-through was collected, and the resin was washed five times with wash buffer. Finally, bound protein was eluted by applying five times 100 μl of elution buffer containing 100 mm Tris, pH 8.0, 150 mm NaCl, and 2.5 mm desthiobiotin. Control samples were cystatin E incubated at 37 °C , cystatin E incubated at 70 °C (to confirm binding of dimeric protein), and cystatin C incubated at 70 °C . Fractions from different stages of purification were analyzed on SDS-PAGE.To test heterodimerization of cystatins E and C, cystatin E carrying a C-terminal Strep-tag was co-incubated with cystatin C at 70 °C for 10 min. Following a further 10-min incubation on ice, the sample was loaded on a Strep-Tactin Sepharose resin  pre-equilibrated in wash buffer  by incubation at 90 °C for 10 min. The insoluble fraction was harvested and washed as described above. Subsequently, the pellet containing hCE amyloid fibrils was resuspended in buffer composed of 20 mm MES, pH 5.5, and 20 mm MgCl2. A 1:10 dilution was prepared and vortexed for 1 min. Subsequently, the solution was centrifuged for 10 min at room temperature, and the supernatant was removed carefully and fixed by the addition of 0.1% glutaraldehyde  for 10 min. 10 μl of sample were incubated on Formvar carbon film-coated 400 mesh copper grids  for 2 min and stained with 2% uranyl formate solution containing 25 mm NaOH for 40 s. TEM imaging was performed using a Philips CM 100 transmission microscope operating at 100 kV. Images were acquired using an AMT 4 × 4 megapixel CCD camera. Imaging was performed at ×28,500 magnification.Cystatin E amyloid fibrils were prepared from monomeric protein at a concentration of 20 mg/ml (in a buffer composed of 50 mE. D., J. C. H., and H. B. designed the experiments. K. H. and H. C. performed the TEM experiments. S. O. D. designed the X-ray experiment to test for fibril formation and contributed with valuable discussions. E. D. and J. C. H. performed all other experiments. E. D. and H. B. wrote the paper, and all authors reviewed the manuscript.


Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC If inhibited below the high critical threshold, the cancerous cell would no longer be malignant as there are no alternative signaling pathways which could by-pass the need for CK2.1.250. Additionally, in one study CX4945 was shown to inhibit CLK2 more potently than CK2 (3.8 nM compared to 14.7 nM).Previous research into CK2 inhibition has focused on small molecules that inhibit the kinase activity at the ATP-binding site.Alternative strategies to selectively inhibit CK2 have recently been explored; these exploit other aspects of the protein unique to CK2. The αD pocket, which is unique to CK2, was discovered near the ATP site. It has been exploited by linking a low affinity ATP site fragment to an elaborated fragment in the αD site. This led to a novel and selective CK2 inhibitor.15Pc, was found to inhibit the interaction between CK2α and CK2β.One of the other areas to probe for non-ATP site inhibition is the protein-protein interaction (PPI) interface between the catalytic and regulatory subunits of CK2: CK2α and CK2β. To date three small molecules and a peptide have been shown to bind in the interface pocket on CK2α. The fragment, DRB, binds at both the ATP and the α/β interface site .16 W16 a1.3−1) to identify starting points against any target of interest.The fragment-based approach to drug discovery utilises small compounds  conducted initial high throughput screening of fragments from their fragment library against CK2 using an FP (fluorescence polarization) assay. This library consisted of over 400 compounds from the Abell Group. The most promising fragment from this screen was NMR154 .24 This A crystal structure was obtained of NMR154L, the ethyl analogue of NMR154, a showed With this crystal structure in hand, it was hoped that the binding could be improved by modifying NMR154 along a series of vectors, utilizing anchoring in the pocket and increasing the interactions outside the pocket with the protein residues .3.2The first iteration explored was substituting the methylamine group 50 ≈ 0.4 μM)ture see . W16  giving an IC50 of 150 μM. Realising this trend additional biaryl compounds were screened against CK2 to give the lead compound, CAM187 (7). These compounds affect their binding by the biaryl part of the molecule filling the PPI pocket on the protein and the pendent ammonium group forming an H-bond with an Asp37 above the pocket.By screening the NMR compound library an initial hit was found for an inhibitor of the PPI between the α-β subunits of CK2 with an ICCAM187 is the first small molecule fragment to inhibit the α-β interface of CK2 without also binding in the ATP site. Due to its fragment-like properties, CAM187 leaves room for further development. These developments could lead to drug-like molecules with high potency and selectivity against CK2 as well as deeper understanding between the structure activity relationship.


Increases in barrel temperature and screw speed decreased the trm, and this was associated with better retentions of antioxidant activity, total polyphenol, betalain contents. The betacyanins k values ranged the −0.0188 to −0.0206/s and for betaxanthins ranged of −0.0122 to −0.0167/s, while aE values were 1.5888 to 6.1815 kJ/mol, respectively. The bioactive compounds retention suggests that encapsulated powder can be used as pigments and to provide antioxidant properties to extruded products.Red cactus pear has significant antioxidant activity and potential as a colorant in food, due to the presence of betalains. However, the betalains are highly thermolabile, and their application in thermal process, as extrusion cooking, should be evaluated. The aim of this study was to evaluate the effect of extrusion conditions on the chemical components of red cactus pear encapsulated powder. Cornstarch and encapsulated powder (2.5% w/w) were mixed and processed by extrusion at different barrel temperatures  and screw speeds  using a twin-screw extruder. Mean residence time ( These pigments are betalamic acid derivatives and are divided into two structural groups: the red-violet betacyanins, e.g., betanin, neobetanin, and isobetanin, and the yellow-orange betaxanthins, e.g., indicaxanthin and vulgaxanthin I, II, and IV [There is a growing interest to include in diets foods that are manufactured with natural functional ingredients. In Mexico, there are many foods that can be considered as functional products; cactus pear is one of these. The fruit is fleshy and appears in various colors, d-purple . In part, and IV . Recent , and IV , among o, and IV ,5. In ad, and IV ,7,8, theRTD), which is a useful tool to study physicochemical changes in feed materials. Additionally, RTD has been used to scale-up operations [d-limonene in the extrusion cooking process has been reported [d-limonene due to the formation of inclusion complexes with starch, obtaining products with lesser components degradation. However, the behavior of the bioactive compounds of encapsulated red cactus pear, e.g., betalains, processed by extrusion cooking has not been investigated, therefore their stability on extruded products should be evaluated due to that the pigments represent a dye alternative and source of antioxidants in the development of food products. The aim of this study was to evaluate the effect of barrel temperature and screw speed during the extrusion cooking process on bioactive compounds of encapsulated red cactus pear powder.Extrusion cooking is a technology that is widely used for the development of new products. During this process the feed is transported, mixed and cooked at a high temperature for a short time to obtain expanded products, provoking some physical and chemical changes in the feed material . The majerations . Recent erations , cerealserations , fruits erations ,18,19, ferations , and vegerations . The reserations ,23, antherations ,20,23,24erations ,23,25, aerations  studied reported , findingRTD of the material in the extruder under different barrel temperature and screw speed conditions is characterized by mtr, which is shown in mtr values obtained for the different extrusion conditions were between 36.62 and 60.13 s. These values represent the average time that the feed material remains in the extruder and time that it is exposed to high temperature and shear stress. The residence time is associated with changes that occur in the feed material and that result in complex formation or degradation of the components of interest.The mtr was significantly affected (p < 0.05) by barrel temperature, screw speed, and the interaction of both factors , a* (greenness-redness), and b* (blueness-yellowness), were significantly affected (p < 0.05) by the process temperature and screw speed, as well as by the interaction of both factors. The extrudates had mean moisture content of 9.26% ± 0.3%. The color analysis results of the extrudates are shown in L* values were obtained when the extrusion was carried out at the highest temperature (140 °C) at 225 and 325 rpm. This result may indicate the formation of some Maillard products, which cause darkening of the extrudates. Others authors observed the same tendency [mtr obtained . These results can be due to the formation of Maillard products by increases in barrel temperature [With regard to the color parameter o yellow . An incrperature .p < 0.05) by the process temperature and screw speed. The highest values of total polyphenol content were obtained at the highest screw speed used (325 rpm); this can be attributed at the decreasing of the residence time reached by screw speed increase. Low values were obtained at the lowest screw speed used (225 rpm), which can be due to the longer exposure time  by barrel temperature and screw speed. Antioxidant activity increased as screw speed and extrusion temperature increased. The highest antioxidant activity values were found in extrudates processed at the highest screw speed (325 rpm) and highest temperature (140 °C). This was associated with a low trm of the material in the extruder  by process temperature, screw speed, and the interaction of these factors. The lowest betaxanthin contents were found under the following conditions: 225 rpm and 140 °C and 275 rpm at 80 and 100 °C. This is related to the mtr of the material in the extruder, i.e., a larger mtr results in a longer exposure time of the components to a high temperature during the process, which causes increases in the degradation of pigments because they are sensitive to temperature [Betalain analyses showed that the betaxanthin content  was signp < 0.05) by the screw speed and the interaction of screw speed and temperature, as shown in The betacyanin content was significantly affected  and at highest temperature (140 °C). This effect is due to the nditions . This reIn addition, pigment loss caused by the extrusion cooking process was observed. The calculated retention values were in the range of 33% to 51%, indicating that more than half of these components were lost during the process. The use of temperature in different process causes a degradation of betalains . During The predominant pigments in red cactus pear are betanin and isobetanin; both pigments belong to the red-purple color betacyanins group. To determine the betacyanin retention, betanin and isobetanin were monitored during the extrusion cooking process. The retention values of both pigments at different extrusion conditions are shown in p < 0.05) by temperature, screw speed, and the interaction of both factors. It was observed that an increase in the barrel temperature caused a decrease in the betanin concentration. The highest retention values were obtained at the lowest temperature (80 °C at any speed screw and 100 °C at 225 rpm). The retention values of betanin ranged between 10.92% and 43.54%. The concentration of this pigment was significantly affected (p < 0.05) by temperature and the interaction of barrel temperature and screw speed; however, it showed a trend opposite to that observed for betanin. An increase in the isobetanin content was observed at all extrusion conditions.The isobetanin concentration was significantly affected  and its respective determination coefficient (R2) for the different extrusion condition were calculated by kinetic analysis; they are shown in The decay constant (k) for betacyanins were not significantly (p > 0.05) affected by the barrel temperatures used in the extruder. However, the k value tended to decrease as temperature was increased. This is due to the mtr decrease of the material in the extruder (p < 0.05) as the temperature applied in the extrusion cooking process increased. This can also be related to the decrease in mtr as the temperature increase (p < 0.05) higher than those obtained for betaxanthins.The degradation rate constants  as carrier agent. The drying process was carried out at 160 °C inlet temperature and 85 °C outlet temperature according to a reported method [L*, a* and b*, respectively. The chemical characteristics: antioxidant activity and total polyphenol, betaxanthin, and betacyanin contents are reported in the Results and Discussion Section. The food matrix for the extrusion cooking process was native cornstarch , which was mixed with 2.5% (w/w) of encapsulated red cactus pear powder.Encapsulated powder of juice of red cactus pear  was obtained by spray drying using soluble fiber [(1→3)(1→4) β-d method . The enc2PO4) were from J. T. Baker . Deionized water was obtained using a deionizer . All other reagents used were analytical grade.2,2-Diphenyl-1-picrylhydrazyl (DPPH), Trolox, Folin-Ciocalteu, sodium carbonate, and gallic acid standard reagent were purchased from Sigma-Aldrich . HPLC-grade methanol, HPLC-grade water, HPLC-grade phosphoric acid and analytical grade methanol and potassium dihydrogen phosphate  with a 600 mm length and L/D = 20. Die diameter was 4 mm, and the screw configuration was selected specifically to create high levels of shear . The mixa* (greenness-redness) in the obtained powders was evaluated using tristimulus colorimetry. The obtained data were used to calculate the residence time distribution E(t) and mtr in the extruder [Cornstarch was mixed with erythrosine (3% w/w) and introduced into the extruder using the pulse technique; effluent samples were taken from the extruder every 20 s, starting from the addition of erythrosine (time = 0). The obtained extrudates were dried at 60 °C in an air convection oven, milled, and passed through a No. 20 mesh (0.85 mm). The color parameter extruder  using thDistribution function:E(t) is the exit age distribution function, C(t) is the tracer concentration (measured as a*) at time t and dt is the time interval between successive sampling.Mean residence time:The moisture content was determined using the gravimetric method 950.02 . The anaL* (lightness), a* (greenness-redness), and b* (blueness-yellowness) values. The determination was carried out in triplicate. The color of the extrudate was determined using a Konica Minolta CR-400/410 colorimeter , calibrated with a tile standard with the following values: X = 94.9, y = 0.3185, and x = 0.3124. The color was expressed as g for 10 min in a Centra CL3R IEC centrifuge ; the procedure was repeated to ensure complete extraction. The supernatants were collected and filtered through a polyethylene filter with a pore size of 0.22 μm , and then stored at −20 °C until analysis.To determine the polyphenol content and antioxidant activity, extracts were obtained according to the reported method , with soThe total polyphenol content was determined spectrophotometrically using the Folin-Ciocalteu method . Mixture• radical. First, extract (0.1 mL) was added to a DPPH• radical solution , shaken, and stored in the dark for 3 h. The absorbance was measured at 517 nm using a Lambda 25 UV-Vis spectrophotometer . A Trolox standard calibration curve (0.08–1.28 mM) was used to calculate the antioxidant activity. Measurements were performed in triplicate and the results were expressed as mmol Trolox equivalent/100 g of extrudate.The antioxidant activity was determined according to the spectrophotometric method . This meg at 10 °C for 40 min in a Centra CL3R IEC centrifuge ; this procedure was repeated to ensure complete extraction. Supernatants were filtered through a 0.45-μm nylon filter . The extractions were performed in triplicate and the extracts were analyzed by photometric and high-performance liquid chromatography methods. Betalain pigments were extracted using a method reported , with soA is the absorption value at the absorption maximum of 535 and 483 nm for betacyanins and betaxanthins, respectively; DF is the dilution factor; dV is the dried pulp solution volume (mL); dW is the dried pulp weight (g); and L is the path length (1 cm) of the cuvette. For the quantification of the betacyanin and betaxanthin contents, we used the molecular weight (Mw) and molar extinction coefficient (ε) of betanin  and indicaxanthin , respectively. The measurements were performed in triplicate and the results were expressed as mg betacyanin/100 g extrudate and mg betaxanthin/100 g extrudate.The betalain content was determined photometrically following a method reported  using a 18 250 × 4.6 mm; 0.5 μm particle size), and a UV detector. The mobile phase A was methanol/0.05 M KH2PO4 (18:82 v/v) adjusted to pH 2.75 using phosphoric acid, and the mobile phase B was methanol. The extracts were analyzed using a gradient that started with 100% A, reaching 80% A and 20% B in 20 min at a flow rate of 1 mL/min. Detection was performed at 536 nm. All determinations were performed in duplicate and reported as retention (%), which was calculated as (0X/X) × 100; where X is the area of the extrudate and 0X is the area obtained for the starch/encapsulated mix before the extrusion-cooking process.The retention of betanin and isobetanin was determined using a method reported . Briefly0C is the initial component content, tC is the component content at the time t, k is the reaction rate constant, and t is the time. The temperature dependence of this constant (k) can be described by the Arrhenius equation:0A is the frequency factor or pre-exponential constant, aE is the activation energy of the reaction, R is the universal gas constant, and T is the absolute temperature.The degradation kinetic of betalains can be described by a general reaction rate expression :ln  and screw speed  were considered as independent variables. The properties of the extrudate were considered as dependent variables. The analyses of variance and the differences between means by Tukey’s test were performed using the software Minitab version 16  at a sigmtr of the material in the extruder. The results show that shorter exposure time of the components of interest to high temperature conditions, providing greater retention of total polyphenol, betacyanins, and betaxanthins, as well as antioxidant activity. The k values of betacyanins were higher than those of betaxanthins, whereas the activation energy for the degradation of betaxanthins was greater than that for the degradation of betacyanins; thus, betaxanthins are more susceptible to temperature increases during the extrusion cooking process. The extrusion cooking process affected the retention of encapsulated red cactus pear pigments; however, the obtained retention values were higher than those reported in other extrusion studies. Higher screw speed and temperature result in a lower Despite the degradation of bioactive compounds contained in red cactus pear, the retention achieved by the extrusion cooking process suggests that encapsulated cactus pear powder is a promising natural pigment that can be used in extruded products. Furthermore, because it has antioxidant activity, it could have beneficial effects on consumers’ health.


Functional designing of nanogels has become an attractive domain of biomedical engineering to develop bioactive materials with innovative features for the human healthcare system. Nanosilver has attracted enormous attention due to its wide antimicrobial spectrum and ability to kill almost all types of bacteria in its vicinity. However, the most crucial challenge for bioscientists is the lack of binding ability of nanosilver with the material surfaces that allow nanosilver to leach out to the surrounding tissue and exert toxicity while the biomaterial is in contact with the living system. Designing nanosilver within a nanogel confinement offers enormous possibilities to develop functional bioactive nanoparticles that may be bonded to any biomaterial surface via the nanogel functionality. This approach requires the proper combination of material science with nanotechnology and biotechnology to innovate interesting domain of functional nanogels with unique features. This work aims at providing a critical review on the current progress, approaches, and vision in designing nanosilver‐entrapped nanogel particles with diverse functionality, and their bioactivity against microorganisms for human healthcare devices. The problem sometimes becomes critical and may eventually lead to the formation of biofilm due to the adherence of bacteria where secretion as well as accumulation of extracellular products takes place Figure1. As aNanotechnology has emerged as a fascinating domain of biomedical engineering where advanced materials and biomedical devices with innovative features may be fabricated.FigureSilver has been very effective against a broad range of aerobic bacteria, anaerobic bacteria, yeast, filamentous fungi, and viruses, and has made significant contribution to developing antimicrobial systems for wound and burn care products.2FigureNanosilver particles may be directly impregnated on a material surface where no interacting site is available for adherence of the particles. Unfortunately, this leads to the weak attachment of silver with the material surface leading to the fast leaching out and its subsequent toxicity to the surroundings Figure3a. In FigureFunctional nanosilver has become the subject of enormous interest in the world of bioactive nanogels.FigureThe bioengineering requires incorporation of specific functional groups that would allow nanogel to anchor and to bind to a substrate to achieve bioactivity. The state of the art in nanogel engineering conceptualizes that the functional groups would act as the anchoring sites either for the immobilization of biomolecules or for the interaction with a material surface by ionic, covalent, coordinate or by hydrogen bonding while protecting the biocompatibility with the surrounding tissue. These unique features of a nanogel therefore open up enormous possibilities in developing sutures, implants, wound dressings, and infection‐resistant surfaces Figure5.28, 23FigureThe realization of bioactive nanogels has become the subject at the forefront of bionanotechnology. The crucial aspect of the nanogel preparation is the size and chemical functionality, where nontoxicity and bioactivity is still protected. Nanoemulsion process has shown enormous potential toward the nanoparticle preparation. However, there are more possibilities of designing the chemistry of nanostructures during the polymerization process. The most appropriate route is the polymerization of a monomer as water phase in water‐in‐oil (w/o) nanoemulsion in the presence of silver nitrate, followed by the reduction of silver ions into nanosilver particle Figure6. Alte3.1FigureRadiation process seems to be the smart approach as compared to the other approaches. The radiation performs three simultaneous processes, i.e., polymerization, crosslinking, and silver reduction, but independent of each other. The radiation initiates polymerization of the monomer in a w/o nanoemulsion where a macromolecular chain is formed. Second, it tends to crosslink the polymer chain as soon as it is formed in the system. Third is the reduction of the silver ions so that the nanosilver formation within the nanogel matrix takes place. In addition to it, the radiation polymerization leads to the extremely pure nanogel without any contamination that may arise due to initiators in a chemical process. These three processes therefore influence the process of nanogel formation Figure7.28 ThE. coli and Staphylococcus aureus (S. aureus).FigureFigureThe synthesis of polymethacrylic acid nanogels with nanosilver core has been carried out by the radiation process where well‐dispersed and stable nanoparticles in the range of 10–25 nm were formed.FigureS. aureus and E. coli.The base matrix, in fact, acts as the carrier for the nanosilver particles along with the possibility of biointeraction with a surface via its functional groups. The antimicrobial efficiency of these nanogels was monitored with varying concentration of nanogels and it was found that a very small amount of bioactive nanogel is sufficient for the bacterial reduction Figure10. TheAloe vera and curcumin as coating material to develop herbal composite dressings where excellent wound healing features were observed. It is interesting to see that nanogel in combination with Aloe vera performs much better than its combination with curcumin  and silver nanogel combination may also be synthesized by gamma radiation where CS helps in the stabilization of these particles.These nanogels may be combined with other biological moieties such as in Figure11. MayE. coli, S. aureus, and MRSA.N,N′‐methylene‐bis‐acrylamide crosslinker, leading to a network structure of the nanogel.The reduction and fragmentation of silver nanoparticles by applying ultraviolet (UV) radiation is another chemical‐free approach, where silver ions are reduced due to the presence of hydrated electrons, produced via UV irradiation. The UV‐radiation source transforms the silver ions into smaller sizes of nanoparticles with uniform distribution with good antimicrobial activity against co‐acryloyl phenylalanine) offers pH‐responsive nature while encapsulating the nanosilver within its domain for antimicrobial action against Bacillus and E. coli.FigureAn additional feature of the micellar polymerization has been the preparation of smart nanogels. By virtue of the presence of the AA and MAA, the nanogels offer pH sensitivity leading to the quick transition at pH > 4.6.3.2FigureAdvancement in the nanogel preparation is the use of polymers instead of using monomers and subsequently polymerizing them. These polymers may range from synthetic ones, such as polyvinyl alcohol (PVA), PVP, and polyethylene glycol (PEG), to biodegradable, such as CS, pectin, dextran, gelatin, alginate, and cellulose.2) as the carrier gas generates carboxyl groups while oxygen plasma creates hydroxyl groups along the macromolecular chains.PEG is an interesting polymer due to its biocompatibility and biofouling features that help in infection control in contaminated wounds. PEG‐nanosilver particles may be prepared by dual action of PEG matrix, i.e., PEG helps in silver reduction into nanosilver while at the same time, it provides hydrogel sheath and stabilizes the nanosilver particles. The resultant nanogel therefore contains a layer of PEG around nanosilver that may offer an anchoring moiety with a biomaterial surface. It is observed that the PEG bioactive nanogels may be immobilized by hydrogen bonding onto plasma‐treated polypropylene or polyester filament surface to develop an antimicrobial suture.The in‐situ preparation of nanosilver within epichlorohydrin crosslinked carboxymethyl chitosan (CMCS) nanogels was reported.E. coli shows good zone of inhibition.S. aureus using the disc diffusion method.Streptococcus mutans.Recently, electrospun polyurethane/keratin and nanosilver have been developed for the wound healing applications, where silver nanoparticles were ex situ grown over the electrospun mat after dipping into ascorbic acid solution.Staphylococcus epidermidis and E. coli suggesting a prominent material within the textile industry as antimicrobial surgeon's care and patient drapings. Similarly this approach has been followed for hybrid core–shell structure where nanosilver remains surrounded by NIPAm‐AA copolymer shell.Smart and responsive nanogels add to the attraction of nanotechnology based healthcare systems, as they exhibit enormous potential to adapt the surrounding environment. Core shell silver nanoparticles have been prepared by using oleic acid and ferric ions using emulsion polymerization.3.3S. aureus, P. aeruginosa, E. coli, and Candida albicans.Some approaches have found way into functional nanosilver preparation in spite of the fact they are very rare in use. CMCS with polyethylene oxide (PEO) was used for the nanosilver composite, in which CMCS played dual functionality of reduction as well as stabilization of nanoparticles. The CMCS‐Ag‐PEO nanocomposite is highly effective antimicrobial material with zone inhibition against 4Aloe vera gel extract has been used for the bioreduction of the silver ions in the presence of PVA as capping and stabilizing agent (FigureCinnamon zeylanicum bark extract was employed for the green mediated synthesis of silver nanoparticles in the presence of PVP gel matrix. In this study, the pre and postaddition of PVP hydrogel in the reaction medium was monitored where the influence of the addition of PVP in the appearance of nanoparticles was observed. When the polymer matrix is added before the reduction process, the core–shell morphology of structure was observed. When the polymer matrix was added after the reduction process, composite structure was observed in TEM images suggesting the presence of nanoparticles within the hydrogel matrix (Figure The reduction of silver ions into nanosilver has led to the development of several commercial products.nt Figure14a. Th5Bioengineering of functional nanogels is the fascinating domain of biomedical engineering with enormous potential to create novel architecture toward dedicated applications in human healthcare systems. Nanosilver has been the material of today for its antimicrobial nature. However it lacks the ability to bind to material surface by chemical and physical bonds. Encapsulating the nanosilver within a nanogel seems to a beautiful idea that may lead to nanoparticles where nanosilver is surrounded by a biocompatible hydrogel matrix. Such a nanoparticle could attach to other surfaces depending on the chemical nature of the hydrogel. State of the art is therefore to design and develop a functional nanosilver nanogel that would be biocompatible and biointeractive in nature. Various routes are available for the encapsulation of silver into nanogels and their composites have been widely used. The nanosilver particles may be encapsulated during the polymerization or in a postpolymerization step to give specific architecture to the bioactive nanogel. It seems that the radiation process is very effective in developing nanogels as this allows the polymerization and the silver reduction simultaneously. Moreover, the nanogel may be chemically designed with very precise functional groups and the base matrix of synthetic in origin. The most innovative feature in today's world is that we have a nanosilver particle that is functional due to the polymer nanolayer around it, which makes it to bind to any surface for infection control. May be the hybrid nanostructure would open up a lot more interesting domain. Therefore, the question is that where are we and where do we have to go? It seems that the functional nanosilver may be the prime requirement and we need to look at the possibilities of creating newer structures with additional features to achieve multifeatured materials. The collaborative efforts of material scientists, bioscientists, and biomedical engineers would continue to develop functional nanomaterials with much wider spectrum of applications. Such biointeractive nanogels would certainly open up a revolution in the world of antimicrobial medical devices leading to enormous control over healthcare expenditures.The authors declare no conflict of interest.


No severe complication was associated with the injectionprocedure.Artificial ascites has been reported as an effective technique to reduce the riskof thermal injury in radiofrequency ablation of liver tumors by increasing thedistance of collateral organs located next to the ablated sites. In this casereport we share our experience with artificial ascites in an attempt to reducethe toxicity of collateral adjacent organs in the setting of re-irradiation forrecurrent cervical cancer. A 52-year-old female who developed local recurrenceafter definitive radiation therapy was treated with interstitial re-irradiationby means of image-guided, (single-implant/multi fraction) high-dose-ratebrachytherapy. Because the sigmoid colon was in close proximity to the recurrenttumor lesion, artificial ascites was generated before each treatment fraction bypercutaneous injection of a defined amount of saline solution through theabdominal wall to create additional space between the two volumes. Artificialascites showed a dosimetric improvement by reducing the sigmoid colonD In addition, there is a significant risk for thermal injury in the case oftumors abutting the diaphragm or close to the gastrointestinal tract, the bile duct,or the gallbladder. For those clinical scenarios, artificial ascites has been provento be an effective method to increase safety space between risk structures and tumorlesions. To the best of our knowledge, there is no published experience describingthe use of artificial ascites in association with RT. In the current report, weutilized it for the safe delivery of interstitial HDR BRT for the re-irradiation ofrecurrent cervical cancer by creating additional space between gastrointestinaltract volumes and the recurrence site.From the standpoint of OARs protection, artificial ascites has been used ininterventional radiology to avoid diaphragm or gastrointestinal tract damage whentreating liver tumors with radiofrequency ablation (RFA).−2) followed by 1 cycle of cisplatin (80 mg m−2) [switch from nedaplatin to cisplatin because of drug induced skinrash] and 50.4 Gy conventionally fractionated whole pelvis EBRT  plus 24 Gy total physical dose HDR intracavitary BRT delivered infour fractions. The patient responded with complete clinical remission.A 52-year-old-female was treated with definitive concurrent chemoradiotherapy forFIGO stage IIB squamous cell uterine cervical cancer. In consequence of multiplethoracic and abdominal aortic dissections for Marfan-Syndrome, radical hysterectomywas not attempted in accordance with the patient´s wish. Chemoradiotherapyconsisted of 2 cycles nedaplatin (100 mg m−2) and cisplatin (50 mg m−2) followed by 6 cycles of bevacizumab (15 mg kg−1) wasadministered resulting in partial response. Considering that recurrent disease waslocally confined, TPE was planned but her cardiovascular comorbidities hindered thisattempt. To this end, she was referred to our department for interstitial BRT asimage-guided re-irradiation method. 11,16 In the current case, treatment consisted of sole HDR BRT with 48 Gy totalphysical dose being delivered in 8 fractions at 6 Gy, applied twice-daily with aninterfractional interval of at least 6 h. No additional EBRT was prescribed. Ⓡ Safety, B. Braun Medical Incorporated, Bethlehem, PA) wasinserted percutaneously by ultrasound-guidance and 500 ml of saline solution wasadministered before every BRT fraction  according to the ethical standards laid down in the Declaration ofHelsinki.8 This approach poses a mutilating surgical procedure with a high incidence ofcomplications and significant impairment of quality of life.10 In unresectable disease or in patients refusing surgery, radicalre-irradiation with EBRT has been tried18 but remains a choice of excessive morbidity which may outweigh the benefitsof therapy. Chemotherapy alone, on the other hand, continues despite its excessiveuse in oncological reality to be of limited benefit with poor response and sobering outcome.20Standard therapy for patients with non-metastatic recurrent cervical cancer who havepast history of pelvic RT is TPE.11 There are, however, no well-defined recommendations for selecting patientsfor interventional radiooncological treatment and in most cases the decision is madeindividually. Notwithstanding this, the rationale for re-irradiation by means of HDRcannot be called into question considering that it offers radiobiological andtechnical advantages. As normal tissue toxicity after repeated full courseconventional EBRT has shown to be significant, it seems reasonable to assume thatfurther improvements in the therapeutic ratio can be generated by escalating thetreatment dose while ameliorating conformity. Interstitial HDR BRT meets thisobjective optimally by exploiting the radiobiological advantage of larger fractionsizes while prospective 3D dosimetry provides anatomy-oriented dose optimization forhighly conformal intensity modulated RT. At this point, the intrinsic characteristicof HDR to generate high intratarget dosing is of particular importance as itfacilitates the application of ablative doses to central tumor volumes that arethought to experience increased radioresistance after previous irradiation.22In this demanding clinical setting, some patients favor re-irradiation andinterstitial BRT has demonstrated effectiveness in the management of locallyrecurrent cervical cancer developing within previously irradiated volumes.9 Therefore, further technical improvements are necessary to facilitate thesafe delivery of cytotoxic doses. One approach could be the avoidance of hot spotsin OARs by increasing the distance between target volume and OARs. In this report,we utilized artificial ascites for the safe delivery of interstitial HDR BRT ofrecurrent cervical cancer by creating additional space between gastrointestinaltract volumes and the recurrence site. It could be demonstrated that the procedureitself is safe and reproducible, confirming the experiences from its use in RFA ofliver tumors.14 From a dosimetric point of view, the displacement of the sigmoid resulted ina decrease of sigmoid D0.1cc from 286y to 189 cGy per fraction.Bearing in mind the predominantly palliative intention of re-irradiation, highergrade toxicity rates are of particular relevance the more so as a balance must beachieved between the probability of LC and the probability of toxic complications.Our group previously reported 2-year local control rates of 51.3% forpatients who received image-guided interstitial BRT for recurrent cervical cancer atthe cost of developing late severe complications greater than Grade 2 in27.8% of patients.To the best of our knowledge, this is the first report on the feasibility and safetyof artificial ascites for OARs sparing in intrapelvic BRT. Concerning its long-termefficacy, further clinical research is needed to define which kind of patients willmost benefit from this technique and whether this method significantly reduces latesevere adverse effects in the re-irradiation settings.Artificial ascites has been reported as an effective technique to reduce therisk of thermal injury in radiofrequency ablation of liver tumors byincreasing the distance of collateral organs located next to the ablatedsites. In this case report it was suggested that a novel technique ofartificial ascites in image-guided interstitial high-dose-rate brachytherapycould be generated safely in order to reduce the radiation exposure oforgans at risk in the case of intrapelvic re-irradiation.


Strontium isotope ratios from 34 archaeological sheep and goat enamel, two archaeological bones and 14 modern tree leaves are presented. The isotopic results suggest that sheep and goats consumed at the site were reared locally (within a few kilometres radius) during the whole period of occupation. The paper discusses the isotopic results in light of the socio-political structure of this period, as complex, strongly territorial societies developed during the Iron Age in the north-east Iberian Peninsula.Animal mobility is a common strategy to overcome scarcity of food and the related over-grazing of pastures. It is also essential to reduce the inbreeding rate of animal populations, which is known to have a negative impact on fertility and productivity. The present paper shows the geographic range of sheep provisioning in different phases of occupation at the Iron Age site of Turó de la Font de la Canya (7 This is reflected in the archaeological record in changing settlement pattern (from open-air sites on the plains to fortified sites on hills), the expansion and progressive complexity of fortifications, and the spread of warrior equipment and weapons in some tombs. These processes thus apparently involved a significant increase in warfare and, probably, in the maintenance of boundaries between territories –8. In thications , 7, 9. Iications –11, was ications –16. Thisattested –14, 16, attested –19.th to 2nd centuries BC, that is, from the spread of iron metallurgy in the area to the period of the Roman conquest.The site named Turó de la Font de la Canya  was an important point for cereal storage and trade with other cultures of the Mediterranean, as suggested by the presence of numerous subterranean ‘silos’ suitable for cereal storage, together with imports of Phoenician, Greek and other origins . Its occThe analysis of strontium isotope ratios from tooth enamel is now a well-established method for exploring human and animal mobility –32. The 87Sr/86Sr) varies in different geological formations according to the age and original rubidium (Rb) /strontium (Sr) ratio of the bedrock, leading to high radiogenic 87Sr/86Sr ratios in old or crustal rocks, and low 87Sr/86Sr ratios in young or mantle rocks [87Sr/86Sr isotope composition of plants reflects the strontium isotopic ratios of the underlying bedrock, as biologic processes involved during plant growth do not entail isotopic fractionation of strontium isotopes [Strontium substitutes for calcium and occurs as a trace element in biogenic tissues, including the hydroxyapatite of teeth and bones , 34–35. le rocks , 36. Theisotopes –38. Otheisotopes  atmospheisotopes –44.87Sr/86Sr isotope composition derives from the food and drink ingested by the animal [In the case of skeletal material, the e animal –46. The e animal –48. There animal –38, 49.The site of Turó de la Font de la Canya is located on a coastal promontory on the south-west margin of the Catalan Coastal Range. From a geological perspective, this mountain range is characterised by fragmented outcrops of mainly Mesozoic and Tertiary sedimentary formations overlying infra-Silurian and Varingian batholith and other Palaeozoic metamorphic rocks . The setth century BC), nine from the Middle Iron Age (6th -5th centuries BC), and 16 from the Late Iron Age (4th -3rd centuries BC). Despite the higher inter-individual variation in the enamel mineralization of the third molar in comparison to the second [The analysed archaeological material totals 30 sheep and four goat teeth (including seven sheep teeth previously reported ) from die second –55, as we second , third m3). Strontium was collected using standard resin columns (Eichrom Sr-Spec resin) and then loaded onto single rhenium filaments with a TaCl5 activator. Total chemistry blanks were less than 20pg and thus negligible for this study. The strontium isotope composition was determined with a solid-source thermal-ionization mass spectrometer at the Labogis of Nîmes University (Thermo Finnigan TRITON TI). Repeated measurements of the international standard for 87Sr/86Sr NBS 987 gave a mean value of 0.710251± 0.000018 for static analysis .The enamel samples were prepared for strontium isotope analysis following standard practices described in previous studies , 33, 57.3) overnight at room temperature. Further acid and a trace of H2O2 were added, before the samples were processed in a microwave oven at 175°C for 20 minutes. The samples obtained were then dried overnight on a hotplate prior to a secondary oxidation stage which repeated the whole process. The samples were converted to chloride in a solution of 6 M HCl, then dried and taken up again in 2N HNO3 prior to strontium separation using standard resin columns (Eichrom Sr-Spec resin).In order to assess the variability of bio-available strontium ratios in the vicinity of the site and surrounding geological areas, 14 modern leaves from oak, pine and strawberry trees growing on different geological formations were also analysed  the Oligocene bedrock displayed values comprised between 0.7093 and 0.7095. All these strontium ratios are consistent with other measurements from similar geological formations in the Iberian Peninsula and elsewhere [lsewhere , 38.87Sr/86Sr isotopic ratios (n = 27), including those for all four goats, range between 0.7091 and 0.7096, compatible with the values attested on the Miocene sediments where the site is located. Six teeth  have values in the range 0.7086–0.7090, which could correspond to Cretaceous sediments from the neighbouring Garraf mountains (circa 4km away). Only one tooth dated to the 7th century BC has a value of 0.7102 , which could be compatible with the Triassic limestone and dolomites present further away in the Garraf (circa 10km from the site).th century BC, SU1087A, 4th century BC and SU1090B, 3rd century BC) have notable differences along the tooth height  as well as SU1087A and SU1090B  are compatible with the geology immediately surrounding the archaeological site. This is consistent with sheep and goats being reared locally in the different phases of occupation, most likely on the Miocene sediments and Pleistocene gravels and clays of the Penedès Valley. Nevertheless, the use of other areas of this valley  cannot be excluded, as the Vallès-Penedès forms a corridor of Plio-Pleistocene alluvial sediments surrounded by older geological formations (see text above and circa 4 km as the crow flies). Overall, the limited variability of strontium ratios points towards local rearing of caprines rather than long-distance trade of animals.The Tanit figurines of Punic origin, a clay mask from the central or eastern Mediterranean, and numerous Campanian A ceramics from Italy in the levels dated from the 4th and 3rd centuries BC among others [The homogeneity of the strontium results contrast with the diversity of imports recovered from the site, which include Cruz del Negro pottery from the Straits of Gibraltar, g others –22. Whilterrestrial movements of livestock were difficult in the north-east Iberian Peninsula at this time.Overall, material cultural data suggest that Turó de la Font de la Canya had an active role in the Mediterranean trade. Conversely, the results from strontium isotopic analysis on sheep and goats suggest that animal were reared locally, and so probably on a small-scale basis, and that caprines slaughtered at the site were mainly reared in the Penedès valley and perhaps also the neighbouring Vallès. This local breeding of livestock may reflect (and be a consequence of) the local socio-political context. The significant change in settlement pattern in the Late Bronze and Iron Ages–from open-air sites located on the plains to fortified settlements located on hills–is thought to reflect increased warfare and territoriality , 7, 66, 87Sr/86Sr) values for the vicinity of the Iron Age site of Turó de la Font de la Canya  and surrounding geological areas, based on analysis of modern leaves from trees growing on different geological substrates around the site and two archaeological bone samples. Modern leaves provided a consistent and fairly narrow range of local strontium ratio values. This new baseline has been central for the interpretation of the strontium isotopic ratios measured in 34 archaeological sheep and goat tooth enamel samples dated from the 7th to 3rd centuries BC, to explore the geographical range of meat provisioning at Iron Age Turó de la Font de la Canya. Seventeen of these samples were sequentially analysed to observe variations along the tooth crown. The investigated archaeological samples indicate limited variability of strontium values throughout the occupation of the site, and 27 of the 34 samples are compatible with the local geology (87Sr/86Sr range: 0.7091–7096), while seven samples may be derived from bedrocks as close as 5 km from the site (87Sr/86Sr: 0.7086–0.7090), and one more (87Sr/86Sr: 0.7102) is compatible with slightly more distant (about 15 km from the site) geological areas. The sequential sampling suggests that most animals were reared locally all the year round, thus supporting the idea of small scale herding rather than long-distance sourcing. Interestingly, some animals display similar patterns of variation along the tooth crown, thus suggesting that some movement of livestock existed. Overall, the archaeological results suggest that sheep and goats slaughtered at the site were mainly reared in the local area, most notably the Miocene clays and Plio-Pleistocene alluvial sediments of the plain. This contrasts with the role of the site as central point for cereal storage (see This study has established baseline strontium isotopic ratio (rage see  as well The low diversity of strontium ratios of sheep and goats at Turó de la Font de la Canya implies herding over a limited geographical range that may partly have been dictated to the difficulty of long-distance terrestrial movements in the Iron Age, as a result of the emergence of the small, strongly defended territories suggested by the settlement pattern, architecture and finds of weapons. Alternatively, the limited geographical scale of herding may primarily reflect the rearing of livestock in only modest numbers. Further zooarchaeological studies, both macroscopic and isotopic, may clarify which of these rival explanations is more plausible.


Pre-treatment with NVP-AEW541 inhibited the FSH-induced MYPT1 and ERK 1/2 phosphorylation, decreased the FSH-dependent Protein kinase B (AKT)308 phosphorylation, but did not affect the FSH-induced AKT473 and JNK phosphorylation rate. It also interfered with the FSH-induced AMH and FSHR down-regulation. No influence was observed on the FSH-stimulated Inhibin B gene expression. Conclusion. These findings support the role of theIGF1R in FSH signaling in porcine SCs. The possible influence of IGF1 stimulation on the FSH-mediated effects on SCs should be further explored.Experimental evidence has shown that the IGF1 receptor (IGF1R) is involved in testicular development during embryogenesis. More recently, data gathered from mice granulosa cells and zebrafish spermatogonia suggest that IGF1R has a role in Follicle-stimulating hormone (FSH) signaling. No evidence has been reported on this matter in Sertoli cells (SCs) so far. The aim of the study was to evaluate the role, if any, of the IGF1R in FSH signaling in SCs. The effects of FSH exposure on myosin-phosphatase 1 (MYPT1), ERK 1/2, AKT Follicle-stimulating hormone (FSH) is required for normal spermatogenesis . A deepeA number of studies have assigned a role in SC function to the insulin-like growth factor 1 receptor (IGF1R), which belongs to the tyrosine kinases receptor family . AccordiThe phosphatidylinositol-3 kinase (PI3K) signaling, involving AKT phosphorylation, is required for cell transcription, translation, proliferation and apoptosis . PI3K, wThe protein phosphatase 1β (PP1β) has been regarded as the possible hub linking between the FSH and the IGF1R signaling in granulosa cells . PP1 is 668, ERK 1/2, AKT308, AKT473, JNK phosphorylation in purified and functional porcine neonatal SCs, with and without pre-treatment with the IGF1R inhibitor NVP-AEW541 and the PP1β inhibitor tautomycin. We also investigated whether the FSH-dependent AMH, Inhibin B and FSHR gene expression was influenced by pre-treatment with the IGF1R inhibitor NVP-AEW541. The role of the IGF1R in FSH signaling has not been investigated in SCs so far. Therefore, this study was undertaken to explore this topic. To accomplish this, we evaluated the effects of FSH on MYPT1This study was conducted in strict compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Perugia University Animal Care. The protocol was approved by the internal Institutional Ethic Committee .6 SCs with a 95% of purity and a negligible percentage of contaminant cells (Leydig and Peritubular cells < 5%), using established methods [2 at 37 °C. After three days in culture, the purity and the functional competence of SC monolayers were performed according to previously established methods [SCs were obtained from neonatal prepubertal Large White pigs at 7–15 days of age. From each testis, we isolated 60 × 10 methods ,15. Brie methods . The res methods .®, IBSA Farmaceutici Srl, Rome, Italy) at the concentration of 50 ng/mL for 15 min; (2) DMSO or 1 µM tautomycin for 5.5 h, followed by vehicle or hpFSH (50 ng/mL) for 15 min; (3) DMSO or 1 µM tautomycin for 5.5 h and NVP-AEW541 for 1 h, followed by vehicle or hpFSH (50 ng/mL) for 15 min, as described elsewhere [2 at 34 °C.When the SC monolayers were confluent (at three days of culture), they underwent the following treatments: (1) dimethyl sulfoxide (DMSO) or NVP-AE541 for 1 h and then incubated with vehicle or urofollitropin (hpFSH)  for 10 min, the supernatant was collected and total protein content was measured by the Bradford method [At the end of the incubation period, total cell lysates were collected in radioimmunoprecipitation assay (RIPA) lysis buffer . The mixture was centrifuged at 1000× d method . Sample Total RNA was extracted and quantified by reading the optical density at 260 nm. In particular, 2.5 μg of total RNA was subjected to reverse transcription  to a final volume of 20 μL. The qPCR was performed using 50 ng of the cDNA prepared by RT and a SYBR Green Master Mix . This was performed in an Mx3000P cycler (Stratagene), using FAM for detection and ROX as the reference dye.The following primers were used for real-time PCR analysis: AMH, forward primers 5′-GCGAACTTAGCGTGGACCTG-3′, revers primers 5′-CTTGGCAGTTGTTGGCTTGATATG-3′; Inhibin B, forward primers 5′-TGGCTGGAGTGACTGGAT-3′, revers primers 5′-CCGTGTGGAAGGATGAGG-3′; FSHR forward primers 5′-TTTCACAGTCGCCCTCTTTCCC-3′, revers primers 5′-TGAGTATAGCAGCCACAGATGACC-3′; actin, forward primers 5′-ATGGTGGGTATGGGTCAGAA-3′, revers primers 5′-CTTCTCCATGTCGTCCCAGT-3′.p value was lower than 0.05.Results are shown as mean ± SD throughout the study. Data were analyzed for statistical significance by one-way ANOVA, followed by Tukey post hoc test using SPSS 9.0 for Windows . A statistically significant difference was accepted when the To elucidate whether the IGF1R and PP1β are involved in FSH signaling, we investigated if the FSH-dependent MYPT1, AKT and JNK phosphorylation was affected by pre-treatment with NPV-AEW541 (an IGF1R inhibitor) and/or tautomycin (a PP1β inhibitor). To further analyze the role of the IGF1R on the FSH-dependent AMH and inhibin B gene expression, we evaluated AMH and inhibin B mRNA levels in the FSH-incubated plates, with and without pre-treatment with NPV-AEW541.308/AKT ratio, but by a lesser extent after pre-treatment with NVP-AEW541 and/or tautomycin (473/AKT phosphorylation ratio. Pre-treatment with NVP-AEW541and/or tautomycin hindered the FSH-stimulated AKT473 phosphorylation rate (Treatment with FSH increased the MYPT1668/MYPT1 phosphorylation ratio. This effect was inhibited by pre-treatment with NVP-AEW541 and/or tautomycin (ion rate .p < 0.01). The extent of this inhibition was lower in pre-treated cultures  (p < 0.01). These effects were not influenced by pre-treatment with NVP-AEW541  (p < 0.01). This was inhibited by NVP-AEW541 pre-treatment   , panel acontrol) , panel bcontrol) , panel cWe have recently reviewed the effects of the IGF system  on testicular differentiation and function in several species including the human one . Altoget308, AKT473, IRS1989 and IGF1RTyr1135/1136 phosphorylation [308, AKT473, IRS1989 phosphorylation, thus suggesting that the serine/threonine (Ser/Thr) PP1 is necessary for the FSH-mediated IRS1 and AKT phosphorylation. Data from zebrafish confirmed such findings. Indeed, incubation with FSH (promoting type A and B spermatogonia proliferation) increased the IGF3 (a fish-specific member of the IGF family) expression by the PKA and ERK pathways. The FSH-induced proliferation was hindered by the incubation with an IGF3R inhibitor in type A spermatogonia [Data from mouse granulosa cells have shown the involvement of the IGF1R in FSH signaling. In greater details, IGF1R was required for the FSH-dependent AKTatogonia .The results of the present study seem to confirm the existence of an interplay between FSH and IGF1R signaling in SCs. Accordingly, we found that both PP1 and IGF1R inhibition resulted in a lack of FSH-mediated MYPT1 phosphorylation in porcine SCs. Therefore, it may be hypothesized that, similarly to what reported in granulosa cells, IGF1R, IRS1, PP1 and MYPT1 gather together in a molecular complex that requires a conserved tyrosine kinase activity of PP1 and IGF1R to achieve a normal MYPT1 phosphorylation rate under FSH stimulation.Moreover, in porcine SCs, the FSH-stimulated ERK1/2 phosphorylation occur with an IGF1R-dependent mechanism. PP1 showed to be replaceable for this outcome. Curiously, the double PP1 and IGF1R inhibition did not affect the FSH ability to phosphorylate ERK1/2. In addition, the FSH-dependent AKT phosphorylation was affected by PP1 or IGF1R inhibition. This was expectable since the phosphorylation of AKT reflects the degree of PI3K activation, which in turn is triggered by the IGF1R ,20.For the first time, we have also observed that a JNK dephosphorylation occurred after the exposure to FSH. This outcome was not affected by PP1 or IGF1R inhibition. Finally, the FSH-induced downregulation of AMH and FSHR gene expression was IGF1R-mediated. By contrast, IGF1R did not interfere with the FSH-mediated enhancement of inhibin B gene expression.Porcine SCs have a high degree of similarity with the human ones. Indeed, they have been used in human transplantation experimental protocols for the treatment of patients with type I diabetes mellitus without the need of immunosuppressive therapy ,22,23. GOur results need to be taken with care since the present experimental model does not resemble the complexity of the testicular tissue. Indeed, being an in-vitro study carried out only on SCs, we do not know how the paracrine cross-talk with Leydig cells might impact the SCs responsiveness to FSH in the presence of IGF1R inhibition in vivo. Second, we referred to protocols adopted in granulosa cells for doses and time of incubation, but dose-response analysis of tautomycin, NVP-AEW541 and FSH incubation in SCs are warranted. All these limitations should be taken into consideration in further experimental studies.In conclusion, the results of this study suggest that IGF1R has a role in the modulation of FSH signaling in porcine SCs. The effects of IGF1 on SC physiology deserve further investigation.