Midpoint Of Thermal Transition Research Articles

Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations.

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day-1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L-1 h-1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of -1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7-9 °C, relative to those at 1-4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14-18% at 25-100 mg/mL, relative to those at 1-4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh.

Disulfide Bond Engineering for Enhancing the Thermostability of the Maltotetraose-Forming Amylase from Pseudomonas saccharophila STB07.

Maltooligosaccharides are a novel type of functional oligosaccharides with potential applications in food processing and can be produced by glycosyl hydrolases hydrolyzing starch. However, the main obstacle in industrial applications is the balance between the high temperature of the process and the stability of enzymes. In this study, based on the structural information and in silico tools (DSDBASE-MODIP, Disulfide by Design2 and FoldX), two disulfide bond mutants (A211C-S214C and S409C-Q412C) of maltotetraose-forming amylase from Pseudomonas saccharophila STB07 (MFAps) were generated to improve its thermostability. The mutation A211C-S214C was closer to the catalytic center and showed significantly improved thermostability with a 2.6-fold improved half-life at 60 °C and the thermal transition mid-point increased by 1.6 °C, compared to the wild-type. However, the thermostability of mutant S409C-Q412C, whose mutation sites are closely to CBM20, did not change observably. Molecular dynamics simulations revealed that both disulfide bonds A211C-S214C and S409C-Q412C rigidified the overall structure of MFAps, however, the impact on thermostability depends on the position and distance from the catalytic center.

Engineering of thermostable phytase-xylanase for hydrolysis of complex biopolymers.

The online version contains supplementary material available at 10.1007/s13205-021-02936-z.

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Site-specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade-off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild-type. The aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para-aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para-cyanophenylalanine (pCNF) and para-nitrophenylalanine (pNTF), and simultaneously modified the hydrogen-bonding potential of the aromatic substituent from accepting to donating. The fine-tuning of residue 385 yielded variants with a 43-fold increase in specific activity for 50mm 3-HBA and 100% increased kcat (pCNF), 290% improvement in Km (pNTF), 240% improvement in kcat /Km (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring-substituted functional group, from donating to accepting, stabilised a helix-turn (D259-H261) through an intersubunit H-bond with G262, to give a 7.8°C increase in the thermal transition mid-point, Tm , and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site-specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs.

Reversible Photocontrol of DNA Melting by Visible-Light-Responsive F4-Coordinated Azobenzene Compounds.

Spatiotemporal control over the regulation of intra- and intermolecular motions in naturally occurring systems is systematically studied to expand the toolbox of mechanical operations in multicomponent nanoarchitectures. DNA is ideally suited for programming light-powered processes that are based on a minimalist molecular design. Here, the noncovalent incorporation of bistable photoswitches into B-like DNA moieties is shown to trigger the thermal transition midpoint of the duplexes by converting visible light into directed mechanical work by orchestrating the collective actions of the photoresponsive chromophores and the host DNA nanostructures. Besides its practical applications, the resulting hybrid nanosystem bears unique features of modulability, biocompatibility, reversibility, and addressability, which are key components for developing molecular photon-controlled programmed materials.

Coupled molecular dynamics mediate long- and short-range epistasis between mutations that affect stability and aggregation kinetics

Multiple mutations are typically required to significantly improve protein stability or aggregation kinetics. However, when several substitutions are made in a single protein, the mutations can potentially interact in a nonadditive manner, resulting in epistatic effects, which can hamper protein-engineering strategies to improve thermostability or aggregation kinetics. Here, we have examined the role of protein dynamics in mediating epistasis between pairs of mutations. With Escherichia coli transketolase (TK) as a model, we explored the epistatic interactions between two single variants H192P and A282P, and also between the double-mutant H192P/A282P and two single variants, I365L or G506A. Epistasis was determined for several measures of protein stability, including the following: the free-energy barrier to kinetic inactivation, ∆∆G‡; thermal transition midpoint temperatures, Tm; and aggregation onset temperatures, Tagg Nonadditive epistasis was observed between neighboring mutations as expected, but also for distant mutations located in the surface and core regions of different domains. Surprisingly, the epistatic behaviors for each measure of stability were often different for any given pairwise recombination, highlighting that kinetic and thermodynamic stabilities do not always depend on the same structural features. Molecular-dynamics simulations and a pairwise cross-correlation analysis revealed that mutations influence the dynamics of their local environment, but also in some cases the dynamics of regions distant in the structure. This effect was found to mediate epistatic interactions between distant mutations and could therefore be exploited in future protein-engineering strategies.

Direct Evidence for the Effect of Glycerol on Protein Hydration and Thermal Structural Transition

The mechanisms of protein stabilization by uncharged solutes, such as polyols and sugars, have been intensively studied with respect to the chemical thermodynamics of molecular crowding. In particular, many experimental and theoretical studies have been conducted to explain the mechanism of the protective action on protein structures by glycerol through the relationship between hydration and glycerol solvation on protein surfaces. We used wide-angle x-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation to quantitatively characterize the hydration and/or solvation shell of myoglobin in aqueous solutions of up to 75% v/v glycerol. At glycerol concentrations below ∼40% v/v, the preservation of the hydration shell was dominant, which was reasonably explained by the preferential exclusion of glycerol from the protein surface (preferential hydration). In contrast, at concentrations above 50% v/v, the partial penetration or replacement of glycerol into or with hydration-shell water (neutral solvation by glycerol) was gradually promoted. WAXS results quantitatively demonstrated the neutral solvation, in which the replacement of hydrated water by glycerol was proportional to the volume fraction of glycerol in the solvent multiplied by an exchange rate (β ≤ 1). These phenomena were confirmed by small-angle neutron scattering measurements. The observed WAXS data covered the entire hierarchical structure of myoglobin, ranging from tertiary to secondary structures. We separately analyzed the effect of glycerol on the thermal stability of myoglobin at each hierarchical structural level. The thermal transition midpoint temperature at each hierarchical structural level was raised depending on the glycerol concentration, with enhanced transition cooperativeness between different hierarchical structural levels. The onset temperature of the helix-to-cross β-sheet transition (the initial process of amyloid formation) was evidently elevated. However, oligomerization connected to fibril formation was suppressed, even at a low glycerol concentration.

Copper, zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692bp with an open reading frame of 459bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with Km of 0.047±0.008μM and Vmax of 1250±24units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80°C with temperature optima at 20°C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80°C for 180min (kd, 6.54±0.17×10−3 min−1; t1/2, 106.07±2.68min), and had midpoint of thermal transition (Tm) of 70.45°C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.

A Single Mutation in Framework 2 of the Heavy Variable Domain Improves the Properties of a Diabody and a Related Single-Chain Antibody

Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

Applications of Differential Scanning Calorimetry for Thermal Stability Analysis of Proteins: Qualification of DSC

Differential scanning calorimetry (DSC) has been used to characterize protein thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical industry. Recently, there have been increased requests from regulatory agencies for the qualification of characterization methods including DSC. Understanding the method precision can help determine what differences between samples are significant and also establish the acceptance criteria for comparability and other characterization studies. In this study, we identify the parameters for the qualification of DSC for thermal stability analysis of proteins. We use these parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable for protein thermal stability analysis for these purposes. Several molecules from different structural families were studied. The experiments and data analyses were performed by different analysts using different instruments at different sites. The results show that the (apparent) thermal transition midpoint (T(m)) values obtained for the same protein by same and different instruments and/or analysts are quite reproducible, and the profile similarity values obtained for the same protein from the same instrument are also high. DSC is an appropriate method for assessing protein thermal stability and conformational changes.

The dipeptidyl carboxypeptidase of Escherichia coli novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The KM and kcat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Porcine pancreatic alpha amylase and its isoforms—Effect of deglycosylation by peptide- N-glycosidase F

Porcine pancreatic α-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide- N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1–2 °C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340 nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils.

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

Increased thermal stability of proteins in the presence of amino acids

This study is a systematic attempt to understand the roles of osmolytes in protecting proteins against denaturing stress. Thermal denaturation of cytochrome c has been studied in the presence of various concentrations of all L-amino acids that are more hydrophobic than glycine and have a solubility of 0.1 M or higher in water at 25 degrees C. The basic observations are as follows. (1) Arginine and histidine destabilize the native protein; both Tm (the midpoint of thermal transition) and delta GDH2O (25 degrees C) (the Gibbs energy of stabilization) decrease with increasing amino acid concentration. (2) Isoleucine, leucine and phenylalanine have no effect on Tm and deltaGDH2O (25 degrees C). (3) Valine and less hydrophobic amino acids stabilize the protein in terms of Tm but deltaGDH2O (25 degrees C) is unchanged. This observation was confirmed by the study of isothermal denaturation of cytochrome c by guanidinium chloride which suggested that delta GDH2O is independent of osmolyte concentration, but Cm (the midpoint of transition) is increased in their presence. (4) In the case of stabilizers, change in Tm/mol of amino acid decreases with increasing hydrophobicity of these osmolytes.

The thermotropic properties of human plasma fibronectin

Thermal denaturation profiles for human plasma fibronectin under a variety of conditions have been determined. Although a single melting curve for this protein, with a thermal transition midpoint of 58.4 ± 1.0 °C and a calorimetric enthalpy change (Δ H c) of 1040 ± 100 kcal/mol, is obtained in dilute neutral salt solutions, it is estimated that a total of seven to eight independent two-state thermal transitions are present in this endotherm. These values are not significantly altered by the presence of Ca 2+, up to levels of at least 20 m m. Upon variation of the pH, no distinct thermal transitions are noted at values below pH 5.0 and above pH 10.0. Between pH 7.0 and 10.0, virtually no alterations in the thermotropic properties of fibronectin are observed, indicating that the individual domains of this protein, which contribute to the thermogram, are preserved in this pH range. Upon alteration of the ionic strength of the buffer, from 0.05 to 0.4 m KCl, small changes are observed in the thermal transition profiles of fibronectin, indicative of conformational changes in the protein resulting in a larger number of cooperative units undergoing the temperature-induced unfolding reaction.

Nucleic Acids of the Marine Ciliate Uronema nigricans, Pc

Nucleic acids of Uronema nigricans, Pc, a small euryhaline hymenostome ciliate isolated from planktonic debris off the coast of Florida, were isolated and partially characterized. DNA was purified from macronuclear preparations and the final product was fibrous, contained less than 1% protein and was essentially free of carbohydrate and RNA. The base composition, determined chromatographically, was (mol% ± s.d.): guanine, 10·8 ± 1·3; adenine, 41·5 ± 1·2; cytosine, 9·0 ± 0·6; thymine, 38·7 ± 1·7. The average molecular weight based on estimates made from sedimentation rate constants determined in sucrose gradients was about 3 × 106. The DNA exhibited a sharp thermal transition over a narrow temperature range accompanied by an increase in absorbance of 42% at 260 nm. The midpoint of thermal transition, Tm , was 77·6°C, corresponding to a guanine + cytosine content of 20·2 mol%, a result supported by the chromatographic studies and among the lowest ever reported for a ciliate. Reassociation of heat-denatured preparations in 1 m-Na+, pH 7·0 at Tm −25 °C followed second-order kinetics for about 30% of the reaction and revealed the presence of two components, a slowly reassociating main component comprising about 96% of the DNA and a rapidly reassociating minor component comprising about 4% of the DNA. The molecular complexity of the main component was 2·4 × 1010 and that of the minor component was about 107. Sucrose gradient analysis of several purified RNA preparations showed three components exhibiting sedimentation coefficients of 24·8 ± 1·2, 17·4 ± 1·0 and 4·0 ± 0·8 S. These values were comparable with those obtained for RNA of the freshwater ciliates Tetrahymena and Paramecium.

Computer analysis for the thermal transition midpoint of DNA

A programmable thermal analyzer system in conjunction with a computer program were used to determine the thermal transition midpoint, T m , and percentage of guanine plus cytosine, % G + C, of a bacterial and two viral DNAs.

Relationship of molecular size of rIn:rCn (poly I:C) to induction of interferon and host resistance.

SummaryPreparations of high molecular weight rIn:rCn were exposed to sonic radiation. A decrease in the average molecular weight of the polynucleotide complexes after treatment was accompanied by an exponential decrease in viscosity of the preparation, an increase in the sensitivity of the polynucleotide complex to degradation by pancreatic ribonuclease, a decrease in the thermal transition midpoint, and a decrease in the hyperchromicity observed during thermal transition.The lower molecular weight rIn:rCn complexes were tested for their capacity to induce interferon in rabbits and resistance against VSV infection in cell culture and PVM infection in mice. Sonic-treated preparations of rIn:rCn reduced to an average molecular weight of approximately 4.6 × 105 were markedly reduced in capacity to protect mice against PVM infection, compared with untreated rIn:rCn preparations containing complexes having an average molecular weight of approximately 7.8 × 106. The capacity of sonic-treated rIn:rCn to induce p...