Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg^2.39, His^2.43 and Glu^3.46, which makes a polar lock with T^6.37. These alignments and models provide useful tools for understanding class B GPCR function.     

Magnesium facilitates the healing of atypical femoral fractures: A single-cell transcriptomic study

Bisphosphonates (BPs)-associated atypical femoral fractures (AFFs) present with impaired fracture healing, yet the underlying mechanism is unclear, which prevents the development of effective therapy. Peripheral sensory nerve has been shown to regulate fracture healing via releasing neuropeptides. Here we show that long-term BPs pre-treatment leads to fracture non-union in rats, characterized by reduced expression of calcitonin gene-related peptide (CGRP, a predominant type of neuropeptides) and abundant fibrous tissues in the non-bridged fracture gap, mimicking clinical AFFs. By using single-cell RNA-sequencing, long-term BPs treatment was identified to promote transition of progenitor cells into a specific cluster of fibroblasts that actively deposit dense extracellular matrix (ECM) to prevent fracture callus bridging. Administration of exogenous CGRP at early stages of fracture repair, in contrast, eliminates the ECM-secreting fibroblast cluster, attenuates fibrogenesis, and facilitates callus bridging, suggesting CGRP is a promising agent to facilitate AFF healing. Accordingly, we have developed an innovative magnesium (Mg) containing hybrid intramedullary nail fixation system (Mg-IMN) to effectively rescue BPs-impaired fracture healing via elevating CGRP synthesis and release. Such device optimizes the fracture healing in BPs-pretreated rats, comparable to direct administration of CGRP. These findings address the indispensable role of CGRP in advancing the healing of AFFs and develop translational strategies to accelerate AFF healing by taking advantage of the CGRP-stimulating effect of Mg-based biodegradable orthopedic implant. The study also indicates fibrosis could be targeted by augmenting CGRP expression to accelerate fracture healing even under challenging scenarios where fibroblasts are aberrantly activated.     

CdS nanocrystal-based electrochemiluminescence biosensor for the detection of low-density lipoprotein by increasing sensitivity with gold nanoparticle amplification

Mercaptoacetic acid (RSH)-capped CdS nanocrystals (NCs) was demonstrated to be electrochemically reduced during potential scan and react with the coreactant S2O8(2-) to generate strong electrochemiluminescence (ECL) in aqueous solution. Based on the ECL of CdS NCs, a novel label-free ECL biosensor for the detection of low-density lipoprotein (LDL) has been developed by using self-assembly and gold nanoparticle amplification techniques. The biosensor was prepared as follows: The gold nanoparticles were first assembled onto a cysteamine monolayer on the gold electrode surface. This gold nanoparticle-covered electrode was next treated with cysteine and then reacted with CdS NCs to afford a CdS NC-electrode. Finally, apoB-100 (ligand of LDL receptor) was covalently conjugated to the CdS NC-electrode. The modification procedure was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy, respectively. The resulting modified electrode was tested as ECL biosensor for LDL detection. The LDL concentration was measured through the decrease in ECL intensity resulting from the specific binding of LDL to apoB-100. The ECL peak intensity of the biosensor decreased linearly with LDL concentration in the range of 0.025-16 ng mL-1 with a detection limit of 0.006 ng mL-1. The CdS NCs not only showed high ECL intensity and good biocompatibility but also could provide more binding sites for apoB-100 loading. In addition, the gold nanoparticle amplification for protein ECL analysis was applied to the improvement of the detection sensitivity. Thus, the biosensor exhibited high sensitivity, good reproducibility, rapid response, and long-term stability.     

Kinetics of antigen-antibody interactions employing a MALDI mass spectrometry immunoassay

Time-course MALDI mass spectrometry immunoassays have been shown to be able to detect differences in the relative rates of binding of peptides, both from within and across epitopic domains, with antibodies in non-competitive and competitive experiments. A monoclonal antibody raised to target the HA1 subunit of the hemagglutinin antigen of type A H3N2 influenza strains is found to recognize two epitopic peptides comprising residues 109-125 and 158-166 that likely form part of an extended discontinuous domain. Time-course experiments show the smaller peptide binds antibody at a rate that is 5-fold faster than that for the larger peptide. A shorter segment of this larger peptide, comprised of residues 119-125, is also found to bind at twice the rate of the extended peptide. Studies of modified peptide variants and synthetic variants of HA peptide 119-125 has enabled important contact residues to be identified whose accessibilities in the native protein are in accord with the mass spectrometry results.     

Analysis of the (Trimethylsilyl)propionic Acid-β(12-28) Peptide Binding Equilibrium with NMR Spectroscopy

The binding of a small molecule, (trimethylsilyl)propionic acid (TSP), to a 17-residue peptide, β(12-28), is examined using (1)H NMR spectroscopy. β(12-28) (VHHQKLVFFAEDVGSNK) is a central fragment of the 40-42-residue Alzheimer's-associated Aβ peptide. This peptide has been previously shown to form soluble aggregates in low-pH aqueous solution. The TSP resonance is broadened appreciably in solutions containing relatively high concentrations (～2 mM) of the peptide. The changes in TSP line width measured by titration of a peptide solution with TSP indicate a 1:1 binding stoichiometry. If the concentrations of both the peptide and TSP are reduced by 1 order of magnitude, the resonances of both species are sharp, suggesting that TSP binds predominately to the aggregated peptide. Nuclear Overhauser effect experiments indicate that the TSP interacts predominately with the side chains of the aliphatic peptide residues Leu(17) and Val(18). Pulsed-field gradient NMR measurements of TSP and peptide diffusion coefficients provide a more quantitative picture of the TSP-peptide binding equilibrium. The measured diffusion coefficients were used to calculate the fractions of the free and bound TSP. These results substantiate the conclusion that the stoichiometry of the TSP-peptide binding equilibrium is essentially 1:1 and further indicate anticooperative behavior in solutions containing an excess of TSP resulting in a dissociation of the peptide aggregates.     

Electrochemiluminescence of luminol in alkaline solution at a paraffin-impregnated graphite electrode

The behavior of luminol electrochemiluminescence (ECL) at a paraffin-impregnated graphite electrode (PIGE) at different applied potentials was studied. Five ECL peaks were observed at 0.31, 0.59, 1.09, 1.54, and -0.58 V versus SCE, respectively, being related to potential scan direction and ranges, N2, O2, pH of the solution, and KCl concentration. The emission spectra of various ECL peaks at different potentials showed that all ECL peaks were initiated by luminol reactions. X-ray diffraction demonstrated that a simple mixture was formed between graphite and paraffin. The fluorescence spectra on the surface of the PIGE suggested that certain groups on the graphite were oxidized when the positive potential was applied to the electrode. In the presence of O2, three main ECL peaks were obtained in 0.1 mol/L KCl at pH 12.2. The ECL peak at 0.59 V with a shoulder is likely due to the reaction of luminol radicals with O2 and further electrooxidation of luminol radicals. The ECL peak at 1.54 V was suggested to be due to the electrooxidation of OH- to HO2- at higher potential and then to O2-, which reacted with luminol to produce light emission. Moreover, the oxygen-containing functional groups formed by the oxidation of the surface of the graphite electrode might enhance the ECL. At -0.58 V, the dissolved oxygen in solution was reduced to HO2-, resulting in light emission. At a potential higher than 1.64 V, ClO- was formed, leading to a broad emission wave and enhancement of the ECL peak at -0.58 V upon the reversal scan. Under nitrogen atmosphere, an ECL peak appeared at 1.09 V. At this potential, OH- was oxidized to O2, followed by the reaction with luminol to generate light emission. At pH 13.2 or 0.5 mol/L KCl, the shoulder of the ECL peak at 0.59 V became an ECL peak at 0.31 V. The conversion of luminol radicals into excited 3-aminophthalate may undergo two routes. Under these conditions, two routes might proceed at a different rate to form another ECL peak. It is concluded that luminol ECL could be readily excited by various oxygen-containing species electrogenerated at different applied potentials. Three strong ECL peaks obtained at different potentials on the PIGE might be of a potential to improve analytical selectivity and sensitivity for the detection of some analytes.     

Evaluation of salmon calcitonin (sCT) enteric-coated capsule for enhanced absorption and GI tolerability in rats

Background: Considering the chronic and repeated nature of salmon calcitonin (sCT) therapy, the oral route is a preferred route of administration. But, the oral bioavailability of sCT is very low due to enzymatic degradation and poor permeation across intestinal epithelial cells. It was the aim of this study to investigate the pharmacodynamic (PD), pharmacokinetic (PK), and mucosal injury characteristic of sCT oral delivery system. Method: In this study, PD experiments were performed to find a suitable releasing region of sCT, an effect absorption enhancer, and an optimal mass ratio of sCT/enhancer. In addition, the PK experiments were designed to validate the absorption enhancement of this oral delivery system. Histopathological evaluations on the intestinal mucosa were carried out to assess any potential toxicity of the absorption enhancer. Results: Through the PD research, we determined that oral sCT enteric-coated capsules containing sCT and citric acid (CA) with a ratio of 1:20 may be an adaptable delivery. PK study further proved that the oral absorption of sCT was enhanced from this delivery system. Finally, no damage on intestinal mucosa was observed when rats received the delivery system containing CA for up to 7 days. Conclusion: These results suggested that enteric-coated capsules with a certain amount of CA might give enhanced oral delivery of peptide drugs like sCT.     

Electrogenerated chemiluminescence. 66. The role of direct coreactant oxidation in the ruthenium tris(2,2')bipyridyl/tripropylamine system and the effect of halide ions on the emission intensity

We describe the electrogenerated chemiluminescence (ECL) processes of the Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl)/ tripropylamine (TPrA) system at glassy carbon, platinum, and gold electrodes. The electrochemical behavior of TPrA on different electrode materials and its influence on the ECL process are demonstrated. At glassy carbon electrodes, the direct oxidation of TPrA began at approximately 0.6 V vs SCE and exhibited a broad irreversible anodic peak. Two ECL waves were observed, one in the potential region more negative than 1.0 V vs SCE and one at more positive potentials. The first ECL process apparently occurs without the electrogeneration of Ru(bpy)3(3+), in contrast to that of the second ECL wave. At Pt and Au electrodes, however, the formation of surface oxides significantly blocked the direct oxidation of TPrA. An ECL wave below 1.0 V did not appear at Pt and was very weak at gold. The ECL peaks at potentials of 1.1-1.2 V were also much weaker than those observed at the glassy carbon electrode. These results showed that the direct oxidation of TPrA played an important role in the ECL processes. Therefore, the enhancement of the TPrA oxidation current might lead to an increase in the ECL intensity. Small amounts of halide species were found to inhibit the growth of surface oxides on Pt and gold electrodes and led to an obvious increase of TPrA oxidation current. The anodic dissolution of gold in halide-containing solution was also important in activating the gold electrode surface. The electrochemical catalytic effect of bromide further promoted the oxidation of TPrA. A halide effect on ECL at Pt and Au electrodes was also evident. The most effective enhancement of ECL was observed at Au electrode in a bromide-containing solution. This effect was also found in an commercial flow-through instrument (IGEN) and provided a simple way to improve the detection sensitivity at low concentrations of Ru(bpy)3(2+).     

Unique electrochemiluminescence behavior of Ru(bpy)3 in a gold/Nafion/Ru(bpy)3 composite

The unique electrochemiluminescence (ECL) behavior of tris(bipyridine) ruthenium(II) (Ru(bpy)₃²⁺) immobilized in a gold/Nafion/Ru(bpy)₃²⁺ composite material was investigated. In this composite, the Ru(bpy)₃²⁺ ECL was found mainly occurred at 0-0.4 V during the cathodic scan process and the ECL peak was at about 0.1 V, which was quite different to the reported Ru(bpy)₃²⁺ ECL. Similar to the generally observed Ru(bpy)₃²⁺ ECL, the present ECL also could be enhanced by tri-n-propylamine (TPA). It is also unique that in the presence of TPA, another ECL peak at about 0.38 V occurred. These two ECL peak potentials all could be used as characteristic potential for the ECL determination of TPA.     

Sensitive electrochemiluminescence detection of c-Myc mRNA in breast cancer cells on a wireless bipolar electrode

We report an ultrasensitive wireless electrochemiluminescence (ECL) protocol for the detection of a nucleic acid target in tumor cells on an indium tin oxide bipolar electrode (BPE) in a poly(dimethylsiloxane) microchannel. The approach is based on the modification of the anodic pole of the BPE with antisense DNA as the recognition element, Ru(bpy)(3)(2+)-conjugated silica nanoparticles (RuSi@Ru(bpy)(3)(2+)) as the signal amplification tag, and reporter DNA as a reference standard. It employs the hybridization-induced changes of RuSi@Ru(bpy)(3)(2+) ECL efficiency for the specific detection of reporter DNA released from tumor cells. Prior to ECL detection, tumor cells are transfected with CdSe@ZnS quantum dot (QD)-antisense DNA/reporter DNA conjugates. Upon the selective binding of antisense DNA probes to intracellular target mRNA, reporter DNA will be released from the QDs, which indicates the amount of the target mRNA. The proof of concept is demonstrated using a proto-oncogene c-Myc mRNA in MCF-7 cells (breast cancer cell line) as a model target. The wireless ECL biosensor exhibited excellent ECL signals which showed a good linear range over 2 × 10(-16) to 1 × 10(-11) M toward the reporter DNA detection and could accurately quantify c-Myc mRNA copy numbers in living cells. C-Myc mRNA in each MCF-7 cell and LO2 cell was estimated to be 2203 and 13 copies, respectively. This wireless ECL strategy provides great promise in a miniaturized device and may facilitate the achievement of point of care testing.     

Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging

The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 microL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K(ads), for the FLAG peptide was measured and found to be 1.5 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).     

Metal binding characterization and conformational studies using Raman microscopy of resin-bound poly(aspartic acid)

The metal binding capacities, conditional stability constants, and secondary structure of immobilized polyaspartic acid (PLAsp) (n = 6, 20, and 30) on TentaGel resin were determined when binding Mg2+, Co2+, Cd2+, and Ni2+. Metal binding to the synthesized peptides was evaluated using breakthrough curves from a packed microcolumn and flame atomic absorption spectrophotometry (FAAS) detection. The metal capacities reached values of 590, 2160, and 3710 mumol of metal/g of resin for the 6-mer, 20-mer, and 30-mer, respectively, and this resulted in 2-3 residues per metal for all peptides and metals tested. Surprisingly, the concentrated environment of the resin along with the spatial distribution of attachment groups allowed for most residues to participate in metal binding regardless of the peptide length. Conditional stability constants calculated using single metal binding isotherms indicated that binding strength decreased as the chain length increased on the resin. Raman microscopy on single beads was used to determine PLAsp secondary structure, and all peptides were of a mixed conformation (i.e., beta-sheets, alpha-helices, random chain, etc.) during neutral conditioning and metal binding. Uniquely, the longer 20-mer and 30-mer peptides showed a distinct change from a mixed conformation to beta-sheets and alpha-helices during metal release with acid. This study confirms that metal release by longer immobilized peptides is often assisted by a conformational change, which easily spoils the binding cavity, while shorter peptides may release metal primarily by H+ displacement.     

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways.

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling is described. Upon binding of insulin to cell-surface insulin receptor, the receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthetic phosphopeptide pY939 derived from the tyrosine phosphorylation domain of IRS-1 and its target protein SH2N containing an N-terminal SH2 domain of PI 3-kinase. The SH2N protein and pY939 phosphopeptide were labeled with fluorescein (F-SH2N) and tetramethylrhodamine (T-pY939), respectively. Formation of a F-SH2N-T-pY939 complex (termed a fluorescence resonance energy-transfer (FRET) pair) was evaluated from a change in a fluorescence emission spectrum based on FRET between the two fluorophores. The FRET pair was formed to dissociate in competition with the unlabeled pY939 phosphopeptide, resulting in a decrease in a pY939 phosphopeptide-dependent FRET emission at 580 nm and causing an increase in emission at 520 nm. Tyrosine phosphorylation by the partially purified insulin receptor of substrate peptide Y939 was detected with this formed FRET pair, and resulting changes in fluorescence emission spectra were observed for insulin concentration from about 1.0 x 10(-9) to 1.0 x 10(-6) M. These results indicated that the FRET pair served as a competitive fluorescent indicator for tyrosine phosphorylation-based insulin signaling.     

Electrochemiluminescence of copper(I) bis(2,9-dimethyl-1,10- phenanthroline)

The electrochemical and electrochemiluminescence (ECL) properties of Cu[dmp]2+ (dmp = 2,9-dimethyl-1,10-phenanthroline) have been investigated. ECL has been observed for Cu(dmp)2+ in aqueous, nonaqueous, and mixed solvent solutions using tri-n-propylamine as an oxidative-reductive coreactant. The ECL intensity peaks at potential corresponding to oxidation of both the coreactant and Cu(dmp)2+. The peak potential corresponding to maximum ECL emission is approximately 500 mV more anodic than corresponding oxidative peak potentials, indicating that the ECL emission may be due to the formation of either the *Cu(dmp)2+ metal-to-ligand charge-transfer excited state or an excited-state product of Cu(dmp)2+ oxidation. ECL efficiencies (phiecl = photons generated per redox event) are solvent-dependent (phiecl (CH3CN) > phiecl (50:50 (v/v) CH3CN:H20) > phiecl (H2O)) and correspond fairly well with photoluminescence efficiencies. Increased ECL efficiencies (> or = 50-fold) are observed in the presence of the nonionic surfactant Triton X-100.     

Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Recruitment of Receptors and Ligands in a Weakly Multivalent System with Omnipresent Signatures of Superselective Binding (Small 23/2023)

Recruitment of receptors at membrane interfaces is essential in biological recognition and uptake processes. The interactions that induce recruitment are typically weak at the level of individual interaction pairs, but are strong and selective at the level of recruited ensembles. Here, a model system is demonstrated, based on the supported lipid bilayer (SLB) that mimics the recruitment process induced by weakly multivalent interactions. The weak (mm range) histidine-nickel-nitrilotriacetate (His₂-NiNTA) pair is employed owing to its ease of implementation in both synthetic and biological systems. The recruitment of receptors (and ligands) induced by the binding of His₂-functionalized vesicles on NiNTA-terminated SLBs is investigated to identify the ligand densities necessary to achieve vesicle binding and receptor recruitment. Threshold values of ligand densities appear to occur in many binding characteristics: density of bound vesicles, size and receptor density of the contact area, and vesicle deformation. Such thresholds contrast the binding of strongly multivalent systems and constitute a clear signature of the superselective binding behavior predicted for weakly multivalent interactions. This model system provides quantitative insight into the binding valency and effects of competing energetic forces, such as deformation, depletion, and entropy cost of recruitment at different length scales.     

Inhibited Ru(bpy)3 2+ electrochemiluminescence related to electrochemical oxidation activity of inhibitors

Electrochemiluminescence (ECL) has been accepted by the analytical chemist as a powerful tool for detection of many inorganic and organic compounds. Ru(bpy)3 2+ has been the most popular ECL system, and many investigations have been focused on the application based on the enhancement or inhibition of Ru(bpy)3 2+ ECL system. However, not much attention has been paid to the theoretical investigation of this ECL system, especially to the inhibiting mechanism for the Ru(bpy)3 2+ ECL system. In the present study, many of the inorganic and organic compounds with electrochemical oxidation activity were found to strongly inhibit Ru(bpy)3 2+ ECL. To explain these inhibited ECL phenomena, a new "electrochemical oxidation inhibiting" mechanism has been proposed via the establishment of a corresponding model. The effects of applied potential, uncompensated resistance, and concentration of inhibitor on the inhibited ECL derived from the model have been verified by experiments. The new ECL inhibition mechanism can be commonly used to explain many kinds of inhibited ECL presently observed, and it is envisioned to result in finding of more inhibitors of this type and establishment of new sensitive ECL detection methods for them.     

Strength and mechanical behavior of soil–cement–lime–rice husk ash (soil–CLR) mixture

This study presents results of geotechnical investigations on treated silty sand soil with cement, lime and rice husk ash (CLR) and cement-lime (CL) admixture. Consolidated undrained triaxial test and unconfined compressive test were performed to estimate the potential of CLR and CL. The study investigates the influence of the amount of CLR%, main effective stress and curing days on soil strength, deformation, post peak behavior and brittleness. The percentages of the additives of CLR and CL varied from 2.5 to 12.5 % by dry weight of the soil with dry densities of 14.5 kN/m³ and the curing times of 3, 7, 28 and 60 days were examined. From the results, the stress–strain response is strongly influenced by the CLR contents and effective confining pressure. Strength and post peak strength of the CLR–soil are greatly improved by an increase in binder content. An increase of the effective cohesion c′ (kPa) and effective friction Φ′ (degree) is observed with increasing the CLR content, consistently. Brittle behavior observed at lower confining pressures and high CLR content. For both CLR and CL additives, linear trend was observed for variation of the q _u (kPa) with respect to the additives percentages. RHA was also found to be effective in increasing the shear strength of CLR–soil mixture.