Performance characteristics of the first Food and Drug Administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR::ABL1 monitoring in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy that accounts for 15-20% of all cases of leukemia. CML is caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR::ABL1. The amount of BCR::ABL1 transcript RNA is a marker of disease progression and the effectiveness of tyrosine kinase inhibitor (TKI) treatment. This study determined the analytical and clinical performance of a droplet digital PCR based assay (QXDx BCR-ABL %IS Kit; Bio-Rad) for BCR::ABL1 quantification. The test has a limit of detection of MR4.7 (0.002%) and a linear range of MR0.3-4.7 (50-0.002%IS). Reproducibility of results across multiple sites, days, instruments, and users was evaluated using panels made from BCR::ABL1 positive patient samples. Clinical performance of the assay was evaluated on patient samples and compared to an existing FDA-cleared test. The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The assay demonstrated excellent clinical correlation with the comparator test using a Deming regression with a Pearson R of 0.99, slope of 1.037 and intercept of 0.1084. This data establishes that the QXDx™ BCR-ABL %IS Kit is an accurate, precise, and sensitive system for the diagnosis and monitoring of CML.

Higher Adalimumab Levels Are Associated with Histologic and Endoscopic Remission in Patients with Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Optimal levels of adalimumab (ADA) have not been defined according to the ultimate goal of inflammatory bowel disease treatment--histologic and/or endoscopic healing. The aim of this study was to assess the relationship between random serum ADA levels and histologic and endoscopic healing in patients with inflammatory bowel disease. METHODS: This was a cross-sectional study including 66 patients receiving maintenance therapy with ADA for Crohn's disease or ulcerative colitis. ADA levels and anti-adalimumab antibodies (AAA) were measured at the time of colonoscopy. The primary outcome was histologic healing (lack of endoscopic and histologic inflammation) and the secondary outcomes were endoscopic healing and serum levels of C-reactive protein, tumor necrosis factor, ICAM, VCAM, and interleukins 1ß, 6, and 8. RESULTS: Sixty-six patients (59 with Crohn's disease) were included. Mean random ADA levels were significantly lower in patients with histologic and endoscopic inflammation (9.2 [SD: 8.4] versus 14.1 [6.4] µg/mL, P = 0.03 and 8.5 [SD: 7.8] versus 13.3 [SD: 7.7], P = 0.02, respectively). The ADA level that was best associated with histologic healing was 7.8 µg/mL (receiver operating characteristic: 0.76 [P = 0.04]), whereas the ADA level that was best associated with endoscopic healing was 7.5 µg/mL (receiver operating characteristic: 0.73 [P = 0.02]). The presence of AAA was associated with lower random ADA levels (5.7 versus 12.5 µg/mL, P = 0.002) and higher C-reactive protein levels (30.3 versus 12.0, P = 0.01). CONCLUSIONS: Achievement of histologic and endoscopic healing may require higher levels of ADA than previously described for endoscopic remission. The measurement of random ADA levels and anti-drug antibodies may guide therapy and edify the course of incomplete responses.

Antibodies to adalimumab are associated with future inflammation in Crohn's patients receiving maintenance adalimumab therapy: a post hoc analysis of the Karmiris trial.

INTRODUCTION: Data on immunogenicity to adalimumab (ADL) therapy in patients with IBD is limited. We performed additional analyses on the Karmiris cohort using the homogeneous mobility shift assay (HMSA) focusing on the inter-relationship of serum ADL concentration, antibodies-to-adalimumab (ATA), inflammatory markers and sustained response. METHODS: 536 prospectively collected serum samples were available for analysis of ADL concentration and ATA using HMSA. We studied the role of week 4 serum ADL concentration and immunomodulator (IMM) use on ATA formation with a Cox proportional hazards model. Mixed model repeated measures analysis was performed to assess the independent effects of serum ADL concentration and ATA on C-reactive protein (CRP) and response. RESULTS: ATA was detected in 20% of patients after a median of 34 (12.4-60.5) weeks. ATA-positive samples correlated with lower serum ADL concentration (p<0.001). Cox regression modelling showed that week 4 ADL concentration of <5 µg/mL significantly increased the future risk of ATA formation (HR=25.1; 95% CI 5.6 to 111.9; p=0.0002) and that IMM co-treatment prevented ATA formation (HR=0.23; 95% CI 0.06 to 0.86; p=0.0293). Regression modelling showed a negative correlation between CRP and ADL concentration (p=0.0001) and a positive one with ATA (p=0.0186). The model revealed that both lower serum ADL concentration and ATA were independently associated with future CRP (p=0.0213 and p=0.0013 respectively). ATA positivity was associated with discontinuation of ADL because of loss or response (OR=3.04; 95% CI 1.039 to 9.093; p=0.034). CONCLUSIONS: ATA were detected in 20% of patients. Risk of ATA formation increased with lower early serum ADL concentration and in patients not on IMM. ATA and ADL were strongly associated with higher future CRP level and discontinuation of ADL.

Concentrations of 6-thioguanine nucleotide correlate with trough levels of infliximab in patients with inflammatory bowel disease on combination therapy.

BACKGROUND & AIMS: In patients with inflammatory bowel diseases, the combination of infliximab and thiopurines (such as 6-thioguanine) is more effective treatment than monotherapy. We assessed the correlation between serum levels of 6-thioguanine (6-TGN) and infliximab levels or antibodies to infliximab (ATI). METHODS: We performed a cross-sectional study of 72 patients receiving maintenance therapy with infliximab and a thiopurine for inflammatory bowel disease at the Crohn's and Colitis Center of the University of Miami, FL. We collected clinical, endoscopic, and biochemical data, and levels of thiopurine metabolites. The primary outcomes were trough level of infliximab and the presence of ATI. RESULTS: Levels of 6-TGN correlated with those of infliximab (ρ, 0.53; P < .0001). The cut-off point of 6-TGN that best predicted a higher level of infliximab was 125 pmol/8 × 10(8) red blood cells (RBCs) (area under receiver operating characteristic, 0.86; P < .001). Patients in the lowest quartile of 6-TGN had infliximab levels that were similar to patients on no thiopurines (4.3 vs. 4.8 mcg/mL, respectively; P = .8). An infliximab level of 8.3 mcg/mL or greater was associated with mucosal healing. Only 8 patients (11%) had detectable ATI. Patients with 6-TGN levels less than 125 pmol/8 × 10(8) RBCs were significantly more likely to have ATI (odds ratio, 1.3; 95% confidence interval, 2.3-72.5; P < .01). CONCLUSIONS: Although 6-TGN levels of greater than 230 pmol/8 × 10(8) RBCs have been associated with improved outcomes in patients on monotherapy, a level of 6-thioguanine of 125 pmol/8 × 10(8) RBCs or greater may be adequate to achieve therapeutic levels of infliximab. In the long term, this may minimize the toxicity for patients on combination therapy.

Antibodies to infliximab are associated with lower infliximab levels and increased likelihood of surgery in pediatric IBD.

BACKGROUND: Adult studies suggest antibodies to infliximab (ATI) correlate with loss of response in inflammatory bowel disease but pediatric data are limited. METHODS: We conducted a cross-sectional study of trough infliximab levels and ATI in 134 pediatric and young adult patients receiving infliximab. At the time serum was obtained demographics, disease phenotype, duration of infliximab therapy, use of combination therapy (methotrexate or 6-mercaptopurine with infliximab), and surgery were recorded. RESULTS: Assays were performed on 134 subjects currently receiving infliximab (85 male; mean age, 17.3 ± 4.3 years; 114 Crohn's disease and 20 ulcerative colitis). Infliximab use ranged from 12 days to 12 years: median 2.0 (interquartile range [1.1-4.3]) years. Twenty-seven of 134 (20%) patients had ATI ≥5 U/mL. Of patients with ATI ≥5 U/mL, 59% had infliximab levels <5 µg/mL, compared with 14% of patients with ATI <5 U/mL (P < 0.001). Ten (7%) patients (9 Crohn's disease, 1 ulcerative colitis) underwent bowel resections after beginning infliximab infusions. Sixty percent who underwent surgery had ATI ≥12 U/mL; in contrast, only 8% of patients who did not undergo surgery had ATI ≥12 U/mL (P = 0.01). At the time of serum sampling, 50 (37%) patients were receiving combination therapy, compared with 84 (63%) on infliximab alone. Combination therapy at the time of serum sampling did not correlate with either increase infliximab levels or lower ATI compared with infliximab monotherapy. However, prior immunomodulator use was associated with lower antibody levels (P = 0.007). CONCLUSIONS: ATI correlates with reduction in infliximab level and a higher risk of surgery in patients with inflammatory bowel disease.

The relationship between infliximab concentrations, antibodies to infliximab and disease activity in Crohn's disease.

OBJECTIVE: Although low infliximab trough concentrations and antibodies to infliximab (ATI) are associated with poor outcomes in patients with Crohn's disease (CD), the clinical relevance of ATI in patients with adequate infliximab concentrations is uncertain. We evaluated this question using an assay sensitive for identification of ATI in the presence of infliximab. DESIGN: In an observational study, 1487 trough serum samples from 483 patients with CD who participated in four clinical studies of maintenance infliximab therapy were analysed using a fluid phase mobility shift assay. Infliximab and ATI concentrations most discriminant for remission, defined as a C-reactive protein concentration of ≤ 5 mg/L, were determined by receiver operating characteristic curves. A multivariable regression model evaluated these factors as independent predictors of remission. RESULTS: Based upon analysis of 1487 samples, 77.1% of patients had detectable and 22.9% had undetectable infliximab concentrations, of which 9.5% and 71.8%, respectively, were positive for ATI. An infliximab concentration of > 2.79 µg/mL (area under the curve (AUC) = 0.681; 95% CI 0.632 to 0.731) and ATI concentration of < 3.15 U/mL (AUC = 0.632; 95% CI 0.589 to 0.676) were associated with remission. Multivariable analysis showed that concentrations of both infliximab trough (OR 1.8; 95% CI 1.3 to 2.5; p < 0.001) and ATI (OR 0.57; 95% CI 0.39 to 0.81; p = 0.002) were independent predictors of remission. CONCLUSIONS: The development of ATI increases the probability of active disease even at low concentrations and in the presence of a therapeutic concentration of drug during infliximab maintenance therapy. Evaluation of strategies to prevent ATI formation, including therapeutic drug monitoring with selective infliximab dose intensification, is needed.

Long-term outcome of patients with Crohn's disease who discontinued infliximab therapy upon clinical remission.

BACKGROUND & AIMS: There are limited data on the effects of discontinuing infliximab therapy for Crohn's disease (CD). We investigated the long-term outcome of patients with CD who discontinued infliximab while in clinical remission, and searched for prognostic markers of continued remission after infliximab cessation. METHODS: We performed a retrospective, single-center study of 100 patients with CD who discontinued infliximab upon achieving clinical remission; 84 patients continued immunomodulator therapy. Clinical and endoscopic data were retrieved from a medical database in Belgium, and patients were followed up through April 2013 (median, 9.7 y; interquartile range, 8-11.5 y). Sustained clinical remission (SCR) was defined as maintenance of disease remission, without escalation in medical therapy or CD-related surgeries, until the end of the follow-up period. We measured trough concentrations of infliximab, antibodies to microbial antigens, and circulating inflammatory markers in serum samples collected before treatment and at the time of infliximab discontinuation. RESULTS: At the end of the follow-up period, 52 patients had SCR. Univariate (log-rank) analysis associated SCR with patient age at diagnosis (≥25 y; P = .012) and disease duration (<1 y; P = .017). Among factors evaluated at the time of infliximab discontinuation, infliximab trough concentrations (<6 µg/mL; P = .031), complete mucosal healing (P = .046), and serum positivity for vascular cell adhesion molecule-1 (>0.67 µg/mL; P = .024) were associated with SCR. In multiple Cox proportional hazards regression analysis, only age at diagnosis of 25 years and older was associated independently with SCR (hazard ratio, 1.83; 95% confidence interval, 1.03-3.25; P = .04). CONCLUSIONS: In a large, real-life study, 52% of patients with CD who discontinued infliximab upon achieving clinical remission remained in SCR after a median period of approximately 10 years; Most patients remained on immunomodulator therapy. Although patients with CD have variable responses to infliximab, a subgroup achieved long-term remission after infliximab discontinuation.

Early trough levels and antibodies to infliximab predict safety and success of reinitiation of infliximab therapy.

BACKGROUND & AIMS: Few agents are available for the treatment of inflammatory bowel diseases, and patients frequently become unresponsive to biologics. We investigated the feasibility of reinitiating infliximab therapy for patients who previously received only episodic therapy with, lost response to, or had infusion reactions to infliximab. We also aimed to identify factors associated with the success and safety of restarting infliximab, such as antibodies to infliximab and trough levels of the drug. METHODS: From the inflammatory bowel disease biobank, we identified 128 consecutive patients (105 patients with Crohn's disease, 23 patients with ulcerative colitis) who restarted infliximab after a median 15-month discontinuation (range, 6-125 mo; 28 patients for loss of response or infusion reactions, 100 patients for remission or pregnancy). We also analyzed serum samples that had been collected during the first period of infliximab therapy (T-1), when therapy was reinitiated (T0), and at later time points (T+1, T+2) for trough levels and antibodies to infliximab. We investigated correlations among response to treatment, infusion reactions, treatment modalities, trough levels, and antibodies to infliximab. RESULTS: Reinitiation of infliximab therapy produced a response in 84.5% of patients at week 14, 70% of patients at 1 year, and in 61% of patients at more than 4 years. Fifteen patients had acute infusion reactions and 10 patients had delayed infusion reactions. The absence of antibodies to infliximab at T+1 (hazard ratio [HR], 0.14; 95% confidence interval [CI], 0.026-0.74; P = .021) and reinitiation with concomitant immunomodulator therapy were associated with short-term responses (HR, 6.0; 95% CI, 1.3-27; P = .019). Pregnancy or remission as reason for discontinuation (HR, 2.70; 95% CI, 1.09-6.67; P = .033) and higher trough levels at T+1 (HR, 2.94; 95% CI, 1.18-7.69; P = .021) were associated with long-term response. Undetectable antibodies to infliximab at T+1 were associated with the safety of reinitiating therapy (HR for infusion reaction with detectable antibodies to infliximab, 7.7; 95% CI, 1.88-31.3; P = .004). CONCLUSIONS: Reinitiating infliximab therapy can be safe and effective for patients with Crohn's disease or ulcerative colitis after a median 15-month discontinuation period.

Monitoring of adalimumab and antibodies-to-adalimumab levels in patient serum by the homogeneous mobility shift assay.

This report describes the analytical validation and application of the homogeneous mobility shift assay (HMSA) method for the measurement of adalimumab and human antibodies-to-adalimumab (ATA) in serum samples from patients who have lost response to adalimumab treatment. Validation of the ATA- and the adalimumab-HMSA revealed a lower limit of detection to be 0.026 U/mL for ATA and 0.018 µg/mL for adalimumab in serum samples. Intra-assay and inter-assay precision determination yielded a coefficient of variation of less than 15%, and the accuracy of both assays was within 20%. Adalimumab drug tolerance in the ATA-HMSA was up to 20 µg/mL in the test serum. Serum samples from 100 drug-naïve healthy subjects were tested to set-up the cut point of 0.55 U/mL for ATA and 0.68 µg/mL for adalimumab. Analysis of 100 serum samples from patients who were losing response to adalimumab showed that 26% had an adalimumab level below the cut point, of these 68% were ATA positive. Overall, 44% of the patients (44/100) were positive for ATA. This study presents evidence that drug and anti-drug antibody levels are important determinants of patient response to therapy.

Antibody response to infliximab and its impact on pharmacokinetics can be transient.

OBJECTIVES: Infliximab (IFX) is successfully used in the treatment of inflammatory bowel diseases but some patients generate antibodies to IFX (ATI) jeopardizing the pharmacokinetics of the drug. Little is known about the factors influencing ATI formation and whether or not this immune reaction is permanent. Our aim was to investigate the kinetics of ATI formation and drug levels in relation to inflammatory markers and the clinical evolution of the patients. METHODS: IFX trough and ATI levels were measured retrospectively in 1,232 consecutive serum samples of 90 (64 Crohn's disease and 26 ulcerative colitis) patients, 57 with previously detected and 33 without antibodies with a new homogenous mobility shift assay. RESULTS: Testing with new assay confirmed ATI in 53/90 patients (59%) and 37/90 patients (41%) were ATI negative. In 15/53 patients (28%), ATI disappeared over time whereas in 38/53 patients (72%) ATI persisted. The 26/38 (68%) patients with sustained ATI needed to discontinue IFX treatment compared with 2/15 (13%) patients with transient ATI (relative risk 5.1; 95% confidence interval 1.4-19.0; P=0.0005). An IFX trough level at week 14<2.2 µg/ml predicted IFX discontinuation due to persistent loss of response (LOR) or hypersensitivity reactions with 74% specificity and 82% sensitivity (likelihood ratio 3.1; P=0.0026). CONCLUSIONS: ATI may be transient and do not always lead to a worse clinical outcome. Sustained high levels of ATI, however, lead to permanent LOR. Patients with low IFX trough levels at week 14 are at risk for ATI formation and IFX discontinuation. Therefore, we recommend to measure IFX trough levels at week 14 and at time of LOR. When undetectable or low, ATI should be determined and if positive followed up on consecutive time points to rule out sustained ATI.

Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum.

Antibody-based drugs such as infliximab (IFX) are effective for the treatment of inflammatory bowel disease (IBD) and other immune-mediated disorders. The development of antibodies against these drugs may result in unfavorable consequences, including the loss of drug efficacy, hypersensitivity reactions, and other adverse events. Therefore, accurate monitoring of serum drug and anti-drug antibody levels should be an important part of therapy for patients being treated with an antibody-based drug. Current methods for the assessment of anti-drug antibodies and drug levels, involving various bridging ELISA and radioimmunoassay techniques, are limited by their sensitivity, interference, and/or complexity. To overcome these limitations, we have developed a non-radiolabeled homogeneous mobility shift assay (HMSA) to measure the antibodies-to-infliximab (ATI) and IFX levels in serum samples. Full method validation was performed on both the ATI- and IFX-HMSA, and the clinical sample test results were also compared with those obtained from a bridging ELISA method to evaluate the difference in performance between the two assays. Validation of the ATI-HMSA revealed a lower limit of quantitation of 0.012 µg/mL in serum. The linear range of quantitation was 0.029-0.54 µg/mL. The intra- and inter-assay precision was less than 20% of coefficient of variation (CV), and the accuracy (% error) of the assay was less than 20%. In serum samples, ATI as low as 0.036 µg/mL can be measured, even in the presence of 60 µg/mL of IFX in the serum. Sera from 100 healthy subjects were tested to determine the cut point of the assay. ATI-positive samples that had been previously analyzed by using a bridging ELISA from 100 patients were also measured by the new method. There was a high correlation between the two methods for ATI levels (p<0.001). Significantly, the new method identified five false-positive samples from the bridging ELISA method. Validation of the mobility shift IFX assay also showed high assay sensitivity, precision and accuracy. The HMSA method may also be applied to other protein-based drugs to accurately detect serum drug and anti-drug antibody levels.

Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei.

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.

The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity.

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.

Shape-selective RNA recognition by cysteinyl-tRNA synthetase.

The crystal structure of Escherichia coli cysteinyl-tRNA synthetase (CysRS) bound to tRNA(Cys) at a resolution of 2.3 A reveals base-specific and shape-selective interactions across an extensive protein-RNA recognition interface. The complex contains a mixed alpha/beta C-terminal domain, which is disordered in the unliganded enzyme. This domain makes specific hydrogen bonding interactions with all three bases of the GCA anticodon. The tRNA anticodon stem is bent sharply toward the enzyme as compared with its conformation when bound to elongation factor Tu, providing an essential basis for shape-selective recognition. The CysRS structure also reveals interactions of conserved enzyme groups with the sugar-phosphate backbone in the D loop, adjacent to an unusual G15.G48 tertiary base pair previously implicated in tRNA aminoacylation. A combined mutational analysis of enzyme and tRNA groups at G15.G48 supports the notion that contacts between CysRS and the sugar-phosphate backbone contribute to recognition by indirect readout.