ABSTRACT
Sustained drug delivery formulations are developed to reduce dose frequency while maintaining efficacy of intravitreal (ITV) administered therapeutics. Available safety data for components novel to the eye's posterior segment may be limited, requiring preclinical assessments to identify potential toxicities. We evaluated the in vivo and in vitro safety of two solvents, acetyl triethyl citrate (ATEC) and benzyl benzoate (BB), as novel sustained delivery formulations for ITV administration. In vivo tolerability was assessed following ITV administration of ATEC and BB to rabbits and cynomolgus monkeys. In rabbits, ITV solvent administration resulted in moderate to severe retinal toxicity characterized by focal retinal necrosis and/or degeneration, sometimes accompanied by inflammation, with a clear association between the physical presence of the solvent and areas of retinal damage. In contrast, solvent administration in monkeys appeared well tolerated, producing no histologic abnormalities. Toxicity in primary human retinal pigment epithelial cells, characterized by cellular toxicity and mitochondrial injury, corroborated the retinal toxicity in rabbits. In conclusion, ITV solvent depots of ATEC or BB result in chemical and focal retinal toxicity in rabbits, but not monkeys. Additional investigation is needed to demonstrate a sufficient margin of safety prior to use of ATEC or BB in ITV drug products.
Subject(s)
Benzoates , Citrates , Animals , Humans , Macaca fascicularis , Rabbits , RetinaABSTRACT
Bruton's tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retained-and in some cases improved-a safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.
ABSTRACT
Micro-RNA (miR)-122 is a promising exploratory biomarker for detecting liver injury in preclinical and clinical studies. Elevations in serum or plasma have been associated with viral and autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma, and drug-induced liver injury (DILI). However, these associations were primarily based upon population differences between the disease state and the controls. Thus, little is known about the variability and subsequent variance components of circulating miR-122 in healthy humans, which has implications for the practical use of the biomarker clinically. To address this, we set out to perform variance components analysis of miR-122 in a cohort of 40 healthy volunteers. Employing a quantitative real-time polymerase chain reaction (qRT-PCR) assay to detect miR-122 and other circulating miRNAs in human serum, the relative expression of miR-122 was determined using two different normalization approaches: to the mean expression of a panel of several endogenous miRNAs identified using an adaptive algorithm (miRA-Norm) and to the expression of an exogenous miRNA control (Caenorhabditis elegans miR-39). Results from a longitudinal study in healthy volunteers (N = 40) demonstrated high variability with 117- and 111-fold 95% confidence reference interval, respectively. This high variability of miR-122 in serum appeared to be due in part to ethnicity, as 95% confidence reference intervals were approximately three-fold lower in volunteers that identified as Caucasian relative to those that identified as Non-Caucasian. Variance analysis revealed equivalent contributions of intra- and inter-donor variability to miR-122. Surprisingly, miR-122 exhibited the highest variability compared to other 36 abundant miRNAs in circulation; the next variable miRNA, miR-133a, demonstrated a 45- to 62-fold reference interval depending on normalization approaches. In contrast, alanine aminotransferase (ALT) activity levels in this population exhibited a 5-fold total variance, with 80% of this variance due to inter-donor sources. In conclusion, miR-122 demonstrated higher than expected variability in serum from healthy volunteers, which has implications for its potential utility as a prospective biomarker of liver damage or injury.
Subject(s)
Biomarkers/blood , MicroRNAs/blood , Racial Groups/genetics , Real-Time Polymerase Chain Reaction/standards , Adult , Female , Genetic Heterogeneity , Healthy Volunteers , Humans , Longitudinal Studies , Male , Middle Aged , Prospective Studies , Reference StandardsABSTRACT
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/chemistry , Sulfonamides/chemistry , Voltage-Gated Sodium Channel Blockers/chemistry , Analgesics/chemistry , Analgesics/metabolism , Analgesics/pharmacology , Analgesics/therapeutic use , Animals , Binding Sites , Cell Line , Cell Survival/drug effects , Chronic Pain/drug therapy , Chronic Pain/pathology , Dogs , Half-Life , Humans , Ligands , Male , Mice , Molecular Docking Simulation , Mutagenesis, Site-Directed , NAV1.7 Voltage-Gated Sodium Channel/genetics , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Rats , Structure-Activity Relationship , Sulfonamides/metabolism , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Voltage-Gated Sodium Channel Blockers/metabolism , Voltage-Gated Sodium Channel Blockers/pharmacology , Voltage-Gated Sodium Channel Blockers/therapeutic useABSTRACT
Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.
Subject(s)
Chemical and Drug Induced Liver Injury/genetics , Colorectal Neoplasms/drug therapy , Liver/drug effects , Liver/pathology , Oxaliplatin/adverse effects , Transcriptome , Aged , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biopsy , Capillaries/drug effects , Capillaries/metabolism , Capillaries/pathology , Colorectal Neoplasms/pathology , Dilatation, Pathologic/chemically induced , Dilatation, Pathologic/genetics , Female , Gene Expression Profiling , Humans , Liver/blood supply , Liver/metabolism , Liver Neoplasms/secondary , Macaca fascicularis , Male , Middle Aged , Neovascularization, Pathologic/chemically induced , Neovascularization, Pathologic/genetics , Oxaliplatin/administration & dosage , Transcriptome/drug effectsABSTRACT
Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Drug Design , Drug-Related Side Effects and Adverse Reactions/diagnosis , Hepatocytes/drug effects , Biomarkers/metabolism , Chemical and Drug Induced Liver Injury/diagnosis , Drug Discovery/methods , Drug-Related Side Effects and Adverse Reactions/pathology , Hepatocytes/pathology , Humans , Inhibitory Concentration 50 , Liver/drug effects , Liver/pathology , Predictive Value of Tests , Risk Assessment/methods , Time FactorsABSTRACT
PURPOSE: High-throughput sequencing (HTS) of immunoglobulin heavy-chain genes (IGH) in unselected clinical samples for minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) has not been tested. As current MRD-detecting methods such as flow cytometry or patient-specific qPCR are complex or difficult to standardize in the clinical laboratory, sequencing may enhance clinical prognostication. EXPERIMENTAL DESIGN: We sequenced IGH in paired pretreatment and day 29 post-treatment samples using residual material from consecutive, unselected samples from the Children's Oncology Group AALL0932 trial to measure MRD as compared with flow cytometry. We assessed the impact of ongoing recombination at IGH on MRD detection in post-treatment samples. Finally, we evaluated a subset of cases with discordant MRD results between flow cytometry and sequencing. RESULTS: We found clonal IGH rearrangements in 92 of 98 pretreatment patient samples. Furthermore, while ongoing recombination of IGH was evident, index clones typically prevailed in MRD-positive post-treatment samples, suggesting that clonal evolution at IGH does not contribute substantively to tumor fitness. MRD was detected by sequencing in all flow cytometry-positive cases with no false-negative results. In addition, in a subset of patients, MRD was detected by sequencing, but not by flow cytometry, including a fraction with MRD levels within the sensitivity of flow cytometry. We provide data that suggest that this discordance in some patients may be due to the phenotypic maturation of the transformed cell. CONCLUSION: Our results provide strong support for HTS of IGH to enhance clinical prognostication in B-ALL.
Subject(s)
High-Throughput Nucleotide Sequencing , Immunoglobulin Heavy Chains/genetics , Leukemia, B-Cell/genetics , Neoplasm, Residual/genetics , Disease-Free Survival , Flow Cytometry , Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Humans , Leukemia, B-Cell/complications , Leukemia, B-Cell/pathology , Neoplasm, Residual/etiology , Neoplasm, Residual/pathology , PrognosisABSTRACT
BACKGROUND: The approaches for shotgun-based sequencing of vertebrate genomes are now well-established, and have resulted in the generation of numerous draft whole-genome sequence assemblies. In contrast, the process of refining those assemblies to improve contiguity and increase accuracy (known as 'sequence finishing') remains tedious, labor-intensive, and expensive. As a result, the vast majority of vertebrate genome sequences generated to date remain at a draft stage. RESULTS: To date, our genome sequencing efforts have focused on comparative studies of targeted genomic regions, requiring sequence finishing of large blocks of orthologous sequence (average size 0.5-2 Mb) from various subsets of 75 vertebrates. This experience has provided a unique opportunity to compare the relative effort required to finish shotgun-generated genome sequence assemblies from different species, which we report here. Importantly, we found that the sequence assemblies generated for the same orthologous regions from various vertebrates show substantial variation with respect to misassemblies and, in particular, the frequency and characteristics of sequence gaps. As a consequence, the work required to finish different species' sequences varied greatly. Application of the same standardized methods for finishing provided a novel opportunity to "assay" characteristics of genome sequences among many vertebrate species. It is important to note that many of the problems we have encountered during sequence finishing reflect unique architectural features of a particular vertebrate's genome, which in some cases may have important functional and/or evolutionary implications. Finally, based on our analyses, we have been able to improve our procedures to overcome some of these problems and to increase the overall efficiency of the sequence-finishing process, although significant challenges still remain. CONCLUSION: Our findings have important implications for the eventual finishing of the draft whole-genome sequences that have now been generated for a large number of vertebrates.
Subject(s)
Genomics/methods , Sequence Analysis, DNA/methods , Vertebrates/genetics , Animals , Chromosome Mapping , Chromosomes, Artificial, Bacterial , GenomeABSTRACT
While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.
Subject(s)
Gene Expression , HIV Infections/virology , HIV/genetics , Mutation , Selection, Genetic , Vaccinia virus/genetics , Base Sequence , Cells, Cultured , Fusion Proteins, gag-pol/genetics , Fusion Proteins, gag-pol/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , HIV/metabolism , Humans , Molecular Sequence Data , Recombination, Genetic , Vaccinia virus/metabolism , pol Gene Products, Human Immunodeficiency Virus/genetics , pol Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
The purpose of the present study was to correlate the in vitro level of HIV Env expression by recombinant modified vaccinia virus Ankara (rMVA) with immunogenicity in mice. A 5-fold difference in Env synthesis was achieved at the translational level by the presence or absence of an out-of-frame initiation codon upstream of the env gene. This perturbation had no effect on the size or processing of Env. In contrast to the variation in Env synthesis, the rMVAs produced similar amounts of HIV Gag, which were expressed from identical cassettes. Mice immunized with the higher Env expressing rMVAs had about 15-fold higher titers of Env antibodies and several fold higher frequencies of Env-specific CD8+ and CD4+ T cells than mice immunized with the low expresser. The greater immune response achieved by high expression was maintained over a 100-fold dose range. Importantly, enhanced Env immune responses did not come at the expense of lower Gag T cell responses. These data suggest that for high immunogenicity, rMVAs should be engineered to produce the most recombinant protein that can be achieved without compromising the growth and stability of the rMVA.
Subject(s)
AIDS Vaccines/genetics , AIDS Vaccines/immunology , HIV Infections/immunology , Vaccination , AIDS Vaccines/administration & dosage , Animals , Antibody Specificity , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cells, Cultured , Cytokines/biosynthesis , Gene Expression , Genetic Vectors , HIV Antibodies/blood , HIV Antibodies/immunology , Humans , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Recombination, Genetic , Spleen/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccinia virus , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/immunology , gag Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
The antidepressant mirtazapine is an optically active drug and currently marketed as a racemic compound consisting of its S(+) and R(-)-enantiomers in a 50:50 mixture. As stereochemistry of antidepressants has become increasingly important to consider for the relevance of their analgesic properties, we investigated the effect of (+/-)-mirtazapine and its enantiomers in an animal model of acute thermal nociception. Wistar rats were injected intrathecal with either (+/-)-mirtazapine, R(-)-mirtazapine, S(+)-mirtazapine from 1 to 0.001 mg/kg and vehicle (0.9% NaCl), respectively. The effects on thermal paw withdrawal thresholds were monitored using the Hargreaves test. (+/-)-Mirtazapine exerted pro- and antinociceptive effects in acute thermal nociception, whereas R(-)-mirtazapine showed solely antinociceptive and S(+)-mirtazapine pronociceptive properties. These results clearly demonstrate a differential effect of (+/-)-mirtazapine and its enantiomers on nociception. As R(-)-mirtazapine exerts the antinociceptive activity of the racemic mixture it may be a putative candidate for an enantioselective use as analgesic.
Subject(s)
Afferent Pathways/drug effects , Mianserin/analogs & derivatives , Nociceptors/drug effects , Pain/drug therapy , Thermosensing/drug effects , Afferent Pathways/physiology , Animals , Antidepressive Agents, Tricyclic/chemistry , Antidepressive Agents, Tricyclic/pharmacology , Antidepressive Agents, Tricyclic/therapeutic use , Central Nervous System/drug effects , Central Nervous System/physiopathology , Disease Models, Animal , Hot Temperature/adverse effects , Injections, Spinal , Isomerism , Male , Mianserin/chemistry , Mianserin/pharmacology , Mianserin/therapeutic use , Mirtazapine , Molecular Conformation , Nociceptors/physiology , Pain/chemically induced , Pain/physiopathology , Pain Measurement , Pain Threshold/drug effects , Pain Threshold/physiology , Rats , Rats, Wistar , Thermosensing/physiology , Treatment OutcomeABSTRACT
We investigated the binding of E. coli RNA polymerase holoenzymes bearing sigma70, sigma(S), sigma32, or sigma54 to the ribosomal RNA operons (rrn) in vivo. At the rrn promoter, we observed "holoenzyme switching" from Esigma70 to Esigma(S) or Esigma32 in response to environmental cues. We also examined if sigma factors are retained by core polymerase during transcript elongation. At the rrn operons, sigma70 translocates briefly with the elongating polymerase and is released stochastically from the core polymerase with an estimated half-life of approximately 4-7 s. Similarly, at gadA and htpG, operons that are targeted by Esigma(S) and Esigma32, respectively, we find that sigma(S) and sigma32 also dissociate stochastically, albeit more rapidly than sigma70, from the elongating core polymerase. Up to approximately 70% of Esigma70 (the major vegetative holoenzyme) in rapidly growing cells is engaged in transcribing the rrn operons. Thus, our results suggest that at least approximately 70% of cellular holoenzymes release sigma70 during transcript elongation. Release of sigma factors during each round of transcription provides a simple mechanism for rapidly reprogramming polymerase with the relevant sigma factor and is consistent with the occurrence of a "sigma cycle" in vivo.
Subject(s)
Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Escherichia coli/physiology , Gene Expression Regulation, Bacterial/physiology , Sigma Factor/metabolism , Transcription, Genetic/physiology , Bacterial Proteins/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Holoenzymes/genetics , Holoenzymes/metabolism , Operon/genetics , Operon/physiology , Promoter Regions, Genetic/physiology , Sigma Factor/genetics , Stochastic ProcessesABSTRACT
We developed an AIDS vaccine for Western and West-Central Africa founded on HIV-1 subtype CRF02_AG. Rhesus macaques were primed with Gag-Pol-Env-expressing plasmid DNA and boosted with a recombinant modified vaccinia virus Ankara (rMVA), expressing matched proteins. Two DNA vaccine constructs (IC1-90 and IC48) that differed by point mutations in gag and pol were compared. IC1-90 produces primarily immature (core comprises unprocessed Pr55Gag) HIV-like particles (VLPs) and IC48 produces mature VLP with processed Pr55Gag, immature VLP, and intracellular protein aggregates. Both vaccines raised significant cellular responses for Gag, Pol, and Env. Approximate twofold higher ELISPOT responses to Gag and Env epitopes were observed for IC48 animals than for IC1-90 animals at the peak post-MVA effector (P = 0.028) and late memory (P = 0.051) phases, respectively. Greater breadth for IC48-primed animals was observed than for IC1-90-primed animals at peak response (P = 0.03). Our results indicated that the vaccines elicited high frequency T cell responses and primed anti-Env antibody. They also suggest that expression of different forms of VLP has a significant effect on elicited cellular and humoral immunity.
Subject(s)
AIDS Vaccines , HIV-1/immunology , Vaccines, DNA , Africa, Western , Animals , Antibody Formation , Cell Line , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Gene Products, env/immunology , Gene Products, gag/immunology , Humans , Kidney/immunology , Kidney/virology , Macaca mulatta , Male , Plasmids , T-Lymphocytes/immunology , TransfectionABSTRACT
Although the cost of generating draft-quality genomic sequence continues to decline, refining that sequence by the process of "sequence finishing" remains expensive. Near-perfect finished sequence is an appropriate goal for the human genome and a small set of reference genomes; however, such a high-quality product cannot be cost-justified for large numbers of additional genomes, at least for the foreseeable future. Here we describe the generation and quality of an intermediate grade of finished genomic sequence (termed comparative-grade finished sequence), which is tailored for use in multispecies sequence comparisons. Our analyses indicate that this sequence is very high quality (with the residual gaps and errors mostly falling within repetitive elements) and reflects 99% of the total sequence. Importantly, comparative-grade sequence finishing requires approximately 40-fold less reagents and approximately 10-fold less personnel effort compared to the generation of near-perfect finished sequence, such as that produced for the human genome. Although applied here to finishing sequence derived from individual bacterial artificial chromosome (BAC) clones, one could envision establishing routines for refining sequences emanating from whole-genome shotgun sequencing projects to a similar quality level. Our experience to date demonstrates that comparative-grade sequence finishing represents a practical and affordable option for sequence refinement en route to comparative analyses.