JMJD7 Antibody

What is JMJD7 and why is it significant for research?

JMJD7 is a member of the Jumonji C domain-containing protein family functioning as a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation, a relatively rare post-translational modification. This protein plays crucial roles in chromatin modification, transcriptional regulation, and protein synthesis regulation. Its significance stems from its unique enzymatic activity, distinctive structural features, and potential involvement in various diseases including cancer and autism, making it an important target for both basic research and therapeutic development .

JMJD7's significance is further highlighted by evolutionary conservation, with functional orthologs present across animal species, suggesting fundamental biological importance. The protein uniquely demonstrates a dimerization mode involving both N- and C-terminal regions with disulfide bond formation, distinguishing it structurally from other JmjC family members .

What applications are JMJD7 antibodies most commonly used for?

JMJD7 antibodies are primarily employed in Western blot (WB) and immunohistochemistry (IHC) applications for detecting and analyzing JMJD7 expression and localization patterns in various tissues and cellular contexts. These antibodies enable researchers to investigate JMJD7's distribution within cellular compartments, protein-protein interactions, and expression changes in response to various stimuli or disease states .

For optimal results in Western blot applications, JMJD7 antibodies like the CAB7408 polyclonal antibody are typically used at dilutions ranging from 1:500 to 1:2000, depending on the specific experimental context and protein expression levels. Additional applications may include immunoprecipitation for studying protein complexes involving JMJD7, ELISA, and potentially chromatin immunoprecipitation (ChIP) for investigating chromatin-associated functions .

How should researchers validate the specificity of JMJD7 antibodies?

Proper validation of JMJD7 antibodies involves a multi-step approach:

• Positive and negative control samples: Use tissue/cell samples known to express (e.g., HeLa, Daudi, mouse brain, rat heart) or not express JMJD7. Validation should include both human and rodent samples if studying cross-species applications .

• Knockdown/knockout controls: Compare antibody reactivity in wildtype cells versus those with JMJD7 knockdown or knockout to confirm specificity. This approach was effectively demonstrated in studies examining endogenous DRG1/2 hydroxylation, where JMJD7 knockout eliminated the hydroxylation signal .

• Peptide competition assay: Pre-incubate the antibody with excess JMJD7 recombinant protein or immunizing peptide prior to sample application to confirm signal abolishment.

• Cross-reactivity assessment: Test against similar Jumonji family members, particularly closely related hydroxylases like FIH, TYW5, JMJD5, MINA53, and NO66, to ensure specificity for JMJD7 .

• Multiple antibody comparison: Use antibodies targeting different epitopes of JMJD7 to validate consistent detection patterns.

How can researchers distinguish between JMJD7 and JMJD7-PLA2G4B fusion protein in experimental systems?

Distinguishing between JMJD7 and the JMJD7-PLA2G4B fusion protein requires careful experimental design:

RT-PCR approach: Design primers spanning the junction between JMJD7 and PLA2G4B genes. Use forward primers within JMJD7 and reverse primers within PLA2G4B to specifically amplify the fusion transcript. Additionally, use primers specific to each individual gene to quantify relative expression levels .

Western blot strategy: Use antibodies targeting:

• N-terminal JMJD7 (detects both JMJD7 and fusion protein)

• C-terminal PLA2G4B (detects both PLA2G4B and fusion protein)

• Junction-specific antibody (if available, detects only fusion protein)

The differential expression pattern observed across these antibodies can help distinguish between the individual proteins and the fusion product .

Mass spectrometry validation: For definitive identification, analyze immunoprecipitated proteins by mass spectrometry to identify peptides specific to the fusion junction region.

Functional studies: Compare phenotypic outcomes between cells with JMJD7-only knockdown versus JMJD7-PLA2G4B knockdown. As demonstrated in HNSCC cells, JMJD7-PLA2G4B ablation produced more significant effects on cell proliferation and apoptosis than JMJD7-only knockdown, providing functional evidence of the fusion protein's distinct roles .

What are the optimal conditions for detecting JMJD7-catalyzed lysyl hydroxylation of DRG1/2?

Detecting JMJD7-catalyzed lysyl hydroxylation requires careful methodological considerations:

Mass spectrometry detection protocol:

• Immunopurify DRG1/2 proteins (either endogenous or epitope-tagged) from cells with and without JMJD7 expression

• Perform trypsin digestion, noting that hydroxylation at K22/K21 positions may result in missed cleavages

• Look for peptides with a characteristic +16 Da mass shift

• Analyze MS/MS fragmentation patterns to confirm the precise hydroxylation site

In vitro enzymatic assay optimization:

• Use peptide substrates spanning the target region (e.g., DRG1 21-40: NKATAHHLGLLKARLAKLRR)

• Include essential cofactors: Fe(II) (50-100 μM), 2OG (100-200 μM)

• Maintain aerobic conditions with buffer at pH 7.2-7.5

• Include controls with enzyme variants (H178A), cofactor omission, and inhibitors (N-oxalylglycine)

Isotopic labeling approaches: For confirmatory studies, conduct reactions under 18O2 atmosphere to demonstrate oxygen incorporation from molecular oxygen rather than water, which is characteristic of 2OG oxygenases .

How should researchers investigate JMJD7 dimerization and its functional significance?

JMJD7 displays a unique dimerization mode involving both N- and C-terminal regions with disulfide bond formation. To investigate this feature:

Biophysical characterization:

• Use size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine the oligomeric state in solution

• Apply analytical ultracentrifugation to verify dimeric state and determine dissociation constants

• Conduct circular dichroism analysis to assess structural changes upon dimerization

Structural mutagenesis approach:

• Perform alanine scanning of residues at the dimerization interface identified by crystallography

• Create variants with disrupted disulfide bonds by mutating key cysteine residues

• Characterize the enzymatic activity and substrate binding of monomeric versus dimeric forms

• Assess cellular localization and interaction patterns of dimerization-defective mutants

Cellular assessment methods:

• Use immunoprecipitation with differentially tagged JMJD7 constructs to confirm dimerization in cells

• Apply proximity ligation assays to visualize dimerization in situ

• Perform Förster resonance energy transfer (FRET) analysis with fluorescently tagged JMJD7 variants to monitor dimerization dynamics

Additionally, researchers should investigate whether dimerization affects substrate specificity, as the unique quaternary structure might create a distinctive substrate binding pocket or influence interaction with DRG1/2 and other potential targets.

How can JMJD7 antibodies be used to investigate its role in cancer progression?

JMJD7 antibodies provide valuable tools for investigating cancer-related mechanisms through several approaches:

Expression profiling in tumor tissues:

• Use immunohistochemistry with validated JMJD7 antibodies to assess expression patterns across cancer types and stages

• Perform tissue microarray analysis to correlate JMJD7 expression with clinicopathological features and patient outcomes

• Compare expression between tumor and adjacent normal tissues to identify cancer-specific alterations

Functional investigations of fusion proteins:

• Apply co-immunoprecipitation studies to identify cancer-relevant JMJD7 interactors

• Use immunofluorescence to determine subcellular localization changes in cancer cells

• Develop specific antibodies against the JMJD7-PLA2G4B fusion junction for detection in head and neck squamous cell carcinoma (HNSCC)

Mechanistic pathway analysis:

• Combine JMJD7 antibodies with phospho-specific antibodies (e.g., phospho-AKT) to investigate signaling pathway modulation in cancer cells

• Perform ChIP-seq to identify JMJD7 genomic binding sites relevant to oncogenic transcriptional programs

• Use proximity ligation assays to visualize interactions between JMJD7 and cancer-relevant proteins in situ

Research has demonstrated that targeting the JMJD7-PLA2G4B fusion gene significantly inhibits proliferation of HNSCC cells by promoting G1 cell cycle arrest and increasing starvation-induced cell death, suggesting potential therapeutic applications .

What is the significance of the R260C mutation in JMJD7 and how can it be studied?

The R260C mutation in JMJD7 has been identified in both endometrial cancer and autism, suggesting potentially broad pathological significance . Investigating this mutation requires:

Structural and enzymatic characterization:

• Compare crystal structures of wild-type and R260C JMJD7 to identify conformational changes

• Assess the impact on catalytic activity using in vitro hydroxylation assays with DRG1/2 peptides

• Evaluate effects on dimerization, 2OG binding, and Fe(II) coordination

Cellular phenotype analysis:

• Generate isogenic cell lines expressing wild-type or R260C JMJD7

• Assess differences in:

  • Substrate hydroxylation efficiency

  • Protein-protein interaction networks

  • Cellular localization patterns

  • Transcriptional profiles

  • Cell growth, survival, and differentiation

Disease model applications:

• For cancer studies: Evaluate the impact on cell transformation, migration, invasion, and response to therapy

• For autism investigations: Examine effects on neuronal development, synaptic function, and relevant signaling pathways

• Create knock-in animal models to study organismal phenotypes

Interestingly, high-resolution (2.2 Å) structural data is available for the R260C variant, providing a foundation for structure-based studies of this mutation's functional consequences . The dual association with different disease contexts suggests that JMJD7 may have tissue-specific functions that are differentially affected by this mutation.

How does JMJD7 interact with the translation machinery and what methodologies can explore this?

JMJD7 has been identified as interacting with and hydroxylating DRG1/2, which are members of the Translation Factor (TRAFAC) family of GTPases, suggesting a role in translation regulation . This can be investigated through:

Co-immunoprecipitation and proximity studies:

• Use JMJD7 antibodies to pull down native complexes from cell lysates

• Perform mass spectrometry to identify translation-related interaction partners

• Confirm interactions through reverse co-IP and proximity ligation assays

• Apply RNA immunoprecipitation to identify associated mRNAs

Ribosome profiling analysis:

• Compare ribosome occupancy patterns in JMJD7 wild-type versus knockout cells

• Analyze translation efficiency of specific mRNAs

• Identify potential transcript-specific translation regulation

Polysome profiling with JMJD7 detection:

• Fractionate polysomes on sucrose gradients

• Use Western blotting with JMJD7 antibodies to determine association with different ribosomal fractions

• Compare profiles with and without translational stressors

In vitro translation systems:

• Supplement rabbit reticulocyte lysate or other cell-free translation systems with recombinant JMJD7

• Assess effects on translation efficiency and accuracy

• Test hypothesis that JMJD7-mediated hydroxylation of DRG1/2 modulates their GTPase activity and thereby affects translation

The interaction between JMJD7 and DRG1/2 appears highly conserved, as even the Drosophila ortholog of JMJD7 (dmJMJD7) can hydroxylate human DRG1/2, suggesting an evolutionarily preserved regulatory mechanism for protein synthesis .

What emerging technologies can enhance JMJD7 research beyond traditional antibody applications?

Several cutting-edge technologies offer new opportunities for JMJD7 research:

CRISPR-based approaches:

• Generate JMJD7 knockout and knock-in cell lines using CRISPR-Cas9

• Apply CRISPRi/CRISPRa for controlled modulation of JMJD7 expression

• Implement CRISPR-based screens to identify synthetic lethal interactions and functional pathways

• Use base editors to introduce specific mutations like R260C for functional studies

Proximity-dependent labeling techniques:

• Develop JMJD7 fusion constructs with BioID, TurboID, or APEX2

• Map the proximate proteome to identify novel interaction partners

• Compare proximal proteins in different cellular contexts or with mutant variants

• Combine with mass spectrometry to identify hydroxylated substrates beyond DRG1/2

Advanced imaging applications:

• Apply live-cell single-molecule tracking of fluorescently tagged JMJD7

• Implement super-resolution microscopy to visualize subcellular localization at nanoscale resolution

• Use FRET-based sensors to monitor JMJD7 activity in real-time

• Develop antibody-based imaging methods to track endogenous JMJD7 dynamics

Hydroxylation-specific detection methods:

• Develop antibodies specifically recognizing hydroxylated K22/K21 in DRG1/2

• Create chemical probes for selective enrichment of hydroxylated peptides

• Implement targeted mass spectrometry methods for sensitive detection of hydroxylation events

These technologies can overcome limitations of traditional antibody applications and provide deeper insights into JMJD7 biology and function.

How can researchers integrate JMJD7 hydroxylation studies with broader epigenetic and translational regulation research?

Integration of JMJD7 research with broader regulatory mechanisms requires:

Multi-omics integration strategies:

• Combine JMJD7 ChIP-seq, RNA-seq, and ribosome profiling to link chromatin regulation with translational outcomes

• Correlate JMJD7-dependent hydroxylation events with changes in the proteome and translatome

• Develop computational frameworks to integrate hydroxylation data with other PTM databases

• Perform network analysis to position JMJD7 within broader regulatory circuits

Comparative studies with other JmjC family members:

• Conduct parallel analysis of JMJD7 with related hydroxylases like FIH, TYW5, JMJD5, MINA53, and NO66

• Identify overlapping and distinct functions in translation regulation

• Investigate potential functional redundancy or cooperation between family members

• Compare structural features determining substrate specificity

Translation regulation studies:

• Assess how JMJD7-mediated hydroxylation of DRG1/2 affects their interactions with ribosomes and translation factors

• Investigate impact on translation initiation, elongation, and termination rates

• Determine whether hydroxylation affects mRNA selection or ribosome recycling

• Explore potential roles in specialized translation mechanisms like IRES-mediated translation

Disease-context integration:

• Examine JMJD7 expression and activity across different disease models

• Correlate hydroxylation patterns with disease progression

• Investigate potential combinatorial therapies targeting JMJD7 and related pathways in cancer

These integrative approaches can position JMJD7 research within the broader context of cellular regulatory mechanisms and disease processes.

What considerations should be made when developing inhibitors or modulators of JMJD7 activity?

Development of JMJD7 inhibitors or modulators requires careful consideration of:

Structural considerations for inhibitor design:

• Target the unique features of JMJD7's active site, including the 4 polar side-chains positioned to interact with the 2OG C5 carboxylate group (Y127, T175, K193, Y186)

• Consider the distinctive 2OG binding mode involving hydrogen bonds with N184 and N289 side-chain amides

• Account for JMJD7's dimeric structure when designing inhibitors to potentially disrupt quaternary structure

• Leverage the available crystal structures for structure-based design

Selectivity challenges:

• Design compounds that distinguish JMJD7 from other 2OG oxygenases

• Consider selectivity against related JmjC hydroxylases versus broader inhibition of the JmjC family

• Develop assays to assess cross-reactivity with other 2OG-dependent enzymes

• Test selectivity against the JMJD7-PLA2G4B fusion protein versus JMJD7 alone

Biological validation approaches:

• Compare phenotypes of genetic knockout versus pharmacological inhibition

• Assess effects on DRG1/2 hydroxylation as a direct measure of target engagement

• Monitor downstream effects on AKT phosphorylation in cancer models

• Evaluate cellular consequences including proliferation, cell cycle progression, and apoptosis sensitivity

Therapeutic potential assessment:

• Evaluate inhibitor effects in models of HNSCC and other cancers where JMJD7 or JMJD7-PLA2G4B may play a role

• Consider potential applications in developmental disorders related to the R260C mutation

• Assess synergy with existing therapeutic approaches

• Develop biomarkers to identify patients most likely to respond to JMJD7-targeted therapies

This strategic approach to inhibitor development requires balancing potency, selectivity, and therapeutic relevance while leveraging the distinctive structural and functional features of JMJD7.

What controls are essential for validating JMJD7 antibody specificity in different experimental contexts?

Ensuring reproducible JMJD7 antibody-based research requires comprehensive controls:

Western blot validation controls:

• Positive control samples with confirmed JMJD7 expression (HeLa, Daudi, mouse brain, rat heart)

• JMJD7 knockout or knockdown samples as negative controls

• Recombinant JMJD7 protein standards at known concentrations

• Related JmjC family members to assess cross-reactivity

• Secondary antibody-only controls to identify non-specific binding

Immunoprecipitation specificity controls:

• IgG isotype control antibodies processed identically to JMJD7 antibodies

• Reverse IP using antibodies against suspected interaction partners

• Competition assays with recombinant JMJD7 or immunizing peptides

• Comparison of results between different JMJD7 antibodies targeting distinct epitopes

• Parallel IP from JMJD7-depleted cells

Immunofluorescence validation controls:

• Peptide competition controls to verify signal specificity

• JMJD7 knockout cells as negative controls

• Co-localization studies with established markers for relevant cellular compartments

• Secondary antibody-only controls to identify autofluorescence or non-specific binding

• Pre-immune serum controls for polyclonal antibodies

Species cross-reactivity considerations:

• Test antibodies against recombinant human, mouse, and rat JMJD7

• Validate in tissues from multiple species if cross-species applications are planned

• Consider sequence homology when interpreting signals across species

• Use ortholog-specific positive controls (e.g., dmJMJD7 for Drosophila studies)

These comprehensive controls ensure reliable interpretation of JMJD7 antibody-based experimental results across diverse research contexts.

How can researchers troubleshoot issues with detecting JMJD7-mediated lysyl hydroxylation?

Detecting JMJD7-catalyzed lysyl hydroxylation presents several challenges that can be addressed through systematic troubleshooting:

Mass spectrometry detection challenges:

• Issue: Missed tryptic cleavages due to hydroxylation
  Solution: Use alternative proteases (e.g., Lys-N, which is also affected by hydroxylation but in a predictable manner) or adjust search parameters to include missed cleavages

• Issue: Low abundance of hydroxylated peptides
  Solution: Implement enrichment strategies, use targeted MS approaches, or increase starting material

• Issue: Difficulty differentiating hydroxylation from other +16 Da modifications
  Solution: Use high-resolution MS/MS to locate the modification precisely; confirm with synthetic hydroxylated peptide standards

In vitro assay optimization:

• Issue: Low enzymatic activity
  Solution: Verify enzyme integrity, optimize cofactor concentrations (Fe(II), 2OG), and ensure aerobic conditions

• Issue: Substrate accessibility
  Solution: Test peptides of varying lengths around the target lysine; consider full-length protein substrates

• Issue: Inefficient detection method
  Solution: Compare direct MS detection with alternate approaches such as oxygen consumption assays or 2OG turnover measurements

Cellular detection strategies:

• Issue: Low endogenous hydroxylation levels
  Solution: Treat cells with DMOG to trap enzyme-substrate complexes; overexpress JMJD7 transiently

• Issue: Interference from other post-translational modifications
  Solution: Use phosphatase treatment or other relevant enzymes to remove potentially interfering modifications

• Issue: Difficulty detecting site-specific hydroxylation
  Solution: Develop site-specific antibodies against hydroxylated K22/K21 in DRG1/2 for immunological detection

These troubleshooting approaches enable more reliable detection and characterization of JMJD7-mediated lysyl hydroxylation.

What recommendations exist for reproducible quantification of JMJD7 expression across diverse experimental systems?

Ensuring reproducible quantification of JMJD7 expression requires standardized approaches:

Western blot quantification best practices:

• Use validated loading controls appropriate for the experimental context (e.g., GAPDH, β-actin, or total protein staining)

• Implement technical replicates (minimum of three) and biological replicates (different passages or samples)

• Ensure signal falls within the linear range of detection by testing serial dilutions

• Include recombinant JMJD7 standards at known concentrations for absolute quantification

• Apply consistent image acquisition settings and use software-based quantification

RT-qPCR standardization approaches:

• Design primers spanning exon-exon junctions to avoid genomic DNA amplification

• Test primer efficiency using standard curves

• Use multiple reference genes validated for stability in the specific experimental system

• For fusion transcript detection, design primers specific to the JMJD7-PLA2G4B junction

• Account for potential splice variants in primer design and data interpretation

Immunohistochemistry quantification methods:

• Implement standardized scoring systems (e.g., H-score, Allred score)

• Use automated image analysis software to reduce subjective bias

• Include positive and negative control tissues in each batch

• Apply consistent antibody concentrations, incubation times, and detection methods

• Conduct blinded scoring by multiple observers

Cross-platform normalization strategies:

• When comparing across different experimental systems or detection methods, use common reference standards

• Normalize to consistent cellular references (per cell number, per unit protein, per unit DNA)

• Include inter-laboratory control samples for multi-center studies

• Apply appropriate statistical methods for handling data from different quantification approaches

Additionally, researchers should maintain detailed records of antibody lots, detection reagents, and instrument settings to facilitate reproducibility and troubleshooting across experiments.

What are the potential additional substrates of JMJD7 beyond DRG1/2?

While DRG1/2 are established substrates for JMJD7-mediated lysyl hydroxylation, several approaches can identify additional targets:

Unbiased proteomics strategies:

• Perform quantitative proteomics comparing hydroxylated lysine-containing peptides in wild-type versus JMJD7 knockout cells

• Implement SILAC labeling to enhance quantitative accuracy

• Develop chemical enrichment methods specific for hydroxylysine-containing peptides

• Apply targeted mass spectrometry to screen candidate substrates with sequence motifs similar to the DRG1/2 hydroxylation sites

Structural and sequence-based prediction:

• Analyze the structural features surrounding K22/K21 in DRG1/2

• Perform bioinformatic screens for proteins containing similar sequence motifs

• Focus on evolutionarily conserved lysine residues in structurally accessible regions

• Prioritize candidates from the TRAFAC family of GTPases and other translation-related factors

Interaction-based approaches:

• Apply proximity labeling (BioID/TurboID) with JMJD7 as the bait to identify physically proximate proteins

• Perform immunoprecipitation followed by mass spectrometry under conditions that preserve enzyme-substrate interactions

• Use JMJD7 catalytic mutants (H178A) that might form more stable complexes with substrates

• Screen interaction partners of DRG1/2 as potential additional substrates

The identification of additional JMJD7 substrates would significantly expand our understanding of its biological roles and potential contributions to disease mechanisms beyond those already established through DRG1/2 hydroxylation and the JMJD7-PLA2G4B fusion protein .

How does JMJD7 activity differ between normal physiological conditions and disease states?

Understanding context-dependent JMJD7 function requires comparative studies across physiological and pathological conditions:

Expression and localization profiling:

• Compare JMJD7 expression levels and subcellular distribution between normal and diseased tissues

• Assess changes in JMJD7-PLA2G4B fusion formation across different cancer types and stages

• Examine JMJD7 expression during development and differentiation

• Analyze potential regulation by microenvironmental factors (hypoxia, nutrient availability, pH)

Functional activity assessment:

• Measure DRG1/2 hydroxylation levels across different physiological and pathological states

• Compare JMJD7 enzymatic activity in normal versus cancer cell lines

• Assess how disease-associated mutations (e.g., R260C) affect function

• Investigate potential context-dependent substrate preferences

Regulatory network analysis:

• Map JMJD7 interactome changes between normal and disease conditions

• Identify condition-specific post-translational modifications of JMJD7 itself

• Analyze transcriptional and post-transcriptional regulation of JMJD7 across contexts

• Examine potential competition or cooperation with other JmjC family members

Disease model comparative studies:

• Compare JMJD7 knockout phenotypes in normal versus disease model systems

• Assess differential sensitivity to JMJD7 inhibition between normal and cancer cells

• Investigate potential synthetic lethal interactions in disease contexts

• Examine how JMJD7 contributes to therapy response or resistance

These comparative approaches can reveal context-specific functions and identify potential therapeutic vulnerabilities in disease states while minimizing impact on normal physiological processes.

What evolutionary insights can be gained from studying JMJD7 orthologs across species?

Investigating JMJD7 orthologs across evolutionary lineages provides valuable insights:

Structural and functional conservation analysis:

• Compare enzymatic activities of JMJD7 orthologs from diverse species (e.g., human JMJD7 versus Drosophila dmJMJD7)

• Assess cross-species substrate recognition (e.g., ability of dmJMJD7 to hydroxylate human DRG1/2)

• Identify structurally conserved regions that likely represent critical functional domains

• Map evolutionary changes onto the three-dimensional structure to identify adaptively evolving sites

Developmental and physiological roles:

• Compare phenotypes of JMJD7 knockdown/knockout across model organisms

• Investigate tissue-specific expression patterns across species

• Assess developmental timing of expression and potential stage-specific functions

• Correlate evolutionary changes with species-specific physiological adaptations

Substrate co-evolution:

• Analyze co-evolutionary patterns between JMJD7 and its substrates (e.g., DRG1/2)

• Identify compensatory mutations that maintain functional interactions

• Examine conservation of the hydroxylation site across DRG1/2 orthologs

• Investigate potential species-specific substrates

Studies in Drosophila have already revealed that knockdown of dmJMJD7 correlates with increased posterior wing compartment size, likely through effects on cell size, suggesting conserved roles in growth regulation. Furthermore, the finding that dmJMJD7 can hydroxylate human DRG1/2 indicates strong conservation of the catalytic mechanism and substrate recognition, despite only 45% sequence identity between human and Drosophila orthologs .

Evolutionary analysis can reveal the fundamental biological importance of JMJD7 and provide insights into which functions represent ancestral roles versus more recent adaptations.