Recombinant Squalus acanthias Creatine kinase B-type

What is the biochemical role of creatine kinase B-type in Squalus acanthias?

Creatine kinase B-type (B-CK) in Squalus acanthias functions as a critical enzyme in cellular energy homeostasis, catalyzing the reversible exchange of high-energy phosphate between ATP and creatine phosphate. In the dogfish shark, B-CK is notably found in the rectal (salt-secreting) gland, which contains high levels of Na+/K+-ATPase, suggesting its specialized role in regenerating ATP for sodium transport processes. The enzyme is particularly important in tissues with fluctuating energy demands, where it helps maintain ATP levels during periods of increased metabolic activity. The colocalization of B-CK with mitochondrial creatine kinase isoenzymes in the shark's rectal gland mirrors a similar arrangement found in mammalian kidneys, indicating evolutionary conservation of this energy shuttle system .

How does shark B-CK compare structurally with other vertebrate B-CK homologs?

Structural analysis of Squalus acanthias B-CK reveals remarkable conservation across evolutionary lineages. Peptide fragment analysis shows that the active site region of shark B-CK is 100% identical to the corresponding region from echinoderm sperm flagellar creatine kinase and 96% homologous with both chicken and rat B-CK subunits. A second fragment, corresponding to a region near the N-terminal of mammalian creatine kinases, demonstrates 89% homology with chicken B-CK . This high degree of structural conservation suggests that the functional domains of B-CK have been maintained throughout vertebrate evolution, reflecting the enzyme's fundamental importance in cellular energy metabolism. The dimeric quaternary structure of B-CK is also preserved across species, with the protein typically functioning as a homodimer in brain and other tissues .

What expression systems are most effective for producing recombinant Squalus acanthias B-CK?

Based on comparative analysis of creatine kinase expression systems, insect cell-based baculovirus expression vectors represent one of the most effective systems for producing functional recombinant Squalus acanthias B-CK. This approach has been successfully employed for human B-CK expression, yielding up to 30% of total soluble cellular protein with excellent enzymatic activity . For shark B-CK expression, the procedure would involve:

• Cloning the full-length Squalus acanthias B-CK cDNA into a baculovirus transfer vector

• Generating recombinant virus through cotransfection with wild-type baculovirus DNA in Sf9 cells

• Purifying and amplifying the recombinant virus through multiple rounds of plaque purification

• Optimizing infection conditions (multiplicity of infection, typically between 1-10)

• Harvesting cells at the appropriate time interval post-infection (typically 48-72 hours)

This system offers advantages including proper folding, dimerization, and post-translational modifications crucial for shark B-CK functionality. Alternative systems such as bacterial expression may yield higher quantities but often produce less active protein due to improper folding or lack of post-translational modifications .

What is the optimal purification protocol for recombinant Squalus acanthias B-CK?

The optimal purification protocol for recombinant Squalus acanthias B-CK would involve a combination of chromatographic techniques similar to those established for other B-CK purifications. Based on available research, a highly efficient protocol would include:

• Initial extraction: Lysis of expression host cells in a buffer containing protease inhibitors, typically at pH 7.4-8.0

• Clarification: Centrifugation to remove cellular debris (typically 12,000-15,000 g for 20-30 minutes)

• First purification step: Anion exchange chromatography using a MonoQ column in FPLC system, which has been shown to achieve >99% homogeneity in a single step for recombinant B-CK

• Elution: Using a linear salt gradient (typically 0-500 mM NaCl)

• Buffer exchange: Dialysis against a storage buffer (typically containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1 mM DTT)

This purification approach typically yields protein with specific activity around 240 U/mg, comparable to that used in crystallization studies . For shark B-CK specifically, chromatographic conditions may need slight modifications due to potential differences in isoelectric point compared to mammalian B-CK. The purified enzyme would be suitable for both structural studies and functional assays .

How can the enzymatic activity of purified recombinant Squalus acanthias B-CK be verified and quantified?

Verification and quantification of recombinant Squalus acanthias B-CK enzymatic activity should employ multiple complementary approaches:

• Spectrophotometric activity assay: The standard method involves coupling the creatine kinase reaction with hexokinase and glucose-6-phosphate dehydrogenase to measure NADPH production at 340 nm. This can be performed in both forward (ATP production) and reverse (creatine phosphate production) directions.

• Zymogram analysis: Non-denaturing polyacrylamide gel electrophoresis followed by activity staining can verify the native state of the enzyme and confirm its migration pattern matches that of authentic B-CK.

• Size exclusion chromatography: Gel filtration can confirm the dimeric state of the active enzyme, which should elute at a molecular weight of approximately 86 kDa (two 43 kDa subunits).

• Immunological verification: Western blot analysis using B-CK specific antibodies, potentially including monoclonal antibodies directed against conserved epitopes in the N-terminus of the protein, can confirm identity.

For quantification, specific activity should be calculated in international units per milligram of protein (U/mg), with optimal preparations yielding approximately 240 U/mg . Temperature and pH optimum determinations are also important, as shark B-CK may have slightly different optimal conditions compared to mammalian homologs due to adaptation to the shark's physiology.

What techniques are most appropriate for structural characterization of Squalus acanthias B-CK?

For comprehensive structural characterization of Squalus acanthias B-CK, multiple complementary techniques should be employed:

• X-ray crystallography: High-resolution structural determination remains the gold standard, with potential resolution of 1.4-2.0 Å as achieved with other B-CK structures . This requires:

  • Highly purified protein (>99% homogeneity)

  • Optimization of crystallization conditions (typically using hanging drop vapor diffusion)

  • Data collection at synchrotron radiation sources

  • Structure solving using molecular replacement with known B-CK structures as templates

• Mass spectrometry: For accurate molecular weight determination and identification of post-translational modifications, utilizing:

  • Intact protein mass analysis by ESI-MS or MALDI-TOF

  • Peptide mapping after enzymatic digestion

  • Targeted analysis of potential phosphorylation sites

• Circular dichroism spectroscopy: To assess secondary structure content and conformational stability under various conditions.

• Analytical ultracentrifugation: To characterize oligomeric state and hydrodynamic properties of the protein.

• Cyanogen bromide peptide analysis: For sequence determination and comparison with other species, particularly focusing on active site regions and N-terminal domains where available shark B-CK data has shown high homology with other vertebrate B-CKs .

These methods collectively provide insight into both the primary sequence and three-dimensional structure of Squalus acanthias B-CK, facilitating comparative analyses with mammalian and other vertebrate B-CK structures.

How can substrate specificity and kinetic parameters of Squalus acanthias B-CK be determined?

Determination of substrate specificity and kinetic parameters for Squalus acanthias B-CK requires systematic biochemical characterization:

• Steady-state kinetics analysis:

  • Measure initial reaction rates at varying concentrations of substrates (ATP, creatine, ADP, phosphocreatine)

  • Plot data using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee transformations

  • Determine Km, Vmax, kcat and kcat/Km values for each substrate

  • Compare with published values for mammalian B-CK

• Alternative substrate analysis:

  • Test activity with structurally related guanidino compounds (e.g., arginine, glycocyamine)

  • Quantify relative activity compared to creatine/phosphocreatine

• Inhibition studies:

  • Evaluate competitive inhibitors (e.g., cyclocreatine phosphate)

  • Determine Ki values and inhibition mechanisms

• pH and temperature profiles:

  • Measure activity across pH range (typically pH 6.0-9.0)

  • Determine temperature optimum and stability

  • These may differ from mammalian B-CK due to the shark's physiological environment

• Metal ion dependency:

  • Assess the requirement for Mg2+ versus other divalent cations

  • Determine optimal metal:ATP ratios

The resulting kinetic parameters would provide insight into potential evolutionary adaptations of shark B-CK to marine environments and specialized tissue functions, particularly in relation to the enzyme's role in sodium transport in the rectal gland .

What are the functional implications of B-CK in osmoregulation of Squalus acanthias?

The presence of B-CK in the rectal gland of Squalus acanthias has significant functional implications for osmoregulation in this marine elasmobranch:

• ATP regeneration for sodium transport: B-CK colocalizes with high levels of Na+/K+-ATPase in the rectal gland, suggesting its primary role is to regenerate ATP from ADP and creatine phosphate to support the energetically demanding process of salt secretion. This functional coupling creates a microcompartment where ATP is rapidly recycled at the site of consumption .

• Creatine phosphate shuttle system: The co-expression of mitochondrial creatine kinase and B-CK in the rectal gland indicates the presence of a complete creatine phosphate shuttle system, similar to that found in mammalian kidney. This system efficiently transfers high-energy phosphates from mitochondria (sites of ATP production) to sites of ATP utilization at the cell membrane .

• Adaptation to fluctuating energy demands: The rectal gland's salt secretion activity fluctuates based on environmental and physiological conditions. The B-CK system provides a buffer for ATP levels, accommodating rapid changes in energy demands without requiring immediate adjustments in mitochondrial oxidative phosphorylation.

• Evolutionary conservation: The similarity in isoform composition between shark rectal gland and mammalian kidney suggests evolutionary conservation of this energy transfer system, highlighting its fundamental importance in tissues with high and fluctuating energy demands related to ion transport .

This specialized energetic role in osmoregulation represents a fascinating example of the adaptation of B-CK function to the unique physiological challenges faced by marine elasmobranchs.

How can site-directed mutagenesis be utilized to investigate structure-function relationships in Squalus acanthias B-CK?

Site-directed mutagenesis provides a powerful approach to investigate structure-function relationships in Squalus acanthias B-CK by enabling the systematic modification of specific amino acid residues:

• Target selection strategy:

  • Active site residues identified through sequence alignment with crystallized B-CKs

  • Regions showing high conservation between shark and other vertebrate B-CKs (e.g., the active site fragment that shows 96% homology with chicken and rat B-CK)

  • Substrate binding domains identified through structural analysis

  • Interface residues involved in dimerization

  • Potential phosphorylation sites that may regulate activity

• Mutation design considerations:

  • Conservative substitutions to test the importance of specific chemical properties

  • Non-conservative substitutions to disrupt function

  • Introduction of reporter groups (e.g., cysteine residues for fluorescent labeling)

  • Creation of phosphomimetic mutations (e.g., serine/threonine to aspartate)

• Expression and functional analysis:

  • Express mutants using the baculovirus system established for wild-type B-CK

  • Assess changes in enzymatic parameters (Km, kcat, substrate specificity)

  • Evaluate structural integrity through circular dichroism and thermal stability assays

  • Examine oligomerization state using gel filtration chromatography

• Specialized applications:

  • Creating chimeric proteins between shark and mammalian B-CK to identify regions responsible for species-specific properties

  • Engineering mutations that enhance stability for biotechnological applications

  • Introducing residues that facilitate crystallization for improved structural studies

This approach would provide molecular-level insights into how Squalus acanthias B-CK has evolved specific structural features adapted to its physiological role in the shark's unique marine environment and osmoregulatory system .

What methodologies can be used to study the interaction of Squalus acanthias B-CK with cellular partners?

Investigating the interaction of Squalus acanthias B-CK with cellular partners requires a multi-faceted approach using various complementary techniques:

• Co-immunoprecipitation studies:

  • Generate specific antibodies against shark B-CK (polyclonal or monoclonal)

  • Prepare native extracts from shark rectal gland tissue

  • Immunoprecipitate B-CK complexes and identify interacting partners using mass spectrometry

  • Verify interactions through reciprocal co-IP with antibodies against identified partners

• Proximity labeling approaches:

  • Create fusion proteins of B-CK with biotin ligases (BioID or TurboID)

  • Express in cell culture systems or through in vivo approaches

  • Identify biotinylated proximal proteins through streptavidin pulldown followed by mass spectrometry

• Fluorescence microscopy:

  • Immunolocalization of B-CK and potential partners (e.g., Na+/K+-ATPase) in shark rectal gland sections

  • Assess colocalization at subcellular resolution using confocal microscopy

  • Implement FRET or FLIM studies for proteins suspected to interact directly

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST):

  • Measure direct binding between purified recombinant B-CK and candidate partners

  • Determine binding affinities and kinetic parameters of interactions

  • Investigate the effect of phosphorylation or other modifications on binding properties

• Functional coupling assays:

  • Design experiments to measure ATP channeling between B-CK and ATP-consuming partners

  • Assess the functional impact of disrupting protein-protein interactions on enzymatic activities

These approaches would provide crucial insights into the molecular organization of energy transfer networks in shark tissues, particularly the functional coupling between B-CK and Na+/K+-ATPase in the rectal gland, which has been proposed based on colocalization studies .

How can recombinant Squalus acanthias B-CK be used as a model system for studying enzyme evolution?

Recombinant Squalus acanthias B-CK offers a valuable model system for studying enzyme evolution due to sharks' position as ancient vertebrates with remarkably conserved biochemistry:

• Comparative sequence-structure-function analysis:

  • Align B-CK sequences from diverse organisms spanning evolutionary history

  • Map conserved and variable regions onto the three-dimensional structure

  • Correlate sequence changes with functional differences in substrate specificity, catalytic efficiency, and regulation

  • The partial sequence data available from shark B-CK already demonstrates high conservation in the active site (96% homology with chicken and rat B-CK) and moderate conservation (89% homology) in N-terminal regions

• Ancestral sequence reconstruction and resurrection:

  • Use phylogenetic methods to infer ancestral B-CK sequences at key evolutionary nodes

  • Express and characterize these reconstructed enzymes

  • Compare properties of extant and ancestral enzymes to understand evolutionary trajectories

• Molecular adaptation studies:

  • Identify residues under positive selection across vertebrate lineages

  • Correlate selective pressure with environmental factors (e.g., temperature, osmotic conditions)

  • Create chimeric enzymes between shark and mammalian B-CKs to isolate regions responsible for adaptive differences

• Structural plasticity and stability analysis:

  • Compare thermal and chemical stability of shark B-CK with homologs from different vertebrate classes

  • Investigate how structure-stability relationships have evolved in different environmental contexts

  • Apply hydrogen-deuterium exchange mass spectrometry to map dynamic regions

This research would contribute to our understanding of protein evolution across 400+ million years of vertebrate history, illuminating how a critical enzyme maintains its core catalytic function while adapting to diverse physiological contexts. The remarkable conservation of B-CK structure and function, despite substantial sequence divergence in some regions, provides insight into evolutionary constraints on metabolic enzymes .

What are common challenges in expressing recombinant Squalus acanthias B-CK and how can they be addressed?

Researchers working with recombinant Squalus acanthias B-CK may encounter several challenges that can be addressed through methodological refinements:

• Low expression levels:

  • Optimize codon usage for the expression host (especially important for shark genes in insect or bacterial systems)

  • Test different promoter strengths in the expression vector

  • Evaluate different expression hosts (Sf9 vs. High Five™ insect cells for baculovirus expression)

  • Optimize infection parameters (MOI, time of harvest) as established for other B-CK expressions

• Protein insolubility:

  • Modify lysis buffer composition (test various salt concentrations, detergents, and pH values)

  • Lower expression temperature to slow folding and prevent aggregation

  • Co-express with molecular chaperones if using bacterial systems

  • Consider fusion tags that enhance solubility (e.g., MBP, SUMO)

• Loss of enzymatic activity:

  • Include protease inhibitors during all purification steps

  • Maintain reducing conditions (add DTT or β-mercaptoethanol) to prevent oxidation of critical cysteines

  • Avoid freeze-thaw cycles; store enzyme with glycerol or lyophilize with appropriate stabilizers

  • Monitor activity throughout purification to identify steps causing activity loss

• Microheterogeneity issues:

  • B-CK is known to exhibit microheterogeneity on two-dimensional gels due to post-translational modifications

  • For crystallization purposes, aim to isolate homogeneous preparations through additional chromatographic steps

  • Consider using phosphatase treatment to remove heterogeneous phosphorylation if present

  • Validate homogeneity through isoelectric focusing or native mass spectrometry

By implementing these strategies, researchers can overcome common technical challenges and obtain functional recombinant Squalus acanthias B-CK suitable for structural and biochemical studies, as has been demonstrated for other B-CK homologs where specific activities of approximately 240 U/mg have been achieved .

How can researchers address the issue of microheterogeneity in purified Squalus acanthias B-CK preparations?

Microheterogeneity in purified Squalus acanthias B-CK presents a significant challenge, particularly for structural studies where homogeneous protein preparations are essential. Based on knowledge from other B-CK studies, this microheterogeneity likely stems from various post-translational modifications, including autophosphorylation and phosphorylation by protein kinase C . To address this issue, researchers can implement several strategies:

• Analytical characterization of heterogeneity:

  • Two-dimensional gel electrophoresis to separate protein species by both isoelectric point and molecular weight

  • Mass spectrometry analysis to identify specific modifications and their sites

  • Isoelectric focusing to resolve charge variants

  • Analytical ion exchange chromatography to quantify the distribution of different protein species

• Enzymatic homogenization approaches:

  • Treatment with lambda phosphatase to remove phosphorylation heterogeneity

  • Application of site-specific proteases to remove modified terminal regions if they're not critical for activity

  • Controlled limited proteolysis to generate stable core domains lacking modified regions

• Advanced purification strategies:

  • Hydrophobic interaction chromatography to separate species based on surface hydrophobicity differences

  • Chromatofocusing to isolate proteins with specific isoelectric points

  • Preparative isoelectric focusing to isolate specific charge variants

  • Affinity chromatography using conformation-specific ligands or antibodies

• Recombinant modification strategies:

  • Site-directed mutagenesis to eliminate potential modification sites (e.g., changing phosphorylatable serine/threonine residues to alanine)

  • Expression in systems with reduced endogenous modification capabilities

  • Co-expression with specific phosphatases to prevent accumulation of phosphorylated species

These approaches can significantly reduce microheterogeneity, yielding more homogeneous preparations suitable for crystallization and high-resolution structural studies, as demonstrated for other members of the creatine kinase family where high-quality crystals have been obtained despite the known tendency for microheterogeneity .

What are the critical factors for successful crystallization of recombinant Squalus acanthias B-CK?

Successful crystallization of recombinant Squalus acanthias B-CK requires careful attention to several critical factors, drawing on experience from other B-CK crystallization studies:

• Protein quality parameters:

  • Achieve exceptional purity (>99% homogeneity), as accomplished for other B-CK crystallization attempts

  • Ensure protein homogeneity by addressing microheterogeneity issues (see FAQ 5.2)

  • Verify proper folding and activity (specific activity of approximately 240 U/mg as reported for crystallization-grade B-CK)

  • Assess monodispersity using dynamic light scattering

  • Confirm protein stability under crystallization conditions through thermal shift assays

• Crystallization strategy:

  • Implement sparse matrix screening to identify initial crystallization conditions

  • Explore crystallization in both apo form and with substrate analogs or inhibitors

  • Test both vapor diffusion (hanging and sitting drop) and microbatch methods

  • Consider crystallization at different temperatures (4°C, 16°C, and 20°C)

  • Implement seeding techniques to improve crystal quality once initial conditions are established

• Optimization approaches:

  • Fine-tune precipitant concentration, pH, and protein concentration

  • Explore the effect of additives (e.g., small molecules, divalent cations)

  • Implement gradient crystallization to establish optimal conditions

  • Consider surface entropy reduction mutants if initial crystallization attempts fail

  • Test the impact of different buffer systems on crystallization outcomes

• Crystal handling and data collection considerations:

  • Develop appropriate cryoprotection protocols to prevent ice formation

  • Test multiple cryoprotectants to minimize crystal damage

  • Implement crystal annealing if necessary to improve diffraction quality

  • Consider room temperature data collection if cryocooling proves problematic

  • Utilize microfocus beamlines at synchrotron facilities for smaller crystals

Through systematic application of these approaches, researchers should be able to obtain diffraction-quality crystals of Squalus acanthias B-CK, potentially reaching atomic resolution comparable to the 1.41 Å achieved with other B-CK structures .

How can molecular dynamics simulations enhance our understanding of Squalus acanthias B-CK function?

Molecular dynamics (MD) simulations offer powerful insights into the dynamic behavior of Squalus acanthias B-CK that cannot be captured by static crystal structures alone:

• Conformational dynamics analysis:

  • Simulate open-to-closed conformational transitions that occur during catalysis

  • Identify potential intermediate states in the catalytic cycle

  • Map energy landscapes associated with substrate binding and product release

  • Compare flexibility profiles with B-CK from other species to identify evolutionary adaptations

• Substrate binding and catalysis simulation:

  • Model interactions between B-CK and its substrates (ATP, creatine, ADP, phosphocreatine)

  • Calculate binding free energies through methods such as thermodynamic integration

  • Simulate the phosphoryl transfer reaction using QM/MM approaches

  • Identify water molecules critical for catalysis and substrate binding

• Environmental adaptation studies:

  • Simulate B-CK behavior under conditions mimicking the shark's physiological environment

  • Compare stability and dynamics at different temperatures and salt concentrations

  • Investigate the impact of pressure on enzyme behavior (relevant to sharks' depth range)

  • Model pH effects on protein dynamics and catalytic activity

• Protein-protein interaction simulations:

  • Model potential interactions between B-CK and Na+/K+-ATPase suggested by colocalization studies

  • Simulate dimerization dynamics and stability

  • Predict potential interaction surfaces for cellular partners

  • Investigate the impact of post-translational modifications on protein-protein interactions

These computational approaches would complement experimental studies by providing atomic-level insights into how Squalus acanthias B-CK functions in its native environment and how it may have adapted to the shark's specific physiological demands. The high sequence similarity with other crystallized B-CKs would facilitate accurate homology modeling as a starting point for simulations .

What potential biotechnological applications exist for recombinant Squalus acanthias B-CK?

Recombinant Squalus acanthias B-CK presents several promising biotechnological applications based on its unique properties and evolutionary adaptations:

• Biocatalytic applications:

  • Development of ATP regeneration systems for in vitro enzymatic processes

  • Creation of biosensors for creatine, creatinine, or ATP detection

  • Use as a component in enzyme cascades for biomanufacturing processes

  • Potential advantages of shark B-CK could include enhanced stability under challenging conditions compared to mammalian enzymes

• Structural biology tools:

  • Serving as a model system for studying protein evolution across vertebrate lineages

  • Use as a scaffold for protein engineering due to its well-characterized structure

  • Development of conformational antibodies for detecting specific metabolic states

  • Creation of fusion proteins for targeting energy metabolism compartments

• Biomedical research applications:

  • Investigating the role of creatine kinase in energy metabolism disorders

  • Developing inhibitors for specific creatine kinase isoforms

  • Creating tools for studying the creatine kinase energy shuttle system

  • Establishing model systems for understanding evolutionary aspects of energy metabolism

• Environmental adaptation studies:

  • Understanding molecular adaptations to marine environments

  • Investigating protein evolution in relation to environmental pressures

  • Exploring potential applications in cold-adapted enzymatic processes if shark B-CK shows adaptation to lower temperature environments

The development of these applications would build upon existing knowledge of B-CK biochemistry and the established recombinant expression systems that have achieved high yields (up to 30% of total soluble protein) and specific activities (approximately 240 U/mg) .

How might studies of Squalus acanthias B-CK contribute to our understanding of metabolic adaptations in marine organisms?

Studies of Squalus acanthias B-CK can provide significant insights into metabolic adaptations in marine organisms, particularly in relation to energy homeostasis in challenging environments:

• Osmoregulatory energy requirements:

  • The localization of B-CK in the rectal gland alongside Na+/K+-ATPase suggests specialized energetic support for osmoregulation

  • Comparative studies between shark B-CK and those from freshwater and terrestrial vertebrates could reveal adaptations specific to marine environments

  • Investigation of how energy metabolism is optimized to support the high energetic costs of maintaining osmotic balance in seawater

• Environmental stress adaptation:

  • Analysis of how B-CK structure and function may be adapted to cold temperatures of marine environments

  • Investigation of potential pressure adaptations relevant to deep-diving shark species

  • Examination of how energy buffering systems help maintain metabolic homeostasis during oxygen limitation or other environmental stressors

• Evolutionary perspectives:

  • Sharks represent ancient vertebrate lineages, providing insight into conserved mechanisms of energy metabolism

  • Comparative analysis with teleost fish, which evolved different osmoregulatory strategies, could highlight alternative solutions to similar energetic challenges

  • Understanding how fundamental energy transfer systems have been maintained or modified across 400+ million years of vertebrate evolution

• Ecological energetics:

  • Investigation of how energy metabolism supports specific behaviors and ecological niches of marine elasmobranchs

  • Understanding how energetic adaptations contribute to sharks' success as apex predators

  • Examination of tissue-specific energy buffering systems in relation to activity patterns and feeding strategies

These studies would contribute to our broader understanding of biochemical adaptation in marine environments and could inform conservation efforts by providing insight into the metabolic requirements and stress responses of shark species facing environmental changes .