Recombinant Salmo salar Pancreatic progenitor cell differentiation and proliferation factor (ppdpf)

What molecular techniques are most effective for identifying and characterizing PPDPF expression in Salmo salar tissues?

For effective characterization of PPDPF in Atlantic salmon tissues, a multi-faceted molecular approach is recommended. Begin with RT-PCR for quantitative expression analysis, as this technique has proven reliable for detecting expression levels of similar factors in various tissues. In HCC studies, RT-PCR successfully revealed PPDPF upregulation in cancerous tissues compared to adjacent normal tissues . For protein-level detection, western blot analysis using antibodies specifically developed against salmon PPDPF or cross-reactive antibodies provides confirmation of translation and relative abundance. Immunohistochemistry should be employed for spatial localization within tissues, particularly focusing on pancreatic regions, similar to the approach used in HCC patient samples where PPDPF showed higher expression (54.07%) compared to adjacent normal tissues (24.17%) .

For functional analysis, both gain-of-function and loss-of-function approaches are valuable. Based on methodologies used in cancer studies, RNA interference using shRNAs targeting PPDPF represents an effective approach. Example target sequences that could be adapted (with appropriate modifications for salmon-specific sequences) include: 5'-CCGGTCCTGACCTGAGCGGTTACCACTCGAGTGGTAACCGCTCAGGTCAGGATTTTT-3' and 5'-CCGGGGGTTCCACTTCCAGCAACACTCGAGTGTTGCTGGAAGTGGAACCCATTTTT-3' .

How should recombinant Salmo salar PPDPF be expressed for structural and functional studies?

Based on established protocols for salmon protein expression, the Drosophila melanogaster S2 cell system presents a promising platform for expressing recombinant Salmo salar PPDPF. This system has been successfully utilized for the expression of Atlantic salmon serum C-type lectin, another cysteine-rich protein requiring proper folding . The molecular approach should involve:

• Cloning the Salmo salar PPDPF coding sequence into a vector under the control of the Drosophila metallothionein promoter

• Including a hexahistidine tag for purification purposes

• Stable transfection into Drosophila S2 cells

• Induction of expression using CdCl₂

• Collection of secreted protein from cell culture medium

For purification, a multi-step process is recommended, similar to the approach used for salmon serum lectin, which involved affinity binding followed by metal-affinity chromatography . The functionality of the purified protein should be verified through appropriate bioassays, such as cell proliferation assays or binding studies with potential interaction partners.

Alternative expression systems to consider include mammalian cell lines for improved post-translational modifications and fish cell lines for a more native environment, though each system presents trade-offs between yield, cost, and proper folding.

What are the key considerations when designing primers and antibodies for Salmo salar PPDPF research?

Designing effective primers and antibodies for Salmo salar PPDPF research requires careful consideration of several factors:

Primer design:

• Conduct comprehensive sequence alignment of PPDPF across fish species to identify conserved regions

• Design primers in exon-exon junctions to prevent genomic DNA amplification

• Validate primer specificity through in silico analysis against the Salmo salar genome

• Include appropriate controls including housekeeping genes validated in salmon tissues

• Consider potential paralogs resulting from the salmonid-specific genome duplication event

Antibody development:

• Select antigenic regions unique to Salmo salar PPDPF based on predicted protein structure

• Consider both polyclonal antibodies for sensitivity and monoclonal for specificity

• Validate antibodies through multiple techniques including western blotting, immunohistochemistry, and immunoprecipitation

• Test cross-reactivity with related proteins, particularly if the structural characteristics of PPDPF include conserved domains

• Ensure antibodies function in different experimental conditions (fixed tissues, protein extracts, etc.)

The research approach should incorporate rigorous validation steps, especially since direct characterization of PPDPF in salmon is limited, requiring careful extrapolation from studies in other species.

How can protein-protein interactions of PPDPF be identified in Salmo salar models?

To identify protein-protein interactions of PPDPF in Salmo salar, several complementary techniques should be employed:

Co-immunoprecipitation (Co-IP): Express tagged versions of salmon PPDPF (such as MYC-PPDPF) and potential interaction partners (Flag-tagged candidates) in appropriate cell systems. The methodology should follow established protocols used for other protein interaction studies, involving:

• Cell lysis in appropriate buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40 with protease and phosphatase inhibitors)

• Immunoprecipitation using antibodies coupled to beads

• Western blot analysis of precipitated complexes

Proximity labeling techniques: BioID or TurboID fusion proteins can be created by fusing the biotin ligase to PPDPF, allowing identification of proximal proteins in living cells. This approach is particularly valuable for detecting transient or weak interactions that might be missed by traditional Co-IP methods.

Yeast two-hybrid screening: Using salmon PPDPF as bait to screen a Salmo salar cDNA library can identify novel interaction partners. This system has the advantage of detecting direct binary interactions, though confirmation in more physiologically relevant systems is necessary.

Based on studies in human cancer cells, PPDPF has been shown to interact with BABAM2, suggesting that homologs of known interaction partners should be prioritized for validation in salmon models . The experimental design should include appropriate controls and validation through multiple independent techniques.

What gene editing approaches can be applied to study PPDPF function in salmon models?

For investigating PPDPF function in Salmo salar through gene editing, several approaches can be considered:

CRISPR-Cas9 system: This represents the most versatile approach for generating targeted modifications in the salmon genome. Implementation should include:

• Design of guide RNAs targeting conserved exons of the PPDPF gene

• Delivery methods including microinjection into salmon eggs or ex vivo editing of primary cells

• Screening strategies to identify successfully edited individuals

• Phenotypic analysis focusing on pancreatic development and function

RNA interference: For more rapid assessment of PPDPF function, shRNA-mediated knockdown provides an alternative to permanent genome editing. Based on cancer research methodologies, design shRNAs targeting specific regions of salmon PPDPF mRNA and deliver via plasmid-based expression systems . This approach is particularly useful for cell culture studies or short-term in vivo experiments.

Overexpression studies: Complementary to loss-of-function approaches, overexpression of wild-type or mutant PPDPF can provide insights into gain-of-function effects. This can be achieved through the generation of transgenic salmon or through transient expression systems.

Each approach has distinct advantages and limitations regarding efficiency, specificity, developmental timing, and phenotypic outcomes. A comprehensive understanding of PPDPF function would benefit from multiple complementary approaches.

How can high-throughput proteomics be leveraged to understand PPDPF networks in salmon?

High-throughput proteomics offers powerful tools for understanding PPDPF networks in Salmo salar:

Interaction network mapping:

• Immunoprecipitation coupled with mass spectrometry (IP-MS) using tagged PPDPF as bait

• Proximity labeling approaches (BioID/TurboID) to identify proteins in spatial proximity to PPDPF

• Quantitative interaction proteomics comparing wild-type vs. mutant forms of PPDPF

Global proteome changes:

• Differential protein expression analysis in PPDPF-knockdown vs. control samples

• Temporal proteomic profiling during pancreatic development or disease progression

• Analysis of subcellular fractions to determine compartment-specific interactions

Post-translational modification analysis:

• Identification of modifications on PPDPF (phosphorylation, ubiquitination, etc.)

• Changes in the phosphoproteome or ubiquitinome in response to PPDPF modulation

The experimental design should include appropriate biological and technical replicates, with careful selection of control samples. Data analysis should incorporate pathway enrichment, protein-protein interaction network analysis, and integration with transcriptomic data when available. This systems-level approach would provide comprehensive insights into PPDPF function beyond what can be achieved through targeted studies alone.

How might PPDPF function in pancreatic development in Salmo salar compared to its role in mammals?

The function of PPDPF in Salmo salar pancreatic development likely shares conserved elements with its mammalian counterparts while exhibiting fish-specific adaptations. To investigate these similarities and differences:

Developmental expression analysis:

• Perform temporal expression profiling throughout salmon development, from embryonic stages through juvenile and adult phases

• Compare expression patterns with known pancreatic development markers

• Conduct in situ hybridization to precisely localize PPDPF expression within developing pancreatic tissue

Functional studies:

• CRISPR/Cas9 knockout or knockdown models to assess effects on pancreatic development

• Analyze the impact on differentiation of endocrine versus exocrine pancreatic cells

• Assess potential compensatory mechanisms through paralogs resulting from the salmonid-specific genome duplication

This comparative approach would not only enhance our understanding of PPDPF in salmon but could also provide evolutionary insights into pancreatic development across vertebrates.

What is the potential role of PPDPF in infectious pancreatic necrosis (IPN) and pancreas disease (PD) in Atlantic salmon?

The potential role of PPDPF in pancreatic diseases of Atlantic salmon warrants investigation given the significant impact of these conditions on aquaculture:

Expression analysis during infection:

• Monitor PPDPF expression changes following experimental infection with IPNV or SAV

• Compare expression patterns in resistant versus susceptible salmon strains

• Correlate expression levels with disease progression and severity

Infectious pancreatic necrosis (IPN) causes cellular necrosis and loss of pancreatic tissue in Atlantic salmon, particularly in the post-smolt stage in seawater . Similarly, pancreas disease (PD) caused by salmonid alphavirus (SAV) induces necrosis in pancreatic tissue along with inflammation in cardiac and skeletal muscles . Given that PPDPF has shown antiapoptotic properties in human cancer cells , it might play a protective role against virus-induced cell death in salmon pancreatic tissue.

Functional investigation:

• Test whether modulation of PPDPF expression affects viral replication or cytopathic effects

• Assess if recombinant PPDPF administration can reduce tissue damage in infection models

• Determine if PPDPF is involved in pancreatic regeneration following disease-induced damage

Understanding PPDPF's role could provide new targets for intervention strategies against these economically important diseases in salmon aquaculture.

How can recombinant PPDPF be incorporated into vaccine strategies for pancreatic diseases in salmon?

The potential application of recombinant PPDPF in vaccine strategies for salmon pancreatic diseases could be approached through several avenues:

As an adjuvant or immunomodulator:
If PPDPF influences cell survival and proliferation pathways, it might serve as an immunomodulator when combined with conventional vaccines. Current vaccination strategies against pancreatic diseases include inactivated virus vaccines and DNA vaccines, with the latter showing promising reductions in PD outbreaks . Recombinant PPDPF could potentially enhance these approaches through:

• Co-administration with viral antigens to promote appropriate immune responses

• Incorporation into delivery systems targeting pancreatic tissue

• Formulation with existing vaccines to enhance duration of immunity

Within expression vectors:
Previous research has demonstrated that a salmonid alphavirus (SAV) replicon expressing IPNV polyprotein (pSAV/PP) induced modest protection against IPN in Atlantic salmon . A similar approach could be explored with PPDPF:

• Creating recombinant viral vectors expressing both viral antigens and PPDPF

• Developing DNA vaccines encoding both protective antigens and PPDPF

• Testing whether co-expression enhances protective immune responses

What structural features of PPDPF are likely conserved between humans and Salmo salar, and how do they relate to function?

Understanding the structural features of PPDPF conserved between humans and Salmo salar requires a detailed comparative analysis:

Sequence analysis:

• Perform multiple sequence alignment of PPDPF proteins across species

• Identify conserved domains, motifs, and critical residues

• Map conservation patterns onto predicted three-dimensional structures

Studies in human cancer models have shown that PPDPF promotes cell growth, colony formation, and invasion while inhibiting apoptosis through mechanisms including BABAM2 stabilization . The conserved structural elements between human and salmon PPDPF would likely mediate similar core functions, while divergent regions might reflect species-specific adaptations.

Functional domains:

• The protein interaction domains responsible for BABAM2 binding in humans should be examined for conservation in salmon

• Post-translational modification sites, including phosphorylation and ubiquitination sites

• Signal peptides or localization sequences that determine subcellular distribution

Experimental validation:

• Generate chimeric proteins combining domains from human and salmon PPDPF to test functional conservation

• Perform site-directed mutagenesis of conserved residues to assess their importance

• Use structural biology approaches (X-ray crystallography, cryo-EM) to determine three-dimensional structures

This structure-function analysis would provide fundamental insights into PPDPF biology across species and guide targeted functional studies.

How can structural biology techniques be applied to determine the three-dimensional structure of Salmo salar PPDPF?

Determining the three-dimensional structure of Salmo salar PPDPF requires a strategic approach utilizing multiple structural biology techniques:

Protein production for structural studies:

• Expression optimization in systems capable of proper folding, such as Drosophila S2 cells that have proven successful for other salmon proteins

• Scale-up to obtain sufficient quantities (milligram amounts) of pure, homogeneous protein

• Assessment of protein stability and monodispersity through techniques like dynamic light scattering

X-ray crystallography approach:

• Crystallization screening using commercial and custom conditions

• Optimization of crystallization conditions to obtain diffraction-quality crystals

• Data collection at synchrotron facilities and structure determination

• Molecular replacement using related structures if available, or experimental phasing

Nuclear magnetic resonance (NMR) spectroscopy:

• For smaller domains or the complete protein if size permits

• Isotopic labeling (¹⁵N, ¹³C) of recombinant protein in suitable expression systems

• Multi-dimensional NMR experiments to assign resonances and determine constraints

• Structure calculation and refinement

Cryo-electron microscopy:

• Particularly valuable if PPDPF forms larger complexes with interaction partners

• Sample preparation on EM grids, vitrification, and data collection

• Image processing and 3D reconstruction

Integrative approaches:

The structural information obtained would provide critical insights into PPDPF function and guide structure-based design of tools to modulate its activity.

What experimental approaches can determine how post-translational modifications affect PPDPF function in salmon?

Post-translational modifications (PTMs) often play crucial roles in regulating protein function. To determine how PTMs affect Salmo salar PPDPF:

Identification of PTM sites:

• Mass spectrometry-based proteomics to identify phosphorylation, glycosylation, ubiquitination, and other modifications

• Enrichment strategies for specific PTMs (phosphopeptide enrichment, ubiquitin remnant profiling)

• Temporal analysis of PTM changes under different physiological conditions

Site-directed mutagenesis:

• Generate point mutations at identified PTM sites (e.g., phosphomimetic mutations)

• Express wild-type and mutant proteins in appropriate cellular contexts

• Compare functional properties including:

  • Protein stability and turnover

  • Subcellular localization

  • Interaction with binding partners

  • Effects on downstream signaling

Kinase/enzyme identification:

• Determine which enzymes are responsible for specific PTMs

• Inhibitor studies to assess the functional consequences of preventing specific modifications

• In vitro enzymatic assays to confirm direct modification

PTM-specific antibodies:

• Develop antibodies recognizing specific PTM states of PPDPF

• Use these for detection of modified forms in different physiological contexts

• Apply in immunoprecipitation to isolate specifically modified subpopulations

This comprehensive approach would provide detailed insights into how PTMs regulate PPDPF function in salmon, potentially revealing mechanisms of regulation that could be targeted for intervention in disease conditions.

How might understanding PPDPF function contribute to improved disease resistance in aquaculture?

Understanding PPDPF function could contribute to improved disease resistance in salmon aquaculture through several pathways:

Disease biomarker development:

• If PPDPF expression changes precede clinical signs of pancreatic diseases, it could serve as an early biomarker

• Monitoring PPDPF levels or modifications might enable early intervention before significant losses occur

• Develop rapid detection methods suitable for field use in aquaculture settings

Genetic selection programs:

• Identify genetic variants in PPDPF or its regulatory regions associated with disease resistance

• Include these markers in selective breeding programs for improved stock resilience

• Use genotyping to identify individuals with favorable PPDPF variants

Therapeutic targets:

• If PPDPF promotes cell survival during infections, enhancing its expression or activity could reduce tissue damage

• Conversely, if certain viruses exploit PPDPF-dependent pathways, targeted inhibition might reduce viral replication

• Development of small molecules or biologics targeting PPDPF or its interaction partners

Pancreatic diseases like IPN and PD remain significant challenges in salmon aquaculture despite vaccination efforts . Current vaccines, including inactivated virus and DNA vaccines, have contributed to reducing disease occurrence, but outbreaks continue to occur frequently . Novel approaches based on understanding host factors like PPDPF could complement existing strategies to further reduce economic losses.

What methodological approaches would determine if PPDPF modulation can enhance vaccine efficacy against pancreatic diseases?

To determine if PPDPF modulation can enhance vaccine efficacy against pancreatic diseases in salmon, a systematic research approach is needed:

In vitro screening studies:

• Develop cell culture models representing salmon pancreatic tissue

• Test how PPDPF modulation affects cellular responses to viral antigens

• Measure parameters including cytokine production, antigen presentation, and cell survival

Recombinant vaccine formulations:

• Develop vaccine constructs co-expressing viral antigens and PPDPF

• Compare against conventional vaccines expressing only viral antigens

• Test in SAV replicon systems, which have shown promise for IPNV antigen delivery

Challenge trial design:

• Vaccinate salmon with conventional vaccines versus PPDPF-enhanced formulations

• Challenge with virulent IPNV or SAV strains following established protocols

• Assess protection through:

  • Survival rates

  • Viral load measurements using RT-PCR

  • Histopathological examination of pancreatic tissue

  • Immune response parameters (antibody titers, cell-mediated responses)

Mechanism investigation:

• Determine whether PPDPF enhances innate immune responses, adaptive immunity, or tissue resilience

• Assess duration of immunity with and without PPDPF inclusion

• Evaluate potential side effects, including impacts on growth performance

This research approach would provide concrete evidence regarding whether PPDPF modulation represents a viable strategy for improving vaccine efficacy against economically important pancreatic diseases in salmon aquaculture.

How can PPDPF research findings in Salmo salar be applied to other commercially important aquaculture species?

Research findings on PPDPF in Salmo salar can be translated to other commercially important aquaculture species through a strategic approach:

Comparative genomics and proteomics:

• Identify PPDPF orthologs in other aquaculture species (trout, sea bass, tilapia, etc.)

• Determine sequence conservation and predicted structural similarities

• Assess expression patterns across species to identify conserved regulatory mechanisms

Cross-species validation:

• Test whether methodologies developed for salmon PPDPF (antibodies, expression systems, functional assays) can be applied to other species

• Validate key findings from salmon models in selected representative species

• Identify species-specific adaptations versus conserved functions

Disease relevance mapping:

• Determine which pancreatic or other diseases in different aquaculture species might involve PPDPF

• Assess whether similar mechanisms operate across species boundaries

• Develop targeted intervention strategies based on disease mechanism commonalities

Technology transfer:

• Adapt recombinant protein production systems established for salmon proteins to other species

• Develop broadly applicable detection methods for PPDPF across multiple species

• Create modular vaccine platforms that can incorporate PPDPF components for different target species

The successful expression of recombinant salmon serum lectin in Drosophila S2 cells provides a technological foundation that could be adapted for PPDPF production across species. Similarly, knowledge gained about protein-protein interactions and functional domains of salmon PPDPF could guide targeted studies in other species, accelerating research progress.

What emerging technologies could accelerate PPDPF research in salmon models?

Several emerging technologies hold promise for accelerating PPDPF research in salmon models:

Single-cell technologies:

• Single-cell RNA sequencing to identify cell-specific expression patterns of PPDPF during development and disease

• Single-cell proteomics to characterize protein expression at cellular resolution

• Spatial transcriptomics to map PPDPF expression in tissue contexts while preserving spatial relationships

Advanced genome editing:

• Prime editing and base editing for precise modifications without double-strand breaks

• Inducible CRISPR systems for temporal control of gene editing

• Tissue-specific promoters for targeted editing in pancreatic cells

Organoid and ex vivo systems:

• Development of salmon pancreatic organoids to model development and disease

• Microfluidic systems for controlled exposure to pathogens and test compounds

• Ex vivo tissue culture systems for short-term experimental manipulations

In vivo imaging:

• Reporter systems for real-time visualization of PPDPF expression

• Intravital microscopy adaptations for live salmon imaging

• PET/CT imaging with labeled antibodies or ligands to track protein distribution

Machine learning applications:

• Prediction of protein-protein interactions involving PPDPF

• Analysis of complex phenotypic data from high-throughput experiments

• Integration of multi-omics datasets to identify regulatory networks

These technologies would enable more precise, efficient, and comprehensive studies of PPDPF biology in salmon, potentially leading to breakthroughs in understanding pancreatic development and disease resistance.

What are the most critical unanswered questions about PPDPF in Salmo salar that require interdisciplinary approaches?

Several critical questions about PPDPF in Salmo salar remain unanswered and require interdisciplinary approaches:

Developmental biology questions:

• How does PPDPF contribute to pancreatic development in salmon, and how does this differ from mammals?

• What signaling pathways interact with PPDPF during organogenesis?

• How is PPDPF expression regulated during the parr-smolt transformation, a critical developmental transition in salmon?

Immunology and disease resistance:

• Does PPDPF play a role in the innate immune response to viral infections like IPNV and SAV?

• How does PPDPF expression change during infection, and does this contribute to pathogenesis or protection?

• Can modulation of PPDPF enhance immune responses to vaccination?

Molecular evolution and adaptation:

• How has PPDPF function evolved in salmonids following the salmonid-specific genome duplication?

• Are there salmon-specific adaptations in PPDPF structure or function?

• How do environmental factors like temperature and salinity affect PPDPF expression and function?

Translational research questions:

• Can PPDPF be targeted to enhance disease resistance in aquaculture?

• Does PPDPF contribute to pancreatic tissue regeneration following disease-induced damage?

• How do different aquaculture conditions affect PPDPF expression and pancreatic health?

Addressing these questions requires collaboration between developmental biologists, immunologists, protein biochemists, evolutionary biologists, and aquaculture specialists. Integrated approaches combining genomics, proteomics, functional studies, and field trials would be necessary to comprehensively understand PPDPF biology in salmon.

How might systems biology approaches enhance our understanding of PPDPF networks in salmon health and disease?

Systems biology approaches offer powerful frameworks for understanding PPDPF within the broader context of salmon health and disease:

Multi-omics integration:

• Combine transcriptomics, proteomics, metabolomics, and epigenomics data to create comprehensive molecular profiles

• Track system-wide changes following PPDPF modulation

• Identify regulatory networks controlling PPDPF expression and mediating its effects

Network analysis:

• Construct protein-protein interaction networks centered on PPDPF

• Identify key hubs and bottlenecks in these networks that might represent intervention targets

• Compare network structures across health and disease states

Mathematical modeling:

• Develop quantitative models of PPDPF-involved pathways

• Simulate perturbations to predict outcomes of experimental interventions

• Refine models iteratively based on experimental validation

Phenomic approaches:

• High-dimensional phenotyping of PPDPF-modified salmon

• Correlation of molecular data with physiological parameters and disease outcomes

• Identification of biomarker signatures predictive of disease resistance or susceptibility

Comparative systems approaches:

• Apply identical analytical pipelines across multiple species to identify conserved systems

• Distinguish species-specific adaptations from core conserved networks

• Leverage insights from model organisms to guide salmon research

These systems-level approaches would place PPDPF within its broader biological context, revealing emergent properties not apparent from reductionist studies alone. This comprehensive understanding could lead to novel strategies for enhancing salmon health in aquaculture settings while advancing fundamental knowledge of pancreatic biology across vertebrate species.