You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction
============

The fibroblast growth factor (FGF) family includes at least 23 members and plays an essential role in regulating the proliferation, differentiation, migration and survival of cells originated from mesoderm and neuroectoderm. FGFs exert their biological activities *via* interactions with a family of FGF receptors (FGFR1--4) on the cell surface \[[@b1]\]. The extracellular ligand binding portion of FGFRs is composed of three immunoglobulin-like (Ig-like) domains (D1, D2 and D3) \[[@b2]--[@b5]\]. Although a highly conserved core region and considerable homology exist between FGFs, they mediate distinct functions.

Basic FGF (bFGF) is an important member of the FGF family. Because bFGF and its receptors are abundantly expressed in malignantly transformed cells and bFGF stimulates tumour angiogenesis and progression \[[@b6]--[@b8]\], antagonists targeting bFGF or its receptors have been considered a potential strategy for inhibiting endothelial cell proliferation and tumour progression \[[@b9]\]. In order to identify peptides that can block bFGF binding to its receptors, we screened a phage display heptapeptide library with bFGF. This led to the isolation of a high-affinity bFGF-binding peptide (referred to as P7) with potent inhibitory activity against bFGF-induced cell proliferation and angiogenesis.

Materials and methods
=====================

Reagents and cell lines
-----------------------

Ph.D.-7™ Phage Display Peptide Library Kit and E.coli ER2738 were purchased from New England Biolabs Inc. (Beverly, MA, USA). Recombinant human bFGF was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA). Recombinant human epidermal growth factor (EGF) was prepared in our laboratory. HRP-anti-M13 mAb was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). BALB/c-derived 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corporation, Carlsbad, CA, USA) with 10% foetal calf serum.

Biopanning of a heptapeptide phage display library with bFGF
------------------------------------------------------------

Petri dishes (35 × 10 mm) were coated with 10 μg bFGF (in 1 ml PBS) overnight at 4°C and then blocked with bovine serum albumin (BSA) at 5 mg/ml in 0.1 M NaHCO~3~ for 2 hrs at room temperature. After the plates were washed 6 times (1 min. each) with 0.05% Tween-20 in PBS (0.05% PBST), the heptapeptide phage library containing 2 × 10^11^ clones was sequentially added and shaken gently at room temperature for 2 hrs. After washing 10 times (1 min. each) with 0.05% PBST, plate-bound phage clones were eluted with Gly-HCl buffer (pH 2.2) and neutralized with 1M Tris-HCl (pH 9.1). The eluate was amplified and purified for the next round of screening. Two additional rounds of selection were performed under more stringent conditions, in which plates were coated with a reduced amount of bFGF and shorter incubation time (5 μg for 1.5 hrs and 2.5 μg for 1 hr in the 2^nd^ and 3^rd^ round, respectively), and washed with a higher concentration of PBST (0.1% and 0.3% for 2^nd^ and 3^rd^ round, respectively) for a longer period (10 × 2 min. and 10 × 3 min. for 2^nd^ and 3^rd^ round, respectively). After the 3^rd^ round of selection, the phage clones were subjected to ELISA analysis.

ELISA assay for selecting positive phages
-----------------------------------------

Microtitre plates were coated at 4°C overnight with bFGF and EGF (as controls), respectively. After the plates were blocked with blocking buffer (*i.e.* PBS with 2% dry milk; PBSM) and washed with 0.05% PBST for three times, phage clones (10^10^ pfu/well) and control phage vcsM13 were added and incubated at room temperature for 1 hr. After washing 3 times with 0.05% PBST, 200 μl of horseradish peroxidase (HRP)-anti-M13 (1:5000) was added and the plates were incubated for another hour at room temperature. The plates were washed again with 0.05% PBST and substrate (50 μl/well of 3,3′,5,5′-tetramethylbenzidine; TMB) was added. The reaction was terminated 20 min. later by adding 50 μl/well of 2M H~2~SO~4~, and the absorbance was measured at 450 and 655 nm.

DNA sequencing and peptide synthesis
------------------------------------

DNA sequences of the positive phage clones were determined performed with an automated DNA sequencer at Shanghai Sangon Company (Shanghai, China) and analysed using the BioEdit Sequence Alignment Editor software and the ProtParam programs (Ibis Biosciences, Carlsbad, CA, USA). The Ph.D.-7 Phage Display Peptide Library consists of phages bearing random heptapeptides fused to the N-terminus of the M13 phage coat protein pIII. The peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then by the wild-type pIII sequence. During panning, the N-terminus of the selected peptide sequence is free and the C-terminus is fused to the phage, having no free negatively charged carboxylate. Given that a synthetic peptide with a free C-terminus will introduce a negatively charged group at a position occupied by a neutral peptide bond, which may influence binding, a spacer sequence Gly-Gly-Gly-Ser was added to the C-terminus and the C-terminal carboxylate was amidated to block the negative charge when synthesizing the peptide corresponding to the selected sequence. The designed peptides were synthesized at SBS Genetech (Beijing, China).

Cell viability assay
--------------------

BALB/c 3T3 cells were seeded in 96-well plates (5 × 10^3^ cells/well) in DMEM containing 10% FCS and incubated for 24 hrs. The cells were washed with DMEM and cultured in DMEM with 0.4% FCS to starve the cells for 24 hrs. The medium was then replaced by DMEM containing 10 ng/ml bFGF alone, or 10 ng/ml bFGF plus serially diluted peptides. After being cultured for 48 hrs, the number of viable cells was determined by the methylthiazoletetrazolium (MTT) method as previously described \[[@b10]\].

Angiogenesis assay using chick embryo chorioallantoic membrane (CAM)
--------------------------------------------------------------------

CAM angiogenesis assay was performed as previously described \[[@b10]\]. Briefly, fifteen 5-day old chicken embryos were selected and randomly divided into three groups, which were treated with 20 μl of 10 ng/ml bFGF, 20 μl of 10 ng/ml bFGF plus 1 μM synthesized peptides, or 20 μl PBS, respectively. After incubation at 37°C for 72 hrs, the blood vessel intensity was recorded.

Results
=======

Isolation of specific bFGF-binding phage clones
-----------------------------------------------

Three cycles of biopanning were performed to select the specific bFGF-binding phages from the heptapeptide library. As shown in [Table 1](#tbl1){ref-type="table"}, phage recovery rate was greatly increased after each round of selection (from 6.50 × 10^−4^% to 4.06 × 10^−3^%). After three rounds of selection, the recovered phage clones were screened by ELISA to identify high-affinity bFGF-biding clones. Phage vcsM13 was used as the control in the ELISA assay. With the exception of its wild-type coat protein pIII, phage vcsM13 coat proteins are identical to those used to construct the heptapeptide library. Phage clones were considered to possess high affinity for bFGF if their O.D. values were two times greater than that of phage vcsM13. We also measured the ability of phage clones to bind EGF in order to determine binding specificities. As shown in [Fig. 1](#fig01){ref-type="fig"}, 11 clones with high affinity and specificity for bFGF were obtained from the recovered phage clones after three cycles of panning.

###### 

Enrichment for bFGF-binding phages

  **Round**   **bFGF (μg)**   **Input phage (pfu)**   **Output phage (pfu)**   **Recovery (%)**
  ----------- --------------- ----------------------- ------------------------ ------------------
  1           10              2.0 × 10^11^            1.3 × 10^6^              6.50 × 10^−4^
  2           5               3.0 × 10^11^            8.0 × 10^6^              2.67 × 10^−3^
  3           2.5             3.2 × 10^11^            1.3 × 10^7^              4.06 × 10^−3^

pfu, plaque forming unit.

![Specific binding of the positive phage clones to bFGF. The binding affinity of the 11 positive phage clones and the control vcsM13 to bFGF and EGF was determined by ELISA assay. Data presented are the mean O.D. values (±S.D.) of triplicate samples.](jcmm0014-0351-f1){#fig01}

Sequence analysis and property prediction of bFGF-binding phage clones
----------------------------------------------------------------------

The amino acid sequences of the peptides displayed on the positive phages were deduced from the DNA sequences and analysed using the BioEdit and ProtParam programs ([Table 2](#tbl2){ref-type="table"}). Phage clone No. 7 (P7) and P11 shared identical sequences P5 and P7 showed the highest sequence similarity to bFGF high-affinity receptors FGFR1 (IIIc) and FGFR2 (IIIc). P5 contains 3 amino acids of the authentic FGFR1 (IIIc) D3 sequence aa 312--318 (KTAGVNT) and 4 amino acids of the authentic FGFR2 (IIIc) D3 sequence aa 313--319 (KAAGVNT). P7 contains 5 amino acids of both the authentic FGFR1 (IIIc) D3 sequence aa 255--261 (PILQAGL) and the authentic FGFR2 (IIIc) D3 sequence aa 256--262 (PILQAGL).

###### 

Properties of peptides displayed by specific bFGF-binding phages

  **Heptapeptide**   **lone**   **Sequence**   **Similarity**   **Theoretical pI**   **GRAVY**
  ------------------ ---------- -------------- ---------------- -------------------- -----------
  P1                 1          RKPGKPV        0.0060901        11.17                −1.671
  P2                 2          TLHSAQA        0.0048721        6.40                 −0.114
  P3                 3          HNRPRNN        0.0060901        12                   −3.471
  P4                 4          RHTHRSH        0.0048721        12                   −2.817
  P5                 5          TAPGVST        0.0073082        5.19                 0.257
  P6                 6          NLTLAWR        0.0036541        9.75                 −0.029
  P7, P11            7, 11      PLLQATL        0.0073082        5.52                 1.057
  P8                 8          HTTHMYL        0.0060827        6.92                 −0.486
  P9                 9          WSAPVPN        0.0048721        5.52                 −0.343
  P10                10         SFRPTPP        0.0036541        9.47                 −1.143

pI, isoelectric point; GRAVY, grand average of hydropathicity.

In the physiological condition, P7 carries negative charges. Since the motif PILQAGL (pI 5.96) in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) also carries negative charges, it is likely that P7 may bind to bFGF *via* electrostatic interaction. However, because P5 carries negative charges and its homologous epitopes in D3 of FGFR1 (IIIc) (aa 312--318; pI 8.75) and FGFR2 (IIIc) (aa 313--319; pI 8.75) have positive charges, P5 cannot interact with bFGF by mimicking the electrostatic interaction of the bFGF-binding motifs of FGFRs. Moreover, P7 is hydrophobic with a grand average of hydropathicity (GRAVY) of 1.057, which is similar to that of its corresponding motifs in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) (GRAVY, 1.200). The mean hydrophobicity profile of P7 calculated using the Kyte & Doolittle scale is also similar to those of its corresponding motifs in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) ([Fig. 2](#fig02){ref-type="fig"}). P5 is also hydrophobic (GRAVY, 0.257), but its corresponding motifs in D3 of FGFR1 (IIIc) (GRAVY, −0.457) and FGFR2 (IIIc) (GRAVY, −0.100) are hydrophilic, suggesting that P5 binding to bFGF in ELISA assay was not mediated by hydrophathic interactions. Taken together, these data suggest that the candidate peptide P7 may bind bFGF *via* both electrostatic and hydrophathic interactions and therefore may have a greater potential to interrupt bFGF binding to its receptors than other identified heptapeptides do.

![Kyte and Doolittle scale mean hydrophobicity profile of the P7 peptide. The hydrophobicity profile was calculated using the Kyte & Doolittle scale. Shown are mean hydrophobicity profile of P7 (top) and the corresponding motif in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) (bottom).](jcmm0014-0351-f2){#fig02}

Heptapeptide-library-derived peptide P7 inhibits the mitogenic activity of bFGF
-------------------------------------------------------------------------------

The ability of P7 to block the biological activity of bFGF was first assessed by measuring its inhibitory effect on bFGF-stimulated proliferation of mouse fibroblast cells. As shown in [Fig. 3](#fig03){ref-type="fig"}, synthetic P7 peptides mediated strong inhibition of BALB/c 3T3 cell proliferation in a dose-dependent manner. The inhibitory effect was clearly detected in the cultures with as low as 0.0625 μM of P7 peptides, and a 50% inhibition (IC50) of cell proliferation was observed at about 1 μM.

![Inhibition of mitogenic activity of bFGF by the synthesized P7 peptides. BALB/c 3T3 cells were incubated with 10 ng/ml bFGF alone or 10 ng/ml bFGF plus P7 at the indicated concentrations, and cell proliferation was determined 48 hrs later. The inhibition rate was calculated as described in the 'Methods' section, and data are presented as the mean (±S.D.) of 3 independent experiments performed in triplicate.](jcmm0014-0351-f3){#fig03}

Heptapeptide-library-derived peptide inhibits angiogenesis *in vivo*
--------------------------------------------------------------------

We next determined the ability of the P7 peptide to inhibit angiogenesis *in vivo* in a chick embryo CAM neovascularization model \[[@b10]\]. Consistent with the known angiogenic activity of bFGF \[[@b1]\], chick embryos treated with bFGF showed significantly enhanced CAM neovasculerization compared to those treated with PBS (*P* \< 0.01; [Fig. 4](#fig04){ref-type="fig"}). However, bFGF-induced angiogenesis was completely blocked by treatment with 1-μM synthetic P7 peptides. The number of CAM blood vessels in chick embryos treated with bFGF plus P7 peptides was markedly reduced compared to those treated with bFGF alone (*P* \< 0.01) and became indistinguishable from the PBS controls.

![Synthesized P7 peptides inhibit bFGF-induced angiogenesis in chick embryo CAM. Data shown are blood vessel numbers (mean ± S.D.) in a 30-mm diameter of CAM (top; *n*= 5 per group) and representative pictures of CAM from each group (bottom).](jcmm0014-0351-f4){#fig04}

Discussion
==========

Tumour vascularization is closely related to tumour growth and metastasis. Therefore, antiangiogenetic therapy is a promising approach for cancer therapy \[[@b11], [@b12]\]. Various angiogenic factors including vascular endothelial growth factor (VEGF), bFGF, hepatocyte growth factor and placenta growth factor have been identified. Among these, VEGF and bFGF were found to be highly important in promoting tumour angiogenesis, and their expression was reported to correlate with vascularity and malignancy of astrocytomas, meningiomas, fibrous histiocytoma and pituitary tumours \[[@b13], [@b14]\]. VEGF promotes tumour angiogenesis through its potent mitogenic effect on the vascular endothelial cells. In addition to its direct activity in stimulating the proliferation of endothelial cells and tumour cells, bFGF can also synergistically enhance the mitogenic effect of VEGF. It has been reported that inhibition of bFGF-induced angiogenesis by anti-bFGF can effectively inhibit tumour cell growth *in vivo*\[[@b15]\].

The phage displaying technology has been widely used in identifying peptides with desirable biological properties. In the present study, we screened a phage-displayed heptapeptide library to identify bFGF antagonists, and obtained 11 phage clones that selectively bind bFGF. Sequence analysis showed that two of these clones (P7 and P11) shared identical sequences, but no consensus motif was identified in the rest of the clones. Alignment of the selected peptide sequences with the primary sequences of bFGF high-affinity receptors, FGFR1 (IIIc) and FGFR2 (IIIc), revealed high sequence homology between P7 and FGFRs. Five amino acids of P7 were identical to its corresponding motif in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) \[[@b2], [@b3], [@b16]\]. Moreover, P7 and the corresponding motif in D3 of FGFR1 (IIIc) and FGFR2 (IIIc) both carry negative charges and share similar hydrophobic profiles.

Given that D3 is involved in ligand biding of FGFR1 (IIIc) and FGFR2 (IIIc) \[[@b2], [@b3], [@b16]\] and both electrostatic and hydropathic complementarities are important in peptide interactions \[[@b17]--[@b22]\], our results suggest that P7 may have the capability to bind bFGF and block the biological activity of bFGF by interrupting its interactions with FGFR1 and FGFR2. Consistent with this possibility, we found that P7 peptides bind with high affinity and specificity to bFGF. Functional analysis demonstrates that synthetic P7 peptides mediate strong inhibition of bFGF-stimulated cell proliferation and angiogenesis. It has been reported that peptides binding to FGFR1 identified by screening phage-epitope libraries with anti-bFGF mAbs have the ability to block high-affinity binding of bFGF to FGFR1, resulting in inhibition of vascular endothelial cell proliferation \[[@b9]\]. Since P7 has the potential to block the interaction of bFGF with multiple FGFRs, it may mediate a stronger antiangiogenetic effect than peptides that bind a single FGFR. Taken together, our results indicate that P7 is a potent bFGF antagonist with strong antiangiogenetic effects. Further characterization of the interaction between the P7 peptide and bFGF may lead to the development of a novel approach towards suppressing tumour angiogenesis.

The work was supported by a grant from the Science Foundation of Zhejiang Province of China (No. Z205755), the Program of New Century Excellent Talents in University (Li, XK), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents (Li, XK) and the 5010 Project of Wenzhou Medical College.

[^1]: These authors contributed equally to the work.