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]


![](jove-43-2198-thumb)

Protocol
========

1. Transfection of Plasmids for Production of the Rev-dependent Lentiviral Particles
------------------------------------------------------------------------------------

Set up: The Rev-dependent GFP lentiviral vector, pNL-GFP-RRE-SA, was described previously^4,5,6^. To assemble into a viral particle, the plasmid was contransfected into HEK293 T cells with an HIV-1 packaging construct, pCMVΔ8.2 (kindly provided by Dr. Dider Trono), and a plasmid carrying the VSV-G glycoprotein (pHCMV-G). The transfection was carried out using the calcium phosphate method.

Preparation of buffers: 10 X HBS (Hepes Buffered Saline) was prepared by dissolving 5 g of Hepes, 8 g of NaCl, 0.37 g of KCl, 1 g of Dextrose, 0.103 g of Na~2~HPO~4~ (anhydrous) in 100 mL of H~2~O. Aliquot the10 X HBS buffer into 1 mL aliquots and store at -20°C. At the time of transfection, convert the 10 X HBS to 2 X HBS with H~2~O (1: 5 dilution), and adjust the pH to between 7.05 - 7.12. (for 10 mL 2 X HBS, it usually requires approximately 50 μL of 1M NaOH). Filter sterilize the 2X HBS buffer by passing through a 0.22 μM Millipore filter. CaCl~2~ buffer was made by dissolving CaCl~2~ in 10 mM Hepes to a final concentration 2M. Adjust pH to 5.8, filter sterilize the buffer and store at 4°C.

Procedure:
----------

1.  One day prior to transfection, seed 2 x 10^6^ HEK293T cells in a 100 mm Petri-dish, and grow cells overnight in 10 mL DMEM + 10% FBS at 37°C, 5% CO~2~.

2.  The next day, remove supernatant from cells and replace with 5 mL fresh DMEM + 10% FBS, continuously culture cells for 4 hours.

3.  In a 5 mL polystyrene tube, add 480 μL 2X HBS buffer.

4.  In a second 5 mL polystyrene tube, add 60 μL 2M CaCl~2~ buffer, the plasmid DNA, and transfection TE buffer (1 mM Tris, 25 mM EDTA, pH 8.0) to a total volume of 480 μL. For each Petri-dish, plasmids should be added in the ratio of 10 μg of pNL-GFP-RRE-SA, 7.5 μg of pCMVΔR8.3, and 2.5 mg of pHCMV-G.

5.  Add the plasmid DNA-CaCl~2~ mixture dropwise to the 2 X HBS tube, and incubate at room temperature for 30 minutes. A fine precipitate will form.

6.  Add the 960 μL of the mixture dropwise by pipette to the cells. Incubate at 37°C, 5% CO~2~ overnight.

7.  The next day, remove the supernatant and add 10 mL fresh DMEM + 10% FBS, and continuously incubate the cells at 37°C, 5% CO~2~ overnight.

8.  At 48 hours post transfection, harvest the virus by removing the supernatant and transferring it into 50 mL sterile tubes. Add fresh 10 mL fresh DMEM + 10% FBS into each Petri-dish and continue to culture overnight. Store the virus supernatant at 4°C.

9.  Continue to harvest at 72 hours post transfection by collecting the supernatant. Combine all the supernatants, and centrifuge the supernatants at 500 x g for 15 minutes to pellet and remove cell debris.

10. The supernatant were collected and filtered through a 0.22 μM Millipore filter. The virus was further concentrated through size-exclusion columns.

2. Concentrate Viral Particles and Determine Viral Titer
--------------------------------------------------------

1.  Viral particles were concentrated by a size-exclusion column (100,000 MW cut off, Centricoin). Viral supernatant was loaded into the column, and centrifuged at 6,000 x g at 4°C for 15 minutes.

2.  Concentrated viral supernatant was collected, aliquoted, and stored at -80°C.

3.  The viral titer was determined by infection of a TNF-treated, HIV-1 positive Jacket cell line, J1.1 7. Cells were cultured in a 96 well plate at 2.5 x 10^5^ cells per mL (100 mL total volume), and infected with 100 mL of serially diluted vNL-GFP-RRE-SA for a week. The titer (TCID50) of the particle was estimated by counting the GFP positive wells following the method of Reed and Muench 8.

3. Marking HIV-1 Positive Cells with the Lentiviral Particle, vNL-GFP-RRE-SA
----------------------------------------------------------------------------

Set up: a human CD4 T cell line, CEM-SS, was first infected with HIV-1. At 48 hours post infection, cells were superinfected with the lentiviral particles, vNL-GFP-RRE-SA. Following superinfection for another two days, GFP-positive cells were analyzed by flow cytometry. As a control, HIV-1 uninfected CEM-SS cells were identically infected with vNL-GFP-RRE-SA. Only the HIV-1-infected CEM-SS cells gave rise to GFP positive cells but not the HIV-1 uninfected CEM-SS cells.

Procedure:
----------

1.  Two hours before infection, add polybrane to a final concentration 4 mg / mL. Count cells and take 2 x 10^5^ cells for each infection. Infect cells with 200 ng (p24) of HIV-1~NL4-3~ for 2 hours.

2.  Washing cells by centrifuge at 300 x g for 10 minutes, resuspend cells into 2 x 10^5^ cells / mL, and culture at 37°C for 48 hours.

3.  Count cells and take 2 x 10^5^ cells per infection with vNL-GFP-RRE-SA. Add 100 mL of vNL-GFP-RRE-SA and incubate at 37°C overnight. Wash cells and resuspend into 2 x 10^5^ cells / mL.

4.  Perform flow cytometry analysis at 48 hours post superinfection. Infected cells were pelleted and resuspended into 500 mL 1% paraformaldehede, incubated at room temperature for 20 minutes, and then analyzed on a FACScan analyzer (Becton Dickenson).

4. Representative Results
-------------------------

If the experiments are successfully performed, a sizable GFP population will be detected in HIV-1-infected CEM-SS cells by the flow cytometer, whereas, in the control, GFP positive cells will not be detected in HIV-1 uninfected CEM-SS cells.

If the experiments are successfully performed, the Rev-dependent lentiviral vector, vNL-GFP-RRE-SA, will permit the highly specific detection of replicating HIV in living cell populations, through measurement of green fluorescence protein (GFP) expression. In this example, harvested CEM-SS cells were stained with a PE-labeled rat monoclonal antibody against mouse CD24, HSA, and then analyzed on a flow cytometer for both HSA and GFP expression.

**Figure 1.** As shown here, a sizable GFP population was detected in HIV-1-infected cells superinfected with vNL-GFP-RRE-SA (Figure 1c). In contrast, GFP positive cells were not detected in either HIV-1 uninfected CEM-SS cells without lentiviral superinfection (Figure 1a) or HIV-1 uninfected cells with lentiviral superinfection (Figure 1b).

Discussion
==========

As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel new approach to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

Disclosures
===========

No conflicts of interest declared.

The work was supported in part by Public Health Service grant 1R01AI081568 from NIAID to Y. W. and by the generous donations of the donors and riders of the 2009 NYCDC AIDS Ride organized by M. Rosen and Day2 Inc.

[^1]: Correspondence to: Yuntao Wu at <<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>>