Innovative Insights into Molecular and Behavioral Research Studies

Genetic Barcoding for Multiplexed Behavioral and Transcriptomic Studies

An emerging challenge in behavioral and molecular studies is the need to streamline the quantification of phenotypes in large cohorts while minimizing hands-on work and cost. Traditional behavioral assays, even when automated, often require the generation of numerous independent samples that must be processed separately. A recent innovative method—referred to as Targeted Genetically-Encoded Multiplexing (TaG-EM)—offers a promising alternative by using deterministic genetic barcoding to label populations of interest. This method enables the pooling of multiple samples prior to single-cell RNA sequencing (scRNA-seq) or behavioral assays such that a large number of experimental conditions can be tracked simultaneously.

Overview of the TaG-EM System

The TaG-EM system relies on a very clever design of a UAS-GFP construct that is modified to include a unique 14-base pair DNA barcode (also known as a “randomer”) immediately upstream of the polyadenylation site. In transgenic Drosophila, this cassette is driven by tissue- or cell-specific Gal4 lines so that the identity of the labeled cells is pre-determined by the unique barcode integrated into their mRNA. By engineering more than 20 uniquely barcoded transgenic fly lines (with over 170 additional lines later generated), researchers can pool flies together and then, following a multiplexed assay, deduce which individual flies or cell populations contributed to the overall behavioral or transcriptomic readouts.

Recent experiments have demonstrated that TaG-EM faithfully recapitulates conventional behavioral measurements—for example, in phototaxis and oviposition assays. In extended work, the system was used to quantify a labor-intensive larval gut motility assay by measuring the food transit time through the gut. Here’s a summary of key aspects of the TaG-EM method:

• Deterministic Barcoding: Instead of relying on stochastic expression as seen in Brainbow or Flybow techniques, TaG-EM uses predetermined barcode sequences. This ensures that a high number of experimental conditions can be encoded with a minimal risk of ambiguity.
• Multiplexing Advantage: By genetically “tagging” samples with distinct barcodes, multiple conditions are mixed and processed together. This allows researchers to reduce the number of separate processing runs, lowering costs and hands-on time dramatically.
• Validation and Enrichment: Although early challenges in detection and amplification existed, researchers developed barcode enrichment protocols to achieve robust detection in the scRNA-seq libraries. With optimized protocols, the reproducibility of barcode recovery has reached approximately 37% detection in combined analyses, with higher correlations when using enriched libraries.
• Cost and Labor Efficiency: A practical application demonstrated labor savings in the measurement of gut transit times with TaG-EM compared to manual scoring. Beyond efficiency, the system provides an inherent blinding of experimental conditions because the multiplexed samples are physically intermingled before sequencing.

The following table summarizes some key parameters and advantages of the TaG-EM system based on the authors’ findings:

This method represents a significant leap forward for high-throughput behavioral and molecular experiments, with potential extensions to other model organisms and tissue systems. Its ability to seamlessly integrate behavioral data with single-cell transcriptomics could ultimately lead to more effective strategies for unpacking complex biological networks and phenotypes.

Psychosocial Impact of Cutaneous Leishmaniasis Lesions and Scars in Southern Ethiopia

Neglected tropical diseases (NTDs) continue to exert a heavy burden on millions of individuals worldwide. In Ethiopia, cutaneous leishmaniasis (CL) is not only a physical health problem but also a significant psychosocial stressor. Recent qualitative research conducted in southern Ethiopia has sought to understand the lived experiences of individuals with active lesions and those burdened with scars. The study employed a descriptive phenomenological design and in-depth interviews to capture how CL affects identity, self-esteem, social interactions, and future prospects.

Key Findings from the Study on CL-Related Stigma

The study, conducted in the rural village of Ochollo, an endemic area in southern Ethiopia, revealed several interrelated themes that collectively depict the profound psychosocial impact of CL:

1. Labeling and Negative Identity Formation
   In local terminology, CL lesions are commonly referred to as “Bolbo” or “Chaja.” These labels, which have been socially constructed over time, contribute to the negative identity assigned to affected individuals. Participants recounted experiences where the mere sight of a lesion attracted derogatory comments and gossip, leading to feelings of humiliation and shame.

2. Traditional Treatments and Their Detrimental Effects
   Many patients seek traditional herbal remedies to treat CL lesions. However, these treatments can lead to pus formation and result in a strong, unpleasant odor. Such symptoms further exacerbate social stigmatization, as community members often react with disgust and ostracism.

3. Unsympathetic External Environment and Family Rejection
   The negative attitudes from peers, neighbors, and even family members profoundly affect those with CL. Affected individuals report being rejected in social settings and sometimes even within their households. This unsupportive environment contributes to feelings of isolation and depression.

4. Low Self-Esteem and Internalized Stigma
   The chronic experience of stigma leads many patients to internalize negative beliefs. Descriptions of low self-worth, persistent anxiety, and emotional distress were common, particularly among females who face additional societal pressures regarding appearance and marriage prospects.

5. Behavioral Changes and Social Withdrawal
   To avoid further stigma, many individuals adopt coping strategies such as self-isolation or reduced participation in community activities. Parents sometimes keep their children at home, and affected individuals may even attempt extreme measures such as self-harm in misguided attempts to rid themselves of the lesion.

6. Impact on Future Life Prospects
   Beyond immediate emotional distress, CL lesions and scars are seen as determinants of social fate. Participants expressed concerns about marriage prospects, job opportunities, and overall happiness, indicating that the scars alter not only their physical appearance but also shape perceptions of their future.

7. Coping Mechanisms and Adaptation
   Despite these challenges, some individuals develop strategies to cope with their condition. Acceptance and self-compassion are cited as ways to mitigate the negative impacts, though such approaches are not uniformly adopted.

The following table provides a concise overview of the psychosocial themes identified in the study:

Implications for Public Health and Policy

The findings of this research underscore the multidimensional burden of CL beyond its physical manifestations. In Ethiopia, and similar settings throughout sub-Saharan Africa, health interventions must address both the medical treatment of CL and its psychosocial repercussions. Public health strategies should include:

• Educational Campaigns: Enhancing awareness to correct misconceptions about the disease and reduce stigma.
• Integrated Health Interventions: Combining clinical care with mental health support to help patients cope with social exclusion and emotional distress.
• Community Engagement: Involving local leaders and community-based organizations to foster an environment of acceptance and support.
• Gender-Sensitive Approaches: Recognizing and addressing the disproportionate psychosocial burden on women, particularly regarding future life prospects and marriage.

The study emphasizes that a holistic approach is essential for mitigating CL’s detrimental psychosocial effects, and these insights will guide future interventions in similar high-burden regions.

Insilico Targeting of NRP1 to Block SARS-CoV-2 Entry

Since the beginning of the COVID-19 pandemic, there has been intense research focus on identifying new targets to block viral entry and reduce infectivity. While angiotensin-converting enzyme 2 (ACE2) remains the primary receptor for SARS-CoV-2, emerging evidence suggests that neuropilin-1 (NRP1) plays an important role in facilitating viral entry. In silico studies have now advanced our understanding of how small molecules can disrupt the interaction between the viral spike protein and NRP1.

The Role of NRP1 in SARS-CoV-2 Infection

NRP1 is a versatile transmembrane receptor that lacks an intracellular kinase domain. It works as a co-receptor for multiple ligands, regulating processes such as angiogenesis, cell migration, and axon guidance. Recent structural studies have revealed that following cleavage of the SARS-CoV-2 spike protein by the host protease furin, a polybasic “C-end rule” (CendR) motif is exposed. This motif, typically marked by an arginine residue at the C-terminus, binds with high affinity to the b1 domain of NRP1. The binding enhances the infectivity of SARS-CoV-2 by facilitating viral entry into host cells. Thus, blocking the NRP1-spike protein interaction presents a novel therapeutic strategy to reduce viral infection.

Virtual Screening and Molecular Dynamics Simulation

The study screened a comprehensive small molecule library of approximately 10,000 compounds derived from multiple sources—including FDA-approved drugs, bioactive compounds, natural organics, and COVID-19–related antivirals—against the b1 domain of human NRP1. The process involved several key steps:

1. Preparation of the Receptor Protein:
   The high-resolution X-ray crystallographic structure of the b1 domain of NRP1 (PDB entry: 7JJC) was prepared by removing water molecules, adding hydrogens, and energy-minimizing the structure. The binding pocket was defined to include crucial residues (such as Tyr297, Trp301, Thr316, Ser346, Thr349, Lys351, and Tyr353) known to be involved in spike protein interaction.

2. Virtual Screening and Docking:
   Using the PyRx virtual screening tool, the researchers docked all compounds into the defined binding site. A grid box was carefully positioned around the binding pocket with precise dimensions and spacing to capture optimal ligand-receptor interactions. From an initial large pool, the top 20 compounds showing high binding affinities were shortlisted.

3. Compound Property Evaluation:
   Candidate compounds underwent detailed property calculations using tools such as SwissADME and Molinspiration. Parameters like molecular weight, topological polar surface area (TPSA), water solubility (LogS), lipophilicity (LogP), blood–brain barrier permeability, and gastrointestinal absorption were critically assessed to ensure drug-likeness and favorable ADMET (absorption, distribution, metabolism, excretion, and toxicity) characteristics.

4. Molecular Dynamics Simulation:
   The top two candidates—AZD3839 and LY2090314—were subjected to 100-nanosecond molecular dynamics simulations using the GROMACS package. These simulations allowed the evaluation of binding stability and the dynamic behavior of the receptor-ligand complex over time. Furthermore, MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) calculations provided estimates of binding free energy, reinforcing the observation that both compounds maintained stable and favorable interactions with the b1 domain.

5. Comparative Docking and Protein-Protein Modelling:
   For further validation, the researchers compared the binding modes of the screening hits with known NRP1 inhibitors such as EG00229 and EG01377. Docking experiments with the spike protein receptor-binding domain were also performed to model the competitive interference by the inhibitors.

Key Findings and Implications

The insilico analysis revealed that both AZD3839 and LY2090314 exhibit strong binding to the b1 domain of NRP1, suggesting that they could effectively disrupt the NRP1–spike protein interaction. The stable binding observed in molecular dynamics simulations, combined with favorable MM/GBSA free energy estimates, underscores the potential of these compounds as drug candidates for reducing SARS-CoV-2 infectivity.

In summary, targeting NRP1 represents a promising therapeutic approach that could complement existing strategies focused on ACE2. With further experimental validation, these findings might pave the way for novel antiviral treatments that help mitigate the ongoing impact of COVID-19.

The following table summarizes the key steps and outcomes from the NRP1 targeting study:

This study’s integrative approach—combining in silico screening with molecular dynamic simulations—exemplifies current trends in computational drug discovery and underscores the potential for rapid development of therapeutic interventions in response to emerging global health threats.

Conclusion

Innovative research continues to provide novel insights and methodologies that bridge molecular precision with behavioral and psychosocial research. The TaG-EM genetic barcoding system stands as a testament to how deterministic barcoding can revolutionize the multiplexing of experimental conditions, streamlining behavioral assays and facilitating high-throughput single-cell transcriptomics. Meanwhile, qualitative studies on cutaneous leishmaniasis in Ethiopia illuminate the deep psychosocial scars—both literal and figurative—that neglected tropical diseases inflict on affected communities. Finally, state-of-the-art in silico strategies targeting NRP1 to block SARS-CoV-2 entry underscore the power of computational simulations to accelerate antiviral drug discovery.

Together, these studies highlight a multidisciplinary approach for resolving challenges in modern biomedical research. From reducing labor and cost in behavioral assays with innovative genetic tools, to addressing the human impact of chronic diseases, and finally to paving the way for next-generation antiviral therapeutics, these research efforts provide comprehensive insights that are crucial for the advancement of science and public health.

Frequently Asked Questions (FAQ)

What is the advantage of deterministic genetic barcoding over traditional fluorescent protein-based methods?
Deterministic genetic barcoding, as implemented in the TaG-EM system, offers the ability to encode hundreds of unique conditions using short DNA sequences. This greatly exceeds the limited number of distinguishable fluorescent proteins while also enabling multiplexing in both behavioral assays and transcriptomic studies. It allows researchers to pool samples and later demultiplex them based on the unique barcode sequences.

How does the TaG-EM system contribute to cost savings in high-throughput experiments?
By pooling multiple samples that have been genetically barcoded with distinct sequences, the TaG-EM system reduces the need for separate processing and manual scoring for each condition. This not only saves on reagent and sequencing costs but also reduces labor-intensive procedures, as evidenced by its success in a larval gut motility assay.

What are the main psychosocial challenges faced by individuals affected by cutaneous leishmaniasis in southern Ethiopia?
The study identified several key challenges including stigma and labeling, negative self-image, social rejection, loss of self-esteem, behavioral changes such as isolation, and long-term impacts on life prospects like marriage and employment. Traditional treatments that produce disfiguring diseases and foul odors further exacerbate these issues.

Why is neuropilin-1 (NRP1) a promising target for blocking SARS-CoV-2 infection?
NRP1 acts as a facilitator for SARS-CoV-2 entry by binding to a CendR motif exposed on the viral spike protein after furin cleavage. Targeting NRP1 can disrupt this interaction and, based on in silico simulations, compounds such as AZD3839 and LY2090314 have shown the potential to bind strongly to its b1 domain and inhibit viral entry.

How are molecular dynamics simulations used to assess potential drug candidates?
Molecular dynamics simulations provide a time-resolved analysis of the interaction between drug candidates and their target proteins. In this study, 100-nanosecond simulations were used to evaluate whether the compounds maintained stable binding to the b1 domain of NRPFree energy calculations (MM/GBSA) were then performed to quantitatively estimate the strength of binding, which is crucial for assessing the inhibitory potential.

References

1. Bibi, N. P., Khan, S., Chohan, M. S., & Kamal, M. A. (2025). Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies. eLife. https://doi.org/10.7554/eLife.88334

2. Merdekios, B., Shewangizaw, M., Ewunetu, E., van Griensven, J., van geertruyden, J.-P., Ceuterick, M., & Bastiaens, H. (2025). Unveiling the hidden burden: Exploring the psychosocial impact of cutaneous leishmaniasis lesions and scars in southern Ethiopia. PLoS ONE. https://doi.org/10.1371/journal.pone.0317576

3. Bibi, N. P., Shah, M., Chohan, M. S., & Kamal, M. A. (2025). Insilico targeting of virus entry facilitator NRP1 to block SARS-CoV-2 entry. PLoS ONE. https://doi.org/10.1371/journal.pone.0310855