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P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase that plays a crucial role in cardiomyocyte survival in response to various stresses. However, the underlying cardioprotective mechanisms of PAK1 remain unclear. Autophagy is a lysosome degradation pathway essential for cellular homeostasis. In the present study, we showed the ability of PAK1 to regulate autophagy in cardiomyocytes and identified p62 as a potential downstream mediator. PAK1 is silenced in H9c2 cardiac myoblasts by siRNA or overexpressed by adenovirus. Autophagy flux was determined by LC3-II protein levels and a fluorescent autophagy reporter with and without the lysosomal protease inhibitors Pepstatin A (pepA) and Aloxistatin (E64d). Downregulation of PAK1 reduced LC3-II levels in whole cell lysates, which was not affected by pepA and E64d, suggesting that PAK1 knockdown reduced autophagy flux. Conversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 is sufficient to promote autophagy. Interestingly, the protein expression of p62, a ubiquitin-binding autophagy adaptor, was increased by PAK1 overexpression and reduced by PAK1 knockdown. Importantly, overexpression of p62 rescued the defective autophagy flux in the absence of PAK1, suggesting that p62 may mediate PAK1-dependent autophagy. RT-qPCR assay showed that the p62 mRNA levels were unchanged by either overexpression or knockdown of PAK1 in vitro and in vivo. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX) and p62 protein levels were measured over time in both PAK1-silencing and overexpressing conditions. The results showed that p62 degradation was mitigated by PAK1 overexpression and accelerated by PAK1 knockdown. Together, these results suggest that PAK1 is sufficient and necessary to maintain autophagy in cardiomyocytes, which is likely mediated by its ability to affect p62 protein stability.

AMA Research Challenge 2024 

PAK1 regulates cardiomyocyte autophagy through p62

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Kv7 channels are potassium ion channels expressed in the nervous system. Kv7 channels function to control the excitability of neurons by reducing the rate of action potential firing. The Kv7.2 channel has been shown to play a particularly crucial role in neurodevelopment. Five different KCNQ genes encode Kv7 subunits, which form a tetrameric Kv7 channel. Loss of function mutations in the KCNQ2 gene have been shown to result in neurologic dysfunction, including neonatal epilepsy. Therefore, regulation of Kv7.2 is essential for normal neuronal function. Calmodulin (CaM) is a calcium (Ca2+)-binding protein shown to interact with the C-terminus of Kv7.2, particularly with its intracellular regulatory domain. This region contains several hotspots for pathological mutations, suggesting their importance in regulating the function of Kv7.2. The regulatory domain of KCNQ2 is composed of two regions of importance to this study: the A helix (Q2A) and B helix (Q2B) (Fig. 1 & 2). CaM binds to each Kv7 subunit by interacting with Q2A and Q2B. The amino acid sequence of Q2B contains phosphorylation sites at serines S520 and S527 which could potentially contribute to the regulation of Kv7.2 function. This study aims to characterize the Ca2+-dependent binding interactions between CaM and the regulatory domain of Kv7.2. It is also investigating whether phosphorylation of Q2B interferes with CaM interactions and how CaM modifies its binding to the regulatory domain. Binding interactions were studied by electrophoretic mobility shift assay (EMSA) and isothermal calorimetry (ITC). Results from the ITC show that CaM has a higher affinity for Q2B (Kd = 0.243 μM) than Q2A (Kd = 5.27 μM). Compared to the binding affinity between CaM and an extended Q2B sequence (Kd = 0.05 μM), the binding affinity is 42x lower when Q2B is phosphorylated at S520 (Kd = 2.1 μM) and only 5x lower when phosphorylated at S527 (Kd = 0.25 μM). EMSA shows a degree of CaM binding to Q2B phosphorylated at S527 but demonstrates no binding when phosphorylated at S520. These findings indicate that Q2B phosphorylation at S520 blocks CaM interactions in the presence of Ca2+.

Phosphorylation of Kv7.2 Potassium Channel's Regulatory Domain Alters Binding Interactions with Calmodulin

Background
An individual’s healthcare costs can provide an objective measure of overall morbidity. Quantifying the impact of genetic variation on healthcare costs can inform public health policies and help identify genetic determinants of mortality. 

Methods
We estimated inpatient, outpatient, primary care and prescription-related costs, from the perspective of healthcare systems, for a total of 1.3 million individuals across 8 different healthcare systems. The median per-patient annual healthcare cost (expressed in 2019 prices) had substantial range across countries, from €483/year in the Estonian Biobank to €458,455/year in the Mass General Brigham Biobank. 

A GWAS meta-analysis identified 27 and 106 significant, independent loci for inpatient and medication costs, respectively. Effects were overall modest, the most significant inpatient signal (rs6938239 near ILRUN) was associated with ~1% increased cost per year, per allele. Following stratified analyses, 33% of inpatient loci had sex-specific effects while 22% had an age-specific effect. E.g. homozygous carriers of variant rs62236881 in the 3’ UTR of ZNRF3 had 9% higher costs in females compared with 4% in males, potentially due to an increased risk of breast cancer.

Results
Despite differences in tariff structure and cost reimbursement across healthcare systems, we observed strong genetic correlations between inpatient studies (median rg=0.88) and 95% of significant loci did not show heterogeneity across studies. PheWAS indicated that significant loci may impact healthcare costs via either common risk factors (e.g. BMI) or chronic diseases risk (e.g. asthma, depression).

Leveraging whole-exome sequencing data from the UK Biobank (UKB), we estimated the cost for putative loss of function (pLOF) burdens for 83 clinically relevant genes. The largest effects were observed for BRCA1/2, MSH2 and APC. Annual inpatient costs were ~1.8 times higher in individuals carrying one pLOF in BRCA2 than in non-carriers. We estimated that the total annual inpatient costs attributed to BRCA2 pLOF mutations amongst those aged 40-69 in the UK would be about €26 million.

Polygenic scores (PGS) for various risk factors and diseases were linked to higher healthcare costs. In UKB, the top 10% with the highest BMI PGS had a median annual cost of €414, compared to €329 for those in the middle 40-60%. Using polygenic weights from GenCost data, PGS for ~11,000 individuals in Genomics England showed that the top 10% incurred ~€250 more than those in the middle 40-60%.

Conclusion
Genetic variation across individuals influences healthcare expenditure. Our findings are applicable across various healthcare systems and help better understanding global morbidity.

Quantifying the impact of genetic variation on healthcare cost across 1.3 million individuals from 11 studies and 8 countries: The GenCost Consortium

Background: Hirschsprung disease is a congenital neurocristopathy that affects 1 in 5000 newborns. The disease involves fetal gut aganglionosis due to the defective migration and differentiation of neural crest cells in the intestines. The condition is associated with intractable constipation, megacolon ileus and the need for colectomy in childhood. While it was previously known that gestational exposures such as maternal vitamin A deficiency, ibuprofen intake, and BP-3 sunscreen use had implications on the development of Hirschsprung, we found that these variables share effects common to the RET proto-oncogene pathway.
Methods: An initial systematic review was done on the NIH library, PubMed and Scorpus databases to evaluate existing literature on maternal gestational exposures tied to Hirschsprung disease. Search queries involved keywords “Hirschsprung disease”, “maternal exposure” and “developmental pathway”. Inclusion criteria for accepted studies were: peer reviewed status, sample size greater than twenty, human models, discussion of developmental mechanisms, and published within the past twenty years. Based on these studies, we categorized the maternal exposure variables and further reviewed the specific biologic pathways impacted. From this list of pathways, the mechanism common between the highest number of categories was selected for this report. 
Results: This study explores how maternal exposures to vitamin A deficiency, BP-3, and ibuprofen increase the risk of Hirschsprung disease by affecting the RET proto-oncogene, which is essential for neural crest cell migration and implicated in at least 20% of all Hirschsprung cases. Retinoic acid, the active form of vitamin A, upregulates RET transcription. As a result, low vitamin A levels can downregulate RET and impair neural crest cell migration. Benzophenone-3 (BP-3), a sunscreen ingredient, reduces RET levels by disrupting fetal enteric neuron development and neural crest cell migration via the SLIT2/ROBO1-miR-218-RET/PLAG1 pathway. While ibuprofen does not directly affect RET, it may affect RET signaling by increasing acetylcholinesterase (AchE) activity, as suggested by studies showing co-expression of RET and choline acetyltransferase in enteric ganglia.
Conclusion: 
Our study found that vitamin A deficiency, BP-3, and ibuprofen increase the risk of Hirschsprung disease via different pathways, with the RET proto-oncogene being the most commonly implicated gene. Vitamin A deficiency and BP-3 exposure both affect the RET signaling pathway, while ibuprofen appears to indirectly increase risk through a separate mechanism. These findings highlight the need to monitor and mitigate maternal exposures to reduce Hirschsprung disease risk and suggest the RET proto-oncogene as a potential target for treatment and prevention.



RET Proto-Oncogene Links Gestational Exposures Implicated in Hirschsprung’s Development: A Review

S. Hamza Naqvi ‡ , Alexandra Yokomizo ‡ , Jonmark Dolendo ‡ , David C. Swinney ¶,§ , Brad A. Haubrich ‡


Background: Human African trypanosomiasis (HAT, also called sleeping sickness) is
caused by the parasite Trypanosoma brucei. HAT is one of three diseases caused by
kinetoplastid protozoa (the TriTryp diseases), alongside Chagas disease and
leishmaniasis. HAT and the other TriTryp diseases are neglected, meaning endemic
populations have less resources, and there is consequently little funding or commercial interest in discovery and development of new drugs. One strategy to expedite drug discovery is to test known compounds for antiparasitic activity. Towards this goal, we have developed in vitro assays for an essential T. brucei MAP kinase and screened a small library of known human protein kinase inhibitors (PKIs).

Methods: PKIs were purchased from chemical and pharmaceutical suppliers, and
TbMAPK6 was produced by Biozilla (Dallas TX). Enzyme assays, in the presence or
absence of PKIs, were run at 37°C and stopped with heat. Reactions were monitored
with ADP-Glo assay kits (Promega, Madison WI). Enzymatic and inhibition data was
analyzed with GraphPad Prism (New York, NY), and protein alignments were conducted with UniProt.

Results: TbMAPK6 phosphorylated myelin basic protein with Michaelis-Menten kinetics, and its activity was linear with respect to both time and enzyme concentration. Kms for MBP and ATP were found to be 47 μM and 7.7 μM, respectively. Pan-kinase inhibitor staurosporine had an IC 50 of 120 nM. Other PKIs tested varied in potency by orders of magnitude. TbMAPK6 aligns with human ERK7/8 with 43.5 percent identity and to several other putative pathogenic MAP kinases at varying identities. PKIs screened included compounds that were not developed for ERK7/8. 

Conclusion:  The differential inhibition among PKIs underscores the potential for future drug discovery in the area of Neglected Tropical Diseases. Furthermore the second key finding was through our percent Identities of TbMAPK6 with MAP kinases from other parasites. This finding will allow additional potential of repurposing for other neglected diseases with higher epidemiological burdens than HAT. Next steps will be to expand our PKI library against TbMAPK6 and expand screening to other pathogenic MAP kinases.

RoadMAP for New TriTryp Therapeutics: Screening of a Trypanosomal
MAP Kinase

Background
The spaceflight environment imposes harmful stressors such as microgravity and ionizing radiation on the human body. The eye is especially susceptible to damage from these stressors due to their effects on mitochondrial function. These stressors result in mitochondrial DNA damage, oxidative stress, impaired oxidative metabolism, and overall decreased mitochondrial function. Mitochondrial DNA acquires mutations more readily than nuclear DNA and has weaker genetic repair mechanisms. The result of these insults is the development of spaceflight-associated ocular pathologies such as Light Flashes (LF), Cataracts, and Spaceflight Associated Neuro-Ocular Syndrome (SANS). Mitochondrial dysfunction is becoming more apparent as a causative factor of these conditions. Therefore, a greater understanding of the pathogenesis is necessary to develop effective countermeasures to preserve ocular health in astronauts as they embark on longer missions.

Methods
A literature review was conducted using online databases such as PubMed and Science Direct. The reference articles were analyzed to develop a narrative.

Results
Conditions that impact astronauts in spaceflight have associations with terrestrial conditions but exhibit key distinctions, pointing to mitochondrial involvement in pathogenesis. Astronauts are more likely to develop cortical cataracts, which occur in the periphery of the lens where mitochondria are abundant. The most common type of cataract on Earth is the nuclear cataract, occurring in the center of the lens where there are no mitochondria. The retina has many mitochondria, and the damage imparted by spaceflight manifests as SANS, characterized by optic disc edema, globe flattening, choroidal retinal folds, and hyperopic refractive error shifts. Recent data point to mitochondrial dysfunction in photoreceptors and supporting structures due to radiation, oxidative stress, and vitamin/metabolic deficiencies as a cause of this unique condition.

Conclusion
Spaceflight damages mitochondria within the eye, contributing to development of ocular diseases such as cataracts and SANS. Astronauts develop cortical cataracts due to the distribution of lens mitochondria and susceptibility to oxidative damage and ionizing radiation. Systemic oxidative stress also contributes to the development, thus antioxidant therapy may be a treatment. Cataracts and SANS development may be connected, as shown by the development of cotton-wool spots experienced by astronauts. Future missions to the Moon and Mars will exacerbate the negative effects of spaceflight experienced in low earth orbit and present novel stressors. Advanced medical care will not be readily accessible during long-duration missions. These demands warrant a greater understanding of the pathogenesis of ocular conditions associated with spaceflight for the development of appropriate countermeasures.


Spaceflight Stressors Impact on Mitochondrial Function and the Development of Ocular Pathology

Background
Despite a decade-long reduction in opioid prescriptions, opioid misuse remains a major health crisis causing most drug overdose fatalities. Meanwhile, gabapentin (Neurontin®) prescriptions have doubled. Gabapentin is present in over 10% of drug overdose fatalities, 90% of which involve opioids. Although reports show gabapentin is prescribed off-label for opioid withdrawal, empirical evidence is lacking regarding its efficacy in mitigating opioid withdrawal symptoms or modulating opioid dependence. It is hypothesized that gabapentin attenuates expression of opioid withdrawal.

Methods
Opioid dependence was established in male Sprague-Dawley rats administered escalating morphine doses (10-40 mg/kg) twice daily for four days. Morphine withdrawal was measured using food-maintained responding, withdrawal signs, and body weight loss in six rats trained to lever press for a sucrose pellet (fixed-ratio 5-response schedule of reinforcement). The operant session comprised five cycles: a 5-minute post-saline timeout and a 10-minute response period. Gabapentin doses (3.2-320 mg/kg) were cumulatively administered for testing and dependence was maintained with a morphine dose (40 mg/kg) after daily operant experiments.

In a separate experiment, seven groups of rats (N=8/group; Groups A - G) were observed for 14 somatic withdrawal signs, and changes in body weight. Opioid dependence was established in Groups A, B, D, and F with morphine doses (10-40 mg/kg), while Group C, E, and G received saline. Subsequently, Group A, the morphine-dependent control group, received saline injections at 7 AM and 5 PM, whereas the rest of the groups received gabapentin (32, 100, or 320 mg/kg) at 7 AM and saline at 5 PM daily. Body weight and rotarod performance were used to quantify effects following morphine discontinuation. 

Results
Following opioid dependence, response rates stabilized just below 35% of baseline, and body weight loss was 20 g greater at 7 AM (14 hours after administration of saline) than at 5PM (10 hours after administration of 40 mg/kg morphine). Morphine and gabapentin significantly increased rate of responding. Only 32 mg/kg gabapentin significantly attenuated body weight loss and withdrawal signs on days 1 and 2. Enhancement of rotarod performance and hypothermia were significantly attenuated by 320 mg/kg gabapentin. No dose of gabapentin significantly attenuated mechanical allodynia. 

Conclusion
These results fail to support the view that gabapentin attenuates opioid withdrawal in male rats. Further research is needed to clarify its role in mitigating opioid withdrawal and dependence, considering potential influences from other factors or perceived benefits.


The Effects of Gabapentin on the Expression of Morphine Withdrawal in Male Rats

Nicotinic receptors (nAChRs) have long been investigated as targets for modulation of pain and inflammation. The α7 subtype of nAChR has more recently been demonstrated to have an important role in pain and inflammation. These receptors are expressed widely in neuronal and immune cells, including myeloid lineage cells, which mediate the endogenous cholinergic anti-inflammatory pathway in cultured immune cells. Complete knock-out of α7-nAChRs in mice results in increased inflammation and hyperalgesia, while α7-nAChR gain of function mice experience attenuation of hyperalgesia and inflammation. However, the mechanism of α7-nAChR attenuation of inflammation is not fully understood. We hypothesize that α7-nAChRs expressed on myeloid lineage cells play an important role in nociception and inflammation in vivo. Conditional knock-out mice deficient in α7-nAChR expression in myeloid cells (α7 cKO) and functionally wild-type (WT) mice were generated using Cre-loxP methodology. Acute inflammation was modeled in the mice using a surgical model in which the hind paw was incised and a chemical model in which mice were injected with LPS. Chronic inflammation was modeled using CFA injected in the dorsal hind paw. Evoked thermal hypersensitivity, mechanical hypersensitivity, and paw edema were measured. Pro-inflammatory cytokine expression in the dorsal root ganglia (DRG) and spinal cord after CFA injection was measured using qRT-PCR. In the surgical and chemical acute inflammatory pain model, all mice in the treatment group experienced increased paw withdrawal latency and increased paw edema, but there was no significant difference in thermal or mechanical hyperalgesia or paw diameter between α7 cKO and WT groups. In the chronic inflammatory pain model, however, a greater degree of hyperalgesia and paw edema was seen in α7 cKO mice compared to WT mice. Additionally, expression of pro-inflammatory cytokines IL-1β and TNF-α was increased in the DRG. In the acute inflammatory pain model, the absence of a significant difference between α7 cKO and WT mice indicates that α7-nAChRs in myeloid lineage cells may not play an important role in the modulation of acute inflammation. In contrast, in the chronic inflammatory model, significantly increased hyperalgesia and paw edema of α7 cKO mice suggests that deficiency of α7-nAChRs in myeloid cells contributes to increased inflammatory response. This is further demonstrated by the elevation of pro-inflammatory cytokine expression in the DRG after CFA, indicating that α7-nAChRs in myeloid lineage cells mediate attenuation of chronic inflammation, making them useful targets for the treatment of chronic pain.

The Role of Alpha-7 Nicotinic Acetylcholine Receptors in Myeloid Lineage Cells in Acute and Chronic Inflammatory Pain

Background:
Psychedelic compounds have long been known for their ability to profoundly alter human consciousness, even at microgram doses. Psilocybin, in particular, has recently garnered significant attention due to its potential therapeutic benefits in treating many mental health disorders. Despite promising clinical results, the neural mechanisms underlying psilocybin's effects remain poorly understood. Psilocybin acts on the brain via multiple neurotransmitters, including serotonin and dopamine. On a whole-brain level, psilocybin decreases activity in the default mode network (DMN) and medial prefrontal cortex (mPFC), while increasing connectivity between the DMN, frontoparietal network, and salience network. These changes are thought to underlie the "ego dissolution" and synesthesia-like effects often reported during psilocybin-induced mystical experiences. However, a major knowledge gap remains in linking these global network effects to the initial receptor-level actions of the drug.
Methods:
To better understand how psilocybin affects the brain, we are combining molecular biology, in vivo imaging, and novel behavioral assays. First, we treated mice with either psilocybin or saline and isolated nuclei from the medial prefrontal cortex (mPFC) 24 hours later for single-nucleus RNA sequencing. This allowed us to identify distinct neuronal cell types (e.g., excitatory vs. inhibitory, different cortical layers) based on their unique gene expression profiles. Next, we performed live 1- and 2-photon microscopy in freely-moving and head-fixed mice with implanted lenses over the mPFC to visualize excitatory neural activity during and after psilocybin administration. Finally, we developed a novel behavioral assay using a high-fidelity camera to record mouse movement on a 2-dimensional linear track, enabling a detailed, AI-driven analysis of kinematic parameters.
Results:
Our molecular profiling experiment revealed that psilocybin treatment upregulated genes involved in excitatory synapse assembly. Excitatory neurons in layers V and VI were particularly affected, including a specific subtype enriched for the serotonin 2C receptor. Our 2-photon imaging data showed that psilocybin decreased mPFC activity in response to learned, positively salient cues. Ongoing analysis of recently acquired 1-photon imaging data will uncover the acute effects of psilocybin in real-time. Behaviorally, acute psilocybin administration led to decreased rearing and grooming, changes in gait and stance, and preserved overall mobility.
Conclusions:
We hope that this work will contribute to the understanding of how psychedelics affect the brain on a molecular, behavioral and neural network level both acutely and longer-term. In doing so, we may begin to elucidate how the natural function of the brain utilizes these pathways for neuroplasticity and consciousness.

Uncovering the molecular, neural circuit, and behavioral mechanisms of psilocybin action

Background : Every year there are more than 15,000 new spinal cord injuries in the United States.1 Of those, a complete spinal cord transection, characterized by a lesion through both the motor and sensory tracts, is one of the most devastating injuries to patients. During the subacute and chronic phases of injury, glial cells in vertebrae species accumulate at the lesion site to provide a protective barrier through scar formation. Although the glial scar is imperative in protecting against additional tissue loss, it blocks axon regeneration, causing additional loss of function. A powerful model that lacks this glial scar formation is the zebrafish (Danio rerio). It’s genetic commonalities to humans and larval translucency make it one of the more resourceful models when studying new pharmacological therapeutics for spinal cord injury.2 4-Aminopyridine (4-AP) is a therapeutic that is inhibitory to voltage-gated potassium channels (Kv) allowing for increasing action potentials across demyelinated axons.3 It is essential to understand 4-AP’s therapeutic effect on glial cells spinal cord injury patients in the future and the zebrafish model allows a window to discover its role in glial cell proliferation, migration, and remodeling.

Methods: A complete spinal cord injury was performed on an Tg(gfap:EGFP) x Tg(elavl3:mcherry) transgenic zebrafish line five days post fertilization. On one day post injury, continuous dosing of E2 recovery buffer supplemented with either DMSO or 4-AP was given to an equal number of transected zebrafish and uninjured zebrafish. Utilizing the Leica Thunder System, Z stack images at 20x were taken from day of injury until five days post injury to analyze pixel intensity of the glial cell bridging to ensure a complete transection and progress of bridging. Zebrafish were fed one day during the recovery period and multi-well plates were replenished with new recovery buffer each day after imaging.

Results: Continuous dosing of 4-AP one-day post injury increased glial cell proliferation at both the rostral and caudal ends of the lesion site. The 4-AP zebrafish also had narrower injury sites by the fifth day post injury. The DMSO zebrafish showed higher proliferation at the rostral lesion site.

Conclusion: In closing, it was found that continuous dosing of 4-AP one day post injury enhances glial cell bridging, promoting regeneration of the spinal cord in the zebrafish model. Varying the dosing schedules and monitoring locomotion behaviors are needed to advance the knowledge of 4-AP's effects on regeneration after spinal cord injury.

References:

(Spinal Cord Injury Prevalence In The U.S. | Reeve Foundation (christopherreeve.org)).

Effectiveness of zebrafish models in understanding human diseases—A review of models - ScienceDirect)

Neuroprotective Properties of 4-Aminopyridine - PMC (nih.gov) Neuroprotective Properties of 4-Aminopyridine - PMC (nih.gov)

Building bridges, not walls: spinal cord regeneration in zebrafish - PMC (nih.gov)


Understanding 4-AP's Capabilites to Enhance Axonal & Glial Spinal Cord Regeneration after Injury Utilizing a Zebrafish Model

As the field of genomics continues to grow, the identification of substantial genes as it relates to rare diseases remains a significant challenge. Traditionally, researchers work backwards starting from the disease to determine associated proteins. Then use expensive and time-consuming methods, such as mass spectroscopy or immunofluorescence staining, to determine genetic codes. While this method may work with single gene disorders, it is lacking when dealing with complex, polygenic diseases. To address this, we aimed to develop a novel method of discovering genes that displayed similar phenotypes when knocked out in mice using data from the International Mouse Phenotyping Consortium (IMPC), streamlining gene identification for complex genetic conditions.
We used the National Organization for Rare Diseases (NORD) to select the Map2K2 gene, associated with cardiofaciocutaneous syndrome, as our starting gene. We then used the IMPC database to identify phenotypes associated with Map2k2 knockout mice. These phenotypes were organized into an Excel spreadsheet. Next, we searched the IMPC database for genes that exhibited the same phenotypes when knocked out in mice. This information was compiled into separate Excel tabs, one for each phenotype. We then applied variations of a conditional format to visualize the frequencies of each gene, assigning specific colors based on the number of hits. 
The IMPC database revealed five phenotypes associated with Map2k2 knockout mice: increased mean corpuscular volume, decreased total body fat, decreased body weight, abnormal gait, and decreased body length. Each phenotype had varying amounts of contributing genes, the most being decreased body weight with 403 associated genes, and the least being abnormal gait with 241 associated genes. Of the 947 unique genes found, 129 genes contributed to two phenotypes, 24 contributed to three phenotypes, and four contributed to four phenotypes. Notably, the gene Slc38a10 was the only gene associated with all five Map2k2 knockout phenotypes.
While the Map2k2 gene’s involvement in the MAPK signaling pathway has been extensively studied, literature regarding interacting genes has been sparse. Our analysis identified Slc38a10 as a potential interacting gene, suggesting interplay between the pathways regulated by Map2k2 and Slc38a10. The Slc38a10 gene encodes an amino acid transporter crucial for metabolism and neurotransmission. Given the role of Map2k2 in the MAPK signaling pathway, it is plausible that these two genes have downstream interactions.
Our gene detection method proved to be inexpensive and quick, offering a valuable tool for future genetic research. We hope that novel approaches like ours will contribute to enhancing the understanding of complex genetic disorders and advancing personalized medicine.


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