Biosketch of Principal Investigator (PI): Prof. Dr. Imasory Shabi is not simply a proper noun. No, he is a verb. An active, ongoing movement that has focused his energies to push the barriers of our understanding of human torment forward into the sunny, happy future. Humble startings with a BMSc. Specialization in Molecular Genetic Ultimatums at Herverd first caught his attention and soon led to a double PhD in Genes and Genetics at MITT. In this short wake lay the first papers to implicate genetic mechanisms into human tendency to buy medium over large, putative molecular therapies to correct this stinginess, and a clear leap over the need for a postdoctoral fellowship. Instead, Shabi  landed right in an Assistant Professorship in the Department of Economic Biology at Sitford. Only as the head of a lab did he find the berth to stretch into genetic origins of the dislike for thinning hair, cargo shorts, and mobile gambling applications. We look forward to his ongoing work with Robert Dockins as to the evolutionary pressures producing these genetic selections. Supported by the Wulson and Rishton professorship for Clarity in a Complicated World, we suspect Shabi will be with us for long to come.

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Introduction

Homelessness is a problem. A recent meta-analytic report published in Nature uncovered that homelessness was “a lot” now and potentially “more” in the near future (Nature, 2022). Historically, however, the study of homelessness has been squandered from science, caught in the feelings and qualities of the social sciences, humanities, and non-academics. These interesting researchers have collectively defined homelessness as the, “lack of living in a home”. Although seemingly satisfactory to most of society, this diagnostic definition suffers from a growing problem in non-medical research, environmental determinism.

To demonstrate the fallacy of environmental determinism, I ask you humbly, do we diagnose cancer simply because someone occupies a bed in a cancer treatment centre? No, no we don’t (we diagnose them because they have cancer). Is blindness diagnosed by one’s simple presence in an optometrist’s office? Again, no (lots of people who can see will occupy the same office). Finally, can we diagnose a heart attack by a rushed moment in an emergency room? Not at all. Following this latest inane assumption, my son Andy, who sliced open his forearm after falling on our push mower one fateful April morning, would be diagnosed as suffering a heart attack. Following this latest inane assumption further, Andy would be receiving heart attack drugs unnecessarily. In summary, environmental determinism is a metaphorical road to wasted money, youth, and insight. Yet environmentally defined homelessness stands strong when the problem needs to be treated the most.

An environmentally deterministic explanation for homelessness also clashes with pre-existing data on the condition. Namely, homeless people tend to exhibit lower IQ (Smort et al., 2021), more body hair (Gilettes et al., 1987), more drug use (Blowe & Meph, 2004), less body weight (Paylayton et al., 2013), and significantly lower Cumulative Health Factor (CHF; Pfizer Pharmaceuticals, personal communication) compared to volunteer undergraduates across North American universities. All of these impacted traits are derived from within the homeless body, not attributed to some imaginary, environmental taint suffered during prolonged exposure to air outside of a house. Accordingly, I propose here to challenge a once un-challengable paradigm with my simple voice and a basic logic: homelessness is a disease, not a condition, environment, or un-fortune. Like all diseases, the issues are trapped inside the body, not outside under a bridge or behind Olive Garden. In this proposal, I outline 3 experimental approaches as vital steps to develop the disease status of homelessness, including a review of preliminary data that has been gathered already for each approach.

With a new, biological locus for research on homelessness comes a new locus for treatment of homelessness. I propose my work will pave novel routes to medical interventions that treat homelessness at its source, the body, revealing unrealistic, non-scientific dreams of affordable housing, incompatible with the beliefs and freedoms of many others, as an ever-impossible solution. I propose a future of housing that is coming without the need for a single sheet of drywall.

Experiment 1 – Establishing a vertebrate model of homelessness

The overly social, environmentally determinant explanation of homelessness referred to above relies on the problematic belief that homelessness is a uniquely human phenomenon. This assumption floats loudly unspoken among decades of research, I believe in part at least, because it has never been tested. Humans have always been the only experimental subject in the study of homelessness.

Not to borrow logic too heavily from my introduction above, but do we assume humans are the only species capable of jumping when we ignore the cricket? The toad? Maybe a deer? We don’t. We recognize the importance of comparative research, where we step away from the university and search the world over, recording the beautiful diversity of animal behaviour the Earth has born to compare to our own. From novel discoveries including the social capacities of spiders (Pruitt et al., 2004) to rhesus monkeys understanding syllabic patterning (Hauser et al., 2008), it is only by reaching far into the feral, untamed wild that we can find homologous behaviours beyond our selfish selves. For this reason, my first experimental approach is to establish a reliable vertebrate model of homelessness: the genetically amicable and convenient lab mouse.

Rationale: Mice live in homes. Whether a hole under a tree in a forest, a hole in a closet owing to ill-fitting floorboards, or the classic cage of the laboratory, mice seemingly love to be housed (perhaps following some natural tendency ingrained in their nature [personal observation]). This tendency supports the study of mice as an opportunity to study the exceptions: the mice refusing to persist in shelter. But do these exceptions exist? Let’s find out.

Methods: Our laboratory mice have been raised for generations to inhabit the cage. Simple, transparent, and reliably produced, the mouse cage presents an ideal shelter for experiments to identify mice that may eschew this very home safety. A clean, sterile testing room is fitted with 25 new, washed mice cages of the same design and dimensions, spaced evenly across the floor. Each cage (house) will be fitted with an exercise wheel, sawdust bedding, a rodent water bottle, and food pellets. Out of a fear that these amenities will confound a test of pure homelessness, we have provisioned similar resources outside of the cages, including water as “puddles” (refilled thrice daily), exercise inherently present in traversing the space between cages, and food pellets provided in taller, narrow food dishes, representative of garbage cans (a vessel consistent with our understanding of human homelessness). Once set up, 50 randomly selected, clonal lab mice will be released into the room and given a week to acclimate to the new, yet familiar, environment. To follow individual mice, their identifier (a name) is written using a permanent marker on their sides. From one week of acclimation onward, mice will be observed via wireless camera and one-way mirror by researchers to score housing status throughout each day. Mice who ultimately spend nights in a cage will be classified as “homed” and mice that spend nights in the “streets” (external to cages) will be classified as “homeless”. As we hypothesize that homeless mice are real, we outnumbered the number of mice to cages as a means of expediting the identification of each group of individuals.

Preliminary results: Using the above methodology and a monitoring period of a week of days (7 days), we successfully categorized upwards of half of our mouse population as “homeless”. Homeless mice were characterized by night behaviour avoidant of any housing/caging throughout most of the week. Interestingly, we noticed significantly higher levels of fatality in the homeless population (homeless deaths throughout the week n = 10; homed deaths throughout the week n = 0), however, elevated fatality during homelessness is completely supported by similar work on humans (Streete et al., 2004). This commonality suggests we were not only accessing a similar behaviour in mice but also that homelessness shares a physiological impact on the lives of mice as it does in humans. In conclusion, our paradigm works to effectively identify homelessness in lab mice. With a homeless sample identified, we next ask if this sample can be used to investigate the biological causes leading to these anti-homing tendencies.

Experiment 2 – Identifying genetic factors associated with mouse homelessness

Rationale: Genes play a role in all biological functions and can be considered to act as a primary mechanism in the production of, among all things, behaviour and cognition (Willson, 1989). Accordingly, we propose to ask if homeless mice identified in our preliminary work in Experiment 1 would exhibit consistent changes in gene expression from homed mice. This approach seems violently at odds with a sociological understanding of homelessness, but in fact will expand our historic understanding of housing deep within the individual, where powerful modern biotechnology aims its medical treatments. Accordingly, we look to use this approach to build a bridge to a new treatment for the homeless.

Methods: At the end of the behavioural observation week, we will sacrifice all mice by flooding the testing room with an overdose concentration of isoflurane. After sacrifice, several researchers enter the room (wearing gas-appropriate PPE) and collect a handful of mice to immediately bring to our surgical suite. Surgical procedures include researchers dissecting out three (3) perfect cubes from each brain. The first cube completely contains the parahippocampal zone, a brain region thought to be involved in homelessness. Supporting this neural function, we have recently reported that the parahippocampal zone is anatomically reduced and functionally silenced in some homeless patients as measured using fMRI approaches (Atome et al., 2022). The remaining two (2) cubes will sample the amygdala and medulla. These are both lower, conserved mammalian brain regions that could reveal neural functions in housing as ancestral vertebrate traits. Cubes are flash frozen on dry ice to preserve RNA prior to storage. Once all brains are frozen and stored at -80 degrees, they are individually dissociated into isolated cells for single-cell RNA sequencing (specific methods available in most papers). Sequencing and expression data are subsequently subjected to classical transcriptomic statistical approaches to produce PCA clustering, volcano plots, and that red to green change-in-transcription kind of grid.

Preliminary Results: We have only run single-cell RNA sequencing and subsequent transcriptomics on a small subset of homeless and homed mouse parahippocampal zones. However, in this small set we see big trends. Following PCA analysis, we found a near significant clustering of cells within the parahippocampal zone unique to homeless mice, suggesting there is a marked difference in the expression of several genes likely to be implicated in homelessness. Elaborating on this result, volcano plots revealed that the most significant (trending) changes in gene expression were found in, unbelievably, housekeeping genes, primarily broom3, windexB2, and swiffer-beta (Fig. 1).  Whereas the history of this gene group points to an ignorable ubiquity (as did misunderstood “junk DNA” beforehand), here we found them as the unique genes underexpressed in homeless mice. This was further confirmed with the red-to-green grid graph, which found these genes are often more red than green in homeless mice. Altogether, we reveal a powerful prescience supporting housekeeping genes as not only no longer ignorable but also well-named. However, despite the expense in this approach, further experimentation is needed to establish statistical significance and a causative role of these genes in homelessness.

Experiment 3: mutation of key “housekeeping” genes

Rationale: Our first two proposed experiments proffer us with the ability to isolate homeless mice (1) and uncover consistent perturbations in gene expression they exhibit within the parahippocampal zone (2). However, even amongst these great advances, science requires the generation of causal relationships, not simply the correlational ones we have observed so far. Accordingly, we finally propose to target mutations to key genes implicated in mouse homelessness in mouse embryos, tracking their housing tendencies beyond development. We hypothesize that by impairing housekeeping gene function, we can induce homelessness in even the most homey mouse.

Methods: To generate mutant mice, we propose to use the latest techniques in targeted mutagenesis centred on the highly popular and accurate cas9 exonuclease (SigmaAldritch-IDT-CRISPoh, technique in preparation). CRISPoh guides will be generated in triplets to induce nonsense/stop mutations in the three housekeeping genes exhibiting the largest decrease in expression in homeless mice identified above: broom3, windexB2, and swiffer-beta, heretofore referred to as the BWS trilogy. Mutations will be induced in one-to-two-cell embryos and confirmed later in development by genetic sequencing of tail samples. We hypothesize that triple mutant mice will overwhelmingly exhibit homeless tendencies as adults.

Preliminary Results: As the final experiment proposed, we have had the least opportunity to produce preliminary mutant results. However, we have generated twenty (20) successful triple mutants that are currently developing to adulthood, when behavioural testing will commence. Our only current finding is that twelves (12) of these triple mutants have already died within the first week of development. Although this may sound problematic to our proposal, we again will highlight that fatality is a trait common to human homelessness. Furthermore, research has consistently demonstrated that all dangers are more likely to lead to fatality in younger, weaker, developing vertebrates compared to mature ones (Matell et al., 2007). Accordingly, we believe this level of fatality (which was not found in controls, singles, or double mutants) validates our proposed approach for more thorough testing. Knowing if these genes play a critical role in homelessness will open the path to gene therapies. Gene therapies enable us to combat homelessness from a perspective too quickly swept under the rug and usher a world into the human body that has been kept outside for far too long.

Conclusion

My proposal is simple. In a world where genetics has reshaped and directed such success over the understanding and treatment of diseases, I reveal that homelessness is a disease ignored for far too long. I propose a means to study the behaviour and genetics of homelessness using a popular lab model, including the establishment of causal mechanisms producing an aversion to the home. While environmental approaches to house provisioning have failed on all fronts (BlacRoc, 2021), I suggest a biological approach holds far more promise. Home is where the parahippocampal zone is.

Equity, Diversity, and Inclusion (EDI) Statement

EDI is central to all of my work. My lab includes all types of people: undergraduates, Masters candidates, PhD candidates, postdocs, technicians, research assistants, and one secretary. I appreciate all lab members and remember to tell them how proud I am of them as often as possible. However, EDI, similar to homelessness as pointed out above, has also been applied to only humans for far too long. Here, I wish to highlight the wholly new ways I apply EDI philosophy to the other biological materials in the lab.

It all began as I watched the caps of the microcentrifuge tubes bob, firmly fit in the foam floaty cutout that resembled a frog silhouette, ever so agitating in the mysterious currents coming and going within a water bath. From the outside, sure, not the most photogenic, but the visual abstract building in my head reminded me that what counts was inside the familiar cell membrane figure I downloaded off of BioRender. Within each tube was a small cube of the parahippocampal zone excised from the brain of either a homed or homeless lab mouse, holding my secrets. The dilute trypsin in the surrounding liquid of each tube was playing the paleontologist, excavating those great unknowns by loosening layer after layer of cells to float freely from the outermost tissue surface.

I would lean in closer. I was liberating these cells, giving them freedom to be themselves. Unjudged. Equal. Diverse. Included in all analysis. No longer were they twisted and tied amongst other neurons or, god forbid, the nearby glia and vasculature. They had been overcommitted to their surroundings and undercommitted to themselves. No, this cabalistic tissue would be dissolved by weak detergents and reasonable incubation temperatures. I imagined every neuron set free in 15-20 minutes, molded by water tension into small, perfect spheres. The liberatory action of detergent did not stop here: we would help each nucleus to even sever its protuberances, axons, dendrites, cutting off these interferences to leave just the essence of life in a million crystal balls. The soma is the brain of the cell, so to speak, these neurites only simple arms, holding nothing new.

With these thoughtful processes, I embrace EDI for both my trainees and our cells to be themselves. Independence, faith, and support have led both to teach me new things: my trainees when I come in each morning to learn of their late night discoveries, and my cells when the transcriptomic graphs come back in. EDI can reach far beyond the boundaries we have given it, if only we are brave enough to let it. 

Conflict of Interest Statements

While I personally believe wishing to see your research through to its most helpful application is simply responsible, I must legally disclose that I am the founder and sitting CEO of The Home Up Top, a private biotechnology start-up corporation seeking to develop genetic therapies to combat homelessness following this research.

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Grant Review Outcome and Recommendations

The study session has met and discussed the proposal, wherein the investigator applied cutting-edge gene-editing technology to study a common disorder of homelessness, the etiology of which remains unknown. All reviewers generally agree with the overall hypothesis and are optimistic about the expected outcome and trust the expertise of Dr. Shabi’s team to complete the study. Furthermore, a few reviewers noted the study’s potential to present a paradigm shift, which sets the foundation for a revolutionary approach to understanding the pressing and unresolved issues of our time, such as hunger, crime, and illiteracy. There are, however, a few minor comments for consideration; however, these do not dampen our enthusiasm and the study session’s recommendation that the study should be funded.

*Author Responses are in the colour blue, but also labeled as “Author Response” for those unable to perceive blue (I apologize for your loss).

Reviewer 1: Homeless humans are typically anonymous. I wonder whether identifying the mice using body marker would change the outcome. A separate control should be added.

Author Response: This ethological aspect of homelessness has rightly been highlighted as a detail alluding our experimental design. However, in lieu of provisioning another control group, we propose to proceed by removing the name-based body marker on each mouse and, instead, add small outfit repositories (structurally reminiscent of dumpsters) filled with tattered clothing, bandanas, and dirty pants outside of the mouse houses (cages). A complementary closet full of new, untattered clothing and jewelry will be introduced inside each cage. This way, we aim to have all mice assign themselves visualized identities, which we suspect will lead our animal models closer to their human homologs while enabling long-term experimental tracking on an individual basis. 

Reviewer 2: The red-to-green-change-in-transcription-kind-of-grid has been challenged by the color-blind, or color-differently-perceived researchers. The field has started to shift toward using yellow-to-blue-absolute-transcription-level-histograms.

Author Response: Firstly, we would like to apologize to the visually difficultied for our ignorance. To repent for our short sight (apologies for that too), we will adopt the yellow-to-blue-absolute-transcription-level-histograms as the field has recently tended. Our red-to-green-change-in-transcription-kind-of-grid will be adapted by transforming all numbers into star ratings (from 10 stars down to “rotten”)  and relegated to supplementary material on future publications.

Reviewer 3: How do you score the anti-homing behavior? An automatic and quantitative methodology should be used to ensure total and complete objectivity of the outcome.

Author Response: This is a thoughtful question. Time in a cage is the primary differentiator between the housed and the homeless mice. We noticed after weeks of hands-on and video observation that homeless mice tend to spend much less time in a cage home. In future work, our interest seeks to subdivide this larger label of homelessness, including by tracking acts in which homeless mice enter cages to steal food, clothing, water, or jewelry (see response to Reviewer 1 above). This theft score will contribute as a sort of severity of homelessness and, we hope, be tied to other genes in the near future.

Reviewer 4: Will you do proteomics to confirm transcription profiles? This would be important as swiffer-beta is typically dominated by swiffer-alpha.

Author Response: …we’ll think about it.

Reviewer 5: Congratulations on achieving such heights as still an Assistant Professor. The founding of a company is no small task and demonstrates your willingness to see this work change the world.

Author Response: Thank you, Robert, this means a lot.