Opioid Abuse in the COVID-19 Era

Somewhat lost in the worldwide COVID-19 health crisis is the continued destruction of lives through opioid abuse. Metaphorically speaking, it is as if the COVID-19 tsunami landed on a beach already flooded by the storm of the opioid abuse epidemic. One crisis does not mitigate the effects of the other; indeed, early data point to increasing opioid use, with already unacceptable consequences only looking to get worse.

Perhaps COVID-19’s effects on substance abuse was predictable – the pandemic has impacted people in numerous ways, many independent of actual viral infection. Social distancing requirements result in increased isolation and alienation. Economic turmoil has caused widespread unemployment (or reduced employment), leading many people to experience deep financial stress and anxiety. For those battling past abusive/addictive behaviors, the pandemic is a relapse catalyst – setting in motion a return to highly destructive actions, attitudes, and decisions. Opportunities to speak with healthcare professionals, therapists, faith-based counselors, or other support personnel are severely curtailed. These conditions facilitate the surge in opioid use and abuse being witnessed across the nation.

Opioids

What could possibly be done when two such health crises collide? We can only begin to attempt an answer here:

First, we must remove the stigma associated with opioid abuse. It should be recognized that opioid and related substance abuse/addiction represents a disease state – involving biological, environmental, and behavioral factors. It is not about moral failings, but neural networks; less about poor decision making, and more about limited perceived options. Individuals experiencing addiction deserve respect and an understanding of the toll of dependency; to marginalize them is demeaning and counterproductive.

COVID-19-related social distancing mandates lead to a reduction of diversions available to potential opioid users/abusers. Fewer people are around to witness and help prevent or treat potential overdoses.

In healthcare, there is an emergent consensus on the effectiveness of medication-assisted treatment (MAT), defined by the Substance Abuse and Mental Health Services Administration (a division of the U.S. Department of Health and Human Services) as “the use of FDA-approved medications, in combination with counseling and behavioral therapies, to provide a “whole-patient” approach to the treatment of substance use disorders.”[1] MAT is not replacing one drug with another – this would be an incomplete understanding of the therapy program. The use of buprenorphine, methadone and naltrexone, in combination with counseling and social support/behavioral interventions, have dramatically altered the landscape of opioid use disorder treatment. MAT works – and works well. The problem is the limited number of physicians trained and licensed to administer MAT. Physicians must receive a Drug Addiction Act of 2000 (DATA) waiver (also known as an “X” waiver) to prescribe the requisite drugs and deliver the appropriate behavioral therapies. Physicians may become waivered after 8 hours of didactic training; medical students require 8 hours of specialized training coupled with a clinical experience demonstrating MAT’s use with opioid use disorder patients. Only a small percentage (<10%) of practicing physicians in this country possess the waiver; of those, less than half actually deliver MAT. Some 40% of counties in the U.S. do not have a waivered physician. Tens of thousands of citizens die from opioid use disorder every year – we must increase the number of X waivered physicians and encourage more to practice the therapy.

If there is any silver lining to the COVID-19 crisis, it is the enabling of telemedicine. For those sheltering in place but requiring access to the health system, telemedicine offers a world of new possibilities. Every attempt must be made to promote digital literacy in vulnerable populations to maximize the impact of this technology.

Recognizing the devastation wrought by COVID-19’s impact on opioid abuse disorders, several local and state jurisdictions across the country are trying to help. As per recommendations made by the American Medical Association[2],[3], several changes are afoot. Buprenorphine may now be prescribed to patients by phone or telemedicine encounter. Methadone is being prescribed in amounts that will last almost a month. These lifesaving drugs are being delivered directly to patients in their homes. The process to have prescriptions refilled has also been streamlined – for example, no toxicology or other testing is required. These developments enable care without the risk of exposure to COVID-19 inherent in in-person visits. Finally, naloxone is being recognized as the true overdose wonder drug – and is being far more liberally distributed.

These are incredibly difficult times – with a global viral-based pandemic intersecting with a devastating substance abuse epidemic. However, as with all crises, good ideas, critical reasoning, and evidence-based decision making will chart a course for real change and true improvement. It cannot happen too quickly for all those affected by opioid use disorders.

 

SRT – July 2020

References:
[1] Medication-Assisted Treatment (MAT). (2020, April 30). Retrieved July 06, 2020, from https://www.samhsa.gov/medication-assisted-treatment

[2] COVID-19 policy recommendations for OUD, pain, harm reduction. (2020, July 2). Retrieved July 06, 2020, from https://www.ama-assn.org/delivering-care/public-health/covid-19-policy-recommendations-oud-pain-harm-reduction

[3] Taking action on opioid use disorder, pain &amp; harm reduction during COVID-19. (2020, July 2). Retrieved July 06, 2020, from https://www.ama-assn.org/delivering-care/opioids/taking-action-opioid-use-disorder-pain-harm-reduction-during-covid-19

Caenorhabditis elegans – the Quintessential Biological Model

The nematode Caenorhabditis elegans (C. elegans) has proven itself time and time again to be an organism of immense value to biomedical researchers. Important studies employing the biological model appear on a regular basis in top tier journals across a wide array of research areas. One perusing the scientific literature is reminded of the worm’s immense power quite regularly. Consider, for example, two related papers that appeared recently in the press; one regarding selective autophagy and lifespan[1], and the other focused on caloric restriction and how its anti-aging effects are elicited from a cellular/metabolic perspective[2].

C. elegans 3D model
The C. elegans 3D model. VirtualWorm project.

In the former, Kumsta and colleagues show that the (C. elegans) protein p62/SQST-1 (~p62) plays an important role in recognizing cellular proteins, macromolecular structures, and even intracellular organelles – e.g., mitochondria, earmarked for destruction. Such recognition leads to trafficking of the p62-substrate to intracellular degradative centers where the actual destruction takes place. Importantly, worms genetically engineered to overexpress p62 enjoy not only an efficiently operating “selective autophagy” pathway, but also a 25% increase in lifespan. Which proteins precisely constitute the repertoire of those recognized by p62 remains to be determined, but the idea that selective autophagy is an extant mechanism in cells suggests myriad potential applications in targeting for destruction those proteins or structures identified as toxic, and associated with human disease. Cellular quality control is critical – and surveillance systems including those mediated by p62 that help maintain proteostasis (i.e., integrity of cell proteome) are essential.

The article by Weir and colleagues suggests that the anti-aging effects of caloric restriction are elicited, at least in part, through maintenance of mitochondrial network integrity, and an interplay with functional (i.e., fatty acid metabolizing) peroxisomes. AMP-activated protein kinase (AMPK) acts similarly to dietary restriction, eliciting many equivalent effects – including those on longevity. These studies beg the follow up question – are anti-aging therapeutics of the future those that both assure structurally sound mitochondria whose metabolic (read: fat metabolizing) functions are carefully coordinated with peroxisomes, and activate appropriate metabolic cascades – including those involving AMPK? The data generated with C. elegans and presented in this interesting (Cell Metabolism) paper certainly supports such conclusions.


A final word or two regarding C. elegans. Dr. Sydney Brenner performed pioneering work in the 1960s and 1970s establishing the organism as a powerful model for biomedical studies. Among the work done was a description of the worm’s neuronal circuitry. For these and related studies, Dr. Brenner, and Drs. H. Robert Horvitz and John Sulston were awarded the 2002 Nobel Prize in Physiology and Medicine. C. elegans was the first multicellular eukaryote to have its genome sequenced; the developmental outcome of every one of its 959 of its cells is known; and all its neural connections are identified. The latter, known as a “connectome”, is available in no other animal at present. The worm has been used in studies involving myriad topics in cell biology, with results impacting all aspects of human health, disease, and aging.

The organism made big news when it was revealed in 2003 that nematodes brought aboard the shuttle Columbia for experimental purposes, had survived the tragic fiery crash of the spacecraft. Upon reentry into the earth’s atmosphere, the creatures were exposed to astonishingly harsh temperatures, centrifugal/gravitational forces, and atmospheric conditions; yet they returned alive. If worms could survive such conditions – could other microorganisms also do so?  Over the course of time, have microorganisms hitched rides on asteroids, comets, meteors and the like and traveled across the heavens – transferring life forms? Hmm…

SRT – January 2020

References:

[1] Kumsta C, Chang JT, Lee R, et al. The autophagy receptor p62/SQST-1 promotes proteostasis and longevity in C. elegans by inducing autophagy. Nat Commun. 2019;10(1):5648. Published 2019 Dec 11. doi:10.1038/s41467-019-13540-4

[2] Weir HJ, Yao P, Huynh FK, et al. Dietary Restriction and AMPK Increase Lifespan via Mitochondrial Network and Peroxisome Remodeling. Cell Metab. 2017;26(6):884–896.e5. doi:10.1016/j.cmet.2017.09.024

A Bioethicist’s Response to “When is a Brain Really Dead?”

Today’s post is a response by Dr. Bryan Pilkington to Dr. Stan Terlecky’s July 15, 2019 Terlecky’s Corner post entitled, “When is a Brain Really Dead?” Dr. Pilkington is an Associate Professor at Seton Hall University.

Last month’s Terlecky’s Corner post raises the question, “When is a brain really dead?” The reanimation – to borrow Terlecky’s phrase and his caution about its use – of porcine brains is both exciting and concerning. The excitement is due, as Terlecky notes well, to the possible benefits of such work: “potentially impacting several health scourges of our time including Alzheimer’s disease, Parkinson’s disease, and other age-related neurodegenerative disorders.”[1] He also notices that ethicists will be busy sorting through these and future studies, especially if the human brain gets involved.

Brain

This is how the usual dialectic goes when addressing ethical questions about emerging medical technologies. Undoubtedly, there will be lectures and papers from ethicists raising the usual kinds of questions, many titled with some version of, “We can, but should we?” These are not endeavors without merit; ethical analysis often lags behind research; in many cases, something has to be created in order to have something about which to raise questions. In fact, one of the important tasks of ethicists in this realm is to serve as watchdogs: to give caution, to aid researchers in thinking through possible concerns, and in some instances – at least with respect to IRB oversight – to say “no.”[2] However, though this is a useful and important role, it comes at a cost. Many times, those raising these kinds of concerns are termed “the ethics police”[3] and this image has hurt collaboration on research and made more challenging the necessary interprofessional tasks that medical research and healthcare practices are.

So what should be said in response to this kind of research? What are the answers to Terlecky’s list of ethical questions? The questions require longer answers than the space of a blog post admits, but I’ll take a shot at two sets of questions, suggesting a model for answering them and those remaining.

The first set comprises a list of real and immediate questions about one of the practical consequences of this research: organ donation. Will donation rates plummet? Will the organ shortage increase because there will be hold outs who think reanimation is possible? Will public trust diminish as this research moves forward?[4] Will families request that additional healthcare resources be spent on loved ones who meet the legal definition of brain death? An answer which avoids the problematic policing approach but takes seriously the importance of these questions is to engage in an extended conversation with researchers, ethicists, healthcare practitioners and administrators, and members of their communities about new technology, how it might be used, and its far-reaching implications. We must not shy away from these hard questions nor from recognizing the potential value of certain kinds of research, but we must also keep in mind the possible negative externalities that could result. This approach raises more questions. Should this research be halted if it damages public trust? Should it be stopped if fewer organs are donated? These questions lead to further questions. If the organ shortage is a primary concern, is it appropriate to connect it to this research? The conversation I am suggesting must consider that, as well. Some have argued that the sale of organs should be allowed and that this would alleviate the shortage[5], others have raised concerns about the commodification of human beings if such sale is legalized[6] – the breadth of the needed conversation is wide, as answers to questions about organ sale are connected to ethical concerns raised about porcine brain experimentation.

A second set of questions hover around the definition of death and the sources upon which we rely to answer those questions. Terlecky helpfully asks about the legal, ethical, and spiritual determinations of death. From where or in what do we root our conceptions of life, of human flourishing, and of death? Are they religious? Are they legalistic? Are they rooted in metaphysical conceptions of the person that we learned in our undergraduate philosophy courses? The kind of conversation I am suggesting is most effectively held when we bring the deep and rich traditions that inform our thought to bear on the subject matter under discussion. It is not a simple task to work through various traditions and try to understand how others think about reanimated porcine brains, human brains, and death, but that is what is needed. As physician and ethicist Lauris Kaldjian recently asked of his colleagues,[7] did you take a course about the existential questions in medical school? Though rhetorical, the suggestion is powerful. How should we respond to this research? In the same way we should respond to all research that raises significant ethical questions, by practically reasoning together.

 

References:

[1] Terlecky, S. 2019, July 15. “When is a Brain Really Dead?

[2] ​Evans, J. 2012. The History and Future of Bioethics: A Sociological Account. Oxford, United​ ​Kingdom: Oxford University Press.

[3] Klitzman, R. 2015. The Ethics Police? The Struggle to Make Human Research Safe. Oxford: Oxford University Press

[4] ​Moschella, M. 2018. Brain death and organ donation: A crisis of public trust. Christian Bioethics 24(2):133–50.

[5] ​Cherry, M. ​2005​. ​K​​idney for Sale by Owner: Human Organs, Transplantation, and the Market. Georgetown University Press.

[6] Pilkington, B. 2018. A Market in Human Flesh: Ramsey’s Argument on Organ Sale, 50 years later. Christian Bioethics 24(2):133–50.

[7] Kaldjian, L. (Personal communication during lecture in Grand Rapids, Michigan, March 25, 2019).

When is a Brain Really Dead?

When is a brain really dead? The answer to this question was made far more complicated by the recent work of Dr. Nenad Sestan and colleagues at Yale University. Their astonishing paper entitled “Restoration of brain circulation and cellular functions post-mortem,” appeared in the journal Nature[1] this April. In it, the authors were able to demonstrate that brains taken from slaughtered animals (pigs in this case), could be “reanimated” 4 hours later in the laboratory – and made at least partially functional for some 6 hours thereafter. I use the word reanimated with some trepidation – it implies the brains were dead and somehow brought back to life. That is not quite the story – rather, the organ turns out to be far more resilient than we previously realized and the research team simply identified a way to tap into that inherent resiliency.

A brief description of the research study: 32 brains from slaughtered pigs were delivered to the research team on ice. Within 4 hours, the scientists carefully perfused the brains using a proprietary surgical procedure, pumping apparatus, and oxygen and nutrient-rich solution collectively termed BrainEx. The team then analyzed the brains for specific cellular, metabolic, and electrical activities. What they found was incredible. The BrainEx perfusion system restored a number of brain functions, including glucose and O2 utilization and concomitant CO2 production (indicative of metabolic function), induced inflammatory responses (suggesting an active immune system), active microcirculation (evidence of structural integrity), and electrical activity (with neuronal firing).

Figure of porcine brain connected to perfusion system
Connection of the porcine brain to the perfusion system via arterial lines. The pulse generator (PG) transforms continuous flow to pulsatile perfusion. Source: Figure 1B, Nature 568, 336–343 (2019).

With respect to the last point, it should be noted the investigators were well aware of the ethical concern that full restoration of brain function could potentially lead to a state of “consciousness.” What it would mean for a disembodied brain to attempt to operate without peripheral sensory input, and what the organ might remember, was simply too much to consider; the brains were treated pharmacologically to assure no coordinated higher level cognitive activity was possible. Said another way, the renewed brains could not begin to think.

Overall, the results suggested to a first approximation, functional activity of the otherwise dead brain had been restored. Importantly from an experimental standpoint, control perfusates were without effect – the brains degenerated much like untreated specimens.

Against the backdrop of these remarkable results are the inevitable ethical questions that follow. Returning to the one posed above – when is a brain really dead? If our understanding of brain death requires a deeper examination, how then do we determine legally, ethically, and/or spiritually, when a person truly dies? What are the implications of this work on the issue of organ donations? Is a brain death as currently defined sufficient to permit the harvesting of a person’s organs? Will (and should) donations ebb as people begin to question the legitimacy of declarations of death. Also, could future brain reanimation experiments include restoration of conscious thought? Certainly the ability to control the brain in this manner permits the testing of drugs in new ways – potentially impacting several health scourges of our time including Alzheimer’s disease, Parkinson’s disease, and other age-related neurodegenerative disorders.

Naturally, science requires replication, expansion, and more careful delineation of what is, and is not possible with the technology described. But what enormous doors have been opened for neuroscientists, neurologists, neuropharmacologists, cognitive scientists, and the many others interested in the structure and function of the brain. I suspect ethicists will also be very busy sorting through these and the inevitable follow-up studies – especially as applications involving the human brain are contemplated.

SRT – July 2019

[1] Vrselja Z, et al., Nature 568, 336-343 (2019). PMID: 30996318

Cardiac Exosomes to the Rescue

In an extremely exciting study that appeared last month in the journal Nature Communications[1], scientists from the University of Alabama at Birmingham, and Huazhong University of Science and Technology in Wuhan, China, showed that after myocardial infarction (~heart attack), heart tissue release tiny membrane-bound vesicles called exosomes, specifically loaded with genetic material designed to promote cardiac tissue repair. Such restoration is mediated by bone marrow progenitor cells that have been released from their sequestered bone-specific existence by the information carried in the newly arrived exosomes.

Several hundred thousand Americans suffer heart attacks each year, making it critical for the medical and scientific communities to understand this potentially transformative study. To begin our analysis, let’s begin with exosomes – tiny membrane-enclosed vesicles released from many cell types. Thought to mediate communication/cell-cell signaling activities, exosomes leave cells loaded with proteins, lipids, DNA, mRNA, and microRNAs among other potentially bioactive molecules. Exosomes are synthesized within the endosome-lysosome system of cells – emerging cargo loaded and poised to deliver their contents locally, or at a distance. It is perhaps not surprising that several pharmaceutical companies – recognizing the potential drug delivery platform that exosomes represent, are pursuing the tiny vesicles in several biomedical contexts.

Once released, how the nascent exosomes hone to specific target tissues and cells is unclear. Perhaps the proteins assembled into their membranes – distinct to each type of exosome – play a role in this discrimination. Also not well understood is the nature of the exosome-target cell interaction. Is it a receptor-ligand-like binding event that triggers signaling events and results in fusion and integration of exosomal contents? Or is it a more basic fusion of two membranes held in close apposition? What about a straightforward endocytic process whereby the exosome is taken up and released within the newly formed endosome?

Back to the study at hand – the investigative team identified the exosomes released from damaged heart muscle as containing specific microRNAs (called myocardial microRNAs or myo-miRs). Recall microRNAs are short, non-coding RNAs with the capacity to (negatively) regulate gene expression. The myo-miRs under consideration here do just that – they very effectively block activity of the chemokine receptor, CXCR4 in bone marrow progenitor cells. Such inhibition allows such stem-like cells to escape their seclusion, enter the bloodstream, and travel to the injured heart…“exosomes to the rescue.” There, repair processes are initiated.

Knowing this cell biology, there must be ways to better harness exosomes to improve cardiac repair. Investigators are sure to pursue this quickly. Almost as assuredly, scientists will also be looking for other examples of exosomes and their bioactive contents as biomarkers for pathology, and as potential rescue mechanisms for disease’s damaging wrath.

SRT – April 2019

[1]Cheng M, et al., Nat Commun. (2019) doi: 10.1038/s41467-019-08895-

Unexpected Role for Mitochondria in Bacterial Killing

Just when we thought we understood the cell biology of antimicrobial resistance in macrophages, a study like the one detailed by Abuaita and colleagues in their Cell Host & Microbe article comes along.[1] Not only are the molecular mechanisms of pathogen metabolism updated, but new cellular signaling and trafficking pathways are implicated. There is a lot of new and important science included in this exciting paper.

Let’s begin by remembering that macrophages are immune cells whose role is to internalize and degrade invading microorganisms. They do so by endocytosing the pathogenic microbes and sequestering them in a newly developed phagosome. It is there that the destructive processes are initiated; final elimination of residual components occurs in the lysosome – a related degradative organelle that works in close concert with the nascent phagosome.

Graphical abstract of Abuaita et al, 2018.

What Abuaita and co-authors describe is a process by which certain bacteria, including the potentially dangerous methicillin-resistant Staphylococcus aureus, trigger more than simply a phagocytic event. Rather, it is clear that the phagosome somehow recognizes the microorganism within its midst, and launches a far more lethal and complex biochemical reaction. The cascade begins with the infected macrophage mounting a stress response – one initiated by the phagosome but further elaborated by the endoplasmic reticulum. Specifically, the endoplasmic reticulum turns on one of the most elegantly choreographed and well-described quality-control systems in the cell, called the unfolded protein response, or UPR. The critical sensor that needs to be tripped to turn on the pathway is the endoplasmic reticulum membrane protein, IRE1a. It is at this point that the cell biology really gets exciting.

The response is not confined to the endoplasmic reticulum and well understood downstream nuclear effector pathways; rather, the signal is somehow transmitted to mitochondria. In response, the organelle is prompted to produce reactive oxygen species – hydrogen peroxide in particular. Mitochondria are known to produce reactive oxygen species, including hydrogen peroxide, as part of their normal energy-generating metabolism. What is different here is that a defense mechanism initiated in the phagosome, signals the endoplasmic reticulum, which then communicates to the mitochondria; a truly elegant and previously unrecognized relay system.

As interesting as the new interorganellar signaling events are – there is more. Specifically, the fascinatingly novel trafficking pathways identified.

The newly synthesized mitochondrial hydrogen peroxide is packaged in vesicles which are shed from the organelle. The authors even identify a critical component of this release step – the ubiquitin ligase, Parkin. These newly created, hydrogen peroxide-loaded mitochondrial vesicles then migrate through the cell, destined to fuse with pathogen-infected phagosomes – releasing their toxic contents and supplementing the already initiated bacterial killing process. Just in case the newly delivered hydrogen peroxide does not provide a sufficient amount of toxic reactive oxygen, some mitochondrial vesicles actually encapsulate the hydrogen peroxide-synthesizing enzyme, superoxide dismutase-2.

What we have here is the cell marshalling its degradative armamentarium in a manner we never imagined to help fight methicillin-resistant Staphylococcus aureus and other invading pathogens. It seems clear that this is only the tip of the iceberg – interorganelle communication/signaling networks and elaborately coordinated effector apparatuses in cells are certain to exist in ways we can only begin to imagine. In this case, the goal is to fight infection; in others, it may very well be to thwart the effects of aging, to counter disease, or neutralize the effects of mutations, toxins, or other environmental insults. The more we know about cell biology, the more we realize how much we do not know; it is amazing how the field continues to captivate our attention.

SRT – February 2019

[1]B.H. Abuaita et al., Cell Host and Microbe (2018) doi: 10.1016/j.chom.2018.10.005. PMID: 30449314 (Request article via ILL).

The Value of Negative Results

Every investigator hopes the results they obtain support the hypothesis they put forth. However, more often than not, this does not happen – the data acquired do not conform to what was expected. What to do then with the “negative” results?

To be clear, the results we are talking about were obtained through carefully considered, well executed, and appropriately controlled (read: presence of positive and negative controls) experiments. They simply do not extend that which was predicted; in some cases, they may even call into question the underlying – often already published – results that led to the hypothesis guiding the study.

Of course, it may be that the work that was to be extended was never solid in the first place. That is, it is possible that the prevailing view in a field is based on incorrect and/or irreproducible results. Indeed, a number of studies are showing an alarmingly large percentage of high-profile published results are not reproducible [1],[2]. In the field of cancer – the pharmaceutical giants Amgen and Bayer Healthcare were unable to replicate the findings included in a large number of studies published in elite journals. The implications are profound; how can companies which rely on such research published in the scientific literature to define molecular targets and develop therapeutic drugs, do so against a backdrop of irreproducibility?

Source: Baker M. 1,500 scientists lift the lid on reproducibility. Nature; 2016.

Why is this happening? Explanations offered include – among others, pressures to publish, financial considerations, poor statistical analyses, insufficiently detailed protocols/technical complexity, selective reporting, and inadequate reagent authentication. The National Institutes of Health is aware of these problems and is calling on investigators seeking support to include in their proposals detailed descriptions of i. the scientific premise of the proposal; ii. experimental design specifications; iii. how biological variability will be considered; and iv. how biological and chemical reagents will be authenticated[3]. This is in addition to newly required statements regarding evidence that a detailed plan for data analysis is in place.

Although the (negative) results obtained do not confirm or extend other studies, the point here is that they are still very much of value. Other scientists in the field would welcome knowing what was done, and what was observed. The obvious benefit is that it will prevent others from wasting time, energy, and resources on approaches that are not fruitful, and would help focus the field by better defining what are, and what are not reliable results/models.

Despite the inherent value of such results, there is the perception of publication bias – the belief that only positive data is worthy of publication. Non-confirmatory or negative results are often not disseminated, at great cost to the scientific community.

One approach to assuring sufficient promulgation of the conclusions of a study is for appropriate journals to agree, in advance, to publish the results regardless of whether they confirm or refute the underlying hypotheses that initiated the study. This would provide a mechanism for a field to enjoy a wealth of otherwise unreported information about what works, and what does not – in particular investigators’ laboratories. Many clinical trials operate this way, with final results disseminated regardless of the effects seen on patients. A second approach would be to develop journals that will consider negative, confirmatory, and non-confirmatory results, data notes, and virtually any valid scientific or technical finding in a particular field. F1000Research is a journal that adheres to just such guidelines. The idea that all results are embraced is extremely attractive; after all, science is, at its core, simply a search for the truth.

SRT – November 2018

[1] M. Baker, Nature (2016) doi: 10.1038/533452a. PMID: 27225100 (article)
[2] C.G. Begley and L.M. Ellis, Nature (2012) doi: 10.1038/483531a. PMID: 22460880 (article)
[3] National Institutes of Health – New Grant Guidelines; what you need to know. https://grants.nih.gov/reproducibility/documents/grant-guideline.pdf

Senolytic Strategies to Combat Aging

Aging is a multifactorial process – with “hallmarks” [1] including genomic instability and altered gene expression, epigenetic effects, telomere erosion, stem cell fatigue, proteostatic errors, senescence, compromised mitochondrial, lysosomal, and peroxisomal function, and disrupted communication networks, among others. Senescence, in particular, is critical as it is thought to constitute a major decision point for cells. With advancing age, cells accumulate sufficient damage and are stressed to the point that they must make a crucial decision – either transform and become cancerous, or pass into senescence.

The anti-tumor senescent state is one of continued metabolic activity, but no cell division. Unfortunately, accompanying the senescent phenotype is cellular release of assorted chemokines, growth factors, interleukins, and proteases. The ultimate effect of this collection of pro-inflammatory mediators is to corrupt the regional cell/tissue milieu. Ironically, this newly created environment is very conducive to cellular transformation and tumor development.  Indeed, investigators in the field refer to the senescence-associated secretory phenotype as “the dark side of tumor suppression”[2].

What if the decision to pass into senescence could be maintained (and thus avoid cancerous transformation), but the senescent cell could somehow be destroyed? Would cancer be averted and cell/tissue integrity maintained?  Could stem cell exhaustion be ameliorated and health span increased?  Could aging, the ultimate risk factor for human disease, be brought under control – at least to some extent? While clear answers to these questions remain elusive, the idea of trying to destroy senescent cells – either through genetic means or specific drugs (called “senolytics”), is a major focus of researchers in the field of cellular aging.

Aged hand with pill
Do senolytics hold the key to healthy aging?

Dr. Van Deursen’s group at the Mayo Clinic College of Medicine developed a brilliant strategy of induced senescent cell suicide. In mouse models, they created a genetic background whereby cells would die the moment they expressed the senescence biomarker p16Ink4A. They showed both in progeroid[3] (i.e., prematurely aged) and wild-type[4] backgrounds, that targeted elimination of senescent cells improved the health of the animals by delaying, or preventing, impaired tissue function. Furthermore, in the wild-type background, the mice lived some 30% longer!

Instead of genetic approaches, investigators from Dr. Robbins’ group at the Scripps Research Institute screened for drugs that would selectively target senescent cells – while not affecting other cells. Interestingly, inhibitors of HSP90 were identified [5] as potent senolytics – with efficacy in both in vitro (i.e., cellular) and in vivo (i.e., whole animal) models. HSP90s are critical cellular chaperones, which facilitate protein folding and stabilize hundreds of molecules involved in a myriad of biochemical and metabolic pathways. The paper’s authors speculate that inhibiting HSP90’s anti-apoptotic (i.e., cell death)/pro-survival activities may be playing some role in selectively targeting senescent cells – a view consistent with the activity of other demonstrated senolytics[6] that are thought to reduce resistance to apoptosis.

How to partner senolytics with other drugs to maximize efficient elimination of senescent cells while limiting toxicity is an important focus going forward. Although the science is not quite there yet – the notion of clinical trials employing senoltyics or other modulators of cell senescence with the goal of thwarting aging’s effects and improving health span are not that far off.

 

SRT – September 2018

[1]C. Lopez-Otin et al., Cell. (2013) doi: 10.1016/j.cell.2013.05.039. (PMID: 23746838)
[2] J-P. Coppe et al., Ann Rev Pathol. (2010) doi: 10.1146/annurev-pathol-121808-102144 (PMID:20078217)
[3] D.J. Baker et al., Nature (2011) doi: 10.1038/nature10600. (PMID: 22048312)
[4] D.J. Baker et al., Nature (2016) doi: 10.1038/nature16932. (PMID: 26840489)
[5] H. Fuhrmann-Stroissnigg et al., Nat Commun. (2017) doi: 10.1038/s41467-017-00314-z. (PMID: 28871086)
[6]A. Hernandez-Segura et al., Trends in Cell Biology (2018) doi: 10.1016/j.tcb.2018.02.001 (PMID: 29477613)

CRISPR-Cas9 and p53

The CRISPR-Cas9 system represents a remarkable breakthrough in genome editing technology. With relative ease and amazing precision, investigators may now alter or replace genes in the genomes of organisms across the evolutionary spectrum. The potential for human disease treatment or prevention is incredible – an observation not lost on the pharmaceutical companies that have invested heavily in the approach.

All the excitement notwithstanding, some problems have arisen with the technology that may limit its usefulness going forward. Specifically, two papers from the journal Nature Medicine[1],[2] suggest that the “genome guardian” protein, p53, is activated in response to employment of CRISPR-Cas9. To understand what p53 is responding to, a brief description of how the CRISPR-Cas 9 system works is warranted.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR associated protein 9) system employs specific enzymes and carefully designed RNA guides to seek out distinct genomic DNA sequences for editing, removal, or replacement. The process involves the cleavage of DNA – an action not unnoticed by the human cell. Indeed, it is at this step that the cell takes exception to indiscriminate cutting of its DNA and turns on the (DNA) damage response pathway – orchestrated by p53. The aforementioned papers document the effects in human cells of such initiation of the p53-mediated reaction to DNA strand breaks.

CRISPR Cas9 system (Source: Marius Walter; Wikimedia Commons)

In the paper by Haapaniemi and colleagues, the authors observe that CRISPR-Cas9 activates the p53 pathway in human cells, and limits the efficiency of the gene editing process. In contrast, inhibiting the damage response pathway allows for greater effectiveness of the CRISPR-Cas9 genomic modification apparatus. The obvious problem is that any attempts to down-regulate p53, or to select for cells that have reduced activity of the pathway, by definition create cells potentially susceptible to cancerous transformation. The authors suggest that in future work, mechanisms by which the DNA repair pathway could be modulated – perhaps turned off during gene editing, but reinitiated shortly thereafter, represent an appropriate work around.

In the study by Ihry and colleagues, attempts are made to employ CRISPR-Cas9 to reprogram pluripotent stem cells. Once again, the gene editing procedure elicits a p53-mediated DNA damage response, and the cells undergo programmed cell death (~apoptosis). Cells that survive are very much at risk as they may harbor down-regulated p53 – potentiating the possibility of an accumulation of offsite mutations. As above, the answer seems to point to modulating p53 activity – turning it off for the editing step, but reactivating immediately following.

Much has been written about these results, both in the scientific literature and popular press. No one is calling for the technique to be abandoned; rather, additional research will be required before it can be expected that the human cell’s sophisticated and evolutionarily developed capacity for protecting itself can be sidestepped. Expect a wealth of research to be published on the topic in coming months. Stay tuned!

SRT – August 2018

References:
[1] E. Haapaniemi et al., Nat Med. (2018) doi: 10.1038/s41591-018-0049-z. [Epub ahead of print] (Link to Text)

[2] R.J. Ihry et al., Nat Med. (2018) doi: 10.1038/s41591-018-0050-6. Epub 2018 Jun 11. (Link to Text).

Targeted Approach to Antimalarial Drug Development

Hello all and welcome to this corner of the Interprofessional Health Sciences Library and Information Commons website, an area devoted to bringing you what we hope you find interesting and exciting stories of innovation in biomedical science.  Thank you for joining us!

A photomicrograph of a blood smear that contains a macro- and microgametocyte of the Plasmodium falciparum parasite. Source: CDC/Dr. Mae Melvin Transwiki

This first installment focuses on malaria – a worldwide health problem that, according to the Centers for Disease Control and Prevention, killed some 445,000 people in 2016.  Malaria is caused by parasites, carried by mosquitos which introduce the microorganisms into humans’ blood. Infection results in flu-like fever/chills, headache, and respiratory problems. Left untreated – progression to more serious and life-threatening complications is rapid. The parasite most responsible for this scourge is the protozoan Plasmodium falciparum. Pharmacological interventions exist – but have been thwarted by the unicellular organisms’ ability to acquire resistance. For example, chloroquine and related compounds were employed for decades to disable the parasite’s lysosome-like digestive vacuole. With the organism unable to completely metabolize the host red blood cells’ hemoglobin, toxic metabolites amass and the parasites die. The more recent drug of choice is artemisinin, a natural product derived from the plant Artemisia annua. Compounds containing artemisinin, or related semi-synthetic derivatives, reduce the number of parasites in the bloodstream through what is thought to involve formation of highly reactive free radical species. Unfortunately, identification of other safe drugs with efficacy in killing the microorganism has not been successful.

Which brings us to the new approach – pioneered by a team of investigators of the University of South Florida and Wellcome Trust Sanger Institute and described in a research article entitled “Uncovering the essential genes of the human malarial parasite Plasmodium falciparum by saturation mutagenesis”[1].  Here, a technique called piggyback transposition mutagenesis was used to insert crippling DNA stretches into genes across the organism’s genome. Non-essential genes were so-identified by their ability to withstand the random insertions. Essential genes, in contrast, resulted in the parasite’s death. Illumina-based deep sequencing allowed for identification of the insertion sites – and the analysis was on!

Of the 5399 genes that exist in the P. falciparum genome, 2680 were found to be essential for the parasite’s disease propagation processes. Powerful bioinformatic analyses revealed that essential gene clusters exist encoding proteins involved in, among other functions – lipid metabolic pathways, proteasomal degradation, cell cycle control, and RNA stability.  Naturally, these genes and their associated proteins/pathways now are the molecular targets for future antimalarial drug development. It is certain that sophisticated drug screens are currently being employed and we can only hope that safe and efficacious compounds are found in a timely manner. With the dearth of currently available antimalarials and the disease’s continued health toll, advances cannot come too soon.

SRT – June 2018

Reference:
[1] M. Zhang et al., Science 360, eaap7847 (2018). DOI: 10:1126/science.aap7847 (Link to Text)