Letter from the Editor
In this special issue of the BPC Newsletter, we present the three winners selected in the inaugural University of Pennsylvania BPC Science Writing Competition. The competition was organized as a joint effort between the University of Pennsylvania BPC Newsletter and Postdoctoral Editors Association, and drew applications from postdoctoral fellows across the University of Pennsylvania, including the School of Medicine, Veterinary Medicine, as well as Wistar and CHOP. The writers’ task? To explain a recent finding in scientific research that’s relevant and interesting, describing the background, findings, and most importantly, relevance to human health. Entries were judged on factors including an engaging writing style, scientific accuracy, and compelling scientific storytelling. Guest judging was provided by Bethany Brookshire, Ph.D., an award-winning science writer with Science News. Dr. Brookshire provided invaluable comments to the finalists, who used her feedback to hone their writing. Additional editorial assistance was provided by Daphne Avgousti, Ph.D., Editor in Chief of the Postdoctoral Editors Association. The top three finalists were recognized at the 2015 Biomedical Postdoctoral Research Symposium; their winning entries are published in this special issue of the BPC Newsletter. We hope that you enjoy these pieces of science writing. I would like to thank Dr. Brookshire for her impeccable and constructive critiques. I would also like to thank Dr. Avgousti for her enthusiasm and collaboration style which made orchestrating this competition so enjoyable. We are excited about this year’s winning entries, and hope to see even more great entries next year.
-Liisa Hantsoo, Ph.D., Editor in Chief, BPC Newsletter
Please access the PDF here: _BPC_Vol_4_Issue_4_10.25.15
1st Place: “Turning Back Time: It’s Never Too Late to Learn Like A Child,” by Jocelyn Lippman-Bell
2nd Place: “A Sexy New Way to Fight Malaria,” by Charitha Gowda
3rd Place: “AMPed and Dangerous,” by Kimberly Malecka
BPC Science Writing Competition 1st Place: “Turning Back Time: It’s Never Too Late to Learn Like a Child”
By Jocelyn Lippman-Bell
If you could take a pill to restore your mind to the flexible state of childhood learning, would you do it?
This is not as far out of the realm of possibility as one might imagine. Children possess an enhanced capacity for learning compared to adults, in part due to the high level of neuronal plasticity, or adaptability, that enables neurons to be especially sensitive and reactive to incoming messages. These periods of high neuronal responsiveness, remodeling, and refinement are called critical periods, and allow environmental cues to mold the developing brain and optimize neural functioning. Critical periods exist for many different forms of learning, including acquiring language and motor skills, developing sharp vision (visual acuity), and learning how to interpret sounds. A mounting body of research aimed at controlling the opening and closing of critical periods is rapidly making the possibility of reestablishing neuronal flexibility in adulthood a reality.
The idea that critical periods can be manipulated emerged largely through research in the developing visual system. Starting with the seminal studies of Hubel and Wiesel, evidence began to build demonstrating that if one eye of an animal remains closed throughout its visual critical period, the deprived eye had forever lost the ability to develop perfect acuity, and connections to the non-deprived eye were persistently much stronger than those to the deprived eye [1-4]. However, raising an animal in complete darkness (i.e. no visual stimulation to either eye) essentially shifted the critical period so that when the lights came back on, regardless of how long the animal had been in the dark, they were able to recover function and normal neural connectivity for both eyes [5, 6]. Because of these observations, scientists realized that the critical period was not an absolute; it could be altered. Thus began the race to uncover the mechanisms governing critical period regulation.
Using clues from the light deprivation experiments, scientists theorized that the key element controlling the critical period time course was the precise balance of excitatory and inhibitory activity at developing local neural circuits. Therefore, researchers began looking to the major mode for neuronal inhibition in the brain, the neurotransmitter gamma-aminobutyric acid, or GABA. Inhibitory circuits come on line later in development than excitatory circuits, and dampen excitation. When Huang and colleagues at MIT examined mice with accelerated onset of inhibitory circuit development, they found not only that the mice developed visual acuity sooner than their unaffected siblings, but also that the critical period for vision closed prematurely in these mice . Conversely, in mice that had genetic modifications that decreased GABA release and therefore inhibition, the critical period never closed throughout the lifespan of the mouse, likely because the neural circuits in these mice never reach the required peak inhibition .
Risk and benefits
It is now clear that critical periods can be experimentally altered, but what is the cost of such manipulation? One must consider that there is an evolutionary advantage underlying the loss of brain flexibility. In the first years of life, an overabundance of neuronal connections is formed, then refined in response to experience and environmental cues to optimize neural circuit function and our perception of our surroundings. What would it be like to be in the throes of a critical period? Imagine being three again. Young children absorb everything around them, everything they see, everything they hear. However, in absorbing everything, the child cannot focus as readily as an adult. They have trouble letting go of unimportant things. Recreating child-like brain flexibility to allow the brain to rewire could result in a loss of memories at best, or loss of sensory mapping or outright brain damage at worse. So for, say, a 50-year-old who wants to master windsurfing, this many not be a viable shortcut.
In contrast to the science fiction-esque idea of creating mind-expanding drugs for personal gain, the real promise of this research lies in its medical implications. Exploiting critical period reopening can open new avenues for cognitive recovery from stroke, enhanced acquisition of motor control of a prosthetic, and reversal of early childhood disorders such as amblyopia (or “lazy eye”, in which vision in one eye is weak despite the eye being perfectly healthy) . Emerging work over the past year from Krishnan, He, and colleagues now also links shifted critical periods to autism and autistic-like disorders such as Rett Syndrome [10, 11], a debilitating neurological disease of early childhood that affects 1 in 10,000 girls worldwide.
While reopening critical periods in humans offers theoretical usefulness, how can we actually accomplish this? Genetically decreasing overall inhibition in the brain tends not to be a viable treatment option. Without inhibition to keep it in check, excitation would overrun the brain, leading to massive seizures and likely death. Thus, therapeutic targeting requires a greater understanding of the specific molecules involved in critical period regulation, and of how to direct therapies only to the desired brain circuits. Though removing inhibition from the brain would be lethal, targeting specific GABA receptors has proven feasible. Luckily, not all GABA receptors regulate critical period closure. Only one subtype of GABA receptor, the GABAA receptor, seems to significantly modify plasticity, but not all the GABAA receptors are the same. GABAA receptors can be made up of different subunits, and the specific combinations of these subunits provide varying levels of influence on critical period closure .
Another promising line of research comes out of the studies of Rett Syndrome, an autistic-like neurodevelopmental disorder. Altered expression of a single gene, MeCP2, causes Rett Syndrome , and the mouse model replicates the human disorder fairly well, providing a straightforward approach for examining functional changes relevant to the disorder. Recent research in a Rett Syndrome mouse model discovered that the main inhibitory neurons involved in critical period plasticity maturated earlier than normal in the visual cortex of animals with MeCP2 gene mutations . Further, the visual critical period opened and closed earlier than normal in these mice, and could be restored by dampening inhibition , reminiscent of the foundational studies of critical period manipulation. Taken together, these data strongly implicate MeCP2 expression, and probably regulation, as a major molecular influence on the visual critical period.
Though the GABAA and MeCP2 pathways are only two of many possible candidates currently emerging as regulators of critical period, a major hurdle remains. Controlling the time course of critical periods depends on more than just altering the molecular pathways. Once the inhibitory circuitry matures, the inhibitory cells (parvalbumin (PV) cells) regulating critical period plasticity become enwrapped in a mesh of extracellular matrix proteins and adhesion molecules that basically form nets around the PV cells [14, 15]. These nets may physically constrict further plasticity of the inhibitory cells, holding the critical period window closed tightly in place. While molecular targets such as GABAA receptors and MeCP2 may represent promising targets for chemically restoring critical period plasticity, research into reversing the structural constraint has proven less fruitful.
Old brain, new tricks
The realization that critical periods could be manipulated and thus reopened brings to the table a new discussion of the mature and immature brain. A child’s brain differs so radically from that of the adult, for example, in receptor expression, in the balance of excitation and inhibition, and in the neuronal capacity for plasticity, that it can never be taken for granted that therapies successful for neuronal conditions in adults would be either safe or effective in children. However, understanding how the brain transitions from immaturity to maturity may blur these lines, holding the key to the possibility of turning back neural time and reopening our minds to cognitive malleability.
- Wiesel, T.N. and D.H. Hubel, Single-cell responses in striate cortex of kittens deprived of vision in one eye. . J Neurophysiol, 1963. 26: p. 1003-17.
- Timney, B., The effects of early and late monocular deprivation on binocular depth perception in cats. Brain Res, 1983. 283(2-3): p. 235-43.
- Guire, E.S., M.E. Lickey and B. Gordon, Critical period for the monocular deprivation effect in rats: assessment with sweep visually evoked potentials. J Neurophysiol, 1999. 81(1): p. 121-8.
- Gordon, J.A. and M.P. Stryker, Experience-dependent plasticity of binocular responses in the primary visual cortex of the mouse. J Neurosci, 1996. 16(10): p. 3274-86.
- Timney, B., D.E. Mitchell and F. Giffin, The development of vision in cats after extended periods of dark-rearing. Exp Brain Res, 1978. 31(4): p. 547-60.
- Fagiolini, M., T. Pizzorusso, N. Berardi, L. Domenici and L. Maffei, Functional postnatal development of the rat primary visual cortex and the role of visual experience: dark rearing and monocular deprivation. Vision Res, 1994. 34(6): p. 709-20.
- Huang, Z.J., A. Kirkwood, T. Pizzorusso, V. Porciatti, B. Morales, M.F. Bear, L. Maffei and S. Tonegawa, BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex. Cell, 1999. 98(6): p. 739-55.
- Fagiolini, M., & Hensch, Inhibitory threshold for critical-period activation in primary visual cortex. Nature, 2000. 404(6774): p. 183-186.
- Hensch, T.K. and P.M. Bilimoria, Re-opening Windows: Manipulating Critical Periods for Brain Development. Cerebrum, 2012. 2012: p. 11.
- Krishnan, K., B.S. Wang, J. Lu, L. Wang, A. Maffei, J. Cang and Z.J. Huang, MeCP2 regulates the timing of critical period plasticity that shapes functional connectivity in primary visual cortex. Proc Natl Acad Sci U S A, 2015. 112(34): p. E4782-91.
- He, L.J., N. Liu, T.L. Cheng, X.J. Chen, Y.D. Li, Y.S. Shu, Z.L. Qiu and X.H. Zhang, Conditional deletion of Mecp2 in parvalbumin-expressing GABAergic cells results in the absence of critical period plasticity. Nat Commun, 2014. 5: p. 5036.
- Hensch, T.K., Critical period plasticity in local cortical circuits. Nat Rev Neurosci, 2005. 6(11): p. 877-888.
- Amir, R.E., I.B. Van den Veyver, M. Wan, C.Q. Tran, U. Francke and H.Y. Zoghbi, Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet, 1999. 23(2): p. 185-8.
- Beurdeley, M., J. Spatazza, H.H. Lee, S. Sugiyama, C. Bernard, A.A. Di Nardo, T.K. Hensch and A. Prochiantz, Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex. J Neurosci, 2012. 32(27): p. 9429-37.
- Mix, A., K. Hoppenrath and K. Funke, Reduction in cortical parvalbumin expression due to intermittent theta-burst stimulation correlates with maturation of the perineuronal nets in young rats. Dev Neurobiol, 2015. 75(1): p. 1-11.
By Charitha Gowda
Having a boy? Or a girl? The odds are 50:50, right? While that may be true for us, it doesn’t have to be the case for some species that preferentially produce more males than females. It is this naturally-occurring imbalance in the sex ratio that scientists are hoping to exploit as part of new malaria eradication efforts.
Scientists at Imperial College London are embarking on an innovative strategy to target the Anopheles gambiae, the main mosquito species that transmits human malaria, by taking advantage of a process known as genetic distortion in sex ratio (1). This was originally described in two different genera of mosquitoes, the Aedes and Culex, where approximately five males to every three females were found when examining natural populations of some species (2). Differences in mortality rates between males and females do not appear to drive the imbalance. Instead, researchers have traced the sex ratio distortion to a bias toward the production of male gametes, or sperm.
Such new approaches to prevent or control malaria are sorely needed. Although substantial progress has been made since 2000, there is still much work to be done. With 650,000 deaths due to malaria each year, this disease remains a major threat to over 3 billion people worldwide who are at risk of contracting malaria. Additionally, the mosquitoes responsible for transmitting malaria are rapidly evolving strategies to overcome or become resistant to our current insecticides and mosquito repellants.
What does sex imbalance have to do with malaria control efforts?
For Anopheles gambiae, only the female mosquitoes bite and pass on the malaria parasite to humans. Additionally, it has been long appreciated that female members of a species are critical for determining ongoing propagation of a population. Thus, inducing a male predominance during sexual reproduction has been explored as an approach to suppress or even eliminate insect populations that transmit disease to humans.
In a study published in the journal Nature Communications in 2014, researchers at Imperial College London described how they genetically modified male mosquitoes of Anopheles gambiae to overwhelmingly produce male offspring (1). When the transgenic male mosquitoes were mated with normal, or wild-type, female mosquitoes, up to 95% of the offspring were male. Furthermore, the scientists found that the sex imbalance trait was inheritable, passed on to sons of the transgenic mosquitoes. Most excitingly, when the scientists released the genetically modified mosquitoes into cages with wild-type mosquito populations, the loss of females over time resulted in loss of the entire population within six generations. The hope is that introducing these genetically modified mosquitoes into the wild could ultimately lead to elimination of the Anopheles gambiae species, taking the deadly malaria parasite with it.
How did the researchers do it?
To describe how the sex imbalance occurs naturally, we first have to understand the genetics of sexual reproduction. During sexual reproduction, a male parent cell divides into two sperm, distributing an X chromosome to one and a Y chromosome to the other. The chance coupling of one of these cells with a female gamete or egg, that by definition has an X chromosome, ultimately leads to either a male (XY) or female (XX) offspring. What happens to preferentially select for males in these mosquitoes? Through a molecular process not fully understood yet, it appears that selective breaks occur in the DNA strands of the X chromosome from the male parent cell, thereby leaving only the sperm with the Y chromosome viable and available for mating.
This process was replicated in the transgenic male mosquitoes by taking advantage of several key features of the Anopheles mosquito’s DNA and sexual reproduction process. Researchers identified an enzyme that specifically cut a DNA sequence found on the Anopheles mosquito’s X chromosome and thereby destroys the chromosome. Introducing this enzyme into male mosquitoes led to loss of the sperm carrying the X chromosome during sperm production. However, the enzyme is also found in the surviving Y chromosome-bearing sperm and can go on to destroy the X chromosome from the maternal gamete after mating has occurred. This leads to a premature halting of sexual reproduction. Indeed, prior efforts by researchers to exploit this specific endonuclease were unsuccessful because the transgenic male mosquitoes were sterile. To tackle this challenge, the scientists set out to create a variant of the endonuclease that works during the first part of sexual reproduction – during sperm production – but not after female fertilization. Now, six years later, introducing this endonuclease variant into male mosquitoes resulted in genetically modified males that were not sterile and favored male offspring.
Toward a brave new world…
While the study has garnered a lot of excitement in the global health and malaria research fields, the study’s authors caution that there are big questions to be answered before implementing this plan. Most importantly, the ethical implications of potentially eradicating another species, even that of just one species of mosquito, are difficult to grasp.
There may be unintended consequences as well. Due to the specificity of the genetic modification for Anopheles gambiae, other mosquito species should not be targeted for elimination, unlike what occurs presently with insecticides. However, loss of even one species may invite different, or new, species to inhabit the empty niche.
That the Anopheles mosquitoes could evolve resistance to the genetic modification is another concern, as this has been seen repeatedly with other malaria control programs. However, the targeted DNA sequence is found within an essential, multi-copy gene, which researchers believe makes the rapid development of resistance unlikely.
The study team is already embarking on new experiments to evaluate the safety and efficacy of the genetically modified mosquitoes on a larger scale. Creating tropical ecosystems in large cages, the team hopes to mimic real-world conditions and results. For now, the researchers are pleased to have taken a novel first step towards envisioning a brave new world, potentially one without the scourge of malaria.
- Galizi R., Doyle, L.A., Menichelli,M., Bernardin, F., Deredec, A., Burt, A. et al. A synthetic sex ratio distortion system for the control of the human malaria mosquito. Nat Commun. 2014;5:3977.
- Hickey, W.A., Craig Jr, G.B. Genetic distortion of sex ratio in a mosquito, Aedes Aegypti. Genetics 1966;53(6):1177-1196.
By Kimberly Malecka
“Antimicrobial resistance poses a catastrophic threat,” declared Dame Sally Davies, Chief Medical Officer for England, in her 2013 annual report (1). She described a post-antibiotic dystopian era in which patients once again succumb to simple infections (1,2). Advanced medical options of routine surgeries, organ transplants and cancer treatments will be nonviable due to our inability to stop infection (1,3). So grim will be the prospects for human health that Davies ranks antimicrobial resistance next to terrorism and global warming as the greatest threats to our current safety (2).
A year later, the World Health Organization (WHO) published its findings from a study of 114 countries worldwide (2). They report that antibiotics are currently failing to treat half of all pneumonia, gonorrhea and simple urinary tract E. coli infections (2). More concerning, however, is the realization that carbapenem, a powerful and “last-resort” antibiotic, is ineffective in more than 50% of patients in some countries (2). The numbers of resistant infectious bacteria and futile antibiotics are only expected to increase alarmingly over the next several decades (2).
A combination of factors is hastening the speed at which microbes grow resistant to medications (2,3,4): overprescription by doctors, failure of patients to finish the prescribed amount, and fast rate of microbe mutation. Unfortunately, development of new antibiotics has been stagnant of late (4) leading to what Davies refers to as the “discovery void (1).” She is imploring scientific researchers to once again focus on antibiotic development in new and innovative ways (1,3).
Researchers in the June 2015 issue of Nature Structural & Molecular Biology heard her rally cry (5,6). These scientists are looking inward at the inherent biology of animals and plants. Upon infection by pathogens, animals and plants induce antimicrobial activities, including expression of antimicrobial peptides (AMPs) (7). AMPs are ancient weapons; these peptides have been used since the beginning of life as part of host immunity and they have remained effective since their inception (8). Interestingly, this fact confounds the current mantra that microbes will eventually develop resistance to any antibiotic. So, the key questions these scientists asked were how do AMPs kill bacteria and why do bacteria not become resistant?
Broadly speaking, AMPs fall into one of four categories, but proline-rich AMPs (PrAMPs) are particularly interesting in terms of antibiotic development. The goal of any antibiotic is for it to get inside and kill a bacterial cell while leaving the host cells untouched. PrAMPs are actively brought inside Gram-negative bacteria, such as E. coli, through specialized proteins on their surface. Mammalian cells lack these specialized proteins so PrAMPs cannot enter (5). As to how the PrAMP kills the bacterial cell after getting inside? The PrAMP binds to the 70S ribosome and blocks the bacterial cell’s ability to make proteins. Proteins are essential to every function of a healthy cell; if protein synthesis is inhibited, then that cell will die almost immediately.
Protein synthesis is a highly choreographed event. It begins at the DNA. Specialized proteins transcribe a gene from our genome into a carrier molecule known as messenger RNA (mRNA). The mRNA then binds to a ribosome. Translation of the genetic material (mRNA) into a string of amino acids (protein) happens at the ribosome. This process is mediated by transfer RNA (tRNA). tRNAs bind to both the mRNA and the ribosome at specific spots, known as A, P and E. Using an analogy, in World War II, if the mRNA is an ecrypted message and protein is the translated message, then the ribosome is the deciphering Enigma machine. Functional ribosomes are essential to proper protein synthesis. Scientists know that ribosomes bound by PrAMPs cannot function, but they didn’t understand why until now.
Two laboratories independently solved the crystal structure of the PrAMP Onc112 bound to the 70s ribosome of Thermus thermophilus to atomic resolutions (5,6). Both structures are similar and together they describe the multi-headed attack by the PrAMP on the ribosome. The Onc112 peptide has four functions. First, residues 1-3 hinder the ability of an incoming tRNA to bind to the A site. Second, residues 5-8 disrupt proper tRNA binding to the P site. Third, residues 9-12 block the ribosomal exit tunnel where the translated protein leaves the ribosome. Fourth, residues 13-19 are necessary for the transport of Onc112 into a Gram-negative bacterium. Removal of those amino acids from Onc112 or the removal of SbmA from baceterial cells results in a peptide that lacks antimicrobial activity.
The areas of contact for the Onc112 peptide within the ribosome are not surprising. Many antibiotics on the market also target these areas individually (5,6). Clindamycin (acne, taxoplasmosis) and thiostrepton (veterinary mastitis) both prevent tRNA accommodation at the A site. Chloramphenicol (eye infections, swimmer’s ear) binds to a similar area as residues 5-8 of Onc112. Erythromycin (acne, chlamydia, syphilis) and azithromycin (tonsillitis, gonorrhea) are part of the macrolide group, which bind within the ribosomal exit tunnel. Thus, while the majority of available antibiotics have uni-modal mechanisms, Onc112 has a tetra-modal (four-moded) mechanism. The first three parts of the peptide bind to and completely shut down a ribosome’s ability to translate proteins; the fourth part ensures that such a powerful peptide is only taken up by infectious bacterial cells. The benefits of a tetra-modal peptide over a uni-modal small molecule are the differences between a short road to antimicrobial resistance and a long road.
These two published structures display why AMPs have been efficacious since the beginning of time: their success is protected by redundancy. A single mutation within the peptide or the ribosome is unlikely to destroy the peptide’s antimicrobial activity. Hurt one area of interaction and there are still others available! This protection is not present for small molecule antibiotics. One nucleotide change within the ribosome can be enough to destabilize the binding of the antibiotic to the ribosome. Bacteria evolve fast. If we show them the same antibiotic enough through over-prescription, then we are hastening the selection of viable bacteria that harbor that key nucleotide change.
Scientists are now trying to capitalize on the secret of AMPs by tweaking current antibiotics to bind mutated or modified ribosomes (11). However, a more effective path is now being pursued based on the research presented here. Scientists are buildling chimeric antibiotics, such as macrolide-chloramphenicol, that show promising results in the clinic (11). Scientists can also successfully replace L-amino acids with D-amino acids in these peptides without disrupting their antibiotic potency (8). Peptides of D-amino acids cannot be broken down mammalian cells. This result can pave the way for peptide-based antibiotics, as well. Seefeldt summarizes that “…it appears likely that [AMPs] represent a vast, untapped resource for the development of new therapeutics.” The past can be our best teacher when it comes to guiding the development of new antimicrobials to assuage the fears of Sally Davies, the WHO, and to protect our future generations.
- Antimicrobial resistance poses ‘catastrophic threat’, says Chief Medical Officer. (2015). <http://bsac.org.uk/news/antimicrobial-resistance-poses-catastrophic-threat-says-chief-medical-officer/>.
- Stephens, P. Antibiotic resistance now ‘global threat’, WHO warns. (2014). <http://www.bbc.com/news/health-27204988>.
- Hampton, T. Novel Programs and Discoveries Aim to Combat Antibiotic Resistance. The Journal of the American Medical Association 313 (2015).
- Hampton, T. Novel Programs and Discoveries Aim to Combat Antibiotic Resistance. The Journal of the American Medical Association 313 (2015).
- Roy, R., Lomakin, I., Gagnon, M. & Steitz, T. The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin. Nature structural & molecular biology 22, 466 – 469 (2015).
- Seefeldt, A. et al. The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex. Nature structural & molecular biology 22, 470 – 475 (2015).
- de Souza Candido, E. et al. The use of versatile plant antimicrobial peptides in agribusiness and human health. Peptides 55, 65 – 78 (2014).
- Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 15, 389 – 395 (2002).
- Li, W. et al. Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria. Amino Acids 46, 2287 – 2294 (2014).
- Krizsan, A. et al. Insect-derived proline-rich antimicrobial peptides kill bacteria by inhibiting bacterial protein translation at the 70S ribosome. Angewandte Chemie 53, 12236 – 12239 (2014).
11. Wilson, D. Ribosome-targeting antibiotics and mechanisms of bacteria resistance. Nature reviews. Microbiolo