Natural and Synthetic Suppressor Mutations Defy Stability-Activity Tradeoffs.

Thermodynamic stability represents one important constraint on protein evolution, but the molecular basis for how mutations that change stability impact fitness remains unclear. Here, we demonstrate that a prevalent global suppressor mutation in TEM ß-lactamase, M182T, increases fitness by reducing proteolysis in vivo. We also show that a synthetic mutation, M182S, can act as a global suppressor and suggest that its absence from natural populations is due to genetic inaccessibility rather than fundamental differences in the protein's stability or activity.

Robust genome and cell engineering via in vitro and in situ circularized RNAs.

Circularization can improve RNA persistence, yet simple and scalable approaches to achieve this are lacking. Here we report two methods that facilitate the pursuit of circular RNAs (cRNAs): cRNAs developed via in vitro circularization using group II introns, and cRNAs developed via in-cell circularization by the ubiquitously expressed RtcB protein. We also report simple purification protocols that enable high cRNA yields (40-75%) while maintaining low immune responses. These methods and protocols facilitate a broad range of applications in stem cell engineering as well as robust genome and epigenome targeting via zinc finger proteins and CRISPR-Cas9. Notably, cRNAs bearing the encephalomyocarditis internal ribosome entry enabled robust expression and persistence compared with linear capped RNAs in cardiomyocytes and neurons, which highlights the utility of cRNAs in these non-dividing cells. We also describe genome targeting via deimmunized Cas9 delivered as cRNA and a long-range multiplexed protein engineering methodology for the combinatorial screening of deimmunized protein variants that enables compatibility between persistence of expression and immunogenicity in cRNA-delivered proteins. The cRNA toolset will aid research and the development of therapeutics.

Analysis of PEG-lipid anchor length on lipid nanoparticle pharmacokinetics and activity in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) affects millions of people worldwide, yet there are currently no therapeutics that address the long-term impairments that develop in a large portion of survivors. Lipid nanoparticles (LNPs) are a promising therapeutic strategy that may address the molecular basis of TBI pathophysiology. LNPs are the only non-viral gene delivery platform to achieve clinical success, but systemically administered formulations have only been established for targets in the liver. In this work, we evaluated the pharmacokinetics and activity of LNPs formulated with polyethylene glycol (PEG)-lipids of different anchor lengths when systemically administered to a mouse model of TBI. We observed an increase in LNP accumulation and activity in the injured brain hemisphere compared to the uninjured contralateral brain hemisphere. Interestingly, transgene expression mediated by LNPs was more durable in injured brain tissue compared to off-target organs when compared between 4 and 24 hours. The PEG-lipid is an important component of LNP formulation necessary for the stable formation and storage of LNPs, but the PEG-lipid structure and content also has an impact on LNP function. LNP formulations containing various ratios of PEG-lipid with C18 (DSPE-PEG) and C14 (DMG-PEG) anchors displayed similar physicochemical properties, independent of the PEG-lipid compositions. As the proportion of DSPE-PEG was increased in formulations, blood circulation times of LNPs increased and the duration of expression increased. We also evaluated diffusion of LNPs after convection enhanced delivery (CED) in healthy brains and found LNPs distributed >1 mm away from the injection site. Understanding LNP pharmacokinetics and activity in TBI models and the impact of PEG-lipid anchor length informs the design of LNP-based therapies for TBI after systemic administration.

Engineered nanomaterials that exploit blood-brain barrier dysfunction for delivery to the brain.

The blood-brain barrier (BBB) is a highly regulated physical and functional boundarythat tightly controls the transport of materials between the blood and the brain. There is an increasing recognition that the BBB is dysfunctional in a wide range of neurological disorders; this dysfunction can be symptomatic of the disease but can also play a role in disease etiology. BBB dysfunction can be exploited for the delivery of therapeutic nanomaterials. Forexample, there can be a transient, physical disruption of the BBB in diseases such as brain injury and stroke, which allows temporary access of nanomaterials into the brain. Physicaldisruption of the BBB through external energy sources is now being clinically pursued toincrease therapeutic delivery into the brain. In other diseases, the BBB takes on new properties that can beleveraged by delivery carriers. For instance, neuroinflammation induces the expression ofreceptors on the BBB that can be targeted by ligand-modified nanomaterials, and theendogenous homing of immune cells into the diseased brain can be hijacked for the delivery ofnanomaterials. Lastly, BBB transport pathways can be altered to increase nanomaterial transport. In this review, we will describe changes that can occur in the BBB in disease, and how these changes have been exploited by engineered nanomaterials forincreased transport into the brain.

TRPA1 modulates noxious odor responses in Lygus hesperus.

Lygus hesperus isa key pest of many economically important crops across western North America. Central to many aspects of the lives of these insects is chemical signalling, with identified roles in host plant selection, aggregation and passive mate guarding. The development of novel monitoring and control approaches for this insect will rely on a sound understanding of how these cues are perceived and processed, and their impact on behavior. Towards this end, we investigated allyl isothiocyanate, cinnamaldehyde and citronellal, compounds that are noxious repellents to other insects. We found that L. hesperus avoided areas containing the three compounds and that exposure induced increases in movement velocity and duration in both nymphs and adults. This suggests these compounds may work as repellents. To better understand the underlying physiology of this response, RNA interference by dsRNA injection was used to inhibit the expression of two chemosensory-associated proteins, the odorant receptor co-receptor (Orco) and the transient receptor potential A (TRPA1) channel. While knockdown of Orco did not change the reaction of adult females to citronellal, TRPA1 silencing effectively eliminated the induced increase to movement, suggesting a chemoperceptory role in citronellal detection.

Regulatory roles of biogenic amines and juvenile hormone in the reproductive behavior of the western tarnished plant bug (Lygus hesperus).

Mating induces behavioral and physiological changes in the plant bug Lygus hesperus Knight (Hemiptera: Miridae). After receiving seminal products, which include the systemic regulator juvenile hormone (JH), females enter a post-mating period lasting several days during which they enhance their oviposition rate and lose interest in remating. To elucidate the regulation of these behavioral changes in L. hesperus, biogenic amines were quantified in the heads of females at 5 min, 1 h and 24 h after copulation and compared to levels in virgins using high-performance liquid chromatography coupled with electrochemical detection. Mating significantly increased dopamine (DA) after 1 and 24 h, and decreased octopamine (OA) after 5 min and 1 h. Serotonin did not change with mating, but tyramine was significantly reduced after 5 min. While injection of amines into virgin females did not influence sexual receptivity, OA caused a decrease in oviposition during the 24 h following injection. Topical application of the JH analog methoprene to virgins caused an increase in DA, and a decline in mating propensity, but did not influence other amines or the oviposition rate. The results suggest the decline in OA observed immediately after mating may promote egg laying, and that male-derived JH may induce an increase in DA that could account for the post-mating loss of sexual receptivity.