Systematic Mutation and Unnatural Base Pair Incorporation Improves Riboswitch-Based Biosensor Response Time.

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.

Reinterpreting the Fate of Iridium(III) Photocatalysts─Screening a Combinatorial Library to Explore Light-Driven Side-Reactions.

Photoredox catalysts are primarily selected based on ground and excited state properties, but their activity is also intrinsically tied to the nature of their reduced (or oxidized) intermediates. Catalyst reactivity often necessitates an inherent instability, thus these intermediates represent a mechanistic turning point that affords either product formation or side-reactions. In this work, we explore the scope of a previously demonstrated side-reaction that partially saturates one pyridine ring of the ancillary ligand in heteroleptic iridium(III) complexes. Using high-throughput synthesis and screening under photochemical conditions, we identified different chemical pathways, ultimately governed by ligand composition. The ancillary ligand was the key factor that determined photochemical stability. Following photoinitiated electron transfer from a sacrificial tertiary amine, the reduced intermediate of complexes containing 1,10-phenanthroline derivatives exhibited long-term stability. In contrast, complexes containing 2,2'-bipyridines were highly susceptible to hydrogen atom transfer and ancillary ligand modification. Detailed characterization of selected complexes before and after transformation showed differing effects on the ground and excited state reduction potentials dependent on the nature of the cyclometalating ligands and excited states. The implications of catalyst stability and reactivity in chemical synthesis was demonstrated in a model photoredox reaction.

Effects of the Response to the COVID-19 Pandemic on Assault-Related Head Injury in Melbourne: A Retrospective Study.

Assault is the leading preventable cause of death, traumatic brain injury (TBI), and associated mental health problems. The COVID-19 pandemic has had a profound impact on patterns of interpersonal violence across the world. In this retrospective cross-sectional study, we analysed medical records of 1232 assault victims (domestic violence: 111, random assault: 900, prison assault: 221) with head injuries who presented to the emergency department (ED) at St Vincent's Hospital in Melbourne, Australia, a city with one of the longest and most severe COVID-19 restrictions worldwide. We examined changes in prevalence in the assault group overall and in domestic violence, random assault, and prison assault victims, comparing data from 19.5 months before and after the first day of COVID-19 restrictions in Melbourne. Moreover, we investigated differences driven by demographic factors (Who: age group, sex, and nationality) and clinical variables (Where: assault location, and When: time of arrival to the ED and time from moment of injury until presentation at ED). Descriptive statistics and chi-square analyses were performed. We found the COVID-19 pandemic significantly affected the Where of assault-related TBI, with a shift in the location of assaults from the street to the home, and the increase at home being driven by random assaults on middle-aged adults. Overall, we observed that 86% of the random assault cases were males, whereas 74% of the domestic assault cases were females. Meanwhile, nearly half (44%) of the random assault victims reported alcohol consumption versus a fifth (20%) of domestic violence victims. These findings will have direct implications for developing screening tools and better preventive and ameliorative interventions to manage the sequelae of assault TBI, particularly in the context of future large-scale health crises or emergencies.

Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter.

Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.

Designing fluorescent biosensors using circular permutations of riboswitches.

RNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology.

Fluorinated N,N'-diarylureas as AMPK activators.

Adenosine monophosphate-activated kinase (AMPK) plays a central role in regulating energy homeostasis in eukaryotic cells. AMPK also regulates lipid synthesis by inhibiting acetyl-CoA carboxylase (ACC) and regulates mTOR signaling by activating TSC2. Due to its important roles in cell metabolism, AMPK is an attractive target for metabolic diseases, such as type II diabetes and obesity. AMPK activators, such as metformin, that are used for diabetes treatment are also effective anticancer agents. However, the efficacies of many known AMPK activators are relatively low. For example, metformin activates AMPK at millimolar levels. In this study, we identified a novel family of AMPK activators, namely fluorinated N,N'-diarylureas, that activate AMPK at 1-3µM concentrations. These novel agents strongly inhibit the proliferation of colon cancer cells. We studied the potential mechanisms of these agents, performed a structure-activity relationship (SAR) study and identified several fluorinated N,N'-diarylureas as potent AMPK activators.