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Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Summary: Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) pose a major healthcare burden. Distinct inflammatory and resolution phases comprise the host immune response to SSTIs. Resolution is a myeloid PPARγ-dependent anti-inflammatory phase that is essential for the clearance of MRSA. However, the signals activating PPARγ to induce resolution remain unknown. Here, we demonstrate that myeloid glucose transporter 1 (GLUT-1) is essential for the onset of resolution. MRSA-challenged macrophages are unsuccessful in generating an oxidative burst or immune radicals in the absence of GLUT-1 due to a reduction in the cellular NADPH pool. This translates in vivo as a significant reduction in lipid peroxidation products required for the activation of PPARγ in MRSA-infected mice lacking myeloid GLUT-1. Chemical induction of PPARγ during infection circumvents this GLUT-1 requirement and improves resolution. Thus, GLUT-1-dependent oxidative burst is essential for the activation of PPARγ and subsequent resolution of SSTIs.

ABSTRACT Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.

After a chemical fire, an investigation assessed health effects by using syndromic surveillance to monitor emergency department (ED) visits, a general health survey to assess the general public, and a first responders health survey to assess first responders. A total of four separate multivariable logistic regression models were developed to examine associations between reported exposure to smoke, dust, debris, or odor with any reported symptom in the general public. Syndromic surveillance identified areas with increased ED visits. Among general health survey respondents, 45.1% (911 out of 2,020) reported at least one symptom. Respondents reporting exposure to smoke, dust, debris, or odor had 4.5 (95% confidence interval (CI) [3.7, 5.5]), 4.6 (95% CI [3.6, 5.8]), 2.0 (95% CI [1.7, 2.5]), or 5.8 (95% CI [4.7, 7.3]) times the odds of reporting any symptom compared with respondents not reporting exposure to smoke, dust, debris, or odor, respectively. First responders commonly reported contact with material and being within 1 mi of the fire [greater than or equal to] 5 hr; 10 out of 31 of first responders reported at least one symptom. There was high symptom burden reported after the fire. Results from our investigation might assist the directing of public health resources to effectively address immediate community needs and prepare for future incidents., Introduction On the morning of June 14, 2021, a fire ignited and spread rapidly through an industrial chemical facility owned by the largest industrial grease manufacturer in the U.S. and [...]

The individual and global burden of migraine is of such significance that there are accelerated efforts to develop new therapies. New migraine therapeutics are needed to address the current deficiencies that exist in the efficacy and adherence rate of approved anti-migraine medications. The recent discovery of the calcitonin gene related peptide as an add-on to the role of serotonin has markedly increased the range of new treatment options for acute and chronic migraine. Despite this, tackling the complexity of migraine disorders requires a complete understanding of its pathophysiology. Preclinical animal models can shed light on disease-related pathophysiology, including migraine. Indeed, the use of animal models has been instrumental in developing many therapeutics. However, an animal model is limited by the predictive and face validity of that model, and this extends to preclinical migraine models. In this review, a summary of the current understanding of the pathophysiology of migraine is given from both a preclinical and clinical perspective, and an emphasis is placed on the animal models of migraine. We will discuss the strengths and pitfalls of common preclinical migraine models as well as experimental research areas to explore further.

For many cancer patients tumor burden negatively impacts quality of life due to associated pain onset. Neuropathic pain is commonly associated with late cancer stages, and is resultant of tumor metastasis to bone, herein referred to as cancer-induced bone pain. Given the severe impact on quality of life and clinical treatment strategies focusing on symptom management, novel therapeutics are needed to alleviate cancer-induced bone pain and/or reduce cancer burden. In the current study we characterized a commercially available murine fibrosarcoma cell line, NCTC-2472 in vitro, which can be used to assess the capacity of novel compounds to impact cellular viability. We found that dimethyl sulfoxide, a known cytotoxic agent and common drug preparation compound, significantly decreased cell viability in a dose-related manner. We then characterized the in vivo tumor development and associated pain behavior characteristics following implantation of NCTC-2472 fibrosarcoma into male and female C3H/HeJ mice. The C3H/HeJ strain was utilized as these mice are syngeneic with NCTC-2472 fibrosarcoma and their use reduces potential implantation failure. We found that tumor development in mice resulted in the development of mechanical allodynia but not thermal hyperalgesia. Gabapentin, a clinically relevant analgesic, produced dose-related mechanical allodynia reversal. These studies provide further characterization of a cancer-induced bone pain model that can be used to examine novel compounds as anti-cancer and analgesic therapeutics.

ABSTRACT Diabetes is associated with several health consequences, including increased susceptibility to more frequent and severe infections. Bacterial infections associated with diabetes are typically polymicrobial, with Staphylococcus aureus and Pseudomonas aeruginosa frequently isolated from the same infection site. S. aureus and P. aeruginosa are frequently found in diabetic skin and soft tissue infections, in the lungs of people with cystic fibrosis, and in indwelling device infections. Numerous studies have investigated interactions between these two pathogens primarily using in vitro systems. These models have several limitations as they do not accurately reflect the complexities of an immune response nor the nutrient dynamics in a diabetic infection microenvironment. Here, we describe a novel murine indwelling device co-infection model that allows us to study the interactions between S. aureus and P. aeruginosa within the context of an immune response during both normal and diabetic infections. Our data shows that P. aeruginosa significantly inhibits S. aureus growth during co-infection in a normal mouse and that inhibition is not dependent on the P. aeruginosa PQS quorum sensing system. Conversely, in a diabetic co-infection, S. aureus overcomes inhibition by P. aeruginosa and this phenotype is reliant on S. aureus glycolysis. We also demonstrate that both organisms display increased virulence potential in a diabetic co-infection as we observe increased dissemination to peripheral tissues. This study revealed novel in vivo interactions between S. aureus and P. aeruginosa and advances our understanding of the complex interactions between microorganisms in polymicrobial infections in clinically relevant infection microenvironments. IMPORTANCE Individuals with diabetes are prone to more frequent and severe infections, with many of these infections being polymicrobial. Polymicrobial infections are frequently observed in skin infections and in individuals with cystic fibrosis, as well as in indwelling device infections. Two bacteria frequently co-isolated from infections are Staphylococcus aureus and Pseudomonas aeruginosa. Several studies have examined the interactions between these microorganisms. The majority of these studies use in vitro model systems that cannot accurately replicate the microenvironment of diabetic infections. We employed a novel murine indwelling device model to examine interactions between S. aureus and P. aeruginosa. Our data show that competition between these bacteria results in reduced growth in a normal infection. In a diabetic infection, we observe increased growth of both microbes and more severe infection as both bacteria invade surrounding tissues. Our results demonstrate that diabetes changes the interaction between bacteria resulting in poor infection outcomes.

Nicotine is the primary psychoactive component responsible for maintaining tobacco dependence in humans. Chronic pain is often a consequence of tobacco-related pathologies, and the development of a dual therapeutic that could treat chronic pain and tobacco dependence would be advantageous. Epibatidine reliably substitutes for nicotine in the drug discrimination assay, and is a potent analgesic, but has a side-effect profile that limits its therapeutic potential. Thus, considerable efforts to produce epibatidine derivatives are underway. Here we tested three epibatidine derivatives, 2′-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102; i.e., RTI-102), 2′-fluorodeschloroepibatidine (RTI-7527-36; i.e., RTI-36), and 3'-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76; i.e., RTI-76) in both the rat nicotine drug discrimination assay as well as in the rat chronic constriction injury (CCI) of the sciatic nerve neuropathic pain model. Male and female Sprague-Dawley rats were trained on a fixed-ratio 10 schedule to discriminate nicotine (0.32 mg/kg base) from vehicle. All compounds dose-dependently substituted for nicotine, without significant decreases in response rates. In the discrimination assay the rank order potency was RTI-36 > nicotine > RTI-102 > RTI-76. Evidence suggests the α4β2* subtype is particularly important to nicotine-related abuse potential. Thus, here we utilized the antagonist dihydro-β-erythroidine (DHβE) to examine relative β2 subunit contribution. DHβE (3.2 mg/kg, s.c.) antagonized the discriminative stimulus effects of nicotine. However, relative to antagonism of nicotine, DHβE produced less antagonism of RTI-102 and RTI-76 and greater antagonism of RTI-36. It is likely that at nicotinic receptor subunits RTI-102, RTI-76 and RTI-36 possess differing activity. To confirm that the full discriminative stimulus of these compounds was due to nAChR activity beyond the β2 subunit, we examined these compounds in the presence of the non-selective nicotinic receptor antagonist mecamylamine. Mecamylamine (0.56 mg/kg, s.c.) pretreatment abolished nicotine-paired lever responding for all compounds. In a separate cohort, male and female Sprague-Dawley rats underwent CCI surgery and tested for CCI-induced mechanical allodynia via the von Frey assay. Each compound produced CCI-induced mechanical allodynia reversal. RTI-36 displayed higher potency than either RTI-102 or RTI-76. These novel epibatidine analogs may prove to be useful tools in the fight against nicotine dependence as well as novel neuropathic pain analgesics.

A global rise in HVAC-R utilization requires a deeper understanding of the industry's effect on electricity consumptions and greenhouse gas emissions. The Total Equivalent Warming Impact (TEWI) methodology was designed to analyze emissions from direct release of refrigerant and indirect emissions through electricity consumption of HVAC-R systems to increase the understanding of system design on emissions, and to guide refrigerant replacement. However, the original TEWI calculation neglects the system degradation due to corrosion. This paper studies on the impact of corrosion and highlights how the original TEWI method underrepresents the lifetime emissions due to energy efficiency decrease and refrigerant release. Corrosion impacts direct emissions by increasing refrigerant leakage rates over time and indirect emissions through heat exchanger efficiency degradation and suboptimal refrigerant level. A modified TEWI equation is proposed to capture the dynamic corrosion impacts over the lifetime of HVAC operations. Three scenarios (low corrosivity, conservative and moderate corrosivity) are examined to analyze different corrosion environments. This analysis indicates 6%-27% increase in TEWI emissions based on a typical residential air conditioner (AC), when the impacts of corrosion are included, with the greatest emissions increase from reduced electrical efficiency. The impact of several current and future corrosion protection scenarios on TEWI are also included. Appropriate corrosion mitigation can reduce total lifecycle emissions of systems by 6% ~ 10%. The proposed modified TEWI method is expected to provide a more accurate emission estimation for AC sustainability and policy making.

Abstract An ongoing healthcare debate is whether controlling hospital-acquired infection (HAI) from methicillin-resistant Staphylococcus aureus (MRSA) will result in lowering the global HAI rate, or if MRSA will simply be replaced by another pathogen and there will be no change in overall disease burden. With surges in drug-resistant hospital-acquired pathogens during the COVID-19 pandemic, this remains an important issue. Using a dataset of more than 1 million patients in 51 acute care facilities across the USA, and with the aid of a threshold model that models the nonlinearity in outbreaks of diseases, we show that MRSA is additive to the total burden of HAI, with a distinct ‘epidemiological position’, and does not simply replace other microbes causing HAI. Critically, as MRSA is reduced it is not replaced by another pathogen(s) but rather lowers the overall HAI burden. The analysis also shows that control of MRSA is a benchmark for how well all non-S. aureus nosocomial infections in the same hospital are prevented. Our results are highly relevant to healthcare epidemiologists and policy makers when assessing the impact of MRSA on hospitalized patients. These findings further stress the major importance of MRSA as a unique cause of nosocomial infections, as well as its pivotal role as a biomarker in demonstrating the measured efficacy (or lack thereof) of an organization’s Infection Control program.

Abstract WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions—that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.

The pathogen Ophidiomyces ophidiicola, widely known as the primary cause of snake fungal disease (SFD) has been detected in Texas’s naïve snakes. Our team set out to characterize O. ophidiicola’s spread in eastern Texas. From December 2018 until November 2021, we sampled and screened with ultraviolet (UV) light, 176 snakes across eastern Texas and detected 27. O. ophidiicola’s positive snakes using qPCR and one snake in which SFD was confirmed via additional histological examination. Upon finding the ribbon snake with clear clinical display, we isolated and cultured what we believe to be the first culture from Texas. This cultured O. ophidiicola TX displays a ring halo formation when grown on a solid medium as well as cellular autofluorescence as expected. Imaging reveals individual cells within the septated hyphae branches contain a distinct nucleus separation from neighboring cells. Overall, we have found over 1/10 snakes that may be infected in East Texas, gives credence to the onset of SFD in Texas. These results add to the progress of the disease across the continental United States.

Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.

ABSTRACT Staphylococcus aureus is a human skin pathogen capable of causing invasive infections in many tissues in the human body. The host of virulence factors, such as toxins and proteases, available to S. aureus contribute to its diverse disease presentations. The majority of these virulence factors are under the control of the Agr quorum sensing system. The interaction between the Agr system and some well-established metabolic regulators has long been noted, but no mechanism has been provided as to these indirect interactions. In this study, we examine the connection between Agr and CcpA, a regulator of central carbon metabolism with a known positive impact on Agr function. We further investigated the interaction of Agr and CodY, a regulator of amino acid metabolism and a member of the stringent response with a known negative impact on Agr function. We show that though there are alterations in intracellular amino acid levels in each of these mutants that are consistent with their effect on Agr, there does not seem to be a direct impact on the translation of the Agr system itself that contributes to the altered expression observed in these mutants. Given the changes in cellular metabolism in a ΔccpA mutant, we find reduced levels of intracellular ATP even in the presence of glucose. This reduction in ATP, combined with the reduced affinity of the AgrC sensor kinase for ATP, explains the reduction in Agr activity long observed in ΔccpA strains. IMPORTANCE The human pathogen Staphylococcus aureus produces a great number of virulence factors that contribute to the pathogen’s ability to cause dangerous, invasive infections. Understanding the full scope of the regulation of these virulence factors can provide us with new information about how to target virulence factor production. For years, researchers in the field have observed an impact of metabolic regulators on virulence factor production with no mechanistic explanation. Here, we describe the role of two of these regulators, CcpA and CodY, in virulence factor expression and provide evidence of indirect mechanisms contributing to the control of the Agr system and virulence factor production by these two metabolic regulators. Our study sheds light on the interplay between metabolism and virulence in S. aureus and provides an explanation as to how these concepts are linked.