P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa . The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthy: The experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa , which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstract: The anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. ( B ) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa . 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage.

An in vitro medium for modeling gut dysbiosis associated with cystic fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation, and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization, and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting nonCF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short-chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but also altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of CF gut dysbiosis. IMPORTANCE Cystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.

Smartphone applications for measuring noise in the intensive care unit: A feasibility study.

PURPOSE: This study aims to explore the suitability of using smartphone applications with low-cost external microphones in measuring noise levels in intensive care units. METHODS: Four apps and two external microphones were tested in a laboratory by generating test signals at five noise levels. The average noise levels were measured using the apps and a professional device (i.e. a sound level meter). A field test was performed in an intensive care unit with two apps and one microphone. Noise levels were measured in terms of average and maximum noise levels according to the World Health Organisation's guidance. All the measurements in both tests were conducted after acoustic calibration using a sound calibrator. RESULTS: Overall, apps with low-cost external microphones produced reliable results of averaged noise levels in both the laboratory and field settings. The differences between the apps and the sound level meter were within ±2 dB. In the field test, the best combination of app and microphone showed negligible difference (< 2 dB) compared to the sound level meter in terms of the average noise level. However, the maximum noise level measured by the apps exhibited significant differences from those measured by the sound level meter, ranging from -0.9 dB to -4.7 dB. CONCLUSION: Smartphone apps and low-cost external microphones can be used reliably to measure the average noise level in the intensive care unit after acoustic calibration. However, professional equipment is still necessary for accurate measurement of the maximum noise level.

APPLICATION OF QUANTILE DISCRETIZATION AND BAYESIAN NETWORK ANALYSIS TO PUBLICLY AVAILABLE CYSTIC FIBROSIS DATA SETS.

The availability of multiple publicly-available datasets studying the same phenomenon has the promise of accelerating scientific discovery. Meta-analysis can address issues of reproducibility and often increase power. The promise of meta-analysis is especially germane to rarer diseases like cystic fibrosis (CF), which affects roughly 100,000 people worldwide. A recent search of the National Institute of Health's Gene Expression Omnibus revealed 1.3 million data sets related to cancer compared to about 2,000 related to CF. These studies are highly diverse, involving different tissues, animal models, treatments, and clinical covariates. In our search for gene expression studies of primary human airway epithelial cells, we identified three studies with compatible methodologies and sufficient metadata: GSE139078, Sala Study, and PRJEB9292. Even so, experimental designs were not identical, and we identified significant batch effects that would have complicated functional analysis. Here we present quantile discretization and Bayesian network construction using the Hill climb method as a powerful tool to overcome experimental differences and reveal biologically relevant responses to the CF genotype itself, exposure to virus, bacteria, and drugs used to treat CF. Functional patterns revealed by cluster Profiler included interferon signaling, interferon gamma signaling, interleukins 4 and 13 signaling, interleukin 6 signaling, interleukin 21 signaling, and inactivation of CSF3/G-CSF signaling pathways showing significant alterations. These pathways were consistently associated with higher gene expression in CF epithelial cells compared to non-CF cells, suggesting that targeting these pathways could improve clinical outcomes. The success of quantile discretization and Bayesian network analysis in the context of CF suggests that these approaches might be applicable to other contexts where exactly comparable data sets are hard to find.

Rocket-miR, a translational launchpad for miRNA-based antimicrobial drug development.

IMPORTANCE: Antimicrobial-resistant infections contribute to millions of deaths worldwide every year. In particular, the group of bacteria collectively known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) pathogens are of considerable medical concern due to their virulence and exceptional ability to develop antibiotic resistance. New kinds of antimicrobial therapies are urgently needed to treat patients for whom existing antibiotics are ineffective. The Rocket-miR application predicts targets of human miRNAs in bacterial and fungal pathogens, rapidly identifying candidate miRNA-based antimicrobials. The application's target audience are microbiologists that have the laboratory resources to test the application's predictions. The Rocket-miR application currently supports 24 recognized human pathogens that are relevant to numerous diseases including cystic fibrosis, chronic obstructive pulmonary disease (COPD), urinary tract infections, and pneumonia. Furthermore, the application code was designed to be easily extendible to other human pathogens that commonly cause hospital-acquired infections.

Trial of Affordable Bone Conduction Headphones to Support a Deaf Child's Education in Malawi.

A 13-year-old child with hearing loss secondary to chronic serous otitis media and bilateral tympanic perforations had been unable to hear the teacher at school and unable to pass end-of-year exams. In 2020, she trialed a bone conduction headset paired by Bluetooth to a remote microphone and used this to support her hearing at school, socializing with friends and in the family home. Due to the COVID (COronaVIrus Disease) pandemic and a cholera epidemic, she was followed up 3 years later. The child (now 16 years old) reported using the headset every day for 3 years. Able to hear the teacher, she reported having passed school exams every year since using the device, and now had ambitions to study medicine.

Analysis of Pseudomonas aeruginosa transcription in an ex vivo cystic fibrosis sputum model identifies metal restriction as a gene expression stimulus.

Chronic Pseudomonas aeruginosa lung infections are a distinctive feature of cystic fibrosis (CF) pathology, that challenge adults with CF even with the advent of highly effective modulator therapies. Characterizing P. aeruginosa transcription in the CF lung and identifying factors that drive gene expression could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa gene expression studies in laboratory culture models designed to model the CF lung environment, we employed an ex vivo sputum model in which laboratory strain PAO1 was incubated in sputum from different CF donors. As part of the analysis, we compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in artificial sputum medium (ASM). Analyses focused on genes that were differentially expressed between sputum and ASM and genes that were most highly expressed in sputum. We present a new approach that used sets of genes with correlated expression, identified by the gene expression analysis tool eADAGE, to analyze the differential activity of pathways in P. aeruginosa grown in CF sputum from different individuals. A key characteristic of P. aeruginosa grown in expectorated CF sputum was related to zinc and iron acquisition, but this signal varied by donor sputum. In addition, a significant correlation between P. aeruginosa expression of the H1-type VI secretion system and corrector use by the sputum donor was observed. These methods may be broadly useful in looking for variable signals across clinical samples.

An In Vitro Medium for Modeling Gut Dysbiosis Associated with Cystic Fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting non-CF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of gut dysbiosis in CF.

Comparative effects of CFTR modulators on phagocytic, metabolic and inflammatory profiles of CF and nonCF macrophages.

Macrophage dysfunction has been well-described in Cystic Fibrosis (CF) and may contribute to bacterial persistence in the lung. Whether CF macrophage dysfunction is related directly to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in macrophages or an indirect consequence of chronic inflammation and mucostasis is a subject of ongoing debate. CFTR modulators that restore CFTR function in epithelial cells improve global CF monocyte inflammatory responses but their direct effects on macrophages are less well understood. To address this knowledge gap, we measured phagocytosis, metabolism, and cytokine expression in response to a classical CF pathogen, Pseudomonas aeruginosa in monocyte-derived macrophages (MDM) isolated from CF F508del homozygous subjects and nonCF controls. Unexpectedly, we found that CFTR modulators enhanced phagocytosis in both CF and nonCF cohorts. CFTR triple modulators also inhibited MDM mitochondrial function, consistent with MDM activation. In contrast to studies in humans where CFTR modulators decreased serum inflammatory cytokine levels, modulators did not alter cytokine secretion in our system. Our studies therefore suggest modulator induced metabolic effects may promote bacterial clearance in both CF and nonCF monocyte-derived macrophages.

Development of the intestinal microbiome in cystic fibrosis in early life.

Cystic fibrosis (CF) is a heritable disease that causes altered physiology at mucosal sites; these changes result in chronic infections in the lung, significant gastrointestinal complications as well as dysbiosis of the gut microbiome, although the latter has been less well explored. Here, we describe the longitudinal development of the gut microbiome in a cohort of children with CF (cwCF) from birth through early childhood (0-4 years of age) using 16S rRNA gene amplicon sequencing of stool samples as a surrogate for the gut microbiota. Similar to healthy populations, alpha diversity of the gut microbiome increases significantly with age, but diversity plateaus at ~2 years of age for this CF cohort. Several taxa that have been associated with dysbiosis in CF change with age toward a more healthy-like composition; notable exceptions include Akkermansia, which decreases with age, and Blautia, which increases with age. We also examined the relative abundance and prevalence of nine taxa associated with CF lung disease, several of which persist across early life, highlighting the possibility of the lung being seeded directly from the gut early in life. Finally, we applied the Crohn's Dysbiosis Index to each sample, and found that high Crohn's-associated dysbiosis early in life (<2 years) was associated with significantly lower Bacteroides in samples collected from 2 to 4 years of age. Together, these data comprise an observational study that describes the longitudinal development of the CF-associated gut microbiota and suggest that early markers associated with inflammatory bowel disease may shape the later gut microbiota of cwCF. IMPORTANCE Cystic fibrosis is a heritable disease that disrupts ion transport at mucosal surfaces, causing a buildup of mucus and dysregulation of microbial communities in both the lungs and the intestines. Persons with CF are known to have dysbiotic gut microbial communities, but the development of these communities over time beginning at birth has not been thoroughly studied. Here, we describe an observation study following the development of the gut microbiome of cwCF throughout the first 4 years of life, during the critical window of both gut microbiome and immune development. Our findings indicate the possibility of the gut microbiota as a reservoir of airway pathogens and a surprisingly early indication of a microbiota associated with inflammatory bowel disease.

PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Recurrent croup is a good indicator of underlying paediatric airway issues: A 10-year retrospective cohort study of airway endoscopy.

OBJECTIVE: Children with a history of recurrent croup alert the ENT clinician to the potential for underlying laryngotracheal pathology. There is equipoise about the likelihood of identifying any underlying structural issues or subglottic stenosis in those children who undergo airway assessment. METHODS: A retrospective cohort study in a tertiary UK paediatric hospital of a decade of children with recurrent croup who underwent a rigid laryngo-tracheo-bronchoscopy (airway endoscopy). MAIN OUTCOME(S): airway pathology seen on endoscopy and need for further airway surgery. RESULTS: In ten years, 139 children underwent airway endoscopy for recurrent croup. Operative findings were abnormal in 62 (45 %) cases. Twelve cases (9%) had subglottic stenosis. Although recurrent croup was more common in males (78% of cases), this was not found to predispose them to operative findings. Children with previous intubations had >2 times the risk of abnormal findings and children born prematurely (<37 wks) had a trend towards abnormal operative findings versus children with no airway findings in our cohort. Even in those patients with abnormal findings, none necessitated further airway surgery. CONCLUSIONS: Surgeons and parents can be reassured that rigid airway endoscopy for children with recurrent croup demonstrated high diagnostic utility but will rarely lead to further surgical intervention. Greater understanding about recurrent croup may require consensus clarification about definitions of recurrent croup and/or a universal adoption of a minimum standard operative record or grading system after rigid endoscopy for recurrent croup.

Foreign body impaction of the hard palate: tabletop party confetti mimicking button batteries in two infants.

We present the first two cases in the literature of tabletop party confetti mimicking button batteries in two infants. Both patients presented to the Emergency Department with an incidentally noticed shiny, metallic appearing, disc-shaped foreign body impacted in the hard palate. Both objects were understandably misdiagnosed as button batteries. The first patient required foreign body retrieval by ENT under general anaesthesia, whilst the second underwent retrieval safely in the Emergency Department. Tabletop party confetti should be considered in patients presenting with a suspected button battery impaction of the hard palate, which will drastically change the approach to clinical management and potentially minimise harms.

CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.