ABSTRACT
For over 15 years, canine genetics research relied on a reference assembly from a Boxer breed dog named Tasha (i.e., canFam3.1). Recent advances in long-read sequencing and genome assembly have led to the development of numerous high-quality assemblies from diverse canines. These assemblies represent notable improvements in completeness, contiguity, and the representation of gene promoters and gene models. Although genome graph and pan-genome approaches have promise, most genetic analyses in canines rely upon the mapping of Illumina sequencing reads to a single reference. The Dog10K consortium, and others, have generated deep catalogs of genetic variation through an alignment of Illumina sequencing reads to a reference genome obtained from a German Shepherd Dog named Mischka (i.e., canFam4, UU_Cfam_GSD_1.0). However, alignment to a breed-derived genome may introduce bias in genotype calling across samples. Since the use of an outgroup reference genome may remove this effect, we have reprocessed 1929 samples analyzed by the Dog10K consortium using a Greenland wolf (mCanLor1.2) as the reference. We efficiently performed remapping and variant calling using a GPU-implementation of common analysis tools. The resulting call set removes the variability in genetic differences seen across samples and breed relationships revealed by principal component analysis are not affected by the choice of reference genome. Using this sequence data, we inferred the history of population sizes and found that village dog populations experienced a 9-13 fold reduction in historic effective population size relative to wolves.
ABSTRACT
This work presents the development and testing of an experimental web-based SDR (software-defined radio) monitoring system for indirect solar activity detection, which has the ability to estimate and potentially predict various events in space and on earth, including solar flares, coronal mass ejections, and geomagnetic storms. The proposed system can be used to investigate the effect of solar activity on the propagation of very-low-frequency (VLF) signals. The advantages and benefits of the given approach are as follows: increasing measurement accuracy and eventual solar activity identification by combining measurements from multiple spatially distributed SDRs. The verification process involves carrying out several experiments comparing data from the GOES satellite system and the Dunksin SuperSID system with information received by the SDR monitoring system. Then, utilizing Pearson correlation coefficients, the measured data from the SDRs, along with those from the GOES satellite system and the Dunsing monitoring station, are investigated. At the time of a solar flare, the correlation value is above 90% for most of the stations used. Combining the signal-to-noise ratio via summation also shows an improvement in the results, with a correlation above 98%.
ABSTRACT
Objective: To identify patients at high risk of delayed in-hospital functional recovery after knee replacement surgery by developing and validating a prediction model, including a combination of preoperative physical fitness parameters and patient characteristics. Design: Retrospective cohort study using binary logistic regression. Setting: University hospital, orthopedic department. Participants: 260 adults (N=260) (≥18y) with knee osteoarthritis awaiting primary unilateral total knee arthroplasty and assessed during usual care between 2016 and 2020. Intervention: Not applicable. Main Outcome Measures: Time to reach in-hospital functional independence (in days), measured by the modified Iowa Level of Assistance Scale. A score of 0 means completely independent. Potential predictor variables are a combination of preoperative physical fitness parameters and patient characteristics. Results: Binary logistic regression modeling was applied to develop the initial model. A low de Morton Mobility Index (DEMMI), walking aid use indoors, and a low handgrip strength (HGS) were the most important predictors of delayed in-hospital recovery. This model was internally validated and had an optimism-corrected R2 of 0.07 and an area under curve of 61.2%. The probability of a high risk of delayed in-hospital recovery is expressed by the following equation:Phighrisk=(1/(1+e(-(2.638-0.193×DEMMI+0.879×indoorwalkingaid-0.007×HGS))))×100%. Conclusions: The model has a low predictive value and a poor discriminative ability. However, there is a positive association between preoperative physical fitness and postoperative recovery of physical function. The validity of our model to distinguish between high and low risk, based on preoperative fitness values and patient characteristics, is limited.
ABSTRACT
Oocyte maturation is accompanied by changes in abundances of thousands of mRNAs, many degraded and many preferentially stabilized. mRNA stability can be regulated by diverse features including GC content, codon bias, and motifs within the 3'-untranslated region (UTR) interacting with RNA binding proteins (RBPs) and miRNAs. Many studies have identified factors participating in mRNA splicing, bulk mRNA storage, and translational recruitment in mammalian oocytes, but the roles of potentially hundreds of expressed factors, how they regulate cohorts of thousands of mRNAs, and to what extent their functions are conserved across species has not been determined. We performed an extensive in silico cross-species analysis of features associated with mRNAs of different stability classes during oocyte maturation (stable, moderately degraded, and highly degraded) for five mammalian species. Using publicly available RNA sequencing data for germinal vesicle (GV) and MII oocyte transcriptomes, we determined that 3'-UTR length and synonymous codon usage are positively associated with stability, while greater GC content is negatively associated with stability. By applying machine learning and feature selection strategies, we identified RBPs and miRNAs that are predictive of mRNA stability, including some across multiple species and others more species-restricted. The results provide new insight into the mechanisms regulating maternal mRNA stabilization or degradation.NEW & NOTEWORTHY Conservation across species of mRNA features regulating maternal mRNA stability during mammalian oocyte maturation was analyzed. 3'-Untranslated region length and synonymous codon usage are positively associated with stability, while GC content is negatively associated. Just three RNA binding protein motifs were predicted to regulate mRNA stability across all five species examined, but associated pathways and functions are shared, indicating oocytes of different species arrive at comparable physiological destinations via different routes.
Subject(s)
MicroRNAs , RNA, Messenger, Stored , Animals , Mammals/genetics , Mammals/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Oocytes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Messenger, Stored/genetics , RNA, Messenger, Stored/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Untranslated Regions , FemaleABSTRACT
BACKGROUND CONTEXT: Prior studies have demonstrated a close association between cervical spine fractures and blunt cerebrovascular injuries (BCVI). Undiagnosed BCVI is a feared complication because of the potentially catastrophic outcomes in a missed posterior circulation stroke. Computed tomography angiography (CTA) is commonly used to screen BCVI in the trauma setting. However, determining which cervical fracture patterns mandate screening is still not clearly known. PURPOSE: The aim of this retrospective review is to further elucidate which fracture patterns are associated with BCVI when using CTA and may mandate screening. STUDY DESIGN/SETTING: Retrospective cohort study. PATIENT SAMPLE: All patients that presented to our trauma and emergency departments with a blunt cervical spine fracture from January 2018 to December 2021. Inclusion criteria included blunt cervical trauma and the use of CTA for BCVI screening. Exclusion criteria included patients under the age of 18, penetrating cervical trauma, and use any imaging modality besides CTA for BCVI screening. OUTCOME MEASURES: Patient demographics (age, gender, Glasgow coma scale, hospital length of stay (LOS), intensive care unit LOS, mechanism of energy of injury, polytrauma status), fracture location, fracture pattern (anterior arch, dens, dislocations/subluxations, facet, hangman, Jefferson, lamina, lateral mass, occipital condyle dissociation, occipital condyle, pedicle, posterior arch, spinous process, transverse process, transverse foramen, and vertebral body), and whether the patient sustained a BCVI or CVA. METHODS: If a patient had multiple fracture levels or fracture patterns, each level and pattern was counted as a separate BCVI. Multilevel fractures were defined as any patient with fractures at two distinct cervical levels. Differences between the patients who had a BCVI and those who did not were analyzed using independent sample t-tests for continuous variables and the chi-square or Fisher exact test for categorical variables. Odds ratios and 95% confidence intervals were calculated to assess likelihood between patient characteristics/fracture characteristics and BCVI. RESULTS: A total of 690 patients were identified as having a blunt cervical spine injury. A total of 453 patients (66%) underwent screening for BCVI with CTA. Among patients who underwent CTA, BCVI was diagnosed in 138 patients (30%), VAI in 119 patients (26%), CAI in 30 patients (7%), and 11 patients were diagnosed with both a VAI and CAI (2%). Overall, among all patients there were 9 strokes, all in patients identified with a BCVI (1%). No individual cervical level was associated with increased risk of BCVI, but when combined, OC-C3 fractures were associated with an increased risk (OR: 1.4, 95% CI: 1.0-1.9, p-value: .006). Multilevel fractures were also associated with an increased risk (OR: 1.7, 95% CI: 1.1-2.3, p-value: .01). The only fracture pattern associated with increased risk of BCVI were fractures associated with a dislocation/subluxation (OR: 3.8, 95% CI: 1.9-7.8, p-value = .0001). CONCLUSIONS: The only fracture pattern associated with an increased risk of BCVI were fractures associated with dislocation/subluxation. The only fracture levels associated with BCVI were combined OC-C3 and multilevel fractures. We recommend that any upper cervical fracture (OC-C3), multilevel fracture, or fracture with dislocation/subluxation undergo screening for BCVI.
Subject(s)
Cerebrovascular Trauma , Joint Dislocations , Spinal Fractures , Stroke , Wounds, Nonpenetrating , Humans , Computed Tomography Angiography/adverse effects , Retrospective Studies , Tomography, X-Ray Computed , Angiography/adverse effects , Wounds, Nonpenetrating/complications , Wounds, Nonpenetrating/diagnostic imaging , Spinal Fractures/diagnostic imaging , Spinal Fractures/epidemiology , Spinal Fractures/etiology , Joint Dislocations/complications , Cerebrovascular Trauma/diagnostic imaging , Cerebrovascular Trauma/epidemiology , Cerebrovascular Trauma/complicationsABSTRACT
In clustered randomized controlled trials (RCTs), sample recruitment is often conducted after cluster randomization. This timing can lead to recruitment bias if access to the intervention affects the composition of study-eligible cluster entrants and study consenters. This article develops a potential outcomes framework in such settings that yields a causal estimand that pertains to the always-recruited in either research condition. A consistent inverse probability weighting (IPW) estimator is developed using data on recruits only, and a generalized estimating equations approach is used to obtain robust clustered SE estimators that adjust for estimation error in the IPW weights. A simple data collection strategy is discussed to improve the predictive accuracy of the logit propensity score models. Simulations show that the IPW estimator achieves nominal confidence interval coverage under the assumed identification conditions. An empirical application demonstrates the methods using data from an RCT testing the effects of a behavioral health intervention in schools. An R program for estimation is available for download.
Subject(s)
Bias , Randomized Controlled Trials as Topic , Humans , Causality , Computer Simulation , Logistic Models , Propensity ScoreABSTRACT
Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreERT2 knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreERT2 transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.
Subject(s)
CRISPR-Cas Systems , Tamoxifen , Mice , Animals , Tamoxifen/pharmacology , Mice, Transgenic , Red Fluorescent Protein , Integrases/genetics , Integrases/metabolismABSTRACT
INTRODUCTION: This study investigated endodontically treated teeth that were replaced by dental implants at the University of North Carolina (UNC) at Chapel Hill School of Dentistry. The primary objective of this study was to determine the reasons leading to the extraction of endodontically treated teeth and their subsequent replacement with dental implants. The secondary objective was to evaluate the proportion of these teeth that, according to experienced endodontists, could have been preserved. METHODS: The UNC-Chapel Hill's dental electronic health records between 2004 and 2019 were probed for implant placement that replaced root canal-treated teeth. Preextraction radiographs and clinical charts were examined to ascertain the primary reason related to the extraction and to compile a profile for each case. In cases in which endodontic failure was the primary reason for extraction, radiographs and clinical findings were evaluated by 2experienced endodontists to assess potential treatment options. RESULTS: Between 2004 and 2019, 29.3% (1564 of 5229) of teeth replaced by dental implants at UNC School of Dentistry had undergone root canal treatment, with the mandibular first molar being the most commonly replaced tooth. The leading reasons for extraction were recurrent caries associated with defective restoration (26.6%), fracture of coronal structure (21.5%), vertical root fracture (20.9%), compromised periodontal condition (13.8%), and endodontic failure (2.4%). Two experienced endodontists evaluated extractions due to endodontic failure and concluded that 61.7% of them could have been candidates for endodontic retreatment. CONCLUSION: Substantial loss of tooth structure was the leading cause of extraction of root canal-treated teeth, followed by vertical root fracture and periodontal disease. Although endodontic failure constituted a minor portion of the reasons for extraction, a considerable number of teeth were extracted due to vertical root fractures following root canal treatment. A significant proportion of the extracted teeth due to endodontic failure could have been considered as potential candidates for endodontic retreatment.
Subject(s)
Dental Caries , Dental Implants , Periodontal Diseases , Tooth, Nonvital , Humans , Tooth, Nonvital/diagnostic imaging , Retrospective Studies , Root Canal Therapy/adverse effectsABSTRACT
To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn2+-dependent gRNA-free DNA cleavage by â¼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.
Subject(s)
DNA Cleavage , Gene Editing , Catalytic Domain , CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems , Endonucleases , Streptococcus pyogenes/geneticsABSTRACT
[This corrects the article DOI: 10.3389/fpubh.2023.1195751.].
ABSTRACT
Background: Vaccine hesitancy has hampered the control of COVID-19 and other vaccine-preventable diseases. Methods: We conducted a national internet-based, quasi-experimental study to evaluate COVID-19 vaccine informational videos. Participants received an informational animated video paired with the randomized assignment of (1) a credible source (differing race/ethnicity) and (2) sequencing of a personal narrative before or after the video addressing their primary vaccine concern. We examined viewing time and asked video evaluation questions to those who viewed the full video. Results: Among 14,235 participants, 2,422 (17.0%) viewed the full video. Those who viewed a personal story first (concern video second) were 10 times more likely to view the full video (p < 0.01). Respondent-provider race/ethnicity congruence was associated with increased odds of viewing the full video (aOR: 1.89, p < 0.01). Most viewers rated the informational video(s) to be helpful, easy to understand, trustworthy, and likely to impact others' vaccine decisions, with differences by demographics and also vaccine intentions and concerns. Conclusion: Using peer-delivered, personal narrative, and/or racially congruent credible sources to introduce and deliver vaccine safety information may improve the openness of vaccine message recipients to messages and engagement.
Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , Ethnicity , Vaccination , IntentionABSTRACT
Recent advances in long-read sequencing have enabled the creation of reference-quality genome assemblies for multiple individuals within a species. In particular, 8 long-read genome assemblies have recently been published for the canine model (dogs and wolves). These assemblies were created using a range of sequencing and computational approaches, with only limited comparisons described among subsets of the assemblies. Here we present 3 high-quality de novo reference assemblies based upon Oxford Nanopore long-read sequencing: 2 Bernese Mountain Dogs (BD & OD) and a Cairn terrier (CA611). These breeds are of particular interest due to the enrichment of unresolved genetic disorders. Leveraging advancement in software technologies, we utilized published data of Labrador Retriever (Yella) to generate a new assembly, resulting in a â¼280-fold increase in continuity (N50 size of 91 kbp vs 25.75 Mbp). In conjunction with these 4 new assemblies, we uniformly assessed 8 existing assemblies for generalized quality metrics, sequence divergence, and a detailed BUSCO assessment. We identified a set of â¼400 conserved genes during the BUSCO analysis missing in all assemblies. Genome-wide methylation profiles were generated from the nanopore sequencing, resulting in broad concordance with existing whole-genome and reduced-representation bisulfite sequencing, while highlighting superior overage of mobile elements. These analyses demonstrate the ability of Nanopore sequencing to resolve the sequence and epigenetic profile of canine genomes.
Subject(s)
Nanopores , Dogs , Animals , Methylation , Genome , Sequence Analysis, DNA , Software , High-Throughput Nucleotide SequencingABSTRACT
Excessive FSH doses during ovarian stimulation in the small ovarian reserve heifer (SORH) cause premature cumulus expansion and follicular hyperstimulation dysgenesis (FHD) in nearly all ovulatory-size follicles with predicted disruptions in cell-signaling pathways in cumulus cells and oocytes (before ovulatory hCG stimulation). These observations support the hypothesis that excessive FSH dysregulates cumulus cell function and oocyte maturation. To test this hypothesis, we determined whether excessive FSH-induced differentially expressed genes (DEGs) in cumulus cells identified in our previously published transcriptome analysis were altered independent of extreme phenotypic differences observed amongst ovulatory-size follicles, and assessed predicted roles of these DEGs in cumulus and oocyte biology. We also determined if excessive FSH alters cumulus cell morphology, and oocyte nuclear maturation before (premature) or after an ovulatory hCG stimulus or during IVM. Excessive FSH doses increased expression of 17 cumulus DEGs with known roles in cumulus cell and oocyte functions (responsiveness to gonadotrophins, survival, expansion, and oocyte maturation). Excessive FSH also induced premature cumulus expansion and oocyte maturation but inhibited cumulus expansion and oocyte maturation post-hCG and diminished the ability of oocytes with prematurely expanded cumulus cells to undergo IVF or nuclear maturation during IVM. Ovarian stimulation with excessive FSH is concluded to disrupt cumulus cell and oocyte functions by inducing premature cumulus expansion and dysregulating oocyte maturation without an ovulatory hCG stimulus yielding poor-quality cumulus-oocyte complexes that may be incorrectly judged morphologically as suitable for IVF during ART.
Subject(s)
Cumulus Cells , Ovarian Reserve , Female , Cattle , Animals , Cumulus Cells/metabolism , Meiosis , Oocytes/metabolism , Follicle Stimulating Hormone/pharmacology , Follicle Stimulating Hormone/metabolism , Ovulation InductionABSTRACT
BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.
Subject(s)
Wolves , Dogs , Animals , Wolves/genetics , Chromosome Mapping , Alleles , Polymorphism, Single Nucleotide , Nucleotides , DemographyABSTRACT
CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)0), with sequences having smaller ΔGNN(17-20)0 showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.
Subject(s)
CRISPR-Cas Systems , RNA , RNA/genetics , Nucleotides , DNA/genetics , Gene Editing/methodsABSTRACT
Introduction: Vaccine hesitancy is a global health threat undermining control of many vaccine-preventable diseases. Patient-level education has largely been ineffective in reducing vaccine concerns and increasing vaccine uptake. We built and evaluated a personalized vaccine risk communication website called LetsTalkShots in English, Spanish and French (Canadian) for vaccines across the lifespan. LetsTalkShots tailors lived experiences, credible sources and informational animations to disseminate the right message from the right messenger to the right person, applying a broad range of behavioral theories. Methods: We used mixed-methods research to test our animation and some aspects of credible sources and personal narratives. We conducted 67 discussion groups (n = 325 persons), stratified by race/ethnicity (African American, Hispanic, and White people) and population (e.g., parents, pregnant women, adolescents, younger adults, and older adults). Using a large Ipsos survey among English-speaking respondents (n = 2,272), we tested animations aligned with vaccine concerns and specific to population (e.g., parents of children, parents of adolescents, younger adults, older adults). Results: Discussion groups provided robust feedback specific to each animation as well as areas for improvements across animations. Most respondents indicated that the information presented was interesting (85.5%), clear (96.0%), helpful (87.0%), and trustworthy (82.2%). Discussion: Tailored vaccine risk communication can assist decision makers as they consider vaccination for themselves, their families, and their communities. LetsTalkShots presents a model for personalized communication in other areas of medicine and public health.
Subject(s)
Communication , Vaccination , Vaccines , Adolescent , Aged , Child , Female , Humans , Pregnancy , Black or African American , Canada , Precision Medicine , Vaccination Hesitancy , Risk , Public Health , Health Promotion , Health Education/methods , Hispanic or Latino , White , Young Adult , ParentsABSTRACT
Gunshot wounds (GSWs) and total knee arthroplasty (TKA) are increasingly common, yet a GSW to a TKA is a rare injury. A 60-year-old man sustained an intra-articular GSW to a prior TKA. The patient was scheduled for irrigation and debridement with polyethylene liner exchange. Intraoperatively, the new polyethylene liner was unable to engage the tibial tray. Damage to the locking mechanism on the tibial tray was suspected so total revision proceeded. Upon inspection of the explanted components, it was noted that a bullet fragment offline from the missile trajectory had blocked the locking of the polyethylene liner in the tibial tray. Expeditious antibiotics should be given and meticulous debridement should be performed to avoid unnecessary total component revision.
ABSTRACT
CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.