CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors

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Surgeon case volume and the risk of complications following surgeries of displaced intra-articular calcaneal fracture

PurposeThis study aimed to address the relationship between surgeon volume and the risk of complications following surgeries of displaced intra-articular calcaneal fractures (DIACFs).MethodsWe retrospectively reviewed the medical records and the follow-up registers for patients who underwent open reduction and internal fixation with plate/screws in our center between January 2015 and June 2020. Surgeon volume was defined as the number of surgically treated calcaneal fractures within the past 12 months, and was dichotomized on basis of the optimal cut-off value. The outcome measure was the documented overall complications within 1 year after surgery. Four logistics regression models were constructed to examine the potential relationship between surgeon volume and complications.ResultsAmong 585 patients, 49 had documented complications, representing an overall rate of 8.4%. The overall complication rate was 20.0% (22/111) in patients operated on by the low-volume surgeons and 5.7% (27/474) by the high-volume surgeons, with a significant difference (p < 0.001). The 4 multivariate analyses showed steady and robust inverse volume-complication relationship, with OR ranging from 3.8 to 4.4. The restricted cubic splines adjusted for total covariates showed the non-linear fitting “L-shape” or “reverse J-shape” curve (p = 0.041), and the OR was reduced until 10 cases, beyond which the curve leveled.ConclusionsOur findings reflected the important role of maintaining necessary operative cases, potentially informing optimized surgical care management.     

Computational analysis of renal artery flow characteristics by modeling aortoplasty and aortic bypass interventions for abdominal aortic coarctation

ObjectiveSuprarenal abdominal aortic coarctation (SAAC) alters flow and pressure patterns to the kidneys and is often associated with severe angiotensin-mediated hypertension refractory to drug therapy. SAAC is most often treated by a thoracoabdominal bypass (TAB) or patch aortoplasty (PA). It is currently unclear what effect these interventions have on renal flow and pressure waveforms. This study, using retrospective data from a patient with SAAC subjected to a TAB, undertook computational modeling to analyze aortorenal blood flow preoperatively as well as postoperatively after a variety of TAB and PA interventions.MethodsPatient-specific anatomic models were constructed from preoperative computed tomography angiograms of a 9-year-old child with an isolated SAAC. Fluid-structure interaction (FSI) simulations of hemodynamics were performed to analyze preoperative renal flow and pressure waveforms. A parametric study was then performed to examine the hemodynamic impact of different bypass diameters and patch oversizing.ResultsPreoperative FSI results documented diastole-dominated renal perfusion with considerable high-frequency disturbances in blood flow and pressure. The postoperative TAB right and left kidney volumes increased by 58% and 79%, respectively, reflecting the increased renal artery blood flows calculated by the FSI analysis. Postoperative increases in systolic flow accompanied decreases in high-frequency disturbances, aortic pressure, and collateral flow after all surgical interventions. In general, lesser degrees of high-frequency disturbances followed PA interventions. High-frequency disturbances were eliminated with the 0% PA in contrast to the 30% and 50% PA oversizing and TAB interventions, in which these flow disturbances remained.ConclusionsBoth TAB and PA dramatically improved renal artery flow and pressure waveforms, although disturbed renal waveforms remained in many of the surgical scenarios. Importantly, only the 0% PA oversizing scenario eliminated all high-frequency disturbances, resulting in nearly normal aortorenal blood flow. The study also establishes the relevance of patient-specific computational modeling in planning interventions for the midaortic syndrome.     

Avoiding iatrogenic vascular injury in tibial external fixation with half pins. An in-vivo study based on CT angiography

BackgroundExternal fixation is an important tool in the management of variety of tibial fractures. Appropriate half pin insertion is important, to provide stable fixation without compromising the surgical field for definitive surgical procedures, and avoiding further damage to the important structures of the traumatized limb. There is paucity of literature about the optimal trajectories and safe corridors for half pins insertion based on in vivo studies. The available studies are based on anatomic atlases, cadaveric studies or half pin related complications.The aim of the current study is to present the findings of CT angiograms, in patients with external fixation of tibia, to enhance our understanding of optimal trajectories in safe corridors for half pins insertion.Material and methodsWe performed a retrospective study of patients with external fixators on the tibia, who had undergone CT angiogram as part of pre-operative planning for orthoplastic reconstructive procedures. The relationship between the tips of the fixator half pins and named vessels of the leg were analyzed, pins within 5 mm of a named vessel were considered to be a risk of causing iatrogenic injury.ResultsA total 51 patients, with in situ temporizing external fixators, with 134 half pins in different segments of the tibia were analyzed. More than 5 mm of penetration beyond the far cortex was noted in 47%, while in another 16% of pins penetration was more than 10 mm beyond the cortex. A tip to vessel distance (TVD) of 5 mm or less was noted in 28/134 (21%) of the pins, which highlights potential risk to the neurovascular bundles of the leg.ConclusionRisk of iatrogenic injury to neurovascular structures from half pin insertion can be reduced by meticulous use of fluoroscopy, by avoiding penetration beyond the far cortex, and avoiding exiting with half pins on the lateral surface in the distal 1/3rd of segment II of tibia. Moreover observing optimal trajectories and safe corridors for pin insertion, and selection of appropriate type of half pin can mitigate the risk to these structures.     

Optimization of backpack loads using gait parameters in school boys

BackgroundBackpacks are an efficient way of manual carriage used by people of all ages, and is commonly used by schoolchildren. Carrying heavy backpacks may result in cumulative trauma later in life due to biomechanical adaptations during gait. Gait parameters are known to be sensitive to force vectors, which can be altered by load carrying. This study attempts to find the most favorable backpack weight using gait changes as an indicator.MethodsThis was an observational study conducted on twenty typically developing boys aged between 9 and 14 years. Gait analysis was done using standard recommendations, with increasing backpack loads with respect to their body weights. Gait cycles were captured using video cameras and analyzed using Kinovea0.8.25 motion analyzing software.ResultsAs the backpack load increased, significant kinematic changes were noted in the child's ankle, knee, and hip joints. These changes were evident when the backpack load increased beyond 15% of their body weight.ConclusionsThis study concludes that the optimum weight that can be carried without having an impact on dynamic posture will be less than 15% of the body weight.     

臭茉莉叶总黄酮提取工艺优化及月周期变化研究

目的:采用超声辅助正交试验法优化臭茉莉叶总黄酮的提取工艺,并进一步研究臭茉莉叶中总黄酮1年内每月含量变化趋势,探讨臭茉莉叶最佳采收时间。方法:以提取液中总黄酮含量为指标,选择乙醇浓度(%)、超声时间(min)、料液比、提取温度(℃)等为考察因素,采用正交试验法L9(34)确定臭茉莉叶提取液中总黄酮的最优工艺。结果:臭茉莉叶所含总黄酮最优提取工艺条件:乙醇浓度70%、超声时间60min、料液比1∶25、提取温度50℃。3月份总黄酮/药材量是0.0235mg·g-1为最高。结论:该提取工艺快捷以及操作简便,适用于臭茉莉叶总黄酮的提取。结合总黄酮月周期变化规律,可以确定1年中5月份和7月份为最佳采摘期。     

Roles of helix H69 of 23S rRNA in translation initiation

Initiation of translation involves the assembly of a ribosome complex with initiator tRNA bound to the peptidyl site and paired to the start codon of the mRNA. In bacteria, this process is kinetically controlled by three initiation factors—IF1, IF2, and IF3. Here, we show that deletion of helix H69 (∆H69) of 23S rRNA allows rapid 50S docking without concomitant IF3 release and virtually eliminates the dependence of subunit joining on start codon identity. Despite this, overall accuracy of start codon selection, based on rates of formation of elongation-competent 70S ribosomes, is largely uncompromised in the absence of H69. Thus, the fidelity function of IF3 stems primarily from its interplay with initiator tRNA rather than its anti-subunit association activity. While retaining fidelity, ∆H69 ribosomes exhibit much slower rates of overall initiation, due to the delay in IF3 release and impedance of an IF3-independent step, presumably initiator tRNA positioning. These findings clarify the roles of H69 and IF3 in the mechanism of translation initiation and explain the dominant lethal phenotype of the ∆H69 mutation.Translation initiation can be divided into two major stages in bacteria. The first stage involves assembly of the 30S initiation complex (30SIC). Facilitated by three initiation factors, initiator tRNA (N-formyl-methionyl-tRNA^fMet, or fMet-tRNA^fMet) binds to the peptidyl (P) site of the 30S subunit and pairs with the start codon on the mRNA. During the second stage, the 50S subunit associates with the 30SIC and triggers dissociation of the initiation factors, leaving the 70S initiation complex (70SIC) with fMet-tRNA^fMet in the P site, ready for elongation. Because initiation is the rate-limiting step of translation and establishes the reading frame, efficient and accurate assembly of the 70SIC is critical for cell survival.30SIC assembly can be considered a largely random-order process, although there is a preferred kinetic pathway of ligand binding. IF2 and IF3 are generally first to bind to the 30S subunit, followed by IF1 and fMet-tRNA^fMet, whereas the timing of mRNA binding depends on its sequence context and cellular concentration (1). The three initiation factors reciprocally stabilize one another in the 30SIC, and their binding induces a conformational change of the subunit, including a clockwise rotation of the head domain (2). IF1 is an 8-kDa protein that binds to helix h44, the 530 loop, and the S12 region and blocks the 30S aminoacyl (A) site (3). IF2 is a multidomain ribosome-dependent GTPase that makes extensive contacts with both the 30S subunit and fMet-tRNA^fMet. Domains G3 and C1 of IF2 bind helix h5 and h14 of 16S rRNA, the N-terminal domain (NTD) interacts with S16 and IF1, and domain C2 recognizes the acceptor stem and fMet moiety of fMet-tRNA^fMet (2, 4). These interactions contribute to functions of IF2 in increasing the on rate of fMet-tRNA^fMet binding and discriminating against elongator tRNAs (5). IF3 consists of two globular domains connected by a flexible linker (6, 7). Based on structural studies of the 30SIC, the C-terminal domain (CTD) binds to the 30S platform near helices h23, h24, and h45 of 16S rRNA (8), whereas the NTD of IF3 has been modeled to interact with the elbow region of fMet-tRNA^fMet (2). The presence of IF3 in the 30SIC induces a conformational change of IF2 and fMet-tRNA^fMet (2, 9) and increases both on and off rates of tRNA binding (5). IF1 enhances the effects of IF2 and IF3 on tRNA stability (5). Together, all three initiation factors tune the kinetics of fMet-tRNA^fMet binding for optimal efficiency and fidelity.Formation of 70SIC is highly dependent on fMet-tRNA^fMet and IF2 (10), which together provide a large surface area complementary to the 50S interface (11, 12). 50S docking stimulates the GTPase activity of IF2 (13), presumably through the interaction between the sarcin–ricin loop and IF2 G domain, and triggers a large conformational change that favors factor dissociation (12). This is accompanied by movement of the initiator tRNA into the P/P site (14). IF3 inhibits subunit joining, an effect more pronounced in the presence of noncanonical codon–anticodon base pairing (5, 15). The mechanism by which IF3 prevents spurious initiation likely involves its interplay with fMet-tRNA^fMet. IF3 and tRNA are mutually destabilizing; thus, noncanonical base pairing delays IF3 dissociation and 70SIC formation (10, 16, 17). More recently, it was shown that the conformation of IF3 in the 30SIC is also sensitive to the identity of the start codon (18).Helix H69 of 23S rRNA is a highly conserved element located at the 50S interface and interacts extensively with helix h44 of 16S rRNA through intersubunit bridge B2a (Fig. 1) (19). In the elongation complex, H69 also makes direct contact with P-site tRNA at nucleotides 11–13 and 24–25 in the D stem-loop region (20). Deletion of H69 (ΔH69, Fig. 1C) has been shown to cause dominant lethality in vivo and a severe subunit association defect in vitro that can only be partially rescued in the presence of mRNA, tRNA, and a high concentration of Mg²⁺ (21). Despite these strong effects, ΔH69 ribosomes are fully competent for in vitro poly-Phe synthesis (21) and are only mildly affected in translocation (22). During subunit association, bridge B2a is among the earliest interactions formed between the two subunits (23), and H69 is in close proximity to the binding sites of initiator tRNA and IF3 (11, 12). Thus, the interplay between H69 and these ligands may be central to the molecular mechanism of 70SIC formation.Open in a separate windowFig. 1.Location and structure of H69 in the ribosome. (A) Secondary structure of H69 and its location in the 23S rRNA [schematic diagram adapted from the Comparative RNA Website (35)]. Nucleotides making contact with P-site tRNA are highlighted in orange, regions predicted to interact with IF2 are marked with a pink line, and regions predicted to clash with IF3 are indicated by a dashed purple line. (B) Tertiary structure of H69 in the 70S ribosome [based on Protein Data Bank ID codes 2WDG and 2WDI (20)]. Atoms involved in interactions between H69 and P-site tRNA or helix h44 of 16S rRNA are shown in spheres. (C) Nucleotides replacing the native sequence (nt 1906–1930) in ΔH69, ΔLoop, and ΔLoop+4 mutants.In the current study, we investigate the effects of H69 deletion on 70SIC formation. We find that the ability of IF3 to regulate 50S docking depends largely on H69. Loss of H69 delays the release of IF3 and inhibits a subsequent conformational change of fMet-tRNA^fMet that leads to formation of elongation-competent 70SIC. Our data support a model in which H69 is critical for coupling IF3 dissociation with subunit joining as well as regulating fMet-tRNA^fMet movement during the second stage of translation initiation.