Non-invasive fractional flow reserve derived from reduced-order coronary model and machine learning prediction of stenosis flow resistance.

BACKGROUND AND OBJECTIVE: Recently, computational fluid dynamics enables the non-invasive calculation of fractional flow reserve (FFR) based on 3D coronary model, but it is time-consuming. Currently, machine learning technique has emerged as an efficient and reliable approach for prediction, which allows saving a lot of analysis time. This study aimed at developing a simplified FFR prediction model for rapid and accurate assessment of functional significance of stenosis. METHODS: A reduced-order lumped parameter model (LPM) of coronary system and cardiovascular system was constructed for rapidly simulating coronary flow, in which a machine learning model was embedded for accurately predicting stenosis flow resistance at a given flow from anatomical features of stenosis. Importantly, the LPM was personalized in both structures and parameters according to coronary geometries from computed tomography angiography and physiological measurements such as blood pressure and cardiac output for personalized simulations of coronary pressure and flow. Coronary lesions with invasive FFR ≤ 0.80 were defined as hemodynamically significant. RESULTS: A total of 91 patients (93 lesions) who underwent invasive FFR were involved in FFR derived from machine learning (FFRML) calculation. Of the 93 lesions, 27 lesions (29.0%) showed lesion-specific ischemia. The average time of FFRML simulation was about 10 min. On a per-vessel basis, the FFRML and FFR were significantly correlated (r = 0.86, p < 0.001). The diagnostic accuracy, sensitivity, specificity, positive predictive value and negative predictive value were 91.4%, 92.6%, 90.9%, 80.6% and 96.8%, respectively. The area under the receiver-operating characteristic curve of FFRML was 0.984. CONCLUSION: In this selected cohort of patients, the FFRML improves the computational efficiency and ensures the accuracy. The favorable performance of FFRML approach greatly facilitates its potential application in detecting hemodynamically significant coronary stenosis in future routine clinical practice.

TREX2 enables efficient genome disruption mediated by paired CRISPR-Cas9 nickases that generate 3'-overhanging ends.

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

Small extrachromosomal circular DNA harboring targeted tumor suppressor gene mutations supports intratumor heterogeneity in mouse liver cancer induced by multiplexed CRISPR/Cas9.

BACKGROUND: Primary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer. METHODS: CRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells. RESULTS: After efficiently inducing autochthonous liver tumors in mice within 30-60 days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells. CONCLUSIONS: Small eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.

A simplified coronary model for diagnosis of ischemia-causing coronary stenosis.

BACKGROUND AND OBJECTIVE: The functional assessment of the severity of coronary stenosis from coronary computed tomography angiography (CCTA)-derived fractional flow reserve (FFR) has recently attracted interest. However, existing algorithms run at high computational cost. Therefore, this study proposes a fast calculation method of FFR for the diagnosis of ischemia-causing coronary stenosis. METHODS: We combined CCTA and machine learning to develop a simplified single-vessel coronary model for rapid calculation of FFR. First, a zero-dimensional model of single-vessel coronary was established based on CCTA, and microcirculation resistance was determined through the relationship between coronary pressure and flow. In addition, a coronary stenosis model based on machine learning was introduced to determine stenosis resistance. Computational FFR (cFFR) was then obtained by combining the zero-dimensional model and the stenosis model with inlet boundary conditions for resting (cFFRr) and hyperemic (cFFRh) aortic pressure, respectively. We retrospectively analyzed 75 patients who underwent clinically invasive FFR (iFFR), and verified the model accuracy by comparison of cFFR with iFFR. RESULTS: The average computing time of cFFR was less than 2 s. The correlations between cFFRr and cFFRh with iFFR were r = 0.89 (p < 0.001) and r = 0.90 (p < 0.001), respectively. Diagnostic accuracy, sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio for cFFRr and cFFRh were 90.7%, 95.0%, 89.1%, 76.0%, 98.0%, 8.7, 0.1 and 92.0%, 95.0%, 90.9%, 79.2%, 98.0%, 10.5, 0.1, respectively. CONCLUSIONS: The proposed model enables rapid prediction of cFFR and exhibits high diagnostic performance in selected patient cohorts. The model thus provides an accurate and time-efficient computational tool to detect ischemia-causing stenosis and assist with clinical decision-making.

Cerebral multi-autoregulation model based enhanced external counterpulsation treatment planning for cerebral ischemic stroke.

Enhanced external counterpulsation (EECP) treatment for cerebral ischemic stroke patients with differing severity of stenosis, is subject to uncertainties due to the varying effects of the cerebral autoregulation mechanism on haemodynamics. The current study reports the development of a cerebral multi-autoregulation (MR) mathematical model, based on cerebral arteriole regulation of neurogenic, vascular smooth muscle reflex and shear stress mechanisms which takes into account the severity of stenosis. The model was evaluated by comparison to authentic clinical measurements of cerebral autoregulatory efficiency. Then it was applied to a 0D/3D geometric multi-scale haemodynamic model of a cerebral artery. Haemodynamic indicators were calculated under different pressurization durations of EECP to evaluate the efficacy for different stenosis lesions. Moderate stenosis of 50% to 60% produced excessive time-averaged wall shear stress in the distal area of the stenosis (>7 Pa) during prolonged pressurization and may result in damage to vascular endothelial cells. However, prolonged pressurization did not result in haemodynamic risk for severe stenosis of 70% to 80%, indicating that the duration of pressurization may be extended with increasing severity of stenosis. The current MR model accurately simulated cerebral blood flow and has relevance to the simulation of cerebral haemodynamics in a clinical setting.

Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.

In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.

Left and right coronary artery blood flow distribution method based on dominant type.

The purpose of the current study was to investigate the effects of left/right coronary artery flow distribution on calculation of fractional flow reserve derived from coronary computed tomography angiography (FFRct) in different dominant types. First, 195 patients were collected to count the distribution ratios of the three categories: right dominance (RD), balanced dominance (BD), and left dominance (LD). Ratios of diameters of the left/right coronary arteries (DLCA :DRCA ) of the three types were calculated and used to represent the ratio of flow distribution (QLCA :QRCA ) in the dominant type method. The other method was known as the fixed ratio method (QLCA :QRCA  = 6:4). Second, a total of 73 patients with coronary artery disease (CAD) were enrolled for numerical calculation. A 0D/3D geometric multiscale model was used for the numerical simulation of FFR and the results of the fixed ratio method and the dominant type method were recorded as F-FFRct and D-FFRct. Lastly, invasive FFR(clinic-FFR)was used as a standard to evaluate the consistency and diagnostic performance of F-FFRct and D-FFRct. Corresponding flow distributions for the dominant type method were QLCA :QRCA  = 5:5 for RD, QLCA :QRCA  = 5.5:4.5 for BD, and QLCA :QRCA  = 6:4 for LD. D-FFRct showed a better correlation than F-FFRct (r = 0.85 vs. r = 0.81, both p < .001); the AUC (95%CI) were 0.974 (0.906-0.997, p < .0001) and 0.960 (0.886-0.992, p < .0001). Accuracy, specificity, sensitivity, positive predictive value (PPV) and negative predictive values (NPV) for D-FFRct and F-FFRct were 94.52%, 93.75%, 94.74%, 83.33%, 98.18% and 90.41%, 87.50%, 91.23%, 73.68%, 96.30%, respectively. Overall, the left/right coronary artery flow distribution was affected by the dominant type and the dominant type method was superior to the fixed ratio method in detecting coronary ischemic lesions.

Effect of the accordion technique on bone regeneration during distraction osteogenesis: A computational study.

BACKGROUND AND OBJECTIVE: Distraction osteogenesis (DO), a bone lengthening technique, is widely employed to treat congenital and acquired limb length discrepancies and large segmental bone defects. However, a major issue of DO is the prolonged consolidation phase (10-36 months) during which patients must wear a cumbersome external fixator. Attempts have been made to accelerate the healing process of DO by an alternating distraction and compression mode (so-called "accordion" technique or AT). However, it remains unclear how varied AT parameters affect DO outcomes and what the most effective AT mode is. METHODS: Based on an experimentally-verified mechanobiological model, we performed a parametric analysis via in silico simulation of the bone regeneration process of DO under different AT modes, including combinations of varied application times (AT began at week 1-8 of the consolidation phase), durations (AT was used continuously for 1 week, 2 weeks or 4 weeks) and rates (distraction or compression at 0.25, 0.5, 0.75, and 1 mm/12 h). The control group had no AT applied during the consolidation phase. RESULTS: Compared with the control group (no AT), AT applied at an early consolidation stage (e.g. week 1 of the consolidation phase) significantly enhanced bone formation and reduced the overall healing time. However, the effect of AT on bone healing was dependent on its duration and rate. Specifically, a moderate rate of AT (e.g. 0.5 mm/12 h) lasting for two weeks promoted blood perfusion recovery and bone regeneration, ultimately shortening the healing time. Conversely, over-high rates (e.g. 1 mm/12 h) and longer durations (e.g. 4 weeks) of AT adversely affected bone regeneration and blood perfusion recovery, thereby delaying bone bridging. CONCLUSIONS: These results suggest that the therapeutic effects of AT on DO are highly dependent of the AT parameters of choice. Under appropriate durations and rates, the AT applied at an early consolidation phase is beneficial for blood recovery and bone regeneration. These results may provide a basis for selecting effective AT modes to accelerate consolidation and reduce the overall treatment period of DO.

Deep learning-based prediction of coronary artery stenosis resistance.

Coronary artery stenosis resistance (SR) is a key factor for noninvasive calculations of fractional flow reserve derived from coronary CT angiography (FFRCT). Existing computational fluid dynamics (CFD) methods, including three-dimensional (3-D) computational and zero-dimensional (0-D) analytical models, are usually limited by high calculation cost or low precision. In this study, we have developed a multi-input back-propagation neural network (BPNN) that can rapidly and accurately predict coronary SR. A training data set including 3,028 idealized anatomic coronary artery stenosis models was constructed for 3-D CFD calculation of SR with specific blood flow boundaries. Based on 3-D calculation results, we established a BPNN whose input is geometric parameters and blood flow, whereas output is SR. Then, a test set (324 cases) was constructed to evaluate the performance of the BPNN model. To verify the validity and practicability of the network, BPNN prediction results were compared with 3-D CFD and 0-D analytical model results from patient-specific models. For test set, the mean square error (MSE) between CFD and prediction results was 2.97%, linear regression analysis indicating a good correlation between the two (P < 0.001). For 30 patient-specific models, the MSE of BPNN and the 0-D model were 3.26 and 9.7%, respectively. The calculation time for BPNN and the 3-D CFD model for 30 cases was about 2.15 s and 2 h, respectively. The present results demonstrate the practicability of using deep learning methods for fast and accurate predictions of coronary artery SR. Our study represents an advance in noninvasive calculations of FFRCT.NEW & NOTEWORTHY This study developed a multi-input back-propagation neural network (BPNN) that can be used to predict coronary artery stenosis resistance by inputting vascular geometric parameters and blood flow. Compared with previous studies, the network developed in this study can accurately and rapidly predict coronary artery stenosis resistance, which can not only meet clinical requirements but also reduce the cost of calculation duration. This study contributes to the noninvasive methods for the numerical calculation of fractional flow reserve derived from coronary CT angiography (FFRCT) and indicates that this technique can potentially be used for evaluating myocardial ischemia.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

DNA nicks induce mutational signatures associated with BRCA1 deficiency.

Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

The quantitative relationship between coronary microcirculatory resistance and myocardial ischemia in patients with coronary artery disease.

It was hypothesized that the microcirculatory resistance of resting state (Rm-res) might be a good predictor for ischemia. In this study, the quantitative relationship between Rm-res and myocardial ischemia in different stenosed degrees was explored and verified through retrospective analysis, and the diagnostic performance was evaluated. 136 patients were screened and divided into a training set (90 patients) and a validation set (46 patients). In the training set, Rm-res was calculated, and thresholds were determined by exploring the relationship between Rm-res and myocardial ischemia in different stenosed degrees. In the validation set, the diagnostic performance of the thresholds was verified. It was found that the 90 data mean difference (95%CI) of Rm-res between the ischemic group and the non-ischemic group was 63.03 (95 %CI: 25.72-100.34), p < 0.05. In the training set with stenosed degree 41-60%, 61-70%, 71-80%, and >81%, the average of Rm-res in the ischemic and non-ischemic groups were (80.79, 136.87), (96.41, 172.62), (128.99, 198.94) and (175.95, 310.79) mmHg/s/ml. The Rm-res thresholds were 87.18, 118.96, 142.35, and 177.39 mmHg/s/ml. In the validation set, the overall sensitivity, specificity, PPV, NPV, and accuracy were 73.3%, 77.4%, 61.1%, 85.7%, and 76.1%. In conclusion, Rm-res had a significant predictor on myocardial ischemia. As a smaller Rm-res represents greater myocardial mass perfusion, it is more likely that a stenosis will have a functional impact. Threshold analysis showed that Rm-res of different stenosed degrees was a quantitative predictor of myocardial ischemia, which could assist physicians with clinical treatment strategies.

Impact of coronary bifurcated vessels flow-diameter scaling laws on fractional flow reserve based on computed tomography images (FFRCT).

OBJECTIVE: To explore the influence of the blood flow-diameter scaling laws of $ \mathrm{Q}\mathrm{\alpha }{\mathrm{D}}^{3} $, $ \mathrm{Q}\mathrm{\alpha }{\mathrm{D}}^{2.7} $ and $ \text{Q}\alpha \text{D}{}^{7}\!\!\diagup\!\!{}_{3}\; $ on the numerical simulation of fraction flow reserve based on CTA images and to find the optimal exponents. METHODS: 1) 26 patients with coronary artery disease were screened according to the inclusion criteria; 2) Microcirculation resistance (Rm) was calculated under the 3, 2.7 and 7/3 power of the flow-diameter scaling law, which were recorded as 3Rm, 2.7Rm and 7/3Rm, respectively; 3) 3Rm, 2.7Rm and 7/3Rm were used as exit boundary conditions to simulate FFRCT, quoted as 3FFRCT, 2.7FFRCT and 7/3FFRCT, respectively; 4) The correlation and diagnostic performance between three kinds of FFRCT and FFR were analyzed. RESULTS: The p-values of comparing 3Rm, 2.7Rm and 7/3Rm with FFR were 0.004, 0.005 and 0.010, respectively; the r value between 7/3FFRCT and FFR (0.96) was better than that of 3FFRCT (0.95) and 2.7FFRCT (0.95); the 95% LoA between 7/3FFRCT and FFR (-0.08~0.11) was smaller than that of 3FFRCT (-0.10~0.12) and 2.7FFRCT (-0.09~0.11); the AUC and accuracy of 7/3FFRCT [0.962 (0.805-0.999), 96.15%] were the same as those of 2.7FFRCT [0.962 (0.805-0.999), 96.15%] and better than those of 3FFRCT [0.944 (0.777-0.996), 92.3%]. The prediction threshold of 7/3FFRCT (0.791) was closer to 0.8 than that of 3FFRCT (0.816) and 2.7FFRCT (0.787). CONCLUSION: The blood flow-diameter scaling law affects the FFRCT simulation by influencing the exit boundary condition Rm of the calculation. With $ Q\alpha D{}^{7}\!\!\diagup\!\!{}_{3}\; $, FFRCT had the highest diagnostic performance. The blood flow-diameter scaling law provides theoretical support for the blood flow distribution in the bifurcated vessel and improves the FFRCT model.

In vivo and in silico monitoring bone regeneration during distraction osteogenesis of the mouse femur.

BACKGROUND AND OBJECTIVE: Distraction osteogenesis (DO) is a mechanobiological process of producing new bone by gradual and controlled distraction of the surgically separated bone segments. Mice have been increasingly used to study the role of relevant biological factors in regulating bone regeneration during DO. However, there remains a lack of in silico DO models and related mechano-regulatory tissue differentiation algorithms for mouse bone. This study sought to establish an in silico model based on in vivo experimental data to simulate the bone regeneration process during DO of the mouse femur. METHODS: In vivo micro-CT, including time-lapse morphometry was performed to monitor the bone regeneration in the distraction gap. A 2D axisymmetric finite element model, with a geometry originating from the experimental data, was created. Bone regeneration was simulated with a fuzzy logic-based two-stage (distraction and consolidation) mechano-regulatory tissue differentiation algorithm, which was adjusted from that used for fracture healing based on our in vivo experimental data. The predictive potential of the model was further tested with varied distraction frequencies and distraction rates. RESULTS: The computational simulations showed similar bone regeneration patterns to those observed in the micro-CT data from the experiment throughout the DO process. This consisted of rapid bone formation during the first 10 days of the consolidation phase, followed by callus reshaping via bone remodeling. In addition, the computational model predicted a faster and more robust bone healing response as the model's distraction frequency was increased, whereas higher or lower distraction rates were not conducive to healing. CONCLUSIONS: This in silico model could be used to investigate basic mechanobiological mechanisms involved in bone regeneration or to optimize DO strategies for potential clinical applications.

The combined effects of dynamization time and degree on bone healing.

Dynamization, increasing the interfragmentary movement (IFM) by reducing the fixation stiffness from a rigid to a more flexible condition, is widely used clinically to promote fracture healing. However, it remains unknown how dynamization degree (relative change in fixation stiffness/IFM from a rigid to a flexible fixation) affects bone healing at various stages. To address this issue, we used a fuzzy logic-based mechano-regulated tissue differentiation algorithm on published experimental data from a sheep osteotomy healing model. We applied a varied degree of dynamization, from 0 (fully rigid fixation) to 0.9 (90% reduction in stiffness relative to the rigid fixation) after 1, 2, 3, and 4 weeks of osteotomy (R1wF, R2wF, R3wF, and R4wF) and computationally evaluated bone regeneration and biomechanical integrity over the healing process of 8 weeks. Compared with the constant rigid fixation, early dynamization (R1wF and R2wF) led to delays in bone bridging and biomechanical recovery of the osteotomized bone. However, the effect of early dynamization on healing was dependent of the degree of dynamization. Specifically, a higher dynamization degree (e.g., 0.9 for R1wF) led to a prolonged delay in bone bridging and largely unrecovered bending stiffness (48% relative to the intact bone), whereas a moderate degree of dynamization (e.g., 0.5 or 0.7) significantly enhanced bone formation and biomechanical properties of the osteotomized bone. These results suggest that dynamization degree and timing interactively affect the healing process. A combination of early dynamization with a moderate degree could enhance the ultimate biomechanical recovery of the fractured bone.

Quercetin Prevents Radiation-Induced Oral Mucositis by Upregulating BMI-1.

Radiation-induced oral mucositis is a major adverse event of radiotherapy. Severe oral mucositis may cause unwanted interruption in radiotherapy and reduce long-term survival in cancer patients receiving radiotherapy, but until now, there have been no effective options for preventing radiation-induced oral mucositis. Quercetin is a flavonoid that is widely found in food species and has anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated a new role of quercetin in preventing radiation-induced oral mucositis. Quercetin exerted preventive effects against radiation-induced oral mucositis induced by single-dose (25 Gy) ionizing radiation or fractionated ionizing radiation (8 Gy × 3) in C57BL/6 mice and maintained the proliferation ability of basal epithelial cells. Quercetin pretreatment alleviated reactive oxygen species generation, NF-κB pathway activation, and downstream proinflammatory cytokine production and reduced DNA double-strand breaks and cellular senescence induced by ionizing radiation. Quercetin also upregulated BMI-1 expression in oral epithelial cells and promoted ulcer repair. In addition, quercetin exerted similar radioprotective effects in irradiated primary cultured normal human keratinocytes, reduced reactive oxygen species generation and proinflammatory cytokine release, and promoted DNA double-strand break repair and wound healing by upregulating the expression of BMI-1, which is a polycomb group protein. Thus, quercetin can block multiple pathological processes of radiation-induced oral mucositis by targeting BMI-1 and may be a potential treatment option for preventing radiation-induced oral mucositis.

Enhancing the Efficiency of Distraction Osteogenesis through Rate-Varying Distraction: A Computational Study.

Distraction osteogenesis (DO) is a mechanobiological process of producing new bone and overlying soft tissues through the gradual and controlled distraction of surgically separated bone segments. The process of bone regeneration during DO is largely affected by distraction parameters. In the present study, a distraction strategy with varying distraction rates (i.e., "rate-varying distraction") is proposed, with the aim of shortening the distraction time and improving the efficiency of DO. We hypothesized that faster and better healing can be achieved with rate-varying distractions, as compared with constant-rate distractions. A computational model incorporating the viscoelastic behaviors of the callus tissues and the mechano-regulatory tissue differentiation laws was developed and validated to predict the bone regeneration process during DO. The effect of rate-varying distraction on the healing outcomes (bony bridging time and bone formation) was examined. Compared to the constant low-rate distraction, a low-to-high rate-varying distraction provided a favorable mechanical environment for angiogenesis and bone tissue differentiation, throughout the distraction and consolidation phase, leading to an improved healing outcome with a shortened healing time. These results suggest that a rate-varying clinical strategy could reduce the overall treatment time of DO and decrease the risk of complications related to the external fixator.

ZNF451 stabilizes TWIST2 through SUMOylation and promotes epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is the process by which epithelial cells lose their tightly packed polarized characteristics and acquire a migratory mesenchymal phenotype. EMT plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. The basic helix-loop-helix (bHLH) transcription factors TWIST1/2 are key EMT-inducing transcription factors that govern transcription of EMT-associated genes. Although regulation of TWIST1 activity and stability has been well studied, little is known about how TWIST2 is post-translationally regulated. Here we have identified ZNF451, a SUMO2/3 specific E3 ligase, as a novel regulator of TWIST2 in promoting its stability. ZNF451 directly binds to and SUMOylates TWIST2 at K129 residue, and consequently blocks ubiquitination and proteasome-dependent degradation of TWIST2. Ectopic expression of ZNF451 increases the protein level of TWIST2 in mammary epithelial cells, leading to increased expression of mesenchymal markers, whereas depletion of ZNF451 suppresses mesenchymal phenotypes. Collectively, our findings demonstrate that ZNF451 plays a vital role in EMT through SUMOylation-dependent stabilization of TWIST2.

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.

BACKGROUND: Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. RESULTS: In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. CONCLUSIONS: NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.