miR-1246-overexpressing exosomes improve UVB-induced photoaging by activating autophagy via suppressing GSK3ß.

Stem cell paracrine has shown potential application in skin wound repair and photoaging treatment. Our previous study demonstrated that miR-1246-overexpressing Exosomes (OE-EXs) isolated from adipose-derived stem cells (ADSCs) showed superior photo-protecting effects on UVB-induced photoaging than that of the vector, however, the underlying mechanism was unclear. The simultaneous bioinformatics analysis indicated that miR-1246 showed potential binding sites with GSK3ß which acted as a negative regulator for autophagy. This study was aimed to explore whether OE-EXs ameliorate skin photoaging by activating autophagy via targeting GSK3ß. The results demonstrated that OE-EXs significantly decreased GSK3ß expression, enhanced autophagy flux and autophagy-related proteins like LC3II, while suppressed p62 expression. Meanwhile, OE-EXs markedly reversed the levels of intracellular ROS, MMP-1, procollagen type I and DNA damage in human skin fibroblasts caused by UVB irradiation, but the ameliorating effects were significantly inhibited when 3-Methyladenine (3-MA) was introduced to block the autophagy pathway. Further, OE-EXs could reverse UVB-induced wrinkles, epidermal hyperplasia, and collagen fibers reduction in Kunming mice, nevertheless, the therapeutical effects of OE-EXs were attenuated when it was combinative treated with 3-MA. In conclusion, OE-EXs could cure UVB induced skin photoaging by activating autophagy via targeting GSK3ß.

Fork coupling directs DNA replication elongation and termination.

DNA replication is initiated at multiple loci to ensure timely duplication of eukaryotic genomes. Sister replication forks progress bidirectionally, and replication terminates when two convergent forks encounter one another. To investigate the coordination of replication forks, we developed a replication-associated in situ HiC method to capture chromatin interactions involving nascent DNA. We identify more than 2000 fountain-like structures of chromatin contacts in human and mouse genomes, indicative of coupling of DNA replication forks. Replication fork interaction not only occurs between sister forks but also involves forks from two distinct origins to predetermine replication termination. Termination-associated chromatin fountains are sensitive to replication stress and lead to coupled forks-associated genomic deletions in cancers. These findings reveal the spatial organization of DNA replication forks within the chromatin context.

CAFs targeted ultrasound-responsive nanodroplets loaded V9302 and GLULsiRNA to inhibit melanoma growth via glutamine metabolic reprogramming and tumor microenvironment remodeling.

Despite rapid advances in metabolic therapies over the past decade, their efficacy in melanoma has been modest, largely due to the interaction between cancer-associated fibroblasts (CAFs) and cancer cells to promote cancer growth. Altering the tumor microenvironment (TME) is challenging and elusive. CAFs is critical for glutamine deprivation survival in melanoma. In this research, we assembled a CAFs-targeted, controlled-release nanodroplets for the combined delivery of the amino acid transporter ASCT2 (SLC1A5) inhibitor V9302 and GLULsiRNA (siGLUL). The application of ultrasound-targeted microbubble disruption (UTMD) allows for rapid release of V9302 and siGLUL, jointly breaking the glutamine metabolism interaction between CAFs and cancer cells on one hand, on the other hand, blocking activated CAFs and reducing the expression of extracellular matrix (ECM) to facilitate drug penetration. In addition, ultrasound stimulation made siGLUL more accessible to tumor cells and CAFs, downregulating GLUL expression in both cell types. FH-V9302-siGLUL-NDs also serve as contrast-enhanced ultrasound imaging agents for tumor imaging. Our study developed and reported FH-NDs as nanocarriers for V9302 and siGLUL, demonstrating that FH-V9302-siGLUL-NDs have potential bright future applications for integrated diagnostic therapy. Graphical Abstract.

Cohesin maintains replication timing to suppress DNA damage on cancer genes.

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.

Smart Biomimetic Nanozymes for Precise Molecular Imaging: Application and Challenges.

New nanotechnologies for imaging molecules are widely being applied to visualize the expression of specific molecules (e.g., ions, biomarkers) for disease diagnosis. Among various nanoplatforms, nanozymes, which exhibit enzyme-like catalytic activities in vivo, have gained tremendously increasing attention in molecular imaging due to their unique properties such as diverse enzyme-mimicking activities, excellent biocompatibility, ease of surface tenability, and low cost. In addition, by integrating different nanoparticles with superparamagnetic, photoacoustic, fluorescence, and photothermal properties, the nanoenzymes are able to increase the imaging sensitivity and accuracy for better understanding the complexity and the biological process of disease. Moreover, these functions encourage the utilization of nanozymes as therapeutic agents to assist in treatment. In this review, we focus on the applications of nanozymes in molecular imaging and discuss the use of peroxidase (POD), oxidase (OXD), catalase (CAT), and superoxide dismutase (SOD) with different imaging modalities. Further, the applications of nanozymes for cancer treatment, bacterial infection, and inflammation image-guided therapy are discussed. Overall, this review aims to provide a complete reference for research in the interdisciplinary fields of nanotechnology and molecular imaging to promote the advancement and clinical translation of novel biomimetic nanozymes.

Change in Eosinophil Count in Patients with Heart Failure Treated with Anakinra.

BACKGROUND: Interleukin-1 blockade with anakinra leads to a transient increase in eosinophil blood count (eosinophils) in patients with acute myocardial infarction. We aimed to investigate the effect of anakinra on changes in eosinophils in patients with heart failure (HF) and their correlation with cardiorespiratory fitness (CRF). METHODS: We measured eosinophils in 64 patients with HF (50% females), 55 (51-63) years of age, before and after treatment, and, in a subset of 41 patients, also after treatment cessation. We also evaluated CRF, measuring peak oxygen consumption (VO2) with a treadmill test. RESULTS: Treatment with anakinra significantly and transiently increased eosinophils, from 0.2 [0.1-0.3] to 0.3 [0.1-0.4] × 103 cells/µL (p < 0.001) and from 0.3 [0.2-0.5] to 0.2 [0.1-0.3] × 103 cells/µL, with suspension (p < 0.001). Changes in eosinophils correlated with the changes in peak VO2 (Spearman's Rho = +0.228, p = 0.020). Eosinophils were higher in patients with injection site reactions (ISR) (n = 8, 13%; 0.5 [0.4-0.6] vs. 0.2 [0.1-0.4] × 103 cells/µL, p = 0.023), who also showed a greater increase in peak VO2 (3.0 [0.9-4.3] vs. 0.3 [-0.6-1.8] mLO2·kg-1·min-1, p = 0.015). CONCLUSION: Patients with HF treated with anakinra experience a transient increase in eosinophils, which is associated with ISR and a greater improvement in peak VO2.

pH-/Redox-Responsive Nanodroplet Combined with Ultrasound-Targeted Microbubble Destruction for the Targeted Treatment of Drug-Resistant Triple Negative Breast Cancer.

Multiple drug resistance (MDR) exists in divergent cancers including triple negative breast cancer (TNBC) and partly results in the resistance to many first-line anti-cancer agents, bringing a big challenge to TNBC management. To develop novel TNBC therapeutics, in our study, a hyaluronic acid (HA)-carboxymethyl chitosan (CMC) conjugate linked via a disulfide-bond (HA-SS-CMC, HSC) was synthesized to fabricate nanodroplets (NDs). The NDs encapsulating doxorubicin (DOX) and perfluorohexane (DOX-HSC-NDs) were prepared via a homogenization/emulsification strategy and exhibited not only high biocompatibility but also noticeable tumor cell targeting ability and dual pH/redox responsiveness. Besides, DOX-HSC-NDs can be used as a contrast-enhanced ultrasound imaging agent for specific tumor imaging. DOX-HSC-NDs in combination with ultrasound targeted microbubble destruction could improve intracellular drug aggregation and retention of MDR cells and work against multiple mechanisms of drug resistance through synergistic strategies, including up-regulating the reactive oxygen species (ROS) level, promoting apoptosis, and scavenging glutathione, while reducing the expression levels of P-glycoprotein and inhibiting the epithelial-mesenchymal transition. This combination strategy showed protective effects against TNBC in both MDA-MB-231/ADR cells and tumor-bearing mice. Our study for the first time developed and reported the ultrasound-augmented HSC-NDs as the DOX nanocarrier and provided scientific evidence to support the future application of DOX-HSC-NDs as a potential TNBC therapy.

Betulinic Acid Improves Cardiac Function in Septic Rats Through AKT / mTOR and AKT / AMPK -modulated Autophagy / 中国生物化学与分子生物学报

Betulinic acid (BA) exerts protective effects on organs in septic animals. However, whether BA can improve cardiac function in sepsis and the underlying mechanism remain unclear. Here, male Sprague-Dawley rats were pretreated with BA (25 mg/ kg/ d, i. g.) for 5 days and then intraperitoneally injected with lipopolysaccharide (LPS, 10 mg/ kg). The rats were anesthetized to determine transthoracic echocardiography using a high-resolution imaging system for small animals after they were treated with LPS for 6 h. Histopathologic alterations were examined by HE staining. Myocardial injury markers (cTnI and CK-MB) and inflammatory factors (TNF-α, IL-1β and IL-6) in the serum were measured by the enzyme-linked immunosorbent assay. Autophagy-related proteins (p62 and LC3 Ⅱ) and AKT-modulated autophagy pathways in the myocardium were determined by Western blotting. Pretreatment with BA markedly improved left ventricular ejection fraction (EF) and fraction shortening (FS) (P<0. 05), improved myocardial histomorphology, and significantly inhibited cTnI, CK-MB, TNF-α, IL-1β and IL-6 (P<0. 05) in the septic rat serum. BA markedly decreased p62 (P<0. 01), increased LC3 Ⅱ (P< 0. 001), and significantly down-regulated p-AKT (Thr308), p-AMPKα (Ser485/ 491), p-mTOR (Ser2448) and p-S6K (Thr389) (P<0. 05), while markedly up-regulated p-AMPKα (Thr172) and pULK1 (Ser317) (P<0. 01) in septic rat hearts. The findings indicate that BA can attenuate sepsis-induced myocardial dysfunctions associated with down-regulating autophagy inhibiting pathways mediated by AKT/ mTOR and AKT/ AMPK pathways.

Dynamic crossover towards energy equipartition in the Fermi-Pasta-Ulam-Tsingou ß model with long-range interactions.

Energy equipartition can be established in short-range systems after the dynamic process of thermalization. However, energy distribution between different degrees of freedom in systems with long-range interactions is unclear. We study the dynamics of energy relaxation in the Fermi-Pasta-Ulam-Tsingou ß model with long-range quartic interactions, which decay as 1/d^{δ} with d being the lattice distance. The dynamic crossover of a mode-energy distribution from localized to equipartitioned with the increase of the power δ is observed. A transition of mode-energy distribution is identified around the value of δ=1, which usually serves as the distinction between strong and weak long-range couplings. We elucidate that the varying frequency overlapping of the mode-energy power spectrum is responsible for this dynamic crossover. Through further calculation of the spectral entropy, the minimum duration of quasistationary states, τ_{QSS}, is found at δ=2, which may provide possible dynamic explanations for the peculiar behavior of heat transport in long-range lattice chains. In addition, the double scaling in τ_{QSS} as a function of energy density is also observed in our long-range lattices. Our results not only contribute to understanding the dynamics of energy relaxation in long-range systems, but also shed light on the longstanding problem of thermalization and low-dimensional heat transport in short-range systems.

What are the factors that may predict the severity of perineal tears in obstetric anal sphincter injuries and how are their outcomes? A 10-year retrospective analysis in a Southeast Asian population.

INTRODUCTION AND HYPOTHESIS: Obstetric anal sphincter injuries (OASIS) complicate around 1-2% of deliveries in low- and middle-income countries. Asians are twice more likely to suffer this complication. The 3c and 4th-degree perineal tears that involve the internal anal sphincter muscle and the anal mucosa have been reported to have a poorer outcome and higher risk of recurrence compared to less severe tears. METHODOLOGY: A retrospective 10-year analysis of third- and fourth-degree perineal tears in a tertiary centre was conducted. The maternal, neonatal factors and their respective outcomes after the repair of an OASI were examined and compared between minor (3a and 3b perineal tears) and major anal sphincter tears (3c and 4th-degree perineal tears). RESULTS: Five hundred twenty patients with OASIS were included into the study. Birthweight ≥ 3.5 kg was significantly associated with having a major anal sphincter tear in this study population, OR 1.91 (95% CI 1.21-3.02), p = 0.006. There was no significant difference in having faecal or flatus incontinence after the repair; however, major anal sphincter tears appeared to be more complicated to repair compared to minor anal sphincter tears, requiring involvement of the consultant, p < 0.001. CONCLUSION: Neonatal birthweight ≥ 3.5 kg was the most significant factor in predicting the severity of OASIS in this study population. After appropriate repair, the rates of reported complications appeared similar between the two groups. However, significantly more expertise and resources were required for the repair of higher degree OASIS.

Patient Perceptions of Exertion and Dyspnea With Interleukin-1 Blockade in Patients With Recently Decompensated Systolic Heart Failure.

Interleukin-1 (IL-1) blockade is an anti-inflammatory treatment that may affect exercise capacity in heart failure (HF). We evaluated patient-reported perceptions of exertion and dyspnea at submaximal exercise during cardiopulmonary exercise testing (CPET) in a double-blind, placebo-controlled, randomized clinical trial of IL-1 blockade in patients with systolic HF (REDHART [Recently Decompensated Heart Failure Anakinra Response Trial]). Patients underwent maximal CPET at baseline, 2, 4, and 12 weeks and rated their perceived level of exertion (RPE, on a scale from 6 to 20) and dyspnea on exertion (DOE, on a scale from 0 to 10) every 3 minutes throughout exercise. Patients also answered 2 questionnaires to assess HF-related quality of life: the Duke Activity Status Index and the Minnesota Living with Heart Failure Questionnaire. From baseline to the 12-week follow-up, IL-1 blockade significantly reduced RPE and DOE at 3- and 6-minutes during CPET without changing values for heart rate, oxygen consumption, and cardiac workload at 3- and 6-minutes. Linear regression identified 6-minute RPE to be a strong independent predictor of both physical symptoms (Minnesota Living with Heart Failure Questionnaire; ß = 0.474, p = 0.002) and perceived exercise capacity (Duke Activity Status Index; ß = -0.443, p = 0.008). In conclusion, patient perceptions of exertion and dyspnea at submaximal exercise may be valuable surrogates for quality of life and markers of response to IL-1 blockade in patients with HF.

Overcoming the inadaptability of sparse group lasso for data with various group structures by stacking.

MOTIVATION: Efficiently identifying genes based on gene expression level have been studied to help to classify different cancer types and improve the prediction performance. Logistic regression model based on regularization technique is often one of the effective approaches for simultaneously realizing prediction and feature (gene) selection in genomic data of high dimensionality. However, standard methods ignore biological group structure and generally result in poorer predictive models. RESULTS: In this article, we develop a classifier named Stacked SGL that satisfies the criteria of prediction, stability and selection based on sparse group lasso penalty by stacking. Sparse group lasso has a mixing parameter representing the ratio of lasso to group lasso, thus providing a compromise between selecting a subset of sparse feature groups and introducing sparsity within each group. We propose to use stacked generalization to combine different ratios rather than choosing one ratio, which could help to overcome the inadaptability of sparse group lasso for some data. Considering that stacking weakens feature selection, we perform a post hoc feature selection which might slightly reduce predictive performance, but it shows superior in feature selection. Experimental results on simulation demonstrate that our approach enjoys competitive and stable classification performance and lower false discovery rate in feature selection for varying sets of data compared with other regularization methods. In addition, our method presents better accuracy in three public cancer datasets and identifies more powerful discriminatory and potential mutation genes for thyroid carcinoma. AVAILABILITY AND IMPLEMENTATION: The real data underlying this article are available from https://github.com/huanheaha/Stacked_SGL; https://zenodo.org/record/5761577#.YbAUyciEwk2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Application of thermoluminescence dosimeter in environmental monitoring of nuclear power plant / 中国辐射卫生

Objective To investigate the effect of gaseous effluent from the six generator sets on the radiation level of the surrounding terrestrial environment in Daya Bay Nuclear Power Base after the operation of Ling’ao Nuclear Power plant. Methods The radiation level in the peripheral environment of the Base was monitored using the thermoluminescence dosimeter （TLD）. Twenty-five monitoring sites were set around the Base to investigate the variation of radiation level over a long period of time by collecting the TLDs every three months. Results From 2011 to 2020, the annual γ dose rate of the 25 sites ranged from 76.7 to 207.1 nGy/h, with an average value of (123.3 ± 5.7) nGy/h and a relative deviation of 2%-12%. The TLD monitoring and instantaneous measuring results of γ dose rate were consistent with the survey of the State Environmental Protection Administration in the 20th century and the baseline level prior to the operation of the nuclear power plant. Conclusion There are great differences in natural environmental radiation level across the TLD monitoring sites. The overall environmental γ radiation level within 50 km of the nuclear power base remains unchanged. The emission of gaseous effluent from the operation of the nuclear power plant does not have a cumulative impact on the radiation level of surrounding environment.

Meta-analysis of clinical outcomes of PCSK9 modulators in patients with established ASCVD.

The advent of monoclonal antibodies targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) ushered in a new era of dyslipidemia pharmacotherapy. The first two antibodies targeting PCSK9 (evolocumab, alirocumab) approved by the United States Food and Drug Administration (FDA) provided significant and sustained reductions in atherogenic lipids and a reduced risk of atherosclerotic cardiovascular disease (ASCVD) events. More recently, phase 3 trials of inclisiran-a small interfering RNA-based agent targeting PCSK9-reported similar lipid-lowering effects and preliminary evidence of ASCVD risk reduction, although significant questions remain regarding the extent of benefits across cardiovascular outcomes. We conducted a systematic review and meta-analysis (random-effects model) of the available data on lipid lowering, incidence of atherosclerotic cardiovascular disease (ASCVD) events, and safety of pharmacologic agents targeting PCSK9. A significant and consistent reduction in low-density lipoprotein cholesterol (LDL-C) was observed across all agents (-51% [95% confidence interval {CI}: -61%, -41%]). Despite the impressive reduction in LDL-C, the individual effects on mortality, cardiovascular death, myocardial infarction (MI), and stroke remained nonsignificant. However, a consistent reduction was observed in the composite outcomes of MI, stroke, and cardiovascular death [relative risk {RR} (95% CI): 0.80 (0.73-0.87)] and MI, stroke, unstable angina (requiring revascularization), and cardiovascular death [RR (95% CI): 0.85 (0.74-0.97)]. In terms of safety outcomes, there was no significant difference in severe adverse events, new onset diabetes, neurocognitive disorders, or myalgia. Meanwhile, injection site reaction was more frequent in patients receiving these agents compared to placebo [RR 2.11 (95% CI): 1.26-3.54]. These findings suggest a class effect for favorable lipid changes and a low risk of serious adverse events among pharmacologic agents targeting PCSK9. Although there is compelling evidence that PCSK9-targeting agents reduce the risk of some cardiovascular outcomes, adequately powered studies with longer follow-up may be needed to fully characterize the magnitude of benefits across the cardiovascular spectrum.

Preservation of Cardiac Reserve and Cardiorespiratory Fitness in Patients With Acute De Novo Versus Acute on Chronic Heart Failure With Reduced Ejection Fraction.

There is limited understanding on the potential differences in the pathophysiology between de novo heart failure with reduced ejection fraction (HFrEF) and acute on chronic HFrEF. The aim of this study was to assess differences in cardiorespiratory fitness (CRF) parameters between de novo heart failure and acute on chronic HFrEF using cardiopulmonary exercise testing (CPX). We retrospectively analyzed CPX data measured within 2 weeks of discharge following acute hospitalization for HFrEF. Data are reported as median and interquartile range or frequency and percentage (%). We included 102 patients: 32 (31%) women, 81 (79%) black, 57 (51 to 64) years of age, BMI of 34 (29 to 39) Kg/m2. Of these, 26 (25%) had de novo HFrEF and 76 (75%) had acute on chronic HFrEF. When compared with acute on chronic, patients with de novo HFrEF had a significantly higher peak oxygen consumption (VO2) (16.5 [12.2 to 19.4] vs 12.8 [10.1 to 15.3] ml·kg-1·min-1, p <0.001), %-predicted peak VO2 (58% [51 to 75] vs 49% [42 to 59]) p = 0.012), peak heart rate (134 [117 to 147] vs 117 [104 to 136] beats/min, p = 0.004), peak oxygen pulse (12.2 [10.5 to 15.5] vs 9.9 [8.0 to 13.1] ml/beat, p = 0.022) and circulatory power (2,823 [1,973 to 3,299] vs 1,902 [1,372 to 2,512] mm Hg·ml·kg-1·min-1, p = 0.002). No significant difference in resting left ventricular ejection fraction was found between groups. In conclusion, patients with de novo HFrEF have better CRF parameters than those with acute on chronic HFrEF. These differences are not explained by resting left ventricular systolic function but may be related to greater preservation in cardiac reserve during exercise in de novo HFrEF patients.

Clinical trial enrollment at a rural satellite hospital during COVID-19 pandemic.

INTRODUCTION: Controlled clinical trials (CCTs) have traditionally been limited to urban academic clinical centers. Implementation of CCTs in rural setting is challenged by lack of resources, the inexperience of patient care team members in CCT conductance and workflow interruption, and global inexperience with remote data monitoring. METHODS: We report our experience during the coronavirus disease 2019 (COVID-19) pandemic in activating through remote monitoring a multicenter clinical trial (the Study of Efficacy and Safety of Canakinumab Treatment for cytokine release syndrome (CRS) in Participants with COVID-19-induced Pneumonia [CAN-COVID] trial, ClinicalTrials.gov Identifier: NCT04362813) at a rural satellite hospital, the VCU Health Community Memorial Hospital (VCU-CMH) in South Hill, VA, that is part of the larger VCU Health network, with the lead institution being VCU Health Medical College of Virginia Hospital (VCU-MCV), Richmond, VA. We used the local resources at the facility and remote guidance and oversight from the VCU-MCV resources using a closed-loop communication network. Investigational pharmacy, pathology, and nursing were essential to operate the work in coordination with the lead institution. RESULTS: Fifty-one patients with COVID-19 were enrolled from May to August 2020, 35 (69%) at VCU-MCV, and 16 (31%) at VCU-CMH. Among the patients enrolled at VCU-CMH, 37.5% were female, 62.5% Black, and had a median age of 60 (interquartile range 56-68) years. CONCLUSION: Local decentralization of this trial in our experience gave rural patients access to a novel treatment and also accelerated enrollment and more diverse participants' representative of the target population.

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

Transcription shapes DNA replication initiation to preserve genome integrity.

BACKGROUND: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. RESULTS: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. CONCLUSION: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.

The outcome of intra-cytoplasmic sperm injection (ICSI): do the sperm concentration and motility matter?

OBJECTIVES: The male-factor subfertility was reported to be 30% globally; thus, the intracytoplasmic sperm injection (ICSI) procedure was implemented to improve the overall in vitro fertilization (IVF) rates. Nevertheless, at least 10% of fertilization failure still occurs. Concerning this issue, we explored the association of sperm concentration and motility with the quality of embryo development and pregnancy outcome in IVF-ICSI cycles. METHODS: Retrospective analysis of 109 couples with male factor were done over 14 months in a tertiary university hospital in Malaysia. The data were divided into four groups; Group I: normal sperm parameters, Group II: normal sperm concentration but reduced total sperm motility, Group III: reduced sperm concentration and motility, Group IV: reduced sperm concentration but normal sperm motility. Only fresh semen samples and fresh embryo transfers were included. The fertilization, cleavage rate, embryo quality and pregnancy outcome were assessed. RESULTS: Overall, group I had the highest oocytes yield and ICSI attempted; (10.12 + 6.50), whereas the lowest was in group IV; (7.00 + 2.82). Group II revealed the highest fertilization and cleavage rates; (54.14 + 25.36), (55.16 + 26.06), thus not surprisingly resulting in the highest number of good embryos and highest clinical pregnancy rates. The lowest cleavage and pregnancy rates were seen in group IV. However, all the outcomes were not statically significant (p>0.05). CONCLUSIONS: Similar fertilization rate and comparable pregnancy outcome was seen among couples with normal and reduced sperm concentration and motility.