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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.

Objective: We present a new artificial intelligence-powered method to predict 3-year hepatocellular carcinoma (HCC) recurrence by analysing the radiomic profile of contrast-enhanced CT (CECT) images that was validated in patient cohorts. Methods: This retrospective cohort study of 224 HCC patients with follow-up for at least 3 years was performed at a single centre from 2012 to 2019. Two groups of radiomic signatures were extracted from the arterial and portal venous phases of pre-operative CECT. Then, the radiological model (RM), deep learning-based radiomics model (DLRM), and clinical & deep learning-based radiomics model (CDLRM) were established and validated in the area under curve (AUC), calibration curve, and clinical decision curve. Results: Comparison of the clinical baseline variables between the non-recurrence (n = 109) and recurrence group (n = 115), three clinical independent factors (Barcelona Clinic Liver Cancer staging, microvascular invasion, and α-fetoprotein) were incorporated into DLRM for the CDLRM construction. Among the 30 radiomic features most crucial to the 3 year recurrence rate, the selection from deep learning-based radiomics (DLR) features depends on CECT. through the Gini index. In most cases, CDLRM has shown superior accuracy and distinguished performance than DLRM and RM, with the 0.98 AUC in the training cohorts and 0.83 in the testing. Conclusion: This study proposed that DLR-based CDLRM construction would be allowed for the predictive utility of 3-year recurrence outcomes of HCCs, providing high-risk patients with an effective and non-invasive method to possess extra clinical intervention. Advances in knowledge: This study has highlighted the predictive value of DLR in the 3-year recurrence rate of HCC.

BACKGROUND Although acute graft-vs-host disease (aGvHD) is a rare complication of liver transplantation, it is poorly understood and has an extremely high mortality rate. No standardized diagnostic criteria or treatment regimens currently exist. CASE SUMMARY The present study investigated the etiology, diagnosis, and treatment of aGvHD following liver transplantation. Presentation, diagnosis, disease course, histology, and treatment of an aGvHD case are reported, and associated literature is reviewed. A 64-year-old female required LTx due to primary biliary cirrhosis. The donor was a 12-year-old male. Three weeks following liver transplantation, the recipient developed pyrexia, diarrhea, rashes, and antibiotic-unresponsive pancytopenia. Clinical symptoms together with laboratory investigations suggested a diagnosis of aGvHD, which was confirmed via peripheral blood fluorescent in situ hybridization. Donor XY chromosome fluorescent in situ hybridization indicating early chimerism achieved 93% sensitivity in the detection of GvHD. Existing immunosuppressants were discontinued, and high-dose intravenous methylprednisolone was initiated along with antibiotics. While diarrhea resolved, the patient’s general condition continued to deteriorate until demise due to multi-system organ failure at 37 d post-liver transplantation. This case illustrates the life-threatening nature of aGvHD. CONCLUSION Herein, we have summarized a post-LTx aGvHD case and reviewed associated literature in order to increase awareness and provide potentially risk-mitigating recommendations.

Jing Xiao,1,2 Wenbo Wan,2 Ying Zhang,1,2 Jun Ma,2 Lin Yan,2 Yukun Luo,1,2 Jie Tang1,2 1School of Medicine, Nankai University, Tianjin, People’s Republic of China; 2Department of Ultrasound, The First Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of ChinaCorrespondence: Jie TangDepartment of Ultrasound, The First Medical Center of Chinese PLA General Hospital, Fuxing Road 28#, Beijing 100853, People’s Republic of ChinaTel +86-10-5549-9056Fax +86-10-5549-9255Email txiner@sina.vip.comBackground: After surgical correction of testicular torsion, up to 68% of ipsilateral testes undergo atrophy due to ischemia-reperfusion injury (IRI). Recent studies have shown that dexmedetomidine (Dex) alleviates IRI in various vital organs. However, those studies evaluated its protective effect on short-term reperfusion.Purpose: We aimed to investigate whether Dex has a long-term protective effect against testicular injury after IRI.Materials and Methods: A total of 24 New Zealand white rabbits were randomly divided into three groups (n = 8/group): the control group (saline-infused rabbits without IRI), the IRI group (saline-injected rabbits with IRI), and the Dex group (Dex-injected rabbits with IRI). The spermatic cord of rabbits in IRI and Dex groups was ligated for 4 h, and 1 h before reperfusion, Dex was administered intraperitoneally at a dose of 50 μg/kg body weight in group Dex, whereas saline was administered at the same dose to the IRI and control groups. Rabbits were kept alive for 4 weeks post reperfusion, then the testes were harvested, and the rabbits were euthanized.Results: Four weeks post reperfusion, testicular volumes of the affected side decreased considerably in the IRI and Dex groups compared to the control group, with no significant difference between the IRI and Dex groups. Compared to the control group, the Johnson score and the mean seminiferous tubular diameters were significantly decreased in the IRI and Dex groups, but no significant differences were observed after administration of Dex. There were no significant differences in malondialdehyde and superoxide dismutase levels between the groups treated with and without Dex.Conclusion: Dex administration 3 h after ischemia and 1 h before reperfusion did not demonstrate a significant protective effect against testicular injury 4 weeks after IRI in rabbits. Further research is needed to confirm the potential therapeutic effects of Dex by varying the experimental conditions.Keywords: dexmedetomidine, testis, reperfusion injury, testicular atrophy, testicular torsion

Given its different target-binding affinity and varying residence time after DNA cleavage, the Cas9-sgRNA complex that remains bound to cleaved DNA may influence DNA double strand break (DSB) repair pathway choices, contributing to highly heterogeneous mutations in genome editing. Here, we found that DSB repair pathway choices vary significantly at different sites for Cas9-induced DSBs. Reduced target-binding affinity of Cas9-sgRNA promotes a bias toward classical NHEJ (c-NHEJ), but inactivation of c-NHEJ aggravates off-target activities. By dislodging Cas9-sgRNA from the cleaved target, local DNA replication suppresses c-NHEJ in favor of other DSB repair pathways by altering DSB configuration and repair timing. It also results in sister chromatid fusion, which may promote chromosomal breakage-fusion-bridge cycles. This suggests that varying Cas9-sgRNA target-binding affinity or residence duration modulate DSB repair pathway choices for Cas9-induced DSBs and widen the mutational spectra 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 in order 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 by different mechanisms may initiate variable cellular DNA damage response, thus 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 non-homologous end joining (c-NHEJ) is not even engaged at some sites. Weakening target interaction of Cas9-sgRNA promotes bias towards c-NHEJ, which is intrinsically accurate for Cas9-induced DSBs. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ however 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 DSB repair pathway choices. 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 interaction 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.

Single crystals of LnRu2Ga8 and LnCo2Al8 (Ln = La and Pr) were grown using a Ga/Al self-flux method. An orthorhombic CaCo2Al8-type structure with space group Pbam (No.55) of them was identified by x-ray diffraction. LaRu2Ga8 and LaCo2Al8 are Pauli paramagnetic down to 2 K, while PrRu2Ga8 and PrCo2Al8 show antiferromagnetic (AFM) order at 2.5 and 5 K, respectively. Strong magnetic anisotropy in PrRu2Ga8 and PrCo2Al8 single crystals was found by an anisotropic magnetic measurement. The field-induced FM state was observed in both PrRu2Ga8 and PrCo2Al8 for H||c. However, in the case of H⊥c, the AFM state is robust. The strong magnetic anisotropy in PrRu2Ga8 FM and PrCo2Al8 is due to their anisotropic magnetic interactions that FM interactions are dominant in the case of H||c while AFM interactions for H⊥c.

Harsh environmental factors have continuous negative effects on plant growth and development, leading to metabolic disruption and reduced plant productivity and quality. However, filamentation temperature-sensitive H protease (FtsH) plays a prominent role in helping plants to cope with these negative impacts. In the current study, we examined the transcriptional regulation of the CaFtsH06 gene in the R9 thermo-tolerant pepper (Capsicum annuum L.) line. The results of qRT-PCR revealed that CaFtsH06 expression was rapidly induced by abiotic stress treatments, including heat, salt, and drought. The CaFtsH06 protein was localized to the mitochondria and cell membrane. Additionally, silencing CaFtsH06 increased the accumulation of malonaldehyde content, conductivity, hydrogen peroxide (H2O2) content, and the activity levels of superoxide dismutase and superoxide (·O2−), while total chlorophyll content decreased under these abiotic stresses. Furthermore, CaFtsH06 ectopic expression enhanced tolerance to heat, salt, and drought stresses, thus decreasing malondialdehyde, proline, H2O2, and ·O2− contents while superoxide dismutase activity and total chlorophyll content were increased in transgenic Arabidopsis. Similarly, the expression levels of other defense-related genes were much higher in the transgenic ectopic expression lines than WT plants. These results suggest that CaFtsH06 confers abiotic stress tolerance in peppers by interfering with the physiological indices through reducing the accumulation of reactive oxygen species, inducing the activities of stress-related enzymes and regulating the transcription of defense-related genes, among other mechanisms. The results of this study suggest that CaFtsH06 plays a very crucial role in the defense mechanisms of pepper plants to unfavorable environmental conditions and its regulatory network with other CaFtsH genes should be examined across variable environments.

Background RNA binding protein motif (RBM3) is associated with radioresistance in nasopharyngeal carcinoma (NPC), and miR-383-5p was predicted to target the 3'-untranslated region (3'UTR) of RBM3 messenger RNA (mRNA). Our study aimed to investigate the role and the mechanisms of miR-383-5p targeting RBM3 in NPC cell proliferation and radioresistance (RR). Methods The expression of miR-383-5p was detected by Real-time quantitative PCR (qRT-PCR) between RS (Radiosensitivity) and RR (Radioresistance) NPC patient- tissue specimens and cell lines. Cell Counting Kit-8 (CCK-8) and Clonogenic survival assay were applied to analyze the effect of miR-383-5p on NPC cell proliferation and radioresistance. Possible downstream target of miR-383-5p in NPC cells, RBM3was evaluated by luciferase assay and qRT-PCR. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3 by rescue experiments. The effect of miR-383-5p on radiation-induced apoptosis was explored through Flow cytometric analysis and Western blotting. Western blotting was analyzed the molecular of RBM3-mediated Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling pathways. Results The expression of miR-383-5p was decreased in radioresistant NPC tissues and cells. miR-383-5p inhibited cell proliferation and radioresistance in CNE1/IR cells. We also observed that therapeutic administration of a miR-383-5p agomir dramatically sensitized NPC xenografts to radiation in a mouse model. Conversely, in the same xenograft model, administration of a miR-383-5p antagomir dramatically increased NPC resistance to radiation. miR-383-5p targeted the 3'UTR of RBM3. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3. Finally, we found that miR-383-5p increased radiation-induced apoptosis, activated JNK signaling, and inhibited ERK signaling. Conclusions Our study revealed that miR-383-5p targeted the 3'UTR of RBM3 and contributed to the efficacy of NPC radiation therapy by altering the RBM3-mediated JNK and ERK signaling pathways.

Heat shock transcription factor (Hsf) plays an important role in regulating plant thermotolerance. The function and regulatory mechanism of CaHsfA1d in heat stress tolerance of pepper have not been reported yet. In this study, phylogenetic tree and sequence analyses confirmed that CaHsfA1d is a class A Hsf. CaHsfA1d harbored transcriptional function and predicted the aromatic, hydrophobic, and acidic (AHA) motif mediated function of CaHsfA1d as a transcription activator. Subcellular localization assay showed that CaHsfA1d protein is localized in the nucleus. The CaHsfA1d was transcriptionally up-regulated at high temperatures and its expression in the thermotolerant pepper line R9 was more sensitive than that in thermosensitive pepper line B6. The function of CaHsfA1d under heat stress was characterized in CaHsfA1d-silenced pepper plants and CaHsfA1d-overexpression Arabidopsis plants. Silencing of the CaHsfA1d reduced the thermotolerance of the pepper, while CaHsfA1d-overexpression Arabidopsis plants exhibited an increased insensitivity to high temperatures. Moreover, the CaHsfA1d maintained the H2O2 dynamic balance under heat stress and increased the expression of Hsfs, Hsps (heat shock protein), and antioxidant gene AtGSTU5 (glutathione S-transferase class tau 5) in transgenic lines. Our findings clearly indicate that CaHsfA1d improved the plant thermotolerance via regulating the expression of stress- and antioxidant-related genes.

Herein, we report a palladium-catalyzed dehydrogenative cross-coupling of indoles with cyclic enones to give β-indolyl cyclic enones under mild and neutral reaction conditions. The key to the success is to explore a mild condition, which ensures the indole C-H activation and subsequent syn β-hydride elimination through rapid enolization isomerization of Pd(II)-enolate while suppressing other undesired side reactions. Synthetic utility has also been demonstrated in the flexible transformation of the coupling products to meta-phenols and benzo[a]carbazoles.

Pepper is a thermophilic crop, shallow-rooted plant that is often severely affected by abiotic stresses such as heat, salt, and drought. The growth and development of pepper is seriously affected by adverse stresses, resulting in decreases in the yield and quality of pepper crops. Small heat shock proteins (s HSPs) play a crucial role in protecting plant cells against various stresses. A previous study in our laboratory showed that the expression level of CaHSP18.1a was highly induced by heat stress, but the function and mechanism of CaHSP18.1a responding to abiotic stresses is not clear. In this study, we first analyzed the expression of CaHSP18.1a in the thermo-sensitive B6 line and thermo-tolerant R9 line and demonstrated that the transcription of CaHSP18.1a was strongly induced by heat stress, salt, and drought stress in both R9 and B6, and that the response is more intense and earlier in the R9 line. In the R9 line, the silencing of CaHSP18.1a decreased resistance to heat, drought, and salt stresses. The silencing of CaHSP18.1a resulted in significant increases in relative electrolyte leakage (REL) and malonaldehyde (MDA) contents, while total chlorophyll content decreased under heat, salt, and drought stresses. Overexpression analyses of CaHSP18.1a in transgenic Arabidopsis further confirmed that CaHSP18.1a functions positively in resistance to heat, drought, and salt stresses. The transgenic Arabidopsis had higherchlorophyll content and activities of superoxide dismutase, catalase, and ascorbate peroxidase than the wild type (WT). However, the relative conductivity and MDA content were decreased in transgenic Arabidopsis compared to the wild type (WT). We further showed that the CaHSP18.1a protein is localized to the cell membrane. These results indicate CaHSP18.1a may act as a positive regulator of responses to abiotic stresses.

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. Electronic supplementary material The online version of this article (10.1186/s13059-018-1518-x) contains supplementary material, which is available to authorized users.

Radioresistance is an important obstacle to nasopharyngeal carcinoma (NPC) therapy. In this study, we explored the role of RNA-binding motif protein 3 (RBM3) in the radioresistance of NPC and its underlying mechanism. We measured the expression of RBM3 in 20 clinical NPC tissues and in NPC cell lines. We found that RBM3 was upregulated in radioresistant NPC tissues and cells. Radioresistant NPC cells (CNE1/IR) and parental NPC cells (CNE1) were subjected to RBM3-shRNA knockdown and RBM3 overexpression, respectively. RBM3 depletion in CNE1/IR cells sensitized cells to radiotherapy, increased DNA damage, and accelerated the rate of apoptosis. In contrast, RBM3 overexpression in CNE1 cells significantly enhanced radioresistance and reduced the rate of apoptosis. Additionally, radioresistance conferred by RBM3 was attributed to the activation of the AKT/Bcl-2 signaling pathway and reduction of caspase 3. Inhibition of AKT signaling attenuated RBM3-mediated radioresistance. Furthermore, RBM3 directly interacted with PI3K subunit p85 in NPC cell lines. Altogether, our data demonstrate that RBM3 enhances radioresistance by inhibiting the apoptotic response to radiotherapy through the PI3K/AKT/Bcl-2 signaling pathway. RBM3 may serve as a novel factor for predicting radioresistance and as a molecular target in the treatment of NPC.

A new class of neutral dicopper(I)–halide complexes with {Cu2(μ-X)} (X = Cl, Br and I) core supported by bispyridylpyrrole ligand have been synthesized and characterized. Replacements of chloride ligand in [Cu2(μ-X)2(PPh3)3] (X = Cl, Br, I) by the anionic PDPH− (PDPH− = 2,5-bis(2′-pyridyl)pyrrole) or PDPBr− (PDPBr− = 2,5-bis(6′-bromo-2′-pyridyl)-pyrrole) ligand gives complexes 1–6. They are [(PDPH)Cu2(μ-X)(PPh3)2] (X = Cl (1); Br (2); I (3)) and [(PDPBr)Cu2(μ-X)(PPh3)2] (X = Cl (4); Br (5); I (6)). The X-ray crystallographic studies reveal these complexes are isostructural. Two phosphine–copper(I) units are linked by one halogen atom and one nitrogen atom from bridging bispyridylpyrrole ligand to form 4-membered Cu2XN core. Each copper atom is located in the center of distorted tetrahedral geometry. The PDPH− and PDPBr− ligands display μ2-κ2(N,N′), κ2(N′,N″) bonding mode. The separation of Cu⋯Cu in 1–6 is in the range of 2.6708(15)–2.7959(6) A. The photophysical properties of 1–6 are described.

The ubiquitin-proteasome system (UPS)-dependent proteolysis plays a major role in the muscle catabolic action of glucocorticoids (GCs). Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1), two E3 ubiquitin ligases, are uniquely expressed in muscle. It has been previously demonstrated that GC treatment induced MuRF1 and atrogin-1 overexpression. However, it is yet unclear whether the higher pharmacological dose of GCs induced muscle protein catabolism through MuRF1 and atrogin-1. In the present study, the role of atrogin-1 and MuRF1 in C2C12 cells protein metabolism during excessive dexamethasone (DEX) was studied. The involvement of Akt/forkhead box O1 (FoXO1) signaling pathway and the cross-talk between anabolic regulator mammalian target of rapamycin (mTOR) and catabolic regulator FoXO1 were investigated. High concentration of DEX increased MuRF1 protein level in a time-dependent fashion (P0.05). FoXO1/3a (Thr24/32) phosphorylation was enhanced (P0.05) by DEX. RU486 treatment inhibited the DEX-induced increase of FoXO1/3a phosphorylation (P0.05), but inhibited the activation of MuRF1 protein induced by DEX (P