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Supplementary Table 2. The percentage of blast in all primary AML patient samples

Supplementary Figure 1. GO/GSEA/pathway analyses with our top candidates identified in the screen revealed that genes regulate cell adhesion are highly enriched. DAVID (the Database for Annotation, Visualization and Integrated Discovery) pathway analysis program was used to perform GO/GSEA/pathway enrichment analysis. The genes with sgRNA enrichment score above 5 were selected as the input for DAVID.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD–positive and –negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402–13. ©2017 AACR.

Background: Colorectal cancer (CRC) is one of malignant tumors that seriously threatening human health. β-catenin is a central hub in Wnt pathway, aberrant activation of Wnt/β-catenin signaling pathway promotes the tumorigenesis/progression of CRC. Methods and Results: Here we found a β-catenin interactor, UXT-V1, could modulate Wnt signaling. The expression of UXT-V1 mRNA was increased in CRC tissues. Overexpression of UXT-V1 increased the canonical Wnt signaling, as evidenced by Wnt reporter systems and the up-regulation of marker genes including Axin, CyclinD1 and c-Myc. While, knockdown of UXT-V1 impaired the expression of these genes and attenuated Wnt signaling. Mechanistically, overexpression of UXT-V1 could inhibit GSK3β mediated β-catenin phosphorylation and degradation. Knockout of UXT-V1 increased β-catenin phosphorylation, prevented CRC cell growth, and inhibited tumorigenesis in NOD-SCID mice. Conclusions: Taken together, our findings revealed that UXT-V1 could control Wnt signaling through targeting GSK3β mediated β-catenin phosphorylation and degradation, providing a molecular basis for CRC treatment.

PS-3-AT as a novel functional microsphere was prepared via grafting process, which exhibited excellent adsorption properties for gold(i)–thiosulfate complex.

Directional rupture is one of the difficult problems in deep rock mechanics and engineering. A directional fracturing method with static expansive agent controlled by dense linear multi boreholes is proposed. A physical experiment is designed and performed to investigate the basic laws of this method. The fracture initiation and propagation process, and the mechanism of directional fracturing are analyzed. The results indicate that a directional fracture is formed along the direction of boreholes layout through directionally fracturing with static expansive agents controlled by the dense linear multi boreholes. According to the variation of strain and the distribution of associated acoustic emission (AE) events and energy, the experiment can be divided into three stages. In the first stage, the static expansive agent expand slowly with no fracturing inside the rock. In the second stage, some initial micro-fracturing occurs inside the rock. In the third stage, a wide range of fracturing occurs inside the sample. The internal micro-fracturing planes are connected to form a macro-fracture. Finally, it propagates to the surface of the sample. The directional fracturing plane presents a relatively smooth plane with little bias but much local fluctuation.

Shuliang Chen,1,2,&ast; Qi Li,1,&ast; Xueliang Zou,3 Zhijun Zhong,1 Qian Ouyang,1 Mengmeng Wang,1 Yaxing Luo,1 Dongyuan Yao1 1Neurological Institute of Jiangxi Province and Department of Neurology, Jiangxi Provincial People’s Hospital and The First Affiliated Hospital of Nanchang Medical College, Jiangxi, People’s Republic of China; 2Queen Mary College, Nanchang University, Jiangxi, People’s Republic of China; 3Jiangxi Mental Hospital, Nanchang University, Jiangxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Dongyuan Yao, Jiangxi Provincial People’s Hospital, 92 Aiguo Road, Nanchang, Jiangxi, 330006, People’s Republic of China, Tel/Fax +86-791-8689-5077, Email yao9000@gmail.comObjective: To investigate whether continuous positive airway pressure (CPAP) treatment would change EEG activities associated with cyclic alternating pattern (CAP subtype A1, A2, and A3) and non-CAP (NCAP) during non-rapid eye movement sleep stage 3 (N3) in patients with obstructive sleep apnea (OSA).Methods: The effects of CPAP treatment on the percentages of sleep stage N3 occupied by the CAP and NCAP, power of EEG waves in the CAP and NCAP were examined in 18 patients with moderate-to-severe OSA undergoing polysomnographic recordings.Results: Apnea and hypopnea index during sleep stage N3 was positively correlated with ratios of phases A2 and A3 duration to total phase A duration [Phase (A2+A3) /Phase A] and negatively correlated with phase A1/phase A. With CPAP treatment, percentages of sleep stage N3 occupied by total CAPs and subtypes A2 and A3, as well as CAP A2 and CAP A3 indexes were significantly decreased while percentages of sleep stage N3 occupied by NCAP (NCAP/N3) and CAP A1 index were significantly increased. In addition, CPAP treatment significantly decreased percentage of respiratory events associated CAPs and increased percentage of non-respiratory related CAPs. Moreover, absolute and relative delta power was significantly increased during phase A1, unchanged during phase A2 and phase B2, and significantly decreased during phases B1, A3 and B3. The absolute power of faster frequency EEG waves in CAPs showed a general trend of decrease. The absolute and relative power of delta waves with amplitudes ≥ 75 μV, but not < 75 μV, was significantly increased.Conclusion: CPAP treatment improves the sleep quality in OSA patients mainly by increasing delta power and decreasing power of higher frequency waves during phase A1, and decreasing CAP A2 and A3 indexes as well as increasing NCAP/N3 and power of delta waves with amplitudes ≥ 75 μV during NCAP.Keywords: obstructive sleep apnea, continuous positive airway pressure, polysomnographic recordings, non-rapid eye movement sleep, cyclic alternating pattern

In the tunneling of frozen soil section of shaft sinking by freezing method, the traditional air pick excavation has large consumption of pick and drill rod, low excavation efficiency, and high lab...

During hydraulic fracturing in a high-methane coal seam, there is a water-displacing-methane effect. A pseudo triaxle experimental system, which is opposite to the name of true triaxial system, for the water-displacing-methane effect was created. First, cylindrical coal samples in a methane adsorption equilibrium state, spontaneously desorbed. And then water was injected into the coal samples. The following was shown: (1) The displacement methane volume gradually rises with an increase of injected water, while the displacement methane rate tends to rise at first before declining later. Simultaneously, the water-displacing-methane process is characterised by a time effect. The methane displacement lags behind water injection. (2) Competitive adsorption and displacement desorption between the water and methane will promote adsorption methane into free methane, while the pore pressure increase caused by water injection will turn free methane into adsorption methane. The net free methane of the combination action provides a methane source for the water-displacing-methane effect. (3) A pore pressure gradient, which provides a power source for the water-displacing-methane effect, is formed and reduces gradually at the front of the water seepage along the seepage direction. The increase in water pressure can rapidly improve the pore pressure gradient and boost the displacement methane volume as well as improve displacement methane efficiency. (4) A starting porosity pressure gradient and limit pore pressure exist in the process of water-displacing-methane. When the pore pressure gradient is less than the starting pore pressure gradient, there is free methane in the coal rock, but it cannot be displaced. When the pore pressure is between the starting pore pressure and the limit pore pressure, the free methane can be displaced. When the pore pressure is greater than the limit pore pressure, the methane is almost completely adsorption methane, and water cannot be used to displace the free methane.

As the first full superconducting tokamak in the world, the equipment and the experimental level on EAST have been constantly improved. A large number of experimental data are produced, and then the high-quality discharge has great significance to EAST project. In order to improve the experimental level of EAST and guide the high-quality discharge, this paper selected the high-quality discharge shots from the historical experimental data of EAST, and statistically analyzed the key physical parameters of this shots. Finally, the standard of high-quality discharge shots has been conclude, so as to guide the experimenter to obtain the optimal parameter configuration and improve the experimental level of EAST.

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.

Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.

In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20–30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3–5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test., Graphical abstract Image 1

ER-phagy keeps cells healthy In eukaryotic cells, about one-third of all proteins are targeted to the endoplasmic reticulum (ER), which serves as a hub for secretory protein traffic and quality control. Cui et al. studied a protein known as Lst1 in yeast and SEC24C in mammalian cells that is involved in loading secretory cargo into vesicles that are delivered to the Golgi complex. In response to stress caused by starvation or misfolded aggregate-prone secretory proteins, Lst1 acted to promote an additional function—ER-phagy. Together with autophagy receptors on the ER, Lst1 targeted ER domains for degradation to avert protein aggregation, thus preserving cellular health. Science , this issue p. 53

Background The chemokine receptor CCR5, which belongs to the superfamily of G protein-coupled receptors, is the major co-receptor for HIV-1 entry. Individuals with a homozygous CCR5Δ32 mutation have a long lasting and increased resistance to HIV-1 infection. Therefore, CCR5 represents an optimal target for HIV-1/AIDS gene therapy. The CRISPR/Cas9 system has been developed as one of the most efficacious gene editing tools in mammalian cells and the small-sized version from Staphylococcus aureus (SaCas9) has an advantage of easier delivery compared to the most commonly used version from Streptococcus pyogenes Cas9 (SpCas9). Results Here, we demonstrated that CCR5 could be specifically and efficiently edited by CRISPR/SaCas9 together with two sgRNAs, which were identified through a screening of 13 sgRNAs. Disruption of CCR5 expression by lentiviral vector-mediated CRISPR/SaCas9 led to increased resistance against HIV-1 infection in human primary CD4+ T cells. Moreover, humanized mice engrafted with CCR5-disrupted CD4+ T cells showed selective survival and enrichment when challenged with CCR5 (R5)-tropic HIV-1 in comparison to mock-treated CD4+ T cells. We also observed CCR5 could be targeted by CRISPR/SaCas9 in human CD34+ hematopoietic stem/progenitor cells without obvious differentiation deficiencies. Conclusions This work provides an alternative approach to disrupt human CCR5 by CRISPR/SaCas9 for a potential gene therapy strategy against HIV-1/AIDS. Electronic supplementary material The online version of this article (10.1186/s12977-019-0477-y) contains supplementary material, which is available to authorized users.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in cells or HIV-1-infected individuals. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in human monocytic cells. Transfection of differentiated monocytic cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered RNA m6A levels by manipulating the expression of the m6A erasers or pharmacological inhibition of m6A addition in virus-producing cells. RNA transfection and viral infection of differentiated monocytic cells demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies revealed that m6A of HIV-1 RNA escaped the RIG-I-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Moreover, RNA of peripheral blood mononuclear cells from HIV-1 viremic patients showed increased m6A levels that correlated with increased IFN-I mRNA expression compared to levels from HIV-1-suppressed patients on antiretroviral therapy. Together, our results suggest that RNA m6A modifications regulate viral replication and antiviral innate immune responses in HIV-1-infected individuals.Author SummaryHIV-1 is known as a weak inducer of antiviral cytokines including IFN-I, but it is unclear how HIV-1 evades innate immunity. Different types of RNA modifications including m6A within the HIV-1 genome modulate viral replication; however, the role of m6A modifications of HIV-1 RNA in regulating innate immune responses remains elusive. In this study, we found that HIV-1 RNA modified with m6A suppresses the expression of IFN-I in differentiated monocytic cells by avoiding innate immune detection of viral RNA mediated by RIG-I, an RNA sensor in host cells. We also observed significantly increased RNA m6A modifications of peripheral blood mononuclear cells from HIV-1 viremic patients compared to virally suppressed patients on combined antiretroviral therapy, suggesting a functional link between m6A modifications and antiretroviral treatment. Investigating the functions of m6A modifications of HIV-1 RNA in regulating innate immune sensing and IFN-I induction in monocytic cells can help understand the mechanisms of HIV-1 persistence.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells., Author summary HIV-1 is known as a weak inducer of antiviral cytokines including IFN-I, but it is unclear how HIV-1 evades innate immunity. Different types of RNA modifications including m6A within the HIV-1 genome modulate viral replication; however, the role of m6A modifications of HIV-1 RNA in regulating innate immune responses remains elusive. Myeloid cells including macrophages are HIV-1 target cells and critical for generating antiviral immunity. In this study, we aimed to investigate the role of m6A modifications of HIV-1 RNA in regulating innate immune responses in myeloid cells. We found that m6A-modified HIV-1 RNA suppresses IFN-I expression in differentiated monocytic cells and primary macrophages. Our data suggest that the cellular protein RIG-I contributes to innate sensing of m6A-defective HIV-1 RNA in differentiated monocytic cells. Our findings provide new insights into the functions and mechanisms of m6A modifications of HIV-1 RNA in regulating innate immune sensing and responses in myeloid cells.

The topology optimization method has become an effective method to solve the structural optimization problem due to its advantages in weight reduction, material saving and cost reduction. Hinge is a commonly used connector with wide application occasions, large demand, and high reliability requirements. It needs to meet the strength requirements while carrying out lightweight design. In this paper, the refrigerator hinge is taken as the object, the topology optimization of the hinge is carried out by introducing manufacturing process constraints, the model is reconstructed according to the result of the topology optimization, and finally the reconstruction model is analyzed by finite element. The results show that under the premise that the hinge meets the requirements of strength, its own weight and maximum stress are reduced, and the weight reduction rate reaches 13.1%, which achieves the purpose of lightweight hinge design. This method can provide a basis for the evaluation and optimization of the initial design scheme of the hinge, and can also provide a way of thinking for the optimization of other structures.

Stroke is the acute onset of neurological deficits and is associated with high morbidity, mortality, and disease burden. In the present study, we aimed to develop a scientific, nomogram for non-invasive predicting risk for early ischemic stroke, in order to improve stroke prevention efforts among high-risk groups. Data were obtained from a total of 2151 patients with early ischemic stroke from October 2017 to September 2018 and from 1527 healthy controls. Risk factors were examined using logistic regression analyses. Nomogram and receiver operating characteristic (ROC) curves were drawn, cutoff values were established. Significant risk factors for early ischemic stroke included age, sex, blood pressure, history of diabetes, history of genetic, history of coronary heart disease, history of smoking. A nomogram predicting ischemic stroke for all patients had an internally validated concordance index of 0.911. The area under the ROC curve for the logistic regression model was 0.782 (95% confidence interval [CI]: 0.766–0.799, P

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

Background The chemokine receptor CCR5 is one of the co-receptor of HIV-1 infection. People with homozygous CCR5Δ32 deletion resist HIV-1 infection, which makes the CCR5 an important target for HIV-1 gene therapy. Although the CRISPR/Cas9 has ever been used for HIV-1 study, the newly developed CRISPR/AsCpf1 has never been utilized in HIV-1 co-receptor disruption. The CRISPR/Cpf1 system shows many advantages over CRISPR/Cas9, such as lower off-target, small size of nuclease, easy sgRNA design for multiplex gene editing, etc. Therefore, the CRISPR/Cpf1 mediated gene editing will confer a more specific and safe strategy in HIV-1 co-receptor disruption. Results Here, we demonstrated that CRISPR/AsCpf1 could ablate the main co-receptor of HIV-1 infection-CCR5 efficiently with two screened sgRNAs via different delivery strategies (lentivirus, adenovirus). The edited cells resisted R5-tropic HIV-1 infection but not X4-tropic HIV-1 infection compared with the control group in different cell types of HIV-1 study (TZM.bl, SupT1-R5, Primary CD4+T cells). Meanwhile, the edited cells exhibited selective advantage over unedited cells while under the pressure of R5-tropic HIV-1. Furthermore, we clarified that the predicted off-target sites of selected sgRNAs were very limited, which is much less than regular using sgRNAs for CRISPR/Cas9, and no evident off-target was observed. We also showed that the disruption of CCR5 by CRISPR/AsCpf1 took no effects on cell proliferation and apoptosis. Conclusions Our study provides a basis for a possible application of CCR5-targeting gene editing by CRISPR/AsCpf1 with high specific sgRNAs against HIV-1 infection.

Autophagy is a conserved process that delivers cytosolic substances to the lysosome for degradation, but its direct role in the regulation of antiviral innate immunity remains poorly understood. Here, through high-throughput screening, we discovered that CCDC50 functions as a previously unknown autophagy receptor that negatively regulates the type I interferon (IFN) signaling pathway initiated by RIG-I-like receptors (RLRs), the sensors for RNA viruses. The expression of CCDC50 is enhanced by viral infection, and CCDC50 specifically recognizes K63-polyubiquitinated RLRs, thus delivering the activated RIG-I/MDA5 for autophagic degradation. The association of CCDC50 with phagophore membrane protein LC3 is confirmed by crystal structure analysis. In contrast to other known autophagic cargo receptors that associate with either the LIR-docking site (LDS) or the UIM-docking site (UDS) of LC3, CCDC50 can bind to both LDS and UDS, representing a new type of cargo receptor. In mouse models with RNA virus infection, CCDC50 deficiency reduces the autophagic degradation of RIG-I/MDA5 and promotes type I IFN responses, resulting in enhanced viral resistance and improved survival rates. These results reveal a new link between autophagy and antiviral innate immune responses and provide additional insights into the regulatory mechanisms of RLR-mediated antiviral signaling.

The C-X-C chemokine receptor type 4 (CXCR4) is one of the major co-receptors for human immunodeficiency virus type 1 (HIV-1) entry and is considered an important therapeutic target. However, its function in maintaining the development of hematopoietic stem cells (HSC) makes it difficult to be used for HIV-1 gene therapy with HSC transplantation. A previous report showed that the natural CXCR4 P191A mutant inhibits HIV-1 infection without any defect in HSC differentiation, which could provide a basis for the development of new approaches for HIV-1 gene therapy. In the present study, we used CRISPR-Cas9 combined with the piggyBac transposon technologies to efficiently induce the expression of the CXCR4 P191A mutant in an HIV-1 reporter cell line, leading to no detectable exogenous sequences. In addition, no off-target effects were detected in the genome-edited cells. The decline of HIV-1 replication in biallelic CXCR4 gene-edited cells suggests that individuals equipped with homologous recombination of the CXCR4 P191A mutant could prevent or reduce HIV-1 infection. This study provides an effective approach to create a CXCR4 mutation with HIV-1 infection inhibition function and without leaving any genetic footprint inside cells, thereby shedding light on an application in HIV-1 gene therapy and avoiding side effects caused by deficiency or destruction of CXCR4 function.

The CRISPR/Cas9 gene-editing approach has been widely used in anti-HIV-1 gene therapy research. However, the major challenges facing the therapeutic application of CRISPR/Cas9 are the precise genome cleavage efficacy and efficient delivery of Cas9/gRNA specifically to the HIV-infected cells. Recently, a small size Cas9 from Staphylococcus aureus (SaCas9) has shown promise in genome editing in eukaryotic cells, suggesting a potential usage in blocking HIV-1 infection by targeting the HIV-1 genome. Here, we designed 43 guide RNAs (gRNAs) against the HIV-1 genome, thereby identifying 8 gRNAs that efficiently and specifically disrupt the target DNA by SaCas9. In addition, we found the selected gRNAs induce SaCas9 to disrupt the latent HIV-1 provirus and suppress HIV-1 proviral reactivation in latently infected Jurkat C11 cells. We further confirmed that the dual or triple gRNAs in an all-in-one lentiviral vector could reduce viral production in TZM-bl cells as well as in Jurkat T cells. Moreover, we did not detect any off-target cleavages in the predicted sites, suggesting that through all-in-one lentiviral vector-mediated HIV-1 genome editing, the selected SaCas9/gRNAs can provide an alternative and flexible strategy for anti-HIV gene therapy.

The synergistic activation mediator (SAM) system can robustly activate endogenous gene expression by a single-guide RNA. This transcriptional modulation has been shown to enhance gene promoter activity and leads to epigenetic changes. Human interferon-γ is a common natural glycoprotein involved in antiviral effects and inhibition of cancer cell growth. Large quantities of high-purity interferon-γ are important for medical research and clinical therapy. To investigate the possibility of employing the SAM system to enhance endogenous human interferon-γ with normal function in innate immunity, we designed 10 single-guide RNAs that target 200 bp upstream of the transcription start sites of the interferon-γ genome, which could significantly activate the interferon-γ promoter reporter. We confirmed that the system can effectively and highly activate interferon-γ expression in several humanized cell lines. Moreover, we found that the interferon-γ induced by the SAM system could inhibit tumorigenesis. Taken together, our results reveal that the SAM system can modulate epigenetic traits of non-immune cells through activating interferon-γ expression and triggering JAK-STAT signaling pathways. Thus, this strategy could offer a novel approach to inhibit tumorigenesis without using exogenous interferon-γ.