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Alveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI). Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a murine model.The effects of Alo in LPS-induced ALI were investigated in C57BL/6 mice. The RIPK1 inhibitor (Nec-1) and the RIPK3 inhibitor (GSK'872) were used to evaluate the relationship of necroptosis, NF-κB activation, and PDC subunits in LPS-treated mouse alveolar epithelial cells (MLE-12). Then the effects of Alo on necroptosis, inflammation, and oxidative stress of LPS-stimulated MLE-12 cells were evaluated.Alo significantly attenuated histopathological lung injuries and reduced lung wet/dry ratio in LPS-induced ALI mice. Alo also remarkedly reduced total protein and neutrophils recruitment in bronchoalveolar lavage fluid of ALI mice. Meanwhile, Alo ameliorated the LPS-induced necroptosis in the lungs of ALI mice. The RIPK3 inhibitor GSK'872, but not the RIPK1 inhibitor Nec-1, reversed LPS-induced p65 phosphorylation and translocation to the nucleus in MLE-12 cells. GSK'872 also reversed the LPS-induced increase in ROS and binding of RIPK3 and PDC subunits in MLE-12 cells. Moreover, Alo down-regulated the levels of p-RIPK1, p-RIPK3, p-MLKL, p-p65, the translocation of p65 to the nucleus, and reduced the expression of IL-6 and IL-8 in LPS-stimulated MLE-12 cells. Alo also inhibited the binding of RIPK3 and PDC-E1α, PDC-E1β, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells.The present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.

Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barre syndrome1,2. While progress has been made in understanding the causal link between ZIKV infection and microcephaly3-9, the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV10-22. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.

Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.

Background CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. Results We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. Conclusion G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

Ozone may lead to DNA breaks in airway epithelial cells. p-ATM (phosphorylated ataxia telangiectasia mutated) plays a pivotal role in DNA repair. Derivatives of NO (nitric oxide) are regulators of the phosphorylation, and NO is increased under oxidative stress. The present study was aimed to study the effect of NO donor L-arg (L-arginine) on DNA damage repair in human bronchial epithelial cells exposed to ozone and the potential mechanisms involved. HBECs (human bronchial epithelial cells) were cultured with or without ozone (1.5 ppm, 30 min), DNA breaks were measured with a comet assay and agarose gel electrophoresis, cell cycling was determined by flow cytometry and p-ATM was measured by immunofluorescence and Western blot. Data were analysed by ANOVA (analysis of variance). P,0.05 was considered as significant. Ozone induced marked DNA breaks, G1-phase arrest and increased expression of p-ATM in HBECs, while wortmannin reduced the levels of p-ATM induced by ozone; the NO donor, L-arg, minimized the effects of ozone-induced DNA breaks and increased the level of p-ATM, while the NO synthase inhibitor, L-NMMA [N(G)-minomethyl-L-arginine], restrained those effects of L-arg. The effect of L-arg on DNA repair is NO-mediated, and p-ATM is implicated in the processes of DNA repair.

To explore the effects of calcitonin-gene-related peptide (CGRP) on LPS-induced MMP-9 secretion by alveolar macrophages (AM) in vitro.The supernatant of LPS-induced Wistar rat AM from different intervention groups were collected to measure the activity by gelatin zymography.(Only secreting a small amount of MMP-9 with unstimulated AM, LPS stimulated MMP-9 production in a concentration-dependent manner (p0.01). (2) The activity of MMP-9 in CGRP intervention groups at different levels were significantly lower than those in non-intervention group (p0.01). (3) The inhibiting effects of CGRP were diminished by H-7 and W-7, an antagonist of protein kinase C (PKC) and calmodulin (CaM) (p0.05).These data suggested that CGRP involved in the MMP-9 secretion by AM, partly, via PKC and CaM pathway.

Vasoactive intestinal peptide (VIP) is one of the most important sensory neuropeptides in respiratory system. We previously reported that VIP enhances wound healing and proliferation of human bronchial epithelial cells (HBECs), and these effects are mediated by intracellular signaling molecules such as protein kinase A (PKA), protein kinase C (PKC), Calmodulin (CaM), and extracellular signal-regulated kinase (ERK). In the present study, we further investigated the role of cAMP Response Element Binding Protein (CREB) in VIP-promoted protective effects in mechanical-damaged HBECs. VIP-mediated wound healing and cell proliferation in HBECs was inhibited by CREB antisense oligonucleotides (ASO) in a time-dependent manner. VIP increased the CREB DNA binding activity and expression of the p-CREB that were inhibited by VIP receptor antagonist. Increased CREB DNA binding activity and expression of the p-CREB were also partially inhibited by PKA and ERK inhibitors. These results suggest that the VIP-mediated wound repair of HBECs is associated with activation of CREB via PKA and ERK dependent pathway.

To examine the expression of matrix metalloproteinase-9 (MMP-9) in human bronchial epithelial cells treated with calcitonin-gene-related peptide (CGRP).RT-PCR and gelatin zymography were performed to examine the dynamic expression and activity of MMP-9 in human bronchial epithelial cells at different doses (10(-10), 10(-9), 10(-8), 10(-7), and 10(-6)mol/L) and different time points (6,12,18,24,36, and 48h) after the stimulation of CGRP.The unstimulated human bronchial epithelial cells only secreted a small amount of MMP-9. After the CGRP stimulation, the expression of MMP-9 presented in a concentration-dependent (10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) and time-dependent (6,12,18,24,36, and 48 h) manners (P0.01) in human bronchial epithelial cells. The effect of CGRP could be diminished by H-7 and W-7, an antagonist of protein kinase C (PKC) and calmodulin (CaM) (P0.05).CGRP can stimulate the secretion and expression of MMP-9 in human bronchial epithelial cells, and the signal transduction is partly via the PKC and CaM pathway.

Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays an important role in maintaining the bronchial tone of the airway and has anti-inflammatory properties. Recently, we reported that VIP enhances wound repair in human bronchial epithelial cells (HBEC). In the present study, we have identified the intracellular signaling molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC. Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and mRNA induced by VIP. These results suggest that intracellular signaling molecules such as PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.