PLAU promotes cell proliferation and migration of head and neck cancer via STAT3 signaling pathway.

It was reported that within the head and neck cancer (HNC) cell line CAL21 the epithelial-mesenchymal transition (EMT) and cell proliferation were promoted by Urokinase-Type Plasminogen Activator (PLAU) proteinase through TNFRSF12A. Additionally, in this paper HNC cell lines refer to Fadu and Tu686. A novel PLAU-STAT3 axis was found to be involved in HNC cell line proliferation and metastasis. PLAU expression in HNC samples was upregulated, besides, the elevated expression of PLAU was linked to the lower overall survival (OS) and disease-free survival (DFS). Ectopic PLAU expression promoted cell proliferation and migration, while PLAU knockdown exhibited opposite results. RNA-seq data identified the JAK-STAT signaling pathway, confirmed by western blotting. A recovery assay using S3I-201, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), indicated that PLAU promoted HNC cell line progression via STAT3 signaling in vitro. The oncogenic role of PLAU in HNC tumor growth in vivo was confirmed using xenograft models. In summary, we identified the tumorigenic PLAU function in the HNC progress. PLAU may represent a potential prognostic biomarker of HNC and the PLAU-STAT3 pathway might be considered a therapeutic target of HNC.

Manual acupuncture ameliorates inflammatory pain by upregulating adenosine A3 receptor in complete Freund's adjuvant-induced arthritis rats.

BACKGROUND: Adenosine A3 receptor (A3R) exerts analgesic, anti-inflammatory, and anti-nociceptive effects. In this study, we determined the analgesic mechanism of manual acupuncture (MA) in rats with complete Freund's adjuvant (CFA)-induced arthritis and explored whether MA ameliorates inflammation in these rats by upregulating A3R. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into the following groups: Control, CFA, CFA + MA, CFA + sham MA, CFA + MA + DMSO, CFA + MA + IB-MECA, and CFA + MA + Reversine groups. The arthritis rat model was induced by injecting CFA into the left ankle joints. Thereafter, the rats were subjected to MA (ST36 acupoint) for 3 days. The clinical indicators paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and open field test (OFT) were used to determine the analgesic effect of MA. In addition, to explore the effect of A3R on inflammation after subjecting arthritis rats to MA, IB-MECA (A3R agonist) and Reversine (A3R antagonist) were injected into ST36 before MA. RESULTS: MA ameliorated the pathological symptoms of CFA-induced arthritis, including the pain indicators PWL and PWT, number of rearing, total ambulatory distance, and activity trajectory. Furthermore, after MA, the mRNA and protein expression of A3R was upregulated in CFA-induced arthritis rats. In contrast, the protein levels of TNF-α, IL-1ß, Rap1, and p-p65 were downregulated after MA. Interestingly, the A3R agonist and antagonist further downregulated and upregulated inflammatory cytokine expression, respectively, after MA. Furthermore, the A3R antagonist increased the degree of ankle swelling after MA. CONCLUSION: MA can alleviate inflammatory pain by inhibiting the NF-κB signaling pathway via upregulating A3R expression of the superficial fascia of the ST36 acupoint site in CFA-induced arthritis rats.

Prediction models for bioavailability of Cu and Zn during composting: Insights into machine learning.

Bioavailability assessment of heavy metals in compost products is crucial for evaluating associated environmental risks. However, existing experimental methods are time-consuming and inefficient. The machine learning (ML) method has demonstrated excellent performance in predicting heavy metal fractions. In this study, based on the conventional physicochemical properties of 260 compost samples, including compost time, temperature, electrical conductivity (EC), pH, organic matter (OM), total phosphorus (TP), total nitrogen, and total heavy metal contents, back propagation neural network, gradient boosting regression, and random forest (RF) models were used to predict the dynamic changes in bioavailable fractions of Cu and Zn during composting. All three models could be used for effective prediction of the variation trend in bioavailable fractions of Cu and Zn; the RF model showed the best prediction performance, with the prediction level higher than that reported in related studies. Although the key factors affecting changes among fractions were different, OM, EC, and TP were important for the accurate prediction of bioavailable fractions of Cu and Zn. This study provides simple and efficient ML models for predicting bioavailable fractions of Cu and Zn during composting, and offers a rapid evaluation method for the safe application of compost products.

Abh, AbrB3, and Spo0A play distinct regulatory roles during polymyxin synthesis in Paenibacillus polymyxa SC2.

IMPORTANCE: Polymyxins are considered the last line of defense against multidrug-resistant bacteria. The regulatory mechanism of polymyxin synthesis is poorly studied in Paenibacillus polymyxa. In this study, we found that Abh and AbrB3 negatively regulated, whereas Spo0A positively regulated polymyxin synthesis in P. polymyxa SC2. In addition, a regulatory relationship between Abh, AbrB3, and Spo0A was revealed, which regulate polymyxin synthesis via multiple regulatory mechanisms in P. polymyxa.

Bioactivities and chemical profiling comparison and metabolomic variations of polyphenolics and steroidal glycoalkaloids in different parts of Solanum nigrum L.

INTRODUCTION: Solanum nigrum L. is a traditional medicinal herb and edible plant. Many studies provide evidence that S. nigrum L. is a nutritious vegetable. Polyphenols and steroidal glycoalkaloids are the main components. OBJECTIVES: This study aimed to systemically evaluate the phytochemical profile, quantification, and bioactivities of polyphenolics and glycoalkaloids in different parts of S. nigrum L. RESULTS: Total polyphenols (TPC) and total glycoalkaloids (TGK) were determined using the Folin-Ciocalteu and acid dye colorimetric methods, respectively. A total of 55 polyphenolic constituents (including 22 phenolic acids and 33 flavonoids) and 24 steroidal glycoalkaloids were identified from different parts using ultrahigh-performance liquid chromatography Q-exactive high-resolution mass spectrometry (UHPLC-QE-HRMS), of which 40 polyphenols (including 15 phenolic acids and 25 flavonoids) and one steroidal glycoalkaloid were characterised for the first time in S. nigrum L. Moreover, typical polyphenols and glycoalkaloids were determined using HPLC-UV and HPLC-evaporative light-scattering detector (ELSD), respectively. In addition, the TPC and TGK and their typical constituents were compared in different anatomical parts. Finally, the antioxidant capacities of polyphenolic extracts from different parts of S. nigrum L. were evaluated by ·OH, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric-reducing antioxidant power (FRAP) assay in vitro. In addition, the antitumour effects of TGK from different parts of S. nigrum L. on the proliferation of PC-3 cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polyphenolic and glycoalkaloid extracts from different parts of S. nigrum L. showed different antioxidant and cytotoxic capacities in vitro. CONCLUSION: This is the first study to systematically differentiate between polyphenolic and glycoalkaloid profiles from different parts of S. nigrum L.

Detection of epistasis interaction loci for fiber quality-related trait via 3VmrMLM in upland cotton.

Cotton fiber quality-related traits, such as fiber length, fiber strength, and fiber elongation, are affected by complex mechanisms controlled by multiple genes. Determining the QTN-by-QTN interactions (QQIs) associated with fiber quality-related traits is therefore essential for accelerating the genetic enhancement of cotton breeding. In this study, a natural population of 1,245 upland cotton varieties with 1,122,352 SNPs was used for detecting the main-effect QTNs and QQIs using the 3V multi-locus random-SNP-effect mixed linear model (3VmrMLM) method. A total of 171 significant main-effect QTNs and 42 QQIs were detected, of which 22 were both main-effect QTNs and QQIs. Of the detected 42 QQIs, a total of 13 significant loci and 5 candidate genes were reported in previous studies. Among the three interaction types, the AD interaction type has a preference for the trait of FE. Additionally, the QQIs have a substantial impact on the enhancement predictability for fiber quality-related traits. The study of QQIs is crucial for elucidating the genetic mechanism of cotton fiber quality and enhancing breeding efficiency.

Aloperine alleviates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome activation.

RATIONALE: Excessive activation of the NLRP3 inflammasome is involved in the pathological progression of acute lung injury (ALI). Aloperine (Alo) has anti-inflammatory effects in many inflammatory disease models; however, its role in ALI remains elusive. In this study, we addressed the role of Alo in NLRP3 inflammasome activation in both ALI mice and LPS-treated RAW264.7 cells. METHODS: The activation of the NLRP3 inflammasome in LPS-induced ALI lungs was investigated in C57BL/6 mice. Alo was administered in order to study its effect on NLRP3 inflammasome activation in ALI. RAW264.7 cells were used to evaluate the underlying mechanism of Alo in the activation of the NLRP3 inflammasome in vitro. RESULTS: The activation of the NLRP3 inflammasome occurs in the lungs and RAW264.7 cells under LPS stress. Alo attenuated the pathological injury of lung tissue as well as downregulates the mRNA expression of NLRP3 and pro-caspase-1 in ALI mice and LPS-stressed RAW264.7 cells. The expression of NLRP3, pro-caspase-1, and caspase-1 p10 were also significantly suppressed by Alo in vivo and in vitro. Furthermore, Alo decreased IL-1ß and IL-18 release in ALI mice and LPS-induced RAW264.7 cells. In addition, ML385, a Nrf2 inhibitor, weakened the activity of Alo, which inhibited the activation of the NLRP3 inflammasome in vitro. CONCLUSION: Alo reduces NLRP3 inflammasome activation via the Nrf2 pathway in ALI mice.

Exploring potential SARS-CoV-2 Mpro non-covalent inhibitors through docking, pharmacophore profile matching, molecular dynamic simulation, and MM-GBSA.

CONTEXT: In the replication of SARS-CoV-2, the main protease (Mpro/3CLpro) is significant. It is conserved in a number of novel coronavirus variations, and no known human proteases share its cleavage sites. Therefore, 3CLpro is an ideal target. In the report, we screened five potential inhibitors (1543, 2308, 3717, 5606, and 9000) of SARS-CoV-2 Mpro through a workflow. The calculation of MM-GBSA binding free energy showed that three of the five potential inhibitors (1543, 2308, 5606) had similar inhibitor effects to X77 against Mpro of SARS-CoV-2. In conclusion, the manuscript lays the groundwork for the design of Mpro inhibitors. METHODS: In the virtual screening phase, we used structure-based virtual screening (Qvina2.1) and ligand-based virtual screening (AncPhore). In the molecular dynamic simulation part, we used the Amber14SB + GAFF force field to perform molecular dynamic simulation of the complex for 100 ns (Gromacs2021.5) and performed MM-GBSA binding free energy calculation according to the simulation trajectory.

Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. Thomsonii in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. Thomsonii (Benth.) Maesen are essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata and P. thomsonii are widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata and P. thomsonii have pharmacological effects in the clinic, such as antipyretic, analgesic, anti-inflammatory and antioxidant effects. These plants are commonly used in the treatment of inflammatory diseases and other related diseases. However, the potential mechanisms of the anti-inflammatory effects of P. lobata and P. thomsonii have not been elucidated. AIM OF THE STUDY: This study aimed to confirm the anti-inflammatory effects of P. lobata and P. thomsonii on inflammatory model diseases and to investigate the mechanism of their anti-inflammatory effects from the perspective of plasma metabolomics. MATERIALS AND METHODS: First, P. lobata and P. thomsonii were identified by high‒performance liquid chromatography (HPLC). Second, we established the following three inflammation models: an acute inflammation model of auricular swelling in mice induced by xylene, an acute inflammation model of foot swelling in rats induced by carrageenan gum, and a chronic inflammation model of cotton ball granuloma in rats. Then we examined the weight and swelling rate of auricular swelling in mice; the residence time, contact area, and mean contact pressure in rats on the gait meter; and the weight of granulomas in rats and the content of IL-1ß and TNF-α in plasma to investigate the anti-inflammatory pharmacodynamics of P. lobata and P. thomsonii. Third, we used LC‒MS‒based plasma metabolomics techniques to obtain potential biomarkers of P. lobata and P. thomsonii related to inflammation. Then, the potential biomarkers were enriched by MetaboAnalyst and KEGG metabolomics analysis tools to obtain metabolic pathways related to inflammation. Finally, we tested the indicators of COX-2, 5-LOX, GSH, GSSG and γ⁃GCL in rat plasma from the granuloma model by enzyme-linked immunosorbent assays (ELISAs) to verify the inflammation-related metabolic pathway. RESULTS: The experimental results showed that P. lobata and P. thomsonii could reduce the swollen weight and swelling rate of the auricle in mice, and could increase the residence time, contact area and mean contact pressure in rats on the gait meter. Moreover, P. lobata and P. thomsonii could inhibit the growth of granulomas and reduce the content of IL-1ß and TNF-α in plasma in rats. The above results preliminarily verified that P. lobata and P. thomsonii have different anti-inflammatory effects. We identified eighteen plasma biomarkers associated with P. lobata and sixteen plasma biomarkers related to P. thomsonii in regulating inflammation by a plasma metabolomics analysis. The following two major metabolic pathways were further screened and enriched: arachidonic acid metabolism and glutathione metabolism. Then we noted that P. lobata and P. thomsonii could reduce the COX-2, 5-LOX and GSSG levels and increase the GSH, GSH/GSSG and γ⁃GCL levels based on the ELISA results, which demonstrated that P. lobata and P. thomsonii affect the anti-inflammatory mechanism through arachidonic acid metabolism and glutathione metabolism. CONCLUSIONS: The results of this study further elucidate the anti-inflammatory mechanism of action of P. lobata and P. thomsonii, providing a scientific basis for developing new drugs for the treatment of inflammation-related diseases and laying a foundation for the development of herbal resources, such as P. lobata and P. thomsonii.

Role of LONP2 in head and neck squamous cell carcinoma.

Lon Peptidase 2, Peroxisomal (LONP2) is a peptidase in peroxisomes that selectively degrades oxidatively damaged proteins in cells and is upregulated in several cancers. In this study, we found both the mRNA and protein level of LONP2 was upregulated in head and neck squamous cell carcinoma (HNSCC) samples. Ectopic overexpressed LONP2 enhanced HNSCC cell growth and migration. Nevertheless, LONP2 silencing suppressed tumor growth in vitro and in vivo. It was also indicated that knockdown of LONP2 also impaired the cell cycle progression and improved apoptosis rate of HNSCC cells. The results of RNA sequencing showed that plastin 3 (PLS3) was heavily downregulated after LONP2 silencing. PLS3 is an actin-bundling protein that functions as an oncogene in several cancers. Furthermore, knockdown of PLS3 inhibited the cell proliferation induced by LONP2 overexpression. In conclusion, we identified an oncogenic effect of LONP2 on cell proliferation and migration in HNSCC via positively regulating the expression of PLS3.

MsmR1, a global transcription factor, regulates polymyxin synthesis and carbohydrate metabolism in Paenibacillus polymyxa SC2.

The multiple-sugar metabolism regulator (MsmR), a transcription factor belonging to the AraC/XylS family, participates in polysaccharide metabolism and virulence. However, the transcriptional regulatory mechanisms of MsmR1 in Paenibacillus polymyxa remain unclear. In this study, knocking out msmR1 was found to reduce polymyxin synthesis by the SC2-M1 strain. Chromatin immunoprecipitation assay with sequencing (ChIP-seq) revealed that most enriched pathway was that of carbohydrate metabolism. Additionally, electromobility shift assays (EMSA) confirmed the direct interaction between MsmR1 and the promoter regions of oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp. MsmR1 stimulates polymyxin biosynthesis by directly binding to the promoter regions of oppC3 and sdr3, while also directly regulating sucA and influencing the citrate cycle (TCA cycle). In addition, MsmR1 directly activates pepF and was beneficial for spore and biofilm formation. These results indicated that MsmR1 could regulate carbohydrate and amino acid metabolism, and indirectly affect biological processes such as polymyxin synthesis, biofilm formation, and motility. Moreover, MsmR1 could be autoregulated. Hence, this study expand the current knowledge of MsmR1 and will be beneficial for the application of P. polymyxa SC2 in the biological control against the certain pathogens in pepper.

Identification and combinatorial engineering of indole-3-acetic acid synthetic pathways in Paenibacillus polymyxa.

BACKGROUND: Paenibacillus polymyxa is a typical plant growth-promoting rhizobacterium (PGPR), and synthesis of indole-3-acetic acid (IAA) is one of the reasons for its growth-promoting capacity. The synthetic pathways of IAA in P. polymyxa must be identified and modified. RESULTS: P. polymyxa SC2 and its spontaneous mutant SC2-M1 could promote plant growth by directly secreting IAA. Through metabonomic and genomic analysis, the genes patA, ilvB3, and fusE in the native IPyA pathway of IAA synthesis in strain SC2-M1 were predicted. A novel strong promoter P04420 was rationally selected, synthetically analyzed, and then evaluated on its ability to express IAA synthetic genes. Co-expression of three genes, patA, ilvB3, and fusE, increased IAA yield by 60% in strain SC2-M1. Furthermore, the heterogeneous gene iaam of the IAM pathway and two heterogeneous IPyA pathways of IAA synthesis were selected to improve the IAA yield of strain SC2-M1. The genes ELJP6_14505, ipdC, and ELJP6_00725 of the entire IPyA pathway from Enterobacter ludwigii JP6 were expressed well by promoter P04420 in strain SC2-M1 and increased IAA yield in the engineered strain SC2-M1 from 13 to 31 µg/mL, which was an increase of 138%. CONCLUSIONS: The results of our study help reveal and enhance the IAA synthesis pathways of P. polymyxa and its future application.

Craspase is a CRISPR RNA-guided, RNA-activated protease.

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.

Isolation and Genome Sequence of a Novel Phosphate-Solubilizing Rhizobacterium Bacillus altitudinis GQYP101 and Its Effects on Rhizosphere Microbial Community Structure and Functional Traits of Corn Seedling.

Bacillus altitudinis is a widely distributed soil bacterium that has various functional activities, including remediation of contaminated soil, degradation of herbicides, and enhancement of plant growth. B. altitudinis GQYP101 was isolated from the rhizosphere soil of Lycium barbarum L. and demonstrated potential as a plant growth-promoting bacterium. In this work, strain GQYP101 could solubilize phosphorus, and increased the stem diameter, maximum leaf area, and fresh weight of corn in a pot experiment. Nitrogen and phosphorus contents of corn seedlings (aerial part) increased by 100% and 47.9%, respectively, after application of strain GQYP101. Concurrently, nitrogen and phosphorus contents of corn root also increased, by 55.40% and 20.3%, respectively. Furthermore, rhizosphere soil nutrients were altered and the content of available phosphorus increased by 73.2% after application of strain GQYP101. The mechanism by which strain GQYP101 improved plant growth was further investigated by whole genome sequence analysis. Strain GQYP101 comprises a circular chromosome and a linear plasmid. Some key genes of strain GQYP101 were identified that were related to phosphate solubilization, alkaline phosphatase, chemotaxis, and motility. The findings of this study may provide a theoretical basis for strain GQYP101 to enhance crop yield as microbial fertilizer.

A chromosome-level genome assembly of Artocarpus nanchuanensis (Moraceae), an extremely endangered fruit tree.

Artocarpus nanchuanensis (Moraceae), which is naturally distributed in China, is a representative and extremely endangered tree species. In this study, we obtained a high-quality chromosome-scale genome assembly and annotation information for A. nanchuanensis using integrated approaches, including Illumina, Nanopore sequencing platform, and Hi-C. A total of 128.71 Gb of raw Nanopore reads were generated from 20-kb libraries, and 123.38 Gb of clean reads were obtained after filtration with 160.34× coverage depth and a 17.48-kb average read length. The final assembled A. nanchuanensis genome was 769.44 Mb with a 2.09 Mb contig N50, and 99.62% (766.50 Mb) of the assembled data was assigned to 28 pseudochromosomes. In total, 39,596 genes (95.10%, 39,596/41,636) were successfully annotated, and 129 metabolic pathways were detected. Plants disease resistance/insect resistance genes, plant-pathogen interaction metabolic pathways, and abundant biosynthesis pathways of vitamins, flavonoid, and gingerol were detected. Unigene reveals the basis of species-specific functions, and gene family in contraction and expansion generally implies strong functional differences in the evolution. Compared with other related species, a total of 512 unigenes, 309 gene families in contraction, and 559 gene families in expansion were detected in A. nanchuanensis. This A. nanchuanensis genome information provides an important resource to expand our understanding of the unique biological processes, nutritional and medicinal benefits, and evolutionary relationship of this species. The study of gene function and metabolic pathway in A. nanchuanensis may reveal the theoretical basis of a special trait in A. nanchuanensis and promote the study and utilization of its rare medicinal value.

DEFB126 polymorphisms and association with idiopathic asthenozoospermia in China.

Idiopathic asthenozoospermia, a common factor in male infertility, is characterized by altered sperm motility function in fresh ejaculate. Although the ß-defensin 126 (DEFB126) protein is associated with asthenozoospermia, DEFB126 gene polymorphisms have not been extensively studied. Therefore, the association between DEFB126 gene polymorphisms and asthenozoospermia requires further investigation. Screening was performed by semen analysis, karyotype analysis, and Y microdeletion detection, and 102 fertile men and 106 men with asthenozoospermia in Chengdu, China, were selected for DEFB126 gene sequence analyses. Seven nucleotide mutations and two nucleotide deletions in the DEFB126 gene were detected. rs11467417 (317-318 del/del), rs11467497 (163-166 wt/del), c.152T>C, and c.227A>G were significantly different between the control and asthenozoospermia groups, likely representing high-risk genetic factors for asthenozoospermia among males. DEFB126 expression was not observed in sperm with rs11467497 homozygous deletion and was unstable in sperm with rs11467417 homozygous deletion. The rs11467497 four-nucleotide deletion leads to truncation of DEFB126 at the carboxy-terminus, and the rs11467417 binucleotide deletion produces a non-stop messenger RNA (mRNA). The above deletions may be responsible for male hypofertility and infertility by reducing DEFB126 affinity to sperm surfaces. Based on in silico analysis, the amino acids 51M and 76K are located in the highly conserved domain; c.152T>C (M51T) and c.227A>G (K76R) are predicted to be damaging and capable of changing alternative splice, structural and posttranslational modification sites of the RNA, as well as the secondary structure, structural stability, and hydrophobicity of the protein, suggesting that these mutations are associated with asthenozoospermia.

Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model.

RATIONALE: 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. METHODS: 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. RESULTS: 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-E1ß, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells. CONCLUSION: The present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

Phage peptides mediate precision base editing with focused targeting window.

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.

KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export.

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.