Elk1 enhances inflammatory cell infiltration and exacerbates acute lung injury/acute respiratory distress syndrome by suppressing Fcgr2b transcription.

OBJECTIVE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant mortality rates. The role of Fcgr2b in the pathogenesis of ALI/ARDS is not fully elucidated. This study aimed to investigate the functions of Fcgr2b in ALI/ARDS and explore its underlying mechanisms. METHODS: Methods: In this study, rat models of ARDS and pulmonary microvascular endothelial cell (PMVEC) injury models were established through the administration of lipopolysaccharide (LPS). The expression levels of Fcgr2b and Elk1 were quantified in both LPS-induced ARDS rats and PMVECs. Subsequent gain- and loss-of-function experiments were conducted, followed by comprehensive assessments of lung tissue for pathomorphological changes, edema, glycogen storage, fibrosis, and infiltration of inflammatory cells. Additionally, bronchoalveolar lavage fluid was analyzed for T-helper 17 (Th17) cell infiltration, inflammatory response, and microvascular permeability to evaluate lung injury severity in ARDS models. Furthermore, the activity, cytotoxicity, apoptosis, and angiogenic potential of PMVECs were assessed to gauge cell injury. The interaction between Elk1 and Fcgr2b was also examined to confirm their regulatory relationship. RESULTS: In the context of LPS-induced ARDS and PMVEC injury, Fcgr2b expression was markedly reduced, whereas Elk1 expression was elevated. Overexpression of Fcgr2b led to a decrease in Th17 cell infiltration and mitigated lung tissue damage in ARDS models, in addition to reducing LPS-induced injury in PMVECs. Elk1 was found to suppress Fcgr2b transcription through the recruitment of histone 3 lysine 9 trimethylation (H3K9me3). Knockdown of Elk1 diminished Th17 cell infiltration and lung tissue damage in ARDS models, and alleviated LPS-induced injury in PMVECs, effects that were reversed upon Fcgr2b upregulation. CONCLUSION: Elk1 negatively regulates Fcgr2b transcription, thereby augmenting the inflammatory response and exacerbating lung injury in LPS-induced ALI/ARDS.

Tracking endogenous proteins based on RNA editing-mediated genetic code expansion.

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection.

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.

Irf7 regulates the expression of Srg3 and ferroptosis axis aggravated sepsis-induced acute lung injury.

OBJECTIVE: To investigate the mechanism of action of Srg3 in acute lung injury caused by sepsis. METHODS: First, a sepsis-induced acute lung injury rat model was established using cecal ligation and puncture (CLP). RNA sequencing (RNA-seq) was used to screen for highly expressed genes in sepsis-induced acute lung injury (ALI), and the results showed that Srg3 was significantly upregulated. Then, SWI3-related gene 3 (Srg3) was knocked down using AAV9 vector in vivo, and changes in ALI symptoms in rats were analyzed. In vitro experiments were conducted by establishing a cell model using lipopolysaccharide (LPS)-induced BEAS-2B cells and coculturing them with phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells to analyze macrophage polarization. Next, downstream signaling pathways regulated by Srg3 and transcription factors involved in regulating Srg3 expression were analyzed using the KEGG database. Finally, gain-of-loss functional validation experiments were performed to analyze the role of downstream signaling pathways regulated by Srg3 and transcription factors involved in regulating Srg3 expression in sepsis-induced acute lung injury. RESULTS: Srg3 was significantly upregulated in sepsis-induced acute lung injury, and knocking down Srg3 significantly improved the symptoms of ALI in rats. Furthermore, in vitro experiments showed that knocking down Srg3 significantly weakened the inhibitory effect of LPS on the viability of BEAS-2B cells and promoted alternative activation phenotype (M2) macrophage polarization. Subsequent experiments showed that Srg3 can regulate the activation of the NF-κB signaling pathway and promote ferroptosis. Specific activation of the NF-κB signaling pathway or ferroptosis significantly weakened the effect of Srg3 knockdown. It was then found that Srg3 can be transcriptionally activated by interferon regulatory factor 7 (Irf7), and specific inhibition of Irf7 significantly improved the symptoms of ALI. CONCLUSIONS: Irf7 transcriptionally activates the expression of Srg3, which can promote ferroptosis and activate classical activation phenotype (M1) macrophage polarization by regulating the NF-κB signaling pathway, thereby exacerbating the symptoms of septic lung injury.

Study on Shrinkage in Alkali-Activated Slag-Fly Ash Cementitious Materials.

Traditional silicate cement materials produce a large amount of CO2 during production, making it urgent to seek alternatives. Alkali-activated slag cement is a good substitute, as its production process has low carbon emissions and energy consumption, and it can comprehensively utilize various types of industrial waste residue while possessing superior physical and chemical properties. However, the shrinkage of alkali-activated concrete can be larger than that of traditional silicate concrete. To address this issue, the present study utilized slag powder as the raw material, sodium silicate (water glass) as the alkaline activator, and incorporated fly ash and fine sand to study the dry shrinkage and autogenous shrinkage values of alkali cementitious material under different content. Furthermore, combined with the change trend of pore structure, the impact of their content on the drying shrinkage and autogenous shrinkage of alkali-activated slag cement was discussed. Based on the author's previous research, it was found that by sacrificing a certain mechanical strength, adding fly ash and fine sand can effectively reduce the drying shrinkage and autogenous shrinkage values of alkali-activated slag cement. The higher the content, the greater the strength loss of the material and the lower the shrinkage value. When the fly ash content was 60%, the drying shrinkage and autogenous shrinkage of the alkali-activated slag cement mortar specimens decreased by about 30% and 24%, respectively. When the fine sand content was 40%, the drying shrinkage and autogenous shrinkage of the alkali-activated slag cement mortar specimens decreased by about 14% and 4%, respectively.

The complete chloroplast genome sequence of Chrysojasminum subhumile and its phylogenetic position within Oleaceae.

We assembled and characterized the complete chloroplast genome sequence of Chrysojasminum subhumile (W.W.Sm.) Banfi & Galasso 2014, a valuable horticultural and medicinal plant species. The total genome size was 159,918 bp in length and the GC content was 37.4%. It displayed a circular structure and could be divided into a large single-copy region, a small single-copy region, and a pair of inverted repeat regions. The genome encoded a total of 131 unique genes, including 82 protein-coding genes, 41 tRNA genes, and eight rRNA genes. Among these genes, 17 contained a single intron, and two genes had two introns. Phylogenetic analysis results showed that C. subhumile was closely related to Jasminum.

Comprehensive analysis illustrating the role of PANoptosis-related genes in lung cancer based on bioinformatic algorithms and experiments.

Background: Recently, PANoptosis has aroused the interest of researchers for its role in cancers. However, the studies that investigated PANoptosis in lung cancer are still few. Methods: The public data were mainly collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database. R software was utilized for the analysis of public data. Quantitative real-time (qRT) polymerase chain reaction (PCR) was used to measure the RNA level of FADD. The cell proliferation ability was evaluated using the CCK8, colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Western blot was used to detect the protein level of specific molecules. Flow cytometry analysis and TUNEL staining were used to evaluate cell apoptosis. Results: In our study, we collected the PANoptosis-related genes from previous studies. Through series analysis, we identified the FADD, an adaptor of PANoptosis and apoptosis, for further analysis. Results showed that FADD is one of the prominent risk factors in lung cancer, mainly localized in nucleoplasm and cytosol. We next performed immune infiltration analysis and biological enrichment to illustrate the underlying cause of FADD in lung cancer. Subsequently, we discovered that the patients with a high level of FADD might respond worse to immunotherapy but better to AICAR, bortezomib, docetaxel, and gemcitabine. In vitro experiments indicated that inhibiting FADD could reduce significantly the ability of cancerous lung cells to proliferate. Meanwhile, we found that the knockdown of FADD promotes the apoptosis and pyroptosis. Ultimately, a prognosis signature was identified based on the FADD-regulated genes, which showed satisfactory prediction efficiency on patients with lung cancer. Conclusion: Our result can provide a novel direction for future studies focused on the role of PANoptosis in lung cancer.

Creation of a Yeast Strain with Co-Translationally Acylated Nucleosomes.

Structurally diverse acylations have been identified as post-translational modifications (PTMs) on histone lysine residues, but their functions and regulations remain largely unknown. Interestingly, in nature, a lysine acylation analog, pyrrolysine, is introduced as a co-translational modification (CTM) through genetic encoding. To explore this alternative life form, we created a model organism Saccharomyces cerevisiae containing site-specific lysine CTMs (acetyl-lysine, crotonyl-lysine, or another synthetic analog) at histone H3K56 using non-canonical amino acid mutagenesis to afford a chemically modified nucleosome in lieu of their own in vivo. We further demonstrated that acetylation of histone H3K56 partly tends to provide a more favorable chromatin environment for DNA repair in yeast compared to crotonylation and crosstalk with other PTMs differently. This study provides a potentially universal approach to decipher the consequences of different histone lysine PTMs in eukaryotes.

Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a.

We recently developed a system to create human chimeric antigen receptor (CAR)-T cells using conjugated Cas12a (cCas12a) in which Cas12a is covalently linked to its CRISPR RNA (crRNA). This protocol describes site-specific modification of Cas12a and the preparation of Cas12a-crRNA complex using bio-orthogonal chemistry, followed by CAR-T cell generation through electroporation and AAV infection. This system shows robust editing efficiency in human cells and can be used for precisely targeted, highly efficient integration of CAR genes into T cell genome. For complete details on the use and execution of this protocol, please refer to Ling et al. (2021).

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates.

The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.

Role of Hydrogen Sulfide in Myocardial Ischemia-Reperfusion Injury.

ABSTRACT: Hydrogen sulfide (H2S), generally known as a new gas signal molecule after nitric oxide and carbon monoxide, has been found as an important endogenous gasotransmitter in the last few decades, and it plays a significant role in the cardiovascular system both pathologically and physiologically. In recent years, there is growing evidence that H2S provides myocardial protection against myocardial ischemia-reperfusion injury (MIRI), which resulted in an ongoing focus on the possible mechanisms of action accounting for the H2S cardioprotective effect. At present, lots of mechanisms of action have been verified through in vitro and in vivo models of I/R injury, such as S-sulfhydrated modification, antiapoptosis, effects on microRNA, bidirectional effect on autophagy, antioxidant stress, or interaction with NO and CO. With advances in understanding of the molecular pathogenesis of MIRI and pharmacology studies, the design, the development, and the pharmacological characterization of H2S donor drugs have made great important progress. This review summarizes the latest research progress on the role of H2S in MIRI, systematically explains the molecular mechanism of H2S affecting MIRI, and provides a new idea for the formulation of a myocardial protection strategy in the future.

The Neutrophil Percentage-to-Albumin Ratio as a New Predictor of All-Cause Mortality in Patients with Cardiogenic Shock.

BACKGROUND: Although the neutrophil percentage-to-albumin ratio (NPAR) has proven to be a robust systemic inflammation-based predictor of mortality in a wide range of diseases, the prognostic value of the NPAR in critically ill patients with cardiogenic shock (CS) remains unknown. This study aimed at investigating the association between the admission NPAR and clinical outcomes in CS patients using real-world data. METHODS: Critically ill patients diagnosed with CS in the Medical Information Mart for Intensive Care-III (MIMIC-III) database were included in our study. The study endpoints included all-cause in-hospital, 30-day, and 365-day mortality in CS patients. First, the NPAR was analyzed as a continuous variable using restricted cubic spline Cox regression models. Second, X-tile analysis was used to calculate the optimal cut-off values for the NPAR and divide the cohort into three NPAR groups. Moreover, multivariable Cox regression analyses were used to assess the association of the NPAR groups with mortality. RESULTS: A total of 891 patients hospitalized with CS were enrolled in this study. A nonlinear relationship between the NPAR and in-hospital and 30-day mortality was observed (all P values for nonlinear trend<0.001). According to the optimal cut-off values by X-tile, NPARs were divided into three groups: group I (NPAR < 25.3), group II (25.3 ≤ NPAR < 34.8), and group III (34.8 ≤ NPAR). Multivariable Cox analysis showed that higher NPAR was independently associated with increased risk of in-hospital mortality (group III vs. group I: hazard ratio [HR] 2.60, 95% confidence interval [CI] 1.72-3.92, P < 0.001), 30-day mortality (group III vs. group I: HR 2.42, 95% CI 1.65-3.54, P < 0.001), and 365-day mortality (group III vs. group I: HR 6.80, 95% CI 4.10-11.26, P < 0.001) in patients with CS. CONCLUSIONS: Admission NPAR was independently associated with in-hospital, 30-day, and 365-day mortality in critically ill patients with CS.

Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion.

Genetic code expansion (GCE) is a powerful technique for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells, which is achieved through evolved aminoacyl-tRNA synthetase mutants. Stability is important for promoting enzyme evolution, and we found that many of the evolved synthetase mutants have reduced thermostabilities. In this study, we characterized two novel pyrrolysyl-tRNA synthetases (PylRSs) derived from thermophilic archaea: Methanosarcina thermophila (Mt) and Methanosarcina flavescens (Mf). Further study demonstrated that the wild-type PylRSs and several mutants were orthogonal and active in both Escherichia coli and mammalian cells and could thus be used for GCE. Compared with the commonly used M. barkeri PylRS, the wild-type thermophilic PylRSs displayed reduced GCE efficiency; however, some of the mutants, as well as some chimeras, outperformed their mesophilic counterparts in mammalian cell culture at 37 °C. Their better performance could at least partially be attributed to the fact that these thermophilic synthetases exhibit a threshold of enhanced stability against destabilizing mutations to accommodate structurally diverse substrate analogues. These were indicated by the higher melting temperatures (by 3-6 °C) and the higher expression levels that were typically observed for the MtPylRS and MfPylRS mutants relative to the Mb equivalents. Using histone H3 as an example, we demonstrated that one of the thermophilic synthetase mutants promoted the incorporation of multiple acetyl-lysine residues in mammalian cells. The enzymes developed in this study add to the PylRS toolbox and provide potentially better scaffolds for PylRS engineering and evolution, which will be necessary to meet the increasing demands for expanded substrate repertoire with better efficiency and specificity in mammalian systems.

Exogenous hydrogen sulfide reduces atrial remodeling and atrial fibrillation induced by diabetes mellitus via activation of the PI3K/Akt/eNOS pathway.

Diabetes mellitus (DM) facilitates atrial fibrosis and increases the risk of atrial fibrillation (AF). The underlying mechanism of DM in causing AF remains mostly unknown and potential therapeutic targets for DM­induced AF are rarely reported. Hydrogen sulfide (H2S) has drawn considerable attention in recent years for its potential as a cardiovascular protector. Thus, the aim of the present study was to investigate the effect of H2S on DM­induced AF and the mechanism of action. Sprague­Dawley rats were divided into four groups, including the control group, the DM group, the H2S group and the DM+H2S group. The DM group and the DM+H2S group were administered streptozotocin to induce DM, whereas the other two groups were given citrate buffer as a control. The H2S group and the DM+H2S group were administered with an intraperitoneal injection of sodium hydrosulfide (precursor of H2S). AF inducibility, AF duration, atrial fibrosis and vital protein expression of oxidative stress were compared among the four groups. The DM group showed significantly higher AF incidence rates and duration (P<0.05). Histology results demonstrated severe atrial fibrosis in the DM group, and the PI3K/Akt/endothelial nitric oxide synthase (eNOS) pathway was significantly downregulated (P<0.05). However, when H2S was administered, the rats showed lower AF incidence and duration compared with the DM group. Additionally, H2S was able to mitigate the atrial fibrosis induced by DM, as well as the proliferation and migration of cardiac fibroblasts, as demonstrated by an MTT assay and real­time cell analyzer migration experiment. Western blotting showed that the expression levels of the PI3K/Akt/eNOS pathway in the DM+H2S group were significantly upregulated compared with those of the DM group (P<0.05). In summary, DM status can lead to the structural remodeling of atrial fibrosis, facilitating AF incidence and persistence. Administration of H2S does not affect the glucose level, but can significantly mitigate atrial fibrosis and reduce the incidence of AF induced by DM, probably via activation of the PI3K/Akt/eNOS pathway.

Rational design of minimum CRISPR guide RNA by site-specific Cas9-RNA conjugation.

The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. We report a Cas9-RNA conjugation strategy to afford minimal crRNA containing only the guide sequence for the target gene, which may simplify and reduce the cost for large-scale and high-throughput crRNA synthesis and lead to new insights into the design of CRISPR family complexes for diverse purposes.

Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9-mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9-based genome-editing systems.

Photocatalytic and Luminescent Sensing Properties of Two New 1D Zn(II)-Based Coordination Polymers.

Two new d10-configuration based Zn(II) coordination polymers with formula [Zn2(L)2(2,2'-bipy)2]n (1) and [Zn2(L)2(4,4'-bipy)]n (2) (H2L = 4,4'-{[1,2-phenylenebis-(methylene)]bis(oxy)}dibenzoic acid, 2,2'-bipy= 2,2'-bipyridine and 4,4'-bipy= 4,4'-bipyridine) have been synthesized and characterized. Both the compounds had been used as possible luminescent sensors for detecting nitroaromatic compounds (NACs) and as photocatalysts to photodegrade methyl violet (MV) under UV irradiation. The sensing experiments indicated that 2 displayed selective sensing for m-nitrophenol (MNP) with lower limit of detection (LOD) of 1.14 ppm while photocatalytic experiments indicated that 1 displayed better photocatalytic performance than 2 in photodegrading MV.