Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

The PERK pathway: beneficial or detrimental for neurodegenerative diseases and tumor growth and cancer.

Protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is one of the three major sensors in the unfolded protein response (UPR). The UPR is involved in the modulation of protein synthesis as an adaptive response. Prolonged PERK activity correlates with the development of diseases and the attenuation of disease severity. Thus, the current debate focuses on the role of the PERK signaling pathway either in accelerating or preventing diseases such as neurodegenerative diseases, myelin disorders, and tumor growth and cancer. In this review, we examine the current findings on the PERK signaling pathway and whether it is beneficial or detrimental for the above-mentioned disorders.

Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

The role of microRNAs in neurodegenerative diseases: a review.

MicroRNAs (miRNAs) are non-coding RNAs which are essential post-transcriptional gene regulators in various neuronal degenerative diseases and playact a key role in these physiological progresses. Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and, stroke, are seriously threats to the life and health of all human health and life kind. Recently, various studies have reported that some various miRNAs can regulate the development of neurodegenerative diseases as well as act as biomarkers to predict these neuronal diseases conditions. Endogenic miRNAs such as miR-9, the miR-29 family, miR-15, and the miR-34 family are generally dysregulated in animal and cell models. They are involved in regulating the physiological and biochemical processes in the nervous system by targeting regulating different molecular targets and influencing a variety of pathways. Additionally, exogenous miRNAs derived from homologous plants and defined as botanmin, such as miR2911 and miR168, can be taken up and transferred by other species to be and then act analogously to endogenic miRNAs to regulate the physiological and biochemical processes. This review summarizes the mechanism and principle of miRNAs in the treatment of some neurodegenerative diseases, as well as discusses several types of miRNAs which were the most commonly reported in diseases. These miRNAs could serve as a study provided some potential biomarkers in neurodegenerative diseases might be an ideal and/or therapeutic targets for neurodegenerative diseases. Finally, the role accounted of the prospective exogenous miRNAs involved in mammalian diseases is described. 1. Listing a large number of neural-related miRNAs and sorting out their pathways. 2. Classify and sort miRNAs according to their mechanism of action. 3. Demonstrating the effects of up-regulation or down-regulation of each miRNAs on the nervous system.

NF-κB Activation Accounts for the Cytoprotective Effects of PERK Activation on Oligodendrocytes during EAE.

Previous studies demonstrate that activation of pancreatic ER kinase (PERK) protects oligodendrocytes against inflammation in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Interestingly, data indicate that the cytoprotective effects of PERK activation on oligodendrocytes during EAE are not mediated by activating transcription factor 4 (ATF4) but are accompanied by activation of nuclear factor κB (NF-κB). NF-κB plays a critical role in MS and EAE; however, the effects of NF-κB activation on oligodendrocytes in these diseases remain elusive. Herein, we generated a mouse model that allow for activation of NF-κB specifically in oligodendrocytes and found that enhanced NF-κB activation in oligodendrocytes had a minimal effect on their viability and function under normal conditions (both male and female mice). Interestingly, we found that enhanced NF-κB activation in oligodendrocytes attenuated EAE disease severity and ameliorated EAE-induced oligodendrocyte loss, demyelination, and axon degeneration, without affecting inflammation (female mice). Moreover, we showed that the detrimental effects of PERK inactivation in oligodendrocytes in EAE were accompanied by impaired NF-κB activation in oligodendrocytes, and were completely rescued by enhanced NF-κB activation in oligodendrocytes (female mice). These findings suggest that NF-κB activation accounts for the cytoprotective effects of PERK activation on oligodendrocytes in MS and EAE.SIGNIFICANCE STATEMENT Nuclear factor κB (NF-κB) is activated in oligodendrocytes in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE); however, the role of NF-κB activation in oligodendrocytes in MS and EAE remains elusive. Herein, we generated a mouse model that allows for activation of NF-κB selectively in oligodendrocytes and demonstrated that NF-κB activation prevented oligodendrocyte death and myelin damage in the EAE model. We further demonstrated that NF-κB activation contributed to the protective effects of pancreatic ER kinase (PERK) activation on oligodendrocytes in the EAE model. As such, this work will facilitate the development of new treatments that enhance oligodendrocyte survival in MS patients by targeting the PERK-NF-κB pathway.

Discovery of novel pan-genotypic HCV NS5A inhibitors containing a novel tetracyclic core.

A series of novel tetracyclic core-containing HCV NS5A inhibitors has been discovered. Incorporation of tetrahydropyran-substituted amino acid moiety improved their potency and yielded HCV NS5A inhibitors with a minimum potency shift from the GT1a strain compared to other genotypes and mutants. Compounds 53 and 54 showed the best potency profile and had reasonable half-times in rat PK studies. However, further optimization of their oral bioavailability is still needed in order to advance them for further development. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.

Resistant cutoff values and optimal scheme establishments for florfenicol against Escherichia coli with PK-PD modeling analysis in pigs.

Florfenicol, a structural analog of thiamphenicol, has broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria. This study was conducted to investigate the epidemiological, pharmacokinetic-pharmacodynamic cutoff, and the optimal scheme of florfenicol against Escherichia coli (E. coli) with PK-PD integrated model in the target infectious tissue. 220 E. coli strains were selected to detect the susceptibility to florfenicol, and a virulent strain P190, whose minimum inhibitory concentration (MIC) was similar to the MIC50 (8 µg/ml), was analyzed for PD study in LB and ileum fluid. The MIC of P190 in the ileum fluid was 0.25 times lower than LB. The ratios of MBC/MIC were four both in the ileum and LB. The characteristics of time-killing curves also coincided with the MBC determination. The recommended dosages (30 mg/kg·body weight) were orally administrated in healthy pigs, and both plasma and ileum fluid were collected for PK study. The main pharmacokinetics (PK) parameters including AUC24 hr , AUC0-∞ , Tmax , T1/2 , Cmax , CLb, and Ke were 49.83, 52.33 µg*h/ml, 1.32, 10.58 hr, 9.12 µg/ml, 0.50 L/hr*kg, 0.24 hr-1 and 134.45, 138.71 µg*hr/ml, 2.05, 13.01 hr, 16.57 µg/ml, 0.18 L/hr*kg, 0.14 hr-1 in the serum and ileum fluid, respectively. The optimum doses for bacteriostatic, bactericidal, and elimination activities were 29.81, 34.88, and 36.52 mg/kg for 50% target and 33.95, 39.79, and 42.55 mg/kg for 90% target, respectively. The final sensitive breakpoint was defined as 16 µg/ml. The current data presented provide the optimal regimens (39.79 mg/kg) and susceptible breakpoint (16 µg/ml) for clinical use, but these predicted data should be validated in the clinical practice.

N-O Reduction and ROS-Mediated AKT/FOXO1 and AKT/P53 Pathways Are Involved in Growth Promotion and Cytotoxicity of Cyadox.

Cyadox is a novel derivative of quinoxaline-1,4-dioxides (QdNOs) with the potential to be developed as a feed additive. However, the pharmacological and toxicological bioactive molecules of cyadox and the molecular mechanism of its pharmacological and toxic actions remain unclear. In the present study, cyadox and its main metabolites of cy1, cy4, cy6, and cy12 were selected; the growth promotion characteristic was indicated by the mRNA level of EGF; and the cytotoxicity of cyadox was determined by methylthiazol tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, and Annexin V-FITC/PI apoptosis detection kit with flow cytometry. The intracellular ROS, cyclin D1, and Akt/P53/FOXO1 signaling pathway were also investigated. Our data suggested that cyadox showed relatively higher activity than its metabolites, and the ROS was generated from N-O reduction of cyadox. Moreover, cyadox (2 µM) activated the Akt and increased the EGF, cyclin D1, and FOXO1 expression levels. Cyadox (100 µM) induced cytotoxicity in L02 cells in a concentration- and time-dependent manner. Additionally, the activated P53 pathway, hyperactivated Akt, and apoptosis were found in L02 cells after incubation with 100 µM cyadox. Our data demonstrated that Akt promoted cell survival when it was mildly activated by cyadox at 2 µM, and Akt leads to apoptosis when it was severely activated by cyadox at 100 µM. Thus, the present study revealed that N-O reduction of cyadox and ROS-mediated AKT/FOXO1 and AKT/P53 pathways were involved in growth promotion and cytotoxicity of cyadox.

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats.

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18-156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

A Radiolabeling-Free, qPCR-Based Method for Locus-Specific Pseudouridine Detection.

Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Methods have been developed for locus-specific Ψ detection; however, they often involve radiolabeling of RNA, require advanced experimental skills, and can be time-consuming. Herein we report a radiolabeling-free, qPCR-based method to rapidly detect locus-specific Ψ. Pseudouridine residues were labeled chemically, and the resulting adducts induced mutation/deletion during reverse transcription (RT) to generate qPCR products with different melting temperatures and hence altered melting curves. We validated our method on known Ψ sites in rRNA and then used it to sensitively detect Ψ residues in lncRNA and mRNA of low abundance. Finally, we applied our method to pseudouridine synthase identification and showed that Ψ616 in PSME2 mRNA is dependent on PUS7. Our facile and cost-effective method takes only 1.5 days to complete, and with slight adjustment it can be applied to the detection of other epitranscriptomic marks.

Sample preparation to observe the straight and flat posture of silkworm embryo under scanning electron microscopy via glycerol substitution method.

In the preparation process for scanning electron microscopy (SEM), flexed silkworm embryos typically assume several curled shapes with irregular postures that obscure morphological details during SEM observation. We describe a preparation technique based on glycerol substitution for better SEM visualization of straight and flat silkworm embryos. Glycerol has high viscosity, low vapor pressure, and sufficient electrical conductivity. Silkworm embryos were infiltrated with glycerol and arranged in a straight posture or flattened using a cover slip. Samples were directly observed by SEM without additional dehydration, drying, or coating procedures. The complete ventral side could be easily viewed in one image. Recoating alleviated the charging phenomenon. This represents a simple method for preparation of straight and flat samples from curled biological specimens for SEM observation.

Mechanisms Governing Oligodendrocyte Viability in Multiple Sclerosis and Its Animal Models.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory demyelinating disease of the central nervous system (CNS), which is triggered by an autoimmune assault targeting oligodendrocytes and myelin. Recent research indicates that the demise of oligodendrocytes due to an autoimmune attack contributes significantly to the pathogenesis of MS and its animal model experimental autoimmune encephalomyelitis (EAE). A key challenge in MS research lies in comprehending the mechanisms governing oligodendrocyte viability and devising therapeutic approaches to enhance oligodendrocyte survival. Here, we provide an overview of recent findings that highlight the contributions of oligodendrocyte death to the development of MS and EAE and summarize the current literature on the mechanisms governing oligodendrocyte viability in these diseases.

Development and challenges of antimicrobial peptide delivery strategies in bacterial therapy: A review.

The escalating global prevalence of antimicrobial resistance poses a critical threat, prompting concerns about its impact on public health. This predicament is exacerbated by the acute shortage of novel antimicrobial agents, a scarcity attributed to the rapid surge in bacterial resistance. This review delves into the realm of antimicrobial peptides, a diverse class of compounds ubiquitously present in plants and animals across various natural organisms. Renowned for their intrinsic antibacterial activity, these peptides provide a promising avenue to tackle the intricate challenge of bacterial resistance. However, the clinical utility of peptide-based drugs is hindered by limited bioavailability and susceptibility to rapid degradation, constraining efforts to enhance the efficacy of bacterial infection treatments. The emergence of nanocarriers marks a transformative approach poised to revolutionize peptide delivery strategies. This review elucidates a promising framework involving nanocarriers within the realm of antimicrobial peptides. This paradigm enables meticulous and controlled peptide release at infection sites by detecting dynamic shifts in microenvironmental factors, including pH, ROS, GSH, and reactive enzymes. Furthermore, a glimpse into the future reveals the potential of targeted delivery mechanisms, harnessing inflammatory responses and intricate signaling pathways, including adenosine triphosphate, macrophage receptors, and pathogenic nucleic acid entities. This approach holds promise in fortifying immunity, thereby amplifying the potency of peptide-based treatments. In summary, this review spotlights peptide nanosystems as prospective solutions for combating bacterial infections. By bridging antimicrobial peptides with advanced nanomedicine, a new therapeutic era emerges, poised to confront the formidable challenge of antimicrobial resistance head-on.

Chemical and Biological Approaches to Interrogate off-Target Effects of Genome Editing Tools.

Various genome editing tools have been developed for programmable genome manipulation at specified genomic loci. However, it is crucial to comprehensively interrogate the off-target effect induced by these genome editing tools, especially when apply them onto the therapeutic applications. Here, we outlined the off-target effect that has been observed for various genome editing tools. We also reviewed detection methods to determine or evaluate the off-target editing, and we have discussed their advantages and limitations. Additionally, we have summarized current RNA editing tools for RNA therapy and medicine that may serve as alternative approaches for genome editing tools in both research and clinical applications.

MicroRNA therapeutics and nucleic acid nano-delivery systems in bacterial infection: a review.

Bacteria that have worked with humans for thousands of years pose a major threat to human health even today, as drug resistance has become a prominent problem. Compared to conventional drug therapy, nucleic acid-based therapies are a promising and potential therapeutic strategy for diseases in which nucleic acids are delivered through a nucleic acid delivery system to regulate gene expression in specific cells, offering the possibility of curing intractable diseases that are difficult to treat at this stage. Among the many nucleic acid therapeutic ideas, microRNA, a class of small nucleic acids with special properties, has made great strides in biology and medicine in just over two decades, showing promise in preclinical drug development. In this review, we introduce recent advances in nucleic acid delivery systems and their clinical applications, highlighting the potential of nucleic acid therapies, especially miRNAs extracted from traditional herbs, in combination with the existing set of nucleic acid therapeutic systems, to potentially open up a new line of thought in the treatment of cancer, viruses, and especially bacterial infectious diseases.

Research progress of nanoparticle targeting delivery systems in bacterial infections.

Bacterial infection is a huge threat to the health of human beings and animals. The abuse of antibiotics have led to the occurrence of bacterial multidrug resistance, which have become a difficult problem in the treatment of clinical infections. Given the outstanding advantages of nanodrug delivery systems in cancer treatment, many scholars have begun to pay attention to their application in bacterial infections. However, due to the similarity of the microenvironment between bacterial infection lesions and cancer sites, the targeting and accuracy of traditional microenvironment-responsive nanocarriers are questionable. Therefore, finding new specific targets has become a new development direction of nanocarriers in bacterial prevention and treatment. This article reviews the infectious microenvironment induced by bacteria and a series of virulence factors of common pathogenic bacteria and their physiological functions, which may be used as potential targets to improve the targeting accuracy of nanocarriers in lesions.

Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.

Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.

Morphological and Transcriptomic Analysis of the Supplemental Boron in the Liver of Ostrich Chicks.

African ostrich chicks (Struthio camelus) were divided into six groups, and each received different levels of boric acid (source of boron) in the drinking water (0, 40, 80, 160, 320, and 640 mg/L respectively) to examine the histological, apoptotic, biochemical, and transcriptomic parameters. Morphological analysis in different groups was assessed by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and terminal deoxynucleotide transferase dUTP Nick-End Labeling (TUNEL) assay. The biochemical profile was evaluated spectrophotometrically. Detailed RNA-Seq of the data was performed using the transcriptomic method. H&E staining showed well-developed liver structure up to the 160 mg/L boric acid (BA) supplement groups, while BA doses (320 mg/L and 640 mg/L) caused changes in hepatocytes and portal triads. PAS staining showed that glycogen levels were optimal in the 80 mg/L BA dose group, but a reduction in glycogen levels was observed after this group, particularly in the 640 mg/L BA supplement group. Cellular apoptosis showed a biphasic pattern, and the BA dose above 160 mg/L enhanced cell death. In addition, serum analysis showed that doses of 80-160 mg BA were beneficial for ostrich liver. Then, the transcriptome analysis of the 80 mg dose also showed mainly positive effects on the liver. These results demonstrated that chronic BA exposure (320-640 mg) can cause significant histological, apoptotic, and biochemical changes in African ostrich liver, while the adequate dose of supplementation (particularly 80 mg BA) promotes liver growth.

Microstructures and Mechanical Properties of Al-Zn-Mg-Cu Alloys under Multi-Directional Severe Strain and Aging.

Microstructure is a significant factor that influences the mechanical properties of alloys. The effect of multiaxial forging (MAF) and subsequent aging treatment on the precipitated phases of Al-Zn-Mg-Cu alloy remains unclear. Therefore, an Al-Zn-Mg-Cu alloy was processed by means of solid solution and aging treatment, and MAF and aging treatment in this work, and the composition and distribution of precipitated phases were characterized in detail. The MAF results for dislocation multiplication and grain refinement were found. The high density of dislocation greatly accelerates the nucleation and growth of precipitated phases. Thus, the GP-zones almost transform into precipitated phases during subsequent aging. The MAF and aging alloy has more precipitated phases than the solid solution and aging treated alloy. The precipitates on the grain boundary are coarse and discontinuously distributed due to dislocation and grain boundary promoting the nucleation, growth and coarsening of the precipitates. The hardness, strength, ductility and microstructures of the alloy have been studied. Without compromising the ductility much, the MAF and aging alloy has higher hardness and strength, with values of 202 HV and 606 MPa, respectively, and an appreciable ductility of 16.2%.