Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

[Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis].

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

Reduction in the occurrence of distressing involuntary memories following propranolol or hydrocortisone in healthy women.

BACKGROUND: Pharmacological treatments targeting the neuroendocrine stress response may hold special promise in secondary prevention of posttraumatic stress disorder (PTSD). However, findings from clinical trials have been inconsistent and the efficacy of specific drugs, their temporal window of efficacy, effective doses and the characteristics of likely treatment responders remain unclear. METHOD: Using an experimental human model of distressing involuntary memory formation, we compare the effects of two drugs that have theoretical or empirical support as secondary preventive agents in PTSD. Eighty-eight healthy women (average age: 23.5 years) received oral propranolol (80 mg), hydrocortisone (30 mg), or matched placebo immediately after viewing a 'trauma film'. They then completed daily, time-stamped intrusion diaries for 1 week, at the end of which, voluntary memory was tested. RESULTS: While neither drug affected voluntary memory for the trauma narrative, propranolol treatment was associated with 42% fewer, and hydrocortisone with 55% fewer intrusions across the week, relative to placebo. Additionally, propranolol reduced general trauma-like symptoms, and post-drug cortisol levels were negatively correlated with intrusion frequency in the hydrocortisone group. CONCLUSIONS: Overall, this study shows substantial reductions in intrusive memories and preserved voluntary narrative-declarative memory following either propranolol or hydrocortisone in an experimental model of psychological trauma. As such, despite some inconsistencies in clinical trials, our findings support continued investigation of propranolol and hydrocortisone as secondary preventive agents for re-experiencing symptoms of PTSD. The findings also suggest that it is critical for future research to identify the conditions governing the preventive efficacy of these drugs in PTSD.

Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum.

Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

Self-renewing human naïve pluripotent stem cells dedifferentiate in 3D culture and form blastoids spontaneously.

Human naïve pluripotent stem cells (hnPSCs) can generate integrated models of blastocysts termed blastoids upon switch to inductive medium. However, the underlying mechanisms remain obscure. Here we report that self-renewing hnPSCs spontaneously and efficiently give rise to blastoids upon three dimensional (3D) suspension culture. The spontaneous blastoids mimic early stage human blastocysts in terms of structure, size, and transcriptome characteristics and are capable of progressing to post-implantation stages. This property is conferred by the glycogen synthase kinase-3 (GSK3) signalling inhibitor IM-12 present in 5iLAF self-renewing medium. IM-12 upregulates oxidative phosphorylation-associated genes that underly the capacity of hnPSCs to generate blastoids spontaneously. Starting from day one of self-organization, hnPSCs at the boundary of all 3D aggregates dedifferentiate into E5 embryo-like intermediates. Intermediates co-express SOX2/OCT4 and GATA6 and by day 3 specify trophoblast fate, which coincides with cavity and blastoid formation. In summary, spontaneous blastoid formation results from 3D culture triggering dedifferentiation of hnPSCs into earlier embryo-like intermediates which are then competent to segregate blastocyst fates.

Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) µg/mL, 1000-fold more sensitive than that reported previously (1 µg/mL). The fusion protein was able to detect fungal concentrations below 1 µg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 µg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity.

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

Colorimetric response of dithizone product and hexadecyl trimethyl ammonium bromide modified gold nanoparticle dispersion to 10 types of heavy metal ions: understanding the involved molecules from experiment to simulation.

A new kind of analytical reagent, hexadecyl trimethyl ammonium bromide (CTAB), and dithizone product-modified gold nanoparticle dispersion, is developed for colorimetric response to 10 types of heavy metal ions (M(n+)), including Cr(VI), Cr(3+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+). The color change of the modified gold nanoparticle dispersion is instantaneous and distinct for Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+). The color change results from the multiple reasons, such as electronic transitions, cation-π interactions, formation of coordination bonds, and M(n+)-induced aggregation of gold nanoparticles (AuNPs). The different combining capacity of heavy metal ions to modifiers results in the different broadening and red-shifting of the plasmon peak of modified AuNPs. In addition, Cr(VI), Cu(2+), Co(2+), Ni(2+), and Mn(2+) cause the new UV-vis absorption peaks in the region of 360-460 nm. The interactions between the modifiers and AuNPs, and between the modifiers and M(n+), are investigated by using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The results confirm that AuNPs are modified by CTAB and dithizone products through electrostatic interactions and Au-S bonds, respectively, and the M(n+)-N bonds form between M(n+) and dithizone products. Furthermore, the experimental and density functional theory calculated IR spectra prove that dithizone reacts with NaOH to produce C6H5O(-) and [SCH2N4](2-). The validation of this method is carried out by analysis of heavy metal ions in tap water.

Coexistence of small gastrointestinal stromal tumor and pancreatic cancer: A case report and literature review.

Small gastrointestinal stromal tumors (GISTs) are rare and malignant tumors that originate in the mesenchymal tissue. Due to their insidious onset and nonspecific symptoms, they are often misdiagnosed, and are generally detected during the diagnosis and treatment of other diseases. The present case report reviewed the treatment process of a patient with a small GIST coexisting with pancreatic cancer who was admitted to the Yiwu Central Hospital (Yiwu, China) in June 2018. The patient was diagnosed and treated comprehensively using a combined approach of urology, and gastrointestinal and hepatobiliary surgery. The present case report provides important clinical insights, which allow for an improved understanding of GIST and provides a reference for clinical treatment.

Atomistic-Continuum Study of an Ultrafast Melting Process Controlled by a Femtosecond Laser-Pulse Train.

In femtosecond laser fabrication, the laser-pulse train shows great promise in improving processing efficiency, quality, and precision. This research investigates the influence of pulse number, pulse interval, and pulse energy ratio on the lateral and longitudinal ultrafast melting process using an experiment and the molecular dynamics coupling two-temperature model (MD-TTM model), which incorporates temperature-dependent thermophysical parameters. The comparison of experimental and simulation results under single and double pulses proves the reliability of the MD-TTM model and indicates that as the pulse number increases, the melting threshold at the edge region of the laser spot decreases, resulting in a larger diameter of the melting region in the 2D lateral melting results. Using the same model, the lateral melting results of five pulses are simulated. Moreover, the longitudinal melting results are also predicted, and an increasing pulse number leads to a greater early-stage melting depth in the melting process. In the case of double femtosecond laser pulses, the pulse interval and pulse energy ratio also affect the early-stage melting depth, with the best enhancement observed with a 2 ps interval and a 3:7 energy ratio. However, pulse number, pulse energy ratio, and pulse interval do not affect the final melting depth with the same total energies. The findings mean that the phenomena of melting region can be flexibly manipulated through the laser-pulse train, which is expected to be applied to improve the structural precision and boundary quality.

Measuring the size and growth of single cells.

The size and growth of a cell can be described by three related physical parameters: volume, density and mass. All the three are coupled to numerous biochemical reactions and biophysical properties of a cell. It is therefore not surprising that cell size and growth pattern are tightly regulated across all kingdoms of life. Indeed, deregulation of cell size and growth has been found to be associated with diseases. Yet, how cells regulate their size and how cell size connects to cell function remain poorly understood, partly due to the difficulties to precisely measure the size and growth of single cells. In this review, we summarize methods of measuring cell volume, density, and mass, and discuss how the new technologies may advance our understanding of cell size control.

Engineering the glyoxylate cycle for chemical bioproduction.

With growing concerns about environmental issues and sustainable economy, bioproduction of chemicals utilizing microbial cell factories provides an eco-friendly alternative to current petro-based processes. Creating high-performance strains (with high titer, yield, and productivity) through metabolic engineering strategies is critical for cost-competitive production. Commonly, it is inevitable to fine-tuning or rewire the endogenous or heterologous pathways in such processes. As an important pathway involved in the synthesis of many kinds of chemicals, the potential of the glyoxylate cycle in metabolic engineering has been studied extensively these years. Here, we review the metabolic regulation of the glyoxylate cycle and summarize recent achievements in microbial production of chemicals through tuning of the glyoxylate cycle, with a focus on studies implemented in model microorganisms. Also, future prospects for bioproduction of glyoxylate cycle-related chemicals are discussed.

The Inhibitory Effect of Pseudomonas stutzeri YM6 on Aspergillus flavus Growth and Aflatoxins Production by the Production of Volatile Dimethyl Trisulfide.

Aspergillus flavus and the produced aflatoxins cause great hazards to food security and human health across all countries. The control of A. flavus and aflatoxins in grains during storage is of great significance to humans. In the current study, bacteria strain YM6 isolated from sea sediment was demonstrated effective in controlling A. flavus by the production of anti-fungal volatiles. According to morphological characteristics and phylogenetic analysis, strain YM6 was identified as Pseudomonas stutzeri. YM6 can produce abundant volatile compounds which could inhibit mycelial growth and conidial germination of A. flavus. Moreover, it greatly prevented fungal infection and aflatoxin production on maize and peanuts during storage. The inhibition rate was 100%. Scanning electron microscopy further supported that the volatiles could destroy the cell structure of A. flavus and prevent conidia germination on the grain surface. Gas chromatography/mass spectrometry revealed that dimethyl trisulfide (DMTS) with a relative abundance of 13% is the most abundant fraction in the volatiles from strain YM6. The minimal inhibitory concentration of DMTS to A. flavus conidia is 200 µL/L (compound volume/airspace volume). Thus, we concluded that Pseudomonas stutzeri YM6 and the produced DMTS showed great inhibition to A. flavus, which could be considered as effective biocontrol agents in further application.

Development of a defined medium for Corynebacterium glutamicum using urea as nitrogen source.

Corynebacterium glutamicum has been widely used for bulk and fine chemicals fermentation these years. In this study, we developed a defined medium for this bacteria based on the widely used CGXII minimal medium. We evaluated the effects of different components in CGXII on cell growth of C. glutamicum ATCC 13032 and improved the medium through single-factor experiment and central composite design (CCD). Urea, K2HPO4 and MgSO4 were found to be significant factors. 7 out of the total 15 components were modified. (NH4)2SO4, KH2PO4, and protocatechuic acid were eliminated. Amounts of urea and MgSO4 were increased, and concentrations of biotin and glucose were reduced. The resulting R2 medium was proved to be more suitable for cell growth, plasmid amplification and protein production than the original recipe. Remarkably, cell biomass accumulation in R2 increased by 54.36% than CGXII. Transcriptome analysis revealed alteration of carbon metabolism, cation transport and energy synthesis, which might be beneficial for cell growth in R2. Considering the high nitrogen content and availability of urea, the new medium is simplified and cost effective, which holds attractive potential for future study. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02959-6.

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D.

Many biological processes happen on a nano- to millimeter scale and within milliseconds. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Multifocal imaging (MFI) allows high-speed 3D imaging but is limited by the compromise between high spatial resolution and large field-of-view (FOV), and the requirement for bright fluorescent labels. Here, we provide an open-source 3D reconstruction algorithm for multi-focal images that allows using MFI for fast, precise, label-free tracking spherical and filamentous structures in a large FOV and across a high depth. We characterize fluid flow and flagellar beating of human and sea urchin sperm with a z-precision of 0.15 µm, in a volume of 240 × 260 × 21 µm, and at high speed (500 Hz). The sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable.

Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility.

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo-electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

Post-traumatic Stress Disorder in Victims of Sexual Assault With Pre-assault Substance Consumption: A Systematic Review.

Background: Post-traumatic stress disorder (PTSD) and substance consumption commonly co-occur in victims of sexual assault. Substance consumption can occur pre- andi/or post-assault. Pre-assault substance consumption may have an impact on the subsequent development of PTSD. This review aims to provide an overview of current understanding of the effects of acute substance intoxication and chronic pre-assault problematic substance use on symptoms of PTSD amongst individuals who were victims of sexual assault. Methods: PsycINFO, EMBASE, and MEDLINE were searched using terms related to PTSD, sexual assault, and substance consumption. These yielded 2,121 articles, 268 of which were retrieved for more detailed evaluation and 13 of these met inclusion criteria and were appraised in full. Results: Overall, the reviewed papers supported our hypothesis that acute substance intoxication and chronic pre-assault problematic substance use are associated with fewer initial PTSD symptoms but less improvement over time, resulting in slower overall PTSD recovery. They also highlighted post-assault characterological self-blame and negative social reactions as mediators of recovery in the context of pre-assault substance consumption. Conclusions: Acute substance intoxication and chronic pre-assault problematic substance use appear to have an impact on the development of PTSD symptoms amongst victims of sexual assault. The importance of developing early interventions and routine screening and assessment for PTSD and pre-assault substance consumption is emphasized. The limited research on male victims and on substances other than alcohol is highlighted.

Inhibitory Effect of Volatiles Emitted From Alcaligenes faecalis N1-4 on Aspergillus flavus and Aflatoxins in Storage.

Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 µL/L, respectively. The effective concentration for MI against A. flavus is 200 µL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.

Aplysin Protects Against Alcohol-Induced Liver Injury Via Alleviating Oxidative Damage and Modulating Endogenous Apoptosis-Related Genes Expression in Rats.

We investigated the protective effects and possible mechanisms of Aplysin against alcohol-induced liver injury. Rats were given daily either alcohol only (alcohol model group; 8 to 12 mL/kg body weight), one of three doses of Aplysin (50, 100, or 150 mg/kg Aplysin) plus alcohol, or volume-matched saline. After 6 weeks, the effects of Aplysin were assessed in terms of changes in histology, biochemical indices, and DNA oxidative damage. Potential mechanisms were analyzed through measurements of lipid peroxidation, antioxidant defense systems, expression of cytochrome P450 2E1, and expression of apoptosis-related genes. We found that Aplysin significantly protected the liver against alcohol-induced oxidative injury, evidenced by improved hepatic histological structure, inhibited alcohol-induced elevation of serum biochemical indices, attenuated extents of hepatocellular DNA damage. At a mechanistic level, Aplysin alleviated alcohol-induced oxidative stress as illustrated by the revivification of erythrocyte membrane fluidity, the attenuation of glutathione depletion, the restoration of antioxidase activities, and reduced malondialdehyde overproduction. Furthermore, the mRNA levels of Bax, cytochrome c, and cytochrome P450 2E1 were significantly down-regulated, whereas those of Bcl-2 and caspase-9 and caspase-3 were markedly up-regulated. These findings suggest that Aplysin provides significant protection against alcohol-induced liver injury, possibly through alleviating oxidative damage and modulating endogenous apoptosis-related genes expression. PRACTICAL APPLICATION: Many natural components derived from alga have been used in the food, cosmetics, and biomedicine industries. Aplysin, a marine bromosesquiterpene, was extracted from the red alga Laurencia tristicha, which could effectively protect against alcohol-induced liver injury, might be a potential natural sources for preventing alcoholic liver damage.