Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

MAIN CONCLUSION: Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Evolutionary genomics of two diploid goat grass species belonging to the section Sitopsis of Aegilops, Aegilops longissima, and Aegilops sharonensis.

Aegilops longissima and Ae. sharonensis, being classified into the Sitopsis section of genus Aegilops, are distinct species both taxonomically and ecologically. Nevertheless, earlier observations indicate that the two species are not reproductively isolated to full extent and can inter-bred upon secondary contact. However, the genomic underpinnings of the morpho-ecological differentiation between the two foci species remained unexplored. Here, we resequenced 31 representative accessions of the two species and conducted in-depth comparative genomic analyses. We demonstrate recurrent and ongoing natural hybridizations between Ae. longissima and Ae. sharonensis, and depict features of genome composition of the resultant hybrids at both individual and population levels. We also delineate genomic regions and candidate genes potentially underpinning the differential morphological and edaphic adaptations of the two species. Intriguingly, a binary morphology was observed in the hybrids, suggesting existence of highly diverged genomic regions that remain uneroded by the admixtures. Together, our results provide new insights into the molding effects of interspecific hybridization on genome composition and mechanisms preventing merge of the two species.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Influences of the Reaction Temperature and Catalysts on the Pyrolysis Product Distribution of Lignocellulosic Biomass (Aspen Wood and Rice Husk).

It is important to clarify the distribution of pyrolysis products from lignocellulosic biomass for its thermal transformation to produce high-quality bio-oil. Influences of the reaction temperature and catalysts on the pyrolysis product distribution from aspen wood (AW) and rice husk (RH) were studied by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). The difference in components from the lignocellulosic biomass results in different pyrolysis characteristics of the biomass raw materials. The reaction temperature significantly influences the product distribution from AW and RH pyrolysis. In all AW catalysis experiments, acids (8.35%), ketones (3.79%), phenols (4.73%), and esters (1.50%) have the lowest content while carbohydrates (48.75%) demonstrate the highest content when taking zinc chloride (ZnCl2) as the catalyst; the HZSM-5 molecular sieve (HZSM-5) promotes the generation of esters (7.97%) and N-compounds (22.43%) while inhibiting production of aldehydes (2.41%); addition of an MCM-41 molecular sieve (MCM-41) is conducive to increasing the contents of aldehydes (21.29%), furans (5.88%), ketones (22.30%), acids (20.46%), and hydrocarbons (4.85%), while reducing the contents of alcohols (0) and carbohydrates (0). In all RH catalysis experiments, the addition of ZnCl2 helps increase the content of carbohydrates (39.16%) and decrease the contents of ketones (3.89%), phenols (5.20%), alcohols (2.34%), esters (1.13%), and N-compounds (3.09%); when applying HZSM-5 as the catalyst, hydrocarbons (18.28%) and alcohols (6.66%) reach their highest content while acids (13.21%) have the lowest content; MCM-41 promotes the generation of aldehydes (25.33%) and furans (5.55%) while inhibiting that of carbohydrates (1.42%).

Genome shock in a synthetic allotetraploid wheat invokes subgenome-partitioned gene regulation, meiotic instability, and karyotype variation.

It is becoming increasingly evident that interspecific hybridization at the homoploid level or coupled with whole-genome duplication (i.e. allopolyploidization) has played a major role in biological evolution. However, the direct impacts of hybridization and allopolyploidization on genome structure and function, phenotype, and fitness remains to be fully understood. Synthetic hybrids and allopolyploids are trackable experimental systems that can be used to address this issue. In this study, we resynthesized a pair of reciprocal F1 hybrids and corresponding reciprocal allotetraploids using the two diploid progenitor species of bread wheat (Triticum aestivum, BBAADD), namely T. urartu (AA) and Aegilops tauschii (DD). By comparing phenotypes related to growth, development, and fitness, and by analysing genome expression in both hybrids and allotetraploids in relation to the parents, we found that the types and trends of karyotype variation in the immediately formed allotetraploids were correlated with both instability of meiosis and chromosome- and subgenome-biased expression. We determined clear advantages of allotetraploids over diploid F1 hybrids in several morphological traits including fitness that mirrored the tissue- and developmental stage-dependent subgenome-partitioning of the allotetraploids. The allotetraploids were meiotically unstable primarily due to homoeologous pairing that varied dramatically among the chromosomes. Nonetheless, the manifestation of organismal karyotype variation and the occurrence of meiotic irregularity were not concordant, suggesting a role of functional constraints probably imposed by subgenome- and chromosome-biased gene expression. Our results provide new insights into the direct impacts and consequences of hybridization and allopolyploidization that are relevant to evolution and likely to be informative for future crop improvement approaches using synthetic polyploids.

Chromosome translocation affects multiple phenotypes, causes genome-wide dysregulation of gene expression, and remodels metabolome in hexaploid wheat.

Chromosomal rearrangements (CRs) may occur in newly formed polyploids due to compromised meiotic fidelity. Moreover, CRs can be more readily tolerated in polyploids allowing their longer-term retention and hence potential spreading/fixation within a lineage. The direct functional consequences of CRs in plant polyploids remain unexplored. Here, we identified a heterozygous individual from a synthetic allohexaploid wheat in which the terminal parts of the long-arms of chromosomes 2D (approximately 193 Mb) and 4A (approximately 167 Mb) were reciprocally translocated. Five homogeneous translocation lines including both unbalanced and balanced types were developed by selfing fertilization of the founder mutant (RT [2DL; 4AL]-ter/1, reciprocal translocation). We investigated impacts of these translocations on phenotype, genome-wide gene expression and metabolome. We find that, compared with sibling wild-type, CRs in the form of both unbalanced and balanced translocations induced substantial changes of gene expression primarily via trans-regulation in the nascent allopolyploid wheat. The CRs also manifested clear phenotypic and metabolic consequences. In particular, the genetically balanced, stable reciprocal translocations lines showed immediate enhanced reproductive fitness relative to wild type. Our results underscore the profound impact of CRs on gene expression in nascent allopolyploids with wide-ranging phenotypic and metabolic consequences, suggesting CRs are an important source of genetic variation that can be exploited for crop breeding.

Astragalus polysaccharides driven stretchable nanofibrous membrane wound dressing for joint wound healing.

Joint wound dressings are currently significantly limited in their clinical applications due to their inferior mechanical properties and single therapeutic effect. Therefore, it is imperative to develop a versatile joint wound dressing that integrates adequate stretchability, desirable biocompatibility, and multiple biological effects into one system. We implemented the electrospinning technique in this study to fabricate a novel nanofibrous membrane (NFM) composed of gelatin (GEL) and astragalus polysaccharides (APS), termed GEL/APS NFM. The selection of GEL and APS confers excellent biocompatibility to GEL/APS NFM. Furthermore, the optimally proportioned GEL/APS NFM exhibits satisfactory stretchability and desirable wound healing efficiency. Furthermore, released APS can exert anti-inflammatory, procollagen deposition, and proangiogenic effects to accelerate epithelial tissue, enhancing joint wound healing. In summary, GEL/APS NFM offers a convenient and effective approach to promoting rapid joint wound healing, providing a novel approach to joint wound care.

The histone H2B ubiquitination regulator Wac is essential for plasma cell differentiation.

Naïve B cells become activated and differentiate into antibody-secreting plasma cells (PCs) when encountering antigens. Here, we reveal that the WW domain-containing adapter protein with coiled-coil (Wac), which is important for histone H2B ubiquitination (ubH2B), is essential for PC differentiation. We demonstrate that B cell-specific Wac knockout mice have severely compromised T cell-dependent and -independent antibody responses. PC differentiation is drastically compromised despite undisturbed germinal center B cell response in the mutant mice. We also observe a significant reduction in global ubH2B in Wac-deficient B cells, which is correlated with downregulated expression of some genes critical for cell metabolism. Thus, our findings demonstrate an essential role of Wac-mediated ubH2B in PC differentiation and shed light on the epigenetic mechanisms underlying this process.

Research on application of tumor treating fields in glioblastoma: A bibliometric and visual analysis.

Background: Glioblastoma, one of the common tumors of the central nervous system (CNS), is prone to recurrence even after standard treatment protocols. As an innovative physiotherapy method emerging in recent years, the tumor treating fields (TTFields) technique has been approved for the treatment of glioblastoma due to its non-invasive and portable features. The purpose of this study is to visualize and analyze the scientific results and research trends in TTFields therapy for glioblastoma. Methods: Publications related to TTFields therapy for glioblastoma were searched in the Web of Science Core Collection (WoSCC) database in September 2022. A bibliometric and visual analysis of publications in this field was performed mainly using CiteSpace and R software for country/region, author, journal, reference and keyword. Results: A total of 618 publications in this field were retrieved, and 248 were finally obtained according to the search criteria, including 159 articles (64.11%) and 89 reviews (37.89%). The cumulative number of publications increased year by year, with an average growth rate (AGR) of 28.50%. The test results of Pearson correlation coefficient showed a high positive correlation between publications and citations (r=0.937, p<0.001). The USA had the largest number of publications (123, 49.60%), followed by Germany (32, 12.90%) and China (30, 12.10%). As for the country/region collaborations, the USA cooperated most closely with other countries/regions, followed by Germany and China. The degree of collaboration (DC) between countries/regions was 25.81%. The institutions with the largest number of publications were Tel Aviv Univ (10), Harvard Med Sch (10) and Novocure Ltd (10). Moreover, Wong E (18) possessed the greatest number of publications, followed by Weinberg U (11) and Kirson E (10). The DC between authors was 97.58%. STUPP R (236) was the most cited author followed by KIRSON ED (164) and GILADI M (104). JOURNAL OF NEURO-ONCOLOGY (22) was the journal with the largest number of published publications (75), followed by FRONTIERS IN ONCOLOGY (15) and CANCERS (13). The top 10 keywords that occurred frequently included glioblastoma (156), tumor treating field (152), temozolomide (134), randomized phase III (48), brain (46), survivor (46), cancer (44), trial (42), alternating electric field (42) and radiotherapy (36). Furthermore, cluster analysis was performed on the basis of keyword co-occurrence, and finally 15 clusters were formed to determine the current research status and future development trend of TTFields therapy for glioblastoma. Conclusion: TTFields has been increasingly known as the fourth novel physical anti-tumor therapy in addition to surgery, radiotherapy and anti-tumor drugs. Cooperation and communication between countries/regions need to be enhanced in future research. Several studies have demonstrated the therapeutic potential of TTFields in glioma, and its application alone or in combination with other treatments has become a current research hotspot.

Chromosomal instability and phenotypic variation in a specific lineage derived from a synthetic allotetraploid wheat.

Newly formed plant allopolyploids usually have meiosis defect, resulting in chromosomal instability manifested as variation in chromosome number and/or structure. However, not all nascent allopolyploids are equally unstable. The wheat group (Aegilops/Triticum) contains 13 diploid species with distinct genome types. Many of these species can be artificially hybridized to produce viable but sterile inter-specific/intergeneric F1 hybrids, which can generate fertile synthetic allotetraploid wheats after whole genome doubling. Compared with synthetic allotetraploid wheats that contain genome combinations of AADD and S*S*DD (S* refers to related S genomes of a different species), those containing an S*S*AA genome are significantly more stable. However, robustness of the relative stability of S*S*AA genomes is unknown, nor are the phenotypic and fitness consequences during occurrences of secondary chromosomal instability. Here, we report a specific lineage originated from a single individual plant of a relatively stable synthetic allotetraploid wheat with genomes S l S l AA (S l and A subgenomes were from Ae. longissima and T. urartu, respectively) that showed a high degree of transgenerational chromosomal instability. Both numerical chromosome variation (NCV) and structural chromosome variation (SCV) occurred widely. While substantial differences in frequencies of both NCV and SCV were detected across the different chromosomes, only NCV frequencies were significantly different between the two subgenomes. We found that NCVs and SCVs occurred primarily due to perturbed meiosis, allowing formation of multivalents and univalents as well as homoeologous exchanges. Thus, the combination of NCVs and SCVs affected multiple phenotypic traits, particularly those related to reproductive fitness.

Human liver organoids for disease modeling of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Computing metasurfaces enabled chiral edge image sensing.

Computing metasurfaces have shown the extraordinary ability to precisely perform optical analog operations to the input light wave, and therefore exhibit greater potentials toward sensing applications. Here, we propose a unique application of computing metasurface for chiral edge sensing by incorporating a weak-value amplification technique. The computing metasurface performs the spatial differentiation operations of phase objects and extracts the edge-enhanced images, because the phase gradient generally occurs at the edge. The chirality-induced polarization rotation acts as the preselection state and the spatial differentiation operations in the metasurface provide weak coupling. The amplified pointer shift related to the tiny polarization rotation will eventually lead to an asymmetric edge-enhanced image. Owing to the high sensitivity of the weak-value amplification, we experimentally demonstrate a high-contrast recognition of chirality by edge detection, which may have potential applications in real-time measurement and separation of chiral enantiomers.

Realization of tunable edge-enhanced images based on computing metasurfaces.

Bright-field imaging and edge imaging can extract different characteristics from objects, and therefore play important roles in image processing and pattern recognition. Here, we propose a fast, convenient, and electrically driven adjustable scheme to achieve tunable edge-enhanced images based on computing metasurfaces. The computing metasurface can perform spatial differential operation as optical waves propagate through it. This optical differential operation is polarization-dependent, thus any desirable contrast can be realized by the interplay between two orthogonal polarization components. By regulating the external voltages applied on the liquid-crystal phase plate, different phase retardances between two orthogonal polarization components are introduced; this allows us to quickly switch between the bright-field image and the edge image.

Visualization of transparent particles based on optical spatial differentiation.

Optical analog computing operates on the amplitude, phase, polarization, and frequency distributions of the electromagnetic field through the interaction of light and matter. The differentiation operation is widely used in all-optical image processing technology, such as edge detection. Here, we propose a concise way to observe transparent particles, incorporating the optical differential operation that occurs on a single particle. The particle's scattering and cross-polarization components combine into our differentiator. We achieve high-contrast optical images of transparent liquid crystal molecules. The visualization of aleurone grains (the structures that store protein particles in plant cells) in maize seed was experimentally demonstrated with a broadband incoherent light source. Avoiding the interference of stains, our designed method provides the possibility to observe protein particles directly in complex biological tissues.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

Electrically driven generation of arbitrary vector vortex beams on the hybrid-order Poincaré sphere.

We propose a simple, efficient, and fast tunable method to generate arbitrary vector vortex beams on the hybrid-order Poincaré sphere in an electrically driven way. The scheme incorporates the tunability and switching capabilities of liquid crystals into dielectric metasurfaces to form an efficient vector vortex beam generator. By applying certain voltages on the liquid crystal phase retarder, the generator converts a linearly polarized Gaussian beam into any desirable vector vortex beams. We demonstrate that the evolution route of the corresponding vector vortex states is just a closed circuit on the hybrid-order Poincaré sphere when the phase retardation varies from 0 to 2π. Several special cases are selected to demonstrate our scheme, and the experimental results coincide well with the theoretical predictions.

Distinguishing between Hepatic Inflammation and Fibrosis with MR Elastography.

Purpose To investigate the utility of magnetic resonance (MR) elastography-derived mechanical properties in the discrimination of hepatic inflammation and fibrosis in the early stages of chronic liver diseases. Materials and Methods All studies were approved by the institutional animal care and use committee. A total of 187 animals were studied, including 182 mice and five pigs. These animals represented five different liver diseases with a varying combination and extent of hepatic inflammation, fibrosis, congestion, and portal hypertension. Multifrequency three-dimensional MR elastography was performed, and shear stiffness, storage modulus, shear loss modulus, and damping ratio were calculated for all animals. Necroinflammation, fibrosis, and portal pressure were either histologically scored or biochemically and physically quantified in all animals. Two-sided Welch t tests were used to evaluate mean differences between disease and control groups. Spearman correlation analyses were used to evaluate the relationships between mechanical parameters and quantitative fibrosis extent (hydroxyproline concentration) and portal pressure. Results Liver stiffness and storage modulus increased with progressively developed fibrosis and portal hypertension (mean stiffness at 80 Hz and 48-week feeding, 0.51 kPa ± 0.12 in the steatohepatitis group vs 0.29 kPa ± 0.01 in the control group; P = .02). Damping ratio and shear loss modulus can be used to distinguish inflammation from fibrosis at early stages of disease, even before the development of histologically detectable necroinflammation and fibrosis (mean damping ratio at 80 Hz and 20-week feeding, 0.044 ± 0.012 in the steatohepatitis group vs 0.014 ± 0.008 in the control group; P < .001). Damping ratio and liver stiffness vary differently with respect to cause of portal hypertension (ie, congestion- or cirrhosis-induced hypertension). These differentiation abilities have frequency-dependent variations. Conclusion Liver stiffness and damping ratio measurements can extend hepatic MR elastography to potentially enable assessment of necroinflammatory, congestive, and fibrotic processes of chronic liver diseases. © RSNA, 2017 Online supplemental material is available for this article.

Hepatic Stellate Cell Selective Disruption of Dynamin-2 GTPase Increases Murine Fibrogenesis through Up-Regulation of Sphingosine-1 Phosphate-Induced Cell Migration.

Dynamin-2 (Dyn2) is implicated in endocytosis of receptor tyrosine kinases, which contribute to hepatic stellate cell (HSC) activation and liver fibrosis. A point mutation converting lysine 44 of Dyn2 to alanine (Dyn2K44A) disrupts its GTPase activity. We hypothesized that Dyn2K44A expression in HSCs would decrease HSC activation and fibrogenesis in vivo by disrupting receptor tyrosine kinase endocytosis and signaling. Dyn2K44Afl/fl mice were crossed with Collagen1-Cre (Col1Cre) mice to generate offspring with HSC selective expression of Dyn2K44A (Col1Cre/Dyn2K44Afl/fl). Contrary to our hypothesis, Col1Cre/Dyn2K44Afl/fl mice showed increased hepatic fibrosis in response to liver injury. To elucidate mechanisms, we conducted in vitro experiments with HSCs infected with adenoviral vectors encoding LacZ, Dyn2K44A, or Dyn2WT. HSC-expressing Dyn2K44A displayed increased mRNA and protein levels of sphingosine kinase-1 (SK1), an enzyme previously implicated in the pathogenesis of fibrosis. To study the functional effects of Dyn2K44A regulation of SK1, we examined effects of AKT signaling and migration in HSCs. Dyn2K44A promoted both AKT phosphorylation and HSC migration in an SK1-dependent manner. Genetic disruption of Dyn2 GTPase activity selectively in HSC enhances fibrogenesis, driven at least in part through up-regulation of the SK1 pathway and cell migration in HSCs.

Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.

BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.