Porous Chitosan/Hydroxyapatite Composite Microspheres for Vancomycin Loading and Releasing.

Porous chitosan/hydroxyapatite (Chi-HAp) composite microspheres were prepared in an aqueous solution containing chitosan, calcium nitrate, and ammonium dihydrogen phosphate by using a hydrothermal method at various temperatures. The investigation indicated that temperature significantly impacted the final product's appearance. Hydroxyapatite (HAp) coupled with dicalcium phosphate dihydrate (DCPD) flakes were obviously found at 65 and 70 °C, while the latter gradually disappeared at higher temperatures. Conversely, synthesis at 90 °C led to smaller particle sizes due to the broken chitosan chains. The microspheres synthesized at 75 °C were selected for further analysis, revealing porous structures with specific surface areas of 36.66 m2/g, pores ranging from 3 to 100 nm, and pore volumes of 0.58 cm3/g. Vancomycin (VCM), an antibiotic, was then absorbed on and released from the microspheres derived at 75 °C, with a drug entrapment efficiency of 20% and a release duration exceeding 20 days. The bacteriostatic activity of the VCM/composite microspheres against Staphylococcus aureus increased with the VCM concentration and immersion time, revealing a stable inhibition zone diameter of approximately 4.3 mm from 24 to 96 h, and this indicated the retained stability and efficacy of the VCM during the encapsulating process.

The Preparation and Characterization of Chitosan/Calcium Phosphate Composite Microspheres for Biomedical Applications.

In this study, we successfully prepared porous composite microspheres composed of hydroxyapatite (HAp), di-calcium phosphate di-hydrated (DCPD), and chitosan through the hydrothermal method. The chitosan played a crucial role as a chelating agent to facilitate the growth of related calcium phosphates. The synthesized porous composite microspheres exhibit a specific surface area of 38.16 m2/g and a pore volume of 0.24 cm3/g, with the pore size ranging from 4 to 100 nm. Given the unique properties of chitosan and the exceptional porosity of these composite microspheres, they may serve as carriers for pharmaceuticals. After being annealed, the chitosan transforms into a condensed form and the DCPD transforms into Ca2P2O7 at 300 °C. Then, the Ca2P2O7 initially combines with HAp to transform into ß tricalcium phosphate (ß-TCP) at 500 °C where the chitosan is also completely combusted. Finally, the microspheres are composed of Ca2P2O7, ß-TCP, and HAp, also making them suitable for applications such as injectable bone graft materials.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Effects of Adding Chitosan on Drug Entrapment Efficiency and Release Duration for Paclitaxel-Loaded Hydroxyapatite-Gelatin Composite Microspheres.

Hydroxyapatite-gelatin microspheres with cone-like pores were synthesized via the wet-chemical method using ammonium dihydrogen phosphate ((NH4)H2PO4) and calcium nitrate (Ca(NO3)2·4H2O) as a source of calcium and phosphate ions with the addition of gelatin, which proved to be more osteoconductive than commercial products, such as fibrin glue and Osteoset® Bone Graft Substitute. Following the method of the previous study for loading paclitaxel (PTX), a drug entrapment efficiency of around 58% was achieved, which is much lower than that of the doxorubicin (DOX)-loaded one. Since PTX is hydrophobic while DOX is hydrophilic, the order of chitosan processing and addition of the solvent were tuned in this study, finally leading to an increase in drug entrapment efficiency of 94%. Additionally, the release duration of PTX exceeded six months. The MTT assay indicated that the effect of drug release on the suppression of cancer cells reached more than 40% after one week, thereby showcasing PTX's capacity to carry out its medicinal functions without being affected by the loading procedures.

Effects of Chitosan on Loading and Releasing for Doxorubicin Loaded Porous Hydroxyapatite-Gelatin Composite Microspheres.

Porous hydroxyapatite-gelatin (Hap-Gel) composite microspheres derived by wet chemical methods were used as carriers of doxorubicin (DOX) coupled with chitosan (Chi) for treating cancers. Through X-ray diffraction, specific surface area porosimetry, chemisorption analysis and inductively coupled plasma mass spectrometry, the crystalline phase, composition, morphology, and pore distribution of HAp-Gel microspheres were all characterized. HAp nanosized crystals and Gel polymers form porous microspheres after blending and exhibit a specific surface area of 158.64 m2/g, pore sizes from 3 to 150 nm, and pore volumes of 0.4915 cm3/g. These characteristics are suitable for carriers of DOX. Furthermore, by the addition of chitosan during drug loading, its drug-entrapment efficiency increases from 70% to 99% and the release duration increases from a 100% burst within a day to only 45% over half a year since the pores in the composite microspheres provide a shielding effect throughout the degradation period of the chitosan. According to the MTT tests, cell viability of DOX-Chi/HAp-Gel is 57.64% on day 5, similar to the result treated with DOX only. It is concluded that under the protection of pores in the microspheres, the chitosan abundant of hydroxyls combining HAp-Gel and DOX by forming hydrogen bonds indeed enhances the entrapment efficiency, prolongs the releasing period and maintains DOX's ability to perform medicine functions unaffected after loading.

Acute depletion of the ARID1A subunit of SWI/SNF complexes reveals distinct pathways for activation and repression of transcription.

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases.

Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.

AIMS: SCUBE1 [signal peptide-CUB-epidermal growth factor (EGF) domain-containing protein 1], expressed in endothelial cells (ECs) and platelets, exists in soluble or membrane forms. We previously showed that soluble SCUBE1 is a biomarker for platelet activation and also an active participant of thrombosis. However, whether the adhesive module of its EGF-like repeats is essential and the specific contribution of SCUBE1 synthesized in ECs or platelets to thrombosis in vivo remain unclear. METHODS AND RESULTS: We generated new mutant (Δ2) mice lacking the entire EGF-like repeats to evaluate the module's functional importance during thrombogenesis in vivo. The Δ2 platelet-rich plasma showed markedly impaired platelet aggregation induced by agonists including adenosine diphosphate, collagen, the thrombin agonist PAR-4 peptide and the thromboxane A2 analogue U46619. Consistently, genetic ablation of the EGF-like repeats diminished arterial thrombosis and protected Δ2 mice against lethal thromboembolism. On flow chamber assay, whole blood isolated from Δ2 or wild-type (WT) mice pre-treated with blocking antibodies against the EGF-like repeats showed a significant decrease in platelet deposition and thrombus formation on collagen-coated surfaces under arterial shear rates. Moreover, we created animals expressing SCUBE1 only in ECs (S1-EC) or platelets (S1-PLT) by reciprocal bone-marrow transplantation between WT and Δ2 mice. The time of carotid arterial thrombosis induced by ferric chloride was normal in S1-PLT chimeric mice but much prolonged in S1-EC animals. CONCLUSIONS: We demonstrate that platelet-derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF-like repeats in vivo and suggest targeting these adhesive motifs of SCUBE1 for potential anti-thrombotic strategy.

Publisher Correction: BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design.

In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.

BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design.

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

Designing Hybrid NiP2/NiO Nanorod Arrays for Efficient Alkaline Hydrogen Evolution.

Transition-metal phosphides (TMPs) have lately drawn intensive attention because of their noble metal-free properties and high catalytic activities for the hydrogen evolution reaction (HER). The current research mainly focuses on the development of TMPs toward the HER in acidic solutions; however, less efforts have been directed to specifically design TMPs for alkaline HER. Here, we design a new bi-functional metal phosphide-oxide catalyst to facilitate the overall multistep HER process in alkaline environments. In this new catalytic system, oxygen-vacancy-rich NiO provides abundant active sites for dissociation of water, and the negatively charged P species in NiP2 facilitate adsorption of hydrogen intermediates. The resulting hybrid NiP2/NiO NRs show excellent alkaline HER catalytic activity and stability. Our work demonstrates that it is highly promising to engineer multiple components in hybrid catalytic systems to enhance the overall reaction kinetics and thus achieve improvements in catalytic performance.

FUT8 promotes breast cancer cell invasiveness by remodeling TGF-ß receptor core fucosylation.

BACKGROUND: Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-ß signaling during breast cancer progression remains largely unknown. METHOD: In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-ß-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-ß1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. RESULTS: Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-ß-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-ß receptor complexes might be core fucosylated by FUT8 to facilitate TGF-ß binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. CONCLUSIONS: Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-ß signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.

Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis.

Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g., 1,4-BDO).

H2B ubiquitylation and the histone chaperone Asf1 cooperatively mediate the formation and maintenance of heterochromatin silencing.

Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin factors results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication and repair by anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications that modify nucleosome dynamics, such as H2B ubiquitylation (H2Bub). We report here that H2Bub and Asf1 cooperatively promote transcriptional silencing at yeast telomeres and mating loci. Through real time monitoring of HML (Hidden MAT Left) locus silencing, we found that transcriptional repression was slowly initiated and never fully established in mutants lacking both Asf1 and H2Bub. These findings are consistent with impaired HML silencer-binding and spreading of repressor proteins, Sir2 and Sir3. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing at mating loci. Thus, the interplay between H2Hbub and Asf1 may fine-tune nucleosome dynamics and SIR protein recruitment, and represent an ongoing requirement for proper formation and maintenance of heterochromatin.

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942.

BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58-0.63 mg/L. CONCLUSIONS: These data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production.

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0µg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

OBJECTIVE: Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. APPROACH AND RESULTS: We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. CONCLUSIONS: We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy.

Tangeretin sensitizes SGS1-deficient cells by inducing DNA damage.

Tangeretin, a polymethoxyflavone found in citrus peel, has been shown to have antiatherogenic, anti-inflammatory, and anticarcinogenic properties. However, the underlying target pathways are not fully characterized. We investigated the tangeretin sensitivity of yeast (Saccharomyces cerevisiae) mutants for DNA damage response or repair pathways. We found that tangeretin treatment significantly reduced (p < 0.05) survival rate, induced preferential G1 phase accumulation, and elevated the DNA double-strand break (DSB) signal γH2A in DNA repair-defective sgs1Δ cells, but had no obvious effects on wild-type cells or mutants of the DNA damage checkpoint (including tel1Δ, sml1Δ mec1Δ, sml1Δ mec1Δ tel1Δ, and rad9Δ mutants). Additionally, microarray data indicated that tangeretin treatment up-regulates genes involved in nutritional processing and down-regulates genes related to RNA processing in sgs1Δ mutants. These results suggest tangeretin may sensitize SGS1-deficient cells by increasing a marker of DNA damage and by inducing G1 arrest and possibly metabolic stress. Thus, tangeretin may be suitable for chemosensitization of cancer cells lacking DSB-repair ability.