DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis.

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway.

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

ARTS and Siah collaborate in a pathway for XIAP degradation.

ARTS (apoptosis-related protein in the TGF-ß signaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction.

Cloning, Expression, and Characterization of a New PL25 Family Ulvan Lyase from Marine Bacterium Alteromonas sp. A321.

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL-1 and 0.11 µmol·min-1·mL-1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.

BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1.

The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.

Virtual screening and biological evaluation of natural products as urate transporter 1 (URAT1) inhibitors.

Hyperuricemia is mainly caused by insufficient renal urate excretion. Urate transporter 1 (URAT1), an organic anion transporter, is the main protein responsible for urate reabsorption. In this study, we utilized artificial intelligence-based AlphaFold2 program to construct URAT1 structural model. After molecular docking and conformational evaluation, four e-pharmacophoric models were constructed based on the complex structures of probenecid-URAT1, benzbromarone-URAT1, lesinurad-URAT1, and verinurad-URAT1. Combining pharmacophore modeling, molecular docking, MM/GBSA calculation and ADME prediction, 25 flavonoids were selected from the natural products database containing 10,968 molecules. Then, a model of HEK-293T cells overexpressing URAT1 was constructed, and the inhibitory activity to URAT1 of 25 flavonoids was evaluated by measuring their effect on cellular uptake of 6-carboxyfluorescein (6-CFL). Fisetin, baicalein, and acacetin showed the best activity with IC50 values of 12.77, 26.71, and 57.30 µM, respectively. Finally, the structure-activity relationship of these three flavonoids was analyzed by molecular docking and molecular dynamics simulations. The results showed that the carbonyl group on C-4 and hydroxyl group on C-7, C-4', and C-5' in flavonoids were conducive for URAT1 inhibitory effects. This study facilitates the application of flavonoids in the development of URAT1 inhibitors.Communicated by Ramaswamy H. Sarma.

Rationale and design of the GOLDEN BRIDGE II: a cluster-randomised multifaceted intervention trial of an artificial intelligence-based cerebrovascular disease clinical decision support system to improve stroke outcomes and care quality in China.

BACKGROUND: Given the swift advancements in artificial intelligence (AI), the utilisation of AI-based clinical decision support systems (AI-CDSSs) has become increasingly prevalent in the medical domain, particularly in the management of cerebrovascular disease. AIMS: To describe the design, rationale and methods of a cluster-randomised multifaceted intervention trial aimed at investigating the effect of cerebrovascular disease AI-CDSS on the clinical outcomes of patients who had a stroke and on stroke care quality. DESIGN: The GOLDEN BRIDGE II trial is a multicentre, open-label, cluster-randomised multifaceted intervention study. A total of 80 hospitals in China were randomly assigned to the AI-CDSS intervention group or the control group. For eligible participants with acute ischaemic stroke in the AI-CDSS intervention group, cerebrovascular disease AI-CDSS will provide AI-assisted imaging analysis, auxiliary stroke aetiology and pathogenesis analysis, and guideline-based treatment recommendations. In the control group, patients will receive the usual care. The primary outcome is the occurrence of new vascular events (composite of ischaemic stroke, haemorrhagic stroke, myocardial infarction or vascular death) at 3 months after stroke onset. The sample size was estimated to be 21 689 with a 26% relative reduction in the incidence of new composite vascular events at 3 months by using multiple quality-improving interventions provided by AI-CDSS. All analyses will be performed according to the intention-to-treat principle and accounted for clustering using generalised estimating equations. CONCLUSIONS: Once the effectiveness is verified, the cerebrovascular disease AI-CDSS could improve stroke care and outcomes in China. TRIAL REGISTRATION NUMBER: NCT04524624.

Tumor-suppressing function of caspase-2 requires catalytic site Cys-320 and site Ser-139 in mice.

The multifunctional caspase-2 protein is involved in apoptosis, NF-κB regulation, and tumor suppression in mice. However, the mechanisms of caspase-2 responsible for tumor suppression remain unclear. Here we identified two sites of caspase-2, the catalytic Cys-320 site and the Ser-139 site, to be important for suppression of cellular transformation and tumorigenesis. Using SV40- and K-Ras-transformed caspase-2 KO mouse embryonic fibroblast cells reconstituted with expression of wild-type, catalytic dead (C320A), or Ser-139 (S139A) mutant caspase-2, we demonstrated that similar to caspase-2 deficiency, when Cys-320 and Ser-139 were mutated, caspase-2 lost its ability to inhibit cellular transformation and tumorigenesis. These mutant cells exhibited enhanced cell proliferation, elevated clonogenic activity, accelerated anchorage-independent growth, and transformation and were highly tumorigenic, rapidly producing large tumors in athymic nude mice. Investigation into the underlying mechanism showed that these two residues are needed for caspase-2 to suppress NF-κB activity, promote apoptosis, and sustain the G(2)/M checkpoint following DNA damage induction. In addition, tumors in nude mice derived from the two mutant cell lines had higher constitutive NF-κB activity and elevated expression of NF-κB targets of antiapoptotic proteins Bcl-xL, XIAP, and cIAP2. A reduction in caspase-2 mRNA was associated with multiple types of cancers in patients. Together, these observations suggest the combined functions of caspase-2 in suppressing NF-κB activation, promoting apoptosis, and sustaining G(2)/M checkpoint contribute to caspase-2 tumor-suppressing function and that caspase-2 may also impact tumor suppression in humans. These findings provide insight into tumor suppression at the cross-roads of apoptosis, cell cycle checkpoint, and NF-κB pathways.

Regulatory effects mediated by ulvan oligosaccharide and its zinc complex on lipid metabolism in high-fat diet-fed mice.

Obesity-induced lipid metabolism disorders are risk factors for hyperlipidemia, atherosclerosis, and non-alcoholic fatty liver disease. Seaweed oligosaccharides and Zn supplements are potential alternatives to alleviate obesity. Herein, ulvan oligosaccharide (UO) was used as a ligand to prepare a novel Zn supplement (UO-Zn). Subsequently, we explored potential mechanisms underlying UO- and UO-Zn-mediated improvements in lipid metabolism in mice fed a high-fat diet. We found that UO enhanced the abundance of key species (Blautia and Turicibacter) and functions (glycolytic, pentose phosphate, and histidine/lysine biosynthesis pathways) in the gut microbiota, thereby increasing the production of short-chain fatty acids and activating AMPK. Accordingly, UO treatment regulated the transcription of lipid metabolism genes, including ACOX1, ACC, and FASN, thereby reducing blood lipid levels and hepatic lipid accumulation. Zn could act synergistically with UO, enhancing the reversal of cholesterol transport and fatty acid ß-oxidation via the MTF1/PPARα pathway, markedly reducing body and adipose tissue weights.

Eugenol exposure inhibits embryonic development and swim bladder formation in zebrafish.

Eugenol is a natural phenolic essential oil extracted from cloves, that has analgesic and anesthetic effects and is widely used in fishery anesthesia. However, the potential safety risks of aquaculture production associated with the massive use of eugenol and its developmental toxicity during early life stages of fish have been overlooked. In this study, zebrafish (Danio rerio) embryos at 24 hours post-fertilization (hpf) were exposed to eugenol at concentrations of 0, 10, 15, 20, 25, or 30 mg/L for 96 h. Eugenol exposure delayed the hatching of zebrafish embryos, and reduced the body length and the inflation rate of the swim bladder. The accumulated number of dead zebrafish larvae in the eugenol-exposed groups was higher than that of the control group, and it was dose-dependent. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the Wnt/ß-catenin signaling pathway that regulates the development of the swim bladder during the hatching and mouth-opening stages was inhibited after eugenol exposure. Specifically, the expression of wif1, a Wnt signaling pathway inhibitor, was significantly up-regulated, whereas the expression of fzd3b, fzd6, ctnnb1, and lef1 involved in the Wnt/ß-catenin pathway was significantly down-regulated. These results suggest that the failure of zebrafish larvae to inflate their swim bladders as a result of eugenol exposure may be caused by the inhibition of the Wnt/ß-catenin signaling pathway inhibited. In addition, the inability to catch food due to the abnormal development of the swim bladder may be the key to the death of zebrafish larvae during the mouth-opening stage.

Genetic ablation of diabetes-associated gene Ccdc92 reduces obesity and insulin resistance in mice.

Multiple genome-wide association studies (GWAS) have identified specific genetic variants in the coiled-coil domain containing 92 (CCDC92) locus that is associated with obesity and type 2 diabetes in humans. However, the biological function of CCDC92 in obesity and insulin resistance remains to be explored. Utilizing wild-type (WT) and Ccdc92 whole-body knockout (KO) mice, we found that Ccdc92 KO reduced obesity and increased insulin sensitivity under high-fat diet (HFD) conditions. Ccdc92 KO inhibited macrophage infiltration and fibrosis in white adipose tissue (WAT), suggesting Ccdc92 ablation protects against adipose tissue dysfunction. Ccdc92 deletion also increased energy expenditure and further attenuated hepatic steatosis in mice on an HFD. Ccdc92 KO significantly inhibited the inflammatory response and suppressed the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome in WAT. Altogether, we demonstrated the critical role of CCDC92 in metabolism, constituting a potential target for treating obesity and insulin resistance.

BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of ß-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis.

BACKGROUND: BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) is a liver tumor suppressor, which is downregulated in a large number of patients with liver diseases. BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine (DEN)-dependent acute liver injury and carcinogenesis. While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma (HCC), DEN exposure alone does not induce robust hepatic fibrosis. Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC. AIM: To investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development. METHODS: Male C57/BL6/J control mice [loxp/Loxp; albumin-cre (Alb-cre)-] and BRUCE Alb-Cre KO mice (loxp/Loxp; Alb-Cre+) were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression. RESULTS: By using a liver-specific BRUCE knockout (LKO) mouse model, we found that BRUCE deficiency, in conjunction with DEN exposure, induced hepatic fibrosis in both premalignant as well as malignant stages, thus recapitulating the chronic fibrosis background often observed in HCC patients. Activated in fibrosis and HCC, ß-catenin activity depends on its stabilization and subsequent translocation to the nucleus. Interestingly, we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity of ß-catenin in the three stages of carcinogenesis: Pre-malignancy, tumor initiation, and HCC. This suggests that BRUCE negatively regulates ß-catenin activity during liver disease progression. ß-catenin can be activated by phosphorylation by protein kinases, such as protein kinase A (PKA), which phosphorylates it at Ser-675 (pSer-675-ß-catenin). Mechanistically, BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level. However, in BRUCE deficient mouse livers or a human liver cancer cell line, both PKA activity and pSer-675-ß-catenin levels were observed to be elevated. CONCLUSION: Our data support a "BRUCE-PKA-ß-catenin" signaling axis in the mouse liver. The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation of ß-catenin. This study implicates BRUCE as a novel negative regulator of both PKA and ß-catenin in chronic liver disease progression. Furthermore, BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA and ß-catenin.

Metal-ion-binding properties of ulvan extracted from Ulva clathrata and structural characterization of its complexes.

The acidic polysaccharide ulvan extracted from the cell wall of the green algae Ulva is a good ligand for metal ions. Therefore, the adsorption properties of the U. clathrata derived ulvan toward Ca(II), Zn(II), Co(II), Cu(II), and Cr(III) were investigated in this study. The results demonstrate that ulvan exhibited good metal ion adsorption capacity at pH 7 and 50 °C. These adsorption processes can largely be explained by the Freundlich isotherm model and the pseudo-second-order kinetic model. The order of the adsorption capacity and affinity is as follows: Cr(III) > Cu(II) > Zn(II) ≈ Co(II) > Ca(II) and Cr(III) > Zn(II) > Co(II) ≈ Cu(II) > Ca(II). Furthermore, structural characterization revealed that the hydroxyl and carboxyl groups were the main functional groups involved in metal ion binding. Unlike the divalent metal ions, Cr(III) can trigger crosslinking of the ulvan chains, and its adsorption capacity was approximately 4.0 mmol/g.

Effects of low molecular weight polysaccharides from Ulva prolifera on the tolerance of Triticum aestivum to osmotic stress.

Polysaccharides derived from seaweeds can be used as biostimulants to enhance plant resistance to different stressors. In this study, we investigated the effects of applying low molecular weight polysaccharides (LPU) derived from Ulva prolifera with 14.2 kDa on the responses of wheat (Triticum aestivum) to osmotic stress. The results showed that osmotic stress simulated using polyethylene glycol inhibited seedling growth, whereas we observed increases in the fresh weights and shoot lengths of seedlings treated with polysaccharide for 120 h. Furthermore, we observed enhanced activities of antioxidant enzymes, and significant reductions in malondialdehyde content of 23.13%, 19.82%, and 20.04% in response treatment for 120 h with 0.01%, 0.03%, and 0.05% LPU, respectively, relative to those in the group treated with polyethylene glycol alone. In all treatments, expression of the P5CS gene was upregulated to promote proline accumulation. Moreover, after 120 h, exogenously applied LPU induced the expression of stress-related genes, including SnRK2, Wabi5, Wrab18, and Wdhn13. Collectively, these findings indicate that LPU might have the effect of regulating the abscisic acid-dependent pathway in wheat, thereby increasing seedling antioxidant capacity and growth. Application of LPU may accordingly represent an effective approach for enhancing the resistance to osmotic stress in wheat.

Intestinal Anti-Inflammatory Effects of Selenized Ulva pertusa Polysaccharides in a Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease Model.

The purpose of this study was to examine the alleviative effects of selenized polysaccharides from Ulva pertusa (ulvan-Se) on inflammatory bowel disease (IBD) in mice. The dextran sulfate sodium (DSS)-induced IBD mouse model was used to explore the protective effects of ulvan-Se on the intestinal mechanical and immune barrier. At doses less than 1208 mg/kg·bw ulvan-Se showed no significant damage to Institute of Cancer Research (ICR) mice in an acute toxicity test. The results showed that DSS destroyed the mechanical barrier, which includes epithelial cells, while ulvan-Se promoted mRNA expression of tight junction proteins (zonula occludens protein 1, occludin, and claudin-1) and inhibited the infiltration of white blood cells into the intestines. At 100 mg/kg·bw, ulvan-Se enhanced the antioxidant capacity of mice more effectively than the 50 mg/kg·bw ulvan-Se. Furthermore, ulvan-Se improved the intestinal immune barrier by increasing immunoglobulin A and immunoglobulin M, while regulating the levels of interleukin (IL)-1ß, interferon-γ, and IL-4. Oral administration of ulvan-Se also suppressed tumor necrosis factor-α, IL-1ß, IL-6, and cyclooxygenase-2 mRNA expression mediated by the nuclear factor kappa B pathway. Taken together, our findings reveal that ulvan-Se could be used as a potential alternative supplement for reducing intestinal inflammation in IBD.

BRUCE preserves genomic stability in the male germline of mice.

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.

Structural characterization of ulvan extracted from Ulva clathrata assisted by an ulvan lyase.

Rhamnan-rich sulfated polysaccharides extracted from green algae (ulvan) constitute potentially useful natural materials for drug development. However, the characterization of their complex structures poses a challenge for their application. In this study, the structure of ulvan extracted from Ulva clathrata was analyzed with the assistance of an ulvan lyase belonging to the PL25 family. According to mass spectrometry and nuclear magnetic resonance analysis of the degraded oligosaccharides, the backbone of such a polysaccharide mainly consisted of →4)-ß-d-GlcA-(1→4)-α-l-Rha3S-(1→ and →4)-ß-d-Xyl-(1→4)-α-l-Rha3S-(1→ disaccharide repeating units, and the ratio is approximately 4:1. In addition, about 4% of the xylose moieties bear sulfate groups. Minor amounts of branches containing hexose and unsaturated glucuronic acid were found during the sequence analysis of hexa- to octasaccharides. These results indicated the presence of a long branch in the ulvan. The clarification of the detailed structure provides a foundation for ulvan modification and its structure-activity relationship studies.