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The blood-brain barrier (BBB) damage has traditionally been considered to determine the occurrence and development of post-stroke brain edema, a devastating and life-threatening complication. However, no treatment strategy targeting BBB damage has been proven clinically effective in ameliorating brain edema. Here, we found that the extravasation of protein-rich fluids into the brain was not temporally correlated with edema formation after middle cerebral artery occlusion (MCAO) in mice, as brain edema reabsorption preceded BBB closure. Strikingly, the time course of edema progression matched well with the glymphatic system (GS) dysfunction after MCAO. Pharmacological enhancement of the GS function significantly alleviated brain edema developed on day 2 after MCAO, accompanied by less deposition of Aβ and better cognitive function. Conversely, functional suppression of the GS delayed the absorption of brain edema on day 7 after MCAO. Moreover, patients with ischemic stroke revealed a consistent trend of GS dysfunction after reperfusion as MCAO mice, which was correlated with the severity of brain edema and functional outcomes. Collectively, these findings indicate that the GS is a key contributor to the formation of brain edema after ischemic stroke, and targeting the GS may be a promising strategy for the treatment of brain edema in ischemic stroke.One Sentence SummaryThe function of the glymphatic system is a key factor in determining the formation or resolution of brain edema after ischemic strokeGraphical Abstract

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

Heparins and sulfated polysaccharides have been recognized as effective clinical anticoagulants for several decades. Heparins exhibit heterogeneity depending on the sources. Meanwhile, the adverse effect in the clinical uses and the adulteration of oversulfated chondroitin sulfate (OSCS) in heparins develop additional attention to analyze the purity of heparins. This review starts with the description of the classification, anticoagulant mechanism, clinical application of heparins and focuses on the existing methods of heparin analysis and detection including traditional detection methods, as well as new methods using fluorescence or gold nanomaterials as probes. The in-depth understanding of these techniques for the analysis of heparins will lay a foundation for the further development of novel methods for the detection of heparins.

Heparan sulfate (HS) interacts with a broad spectrum of inflammatory cytokines, thereby modulating their biological activities. It is believed that there is a structural‐functional correlation between each protein and sugar sequences in the HS polysaccharides, however, the information in this regard is limited. In this study, we compared the binding of four inflammatory cytokines (CCL8, IL‐1beta, IL‐2 and IL‐6) to immobilized heparin by an SPR analysis. To define the molecular base of the binding, we used a heparin pentasaccharide as representative structure to dock into the 3D‐molecular structure of the cytokines. The results show a discrepancy in KD values obtained by SPR analysis and theoretical calculation, pointing to the importance to apply more than one method when describing affinity between proteins and HS. By cluster analysis of the complex formed between the pentasaccharide and cytokines, we have identified several groups in heparin forming strong hydrogen bonds with all four cytokines, which is a significant finding. This molecular and conformational information should be valuable for rational design of HS/heparin‐mimetics to interfere cytokine‐HS interactions., A novel binding mode between heparin and inflammatory cytokines was discovered by both SPR analysis and theoretical calculations. Several key groups responsible for heparin forming strong hydrogen bond with all four cytokines have been identified. This molecular and conformational information should be valuable for rational design of HS/heparin‐mimetics to interfere with cytokine‐HS interactions.

NEMO (NF-κB essential modulator) is one of the important regulatory subunits of the IκB kinase (IκK) complex that controls the activation of the NF-κB signaling pathway. Here, we have identified the homolog of NEMO from the pacific oyster Crassostrea gigas. CgNEMO harbors the conserved the IκK binding region, NEMO ubiquitin binding domain and Zinc finger domain. In terms of tissue distribution, CgNEMO is expressed in various tissues with an observed highest expression in the hemocytes. Furthermore, infection by two related Vibrio strains significantly increased CgNEMO expression in the hemocytes. Cell culture based luciferase reporter assays showed that CgNEMO activates the NF-κB reporter in a dose-pendent manner. Moreover, CgNEMO was also found to counter the IkB-dependent inhibitory effect on NF-κB activation, providing a plausible mechanism of NF-κB activation by CgNEMO. Meanwhile, site-directed mutagenesis demonstrated that the putative ubiquitination site K535 is required for the activation of NF-κB, implying that ubiquitination of NEMO may be involved in regulating its activity. Finally, RNAi mediated knockdown of CgNEMO in vivo significantly compromised the bacterial induction of key cytokines TNF-α and IL-17, strongly suggesting a role for CgNEMO in acute immune defense in oyster. In conclusion, this study provides new insights into our understanding about the evolution of NEMO mediated NF-κB activation and the induction of cytokine. Our findings may provide valuable information about diseases control and management in oyster aquaculture.

In bivalve mollusks, circulating hemocyte mediated phagocytosis is one of the primary ways to eliminate invading microbes. Here, we have identified one CgLRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) in the Crassostrea gigas as a novel transmembrane LRR (Leucine-rich repeat) domain containing protein in C. gigas, homologous to the jawless fish VLR protein, that plays an important role in recognizing bacteria and promoting hemocytic phagocytosis. Tissue distribution analysis of CgLRFN in Pacific oyster showed that it is widely expressed in various tissues like the gills, adductor muscles, digestive glands, gonads, heart and in the hemocytes. Furthermore, infection of Pacific oysters with two marine Vibrio strains V. alginolyticus and V. parahaemolyticus was found to significantly increase CgLRFN expression in the hemocytes. Analysis of subcellular localization showed that CgLRFN is primarily localized in the cell membrane. Additionally, CgLRFN was found to be able to bind both the bacterial strains, indicating its possible role as a cell surface receptor. Flow cytometry analysis revealed that CgLRFN coated bacteria was phagocytosed by oyster hemocytes at a significantly higher rate compared to the uncoated bacteria. Finally, RNAi mediated knockdown of CgLRFN in vivo resulted in reduced clearance of both the bacterial strains from the oyster hemolymph. Overall, our study demonstrates that CgLRFN acts as a pattern recognition receptor for Vibrio spp. and promotes hemocytic phagocytosis in the Pacific oyster, which is critical for understanding the mechanism of bacterial infection in lower invertebrates, and also contributes to disease management of this economically and ecologically important marine mollusk.

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26–38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people’s view on inhibitor discovery for the checkpoint pair.

Chlorella minutissima is considered to be one of the promising feedstocks for biofuels in the future. In this study, the transcriptome from the oil-rich strain UTEX2341 of C. minutissima was generated based on Illumina paired-end sequencing. Through de-novo assembly, a total of 14,905 isogenes were obtained and compacted into 6216 unigenes. A total of 80% of the unigenes were assigned with GO terms and were further subdivided into 55 sub-categories. KEGG analysis demonstrated that 37.2% of the unigenes could be accessed and mapped into 278 pathways. Interestingly, the genes that encoded key enzymes that are involved in the biosynthesis, elongation, and metabolism of fatty acids were identified, including malonyl-CoA-ACP transacylase, 3-ketoacyl-ACP synthase, 3-ketoacyl-ACP reductase, and others. Moreover, the genes that are involved in triacylglycerol (TAG) biosynthesis and metabolism were also observed. Therefore, the transcriptome analysis of C. minutissima UTEX2341 not only supplies comprehensive insight into the molecular pathway that is involved in the biosynthesis of biofuel precursors but also provides substantial valuable genomic resources to accelerate the further development and utilization of biofuels.

Spätzle, is the only identified endogenous Toll receptor ligand, plays a critical role in initiatinge innate immune responses and controlling dorsal-ventral axis formation in Drosophila. Here we identified the first spätzle gene homolog, Pu-Spz, in the marine mollusk Paphia undulate. The full-length of Pu-Spz cDNA is 1248 bp, including an open reading frame (ORF) of 702 bp, a 5'-untranslated region (UTR) of 26 bp and a 3'-UTR of 203 bp. The ORF encodes a 233-amino-acid protein with conserved domains; it includes a putative signal peptide and a C-terminal cystine-knot. Sequence alignment revealed that the cystine-knot domain of Pu-Spz contains six highly conserved Cys residues, which maintain a molecular conformation suitable for Toll receptor binding. Unlike Spätzle, Pu-Spz lacks a seventh Cys residue, which is essential for forming intermolecular disulfide bridge. Phylogenetic analysis revealed that Pu-Spz is closer to the homologs found in crustaceans than to those in the insect branch. Transcript abundance of Pu-Spz was increased after challenging P. undulate with either heat-killed Listeria monocytogenes or heat-killed Vibrio alginolyticus, suggesting Spätzle is involved in P. undulate host defense. Our results demonstrate convergent evolution of the spätzle-Toll system between the mollusk and arthropod lineages.

Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

NF-κB signaling pathway is an evolutionarily conserved pathway that plays highly important roles in several developmental, cellular and immune response processes. With the recent release of the draft Pacific oyster (Crassostra gigas) genome sequence, we have sought to identify the various components of the NF-κB signaling pathway in these mollusks and investigate their gene structure. We further constructed phylogenetic trees to establish the evolutionary relationship of the oyster proteins with their homologues in vertebrates and invertebrates using BLASTX and neighbor-joining method. We report the presence of two classic NF-κB/Rel homologues in the pacific oyster namely Cgp100 and CgRel, which possess characteristic RHD domain and a consensus nuclear localization signal, similar to mammalian homologues and an additional CgRel-like protein, unique to C. gigas. Further, in addition to two classical IκB homologues, CgIκB1 and CgIκB2, we have identified three atypical IκB family members namely CgIκB3, CgIκB4 and CgBCL3 which lack the IκB degradation motif and consist of only one exon that might have arisen by retrotransposition from CgIκB1. Finally, we report the presence of three IKKs and one NEMO genes in oyster genome, named CgIKK1, CgIKK2, CgIKK3 and CgNEMO, respectively. While CgIKK1 and CgIKK3 domain structure is similar to their mammalian homologues, CgIKK2 was found to lack the HLH and NBD domains. Overall, the high conservation of the NF-κB/Rel, IκB and IKK family components in the pacific oyster and their structural similarity to the vertebrate and invertebrate homologues underline the functional importance of this pathway in regulation of critical cellular processes across species.

Dietzia sp. 111N12-1, isolated from the seawater of South China Sea, shows strong petroleum hydrocarbons degradation activity. Here, we report the draft sequence of approximately 3.7-Mbp genome of this strain. To the best of our knowledge, this is the first genome sequence of Dietzia strain isolated from the sea. The genome sequence may provide fundamental molecular information on elucidating the metabolic pathway of hydrocarbons degradation in this strain.

The full-length cDNA that encodes the MRE-binding transcription factor (MTF) was cloned from the Pacific oyster (Crassostrea gigas) using reverse transcription polymerase chain reaction and the rapid amplification of cDNA ends. The cgMTF cDNA sequence is 2892 bp long, with a 2508 bp open reading frame that encodes an 835-amino acid polypeptide. Multiple alignment revealed that cgMTF has four putative zinc finger-like regions in cgMTF with three C2C2-type zinc fingers and one C2H2-type zinc finger. After 12 h of exposure to Cd(2+), the cgMTF mRNA level was increased in a dose-dependent manner, which then subsided with time. cgMTF stimulates the cgMT promoter reporter in the HEK293 cell line in a dose-dependent manner. When either of the metal-responsive elements (MRE1 or MRE2) of the cgMT promoter was mutated, the cgMT promoter reporter activity was significantly reduced. After the two MREs were mutated simultaneously, the promoter activity was completely abolished. In conclusion, we identified an MTF in C. gigas and revealed the presence of an evolutionarily conserved molecular mechanism for coping with environmental metal stress.

Iron homeostasis is essential to almost all organisms. In this study, we identified the putative homolog of the iron-storage protein-encoding gene, dpsL, in the intracellular pathogen Legionella pneumophila and demonstrated its expression under iron-limited conditions and its responses to multiple stresses. Quantitative real-time PCR analysis indicated that the expression of dpsL was enhanced under iron limitation regardless of the growth phase. Compared with the wild-type cells, the cells devoid of dpsL were heat and H2O2-sensitive. In contrast to the dps mutants of other bacteria, the growth of the dpsL mutant in an iron-deprived medium was delayed but finally reached the same cell density as wild-type cells during the stationary phase of growth. The finding that the dpsL mutant is salt resistant suggested the involvement of DpsL in virulence. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

As one of the surface membrane proteins of tetraspanin family, CD63 plays a crucial role in cellular trafficking and endocytosis, which also is associated with activation of a wide variety of immune cells. Here, the homolog of CD63 was characterized from one marine mollusk,Paphia undulata, which is designated as Pu-CD63. The complete cDNA of Pu-CD63 is 1,738 bp in length with an open reading frame (ORF) of 849 bp, encoding a 282 amino acid protein with four putative hydrophobic transmembrane helixes. Bioinformatic analysis revealed that Pu-CD63 contains one putative YXXØ consensus motif of “110-YVII-113” and one N-glycosylation site “155-NGT-157” within the large extracellular loop (LEL) region, supporting its conserved function in plasma membrane and endosomal/lysosomal trafficking. Moreover, temporal expression profile analysis demonstrates a drastic induction in the expression of CD63 in hemocytes after pathogenic challenge with eitherV. parahaemolyticusorV. alginolyticus. By performing dsRNA-mediate RNAi knockdowns of CD63, a dramatic reduction in hemocytes phagocytic activity to pathogenicVibriois recorded by flow cytometry, revealing the definite role of Pu-CD63 in promoting hemocyte-mediated phagocytosis. Therefore, our work has greatly enhanced our understanding about primitive character of innate immunity in marine mollusk.

Currently, corticosteroids are the most potent anti-inflammatory drugs on the market. Here, we announce the draft genome sequence of the marine-derived Streptomyces sp. strain CNQ431, which produces cytokine inhibitors, termed splenocins, which display potent suppression of cytokine production at a comparable level to that of corticosteroids. The genome is approximately 498,750 bp with 72.03% G+C content.

Vibrio harveyi strain E385 was isolated from a diseased cage-cultured grouper in Daya Bay, China. Phylogenetic analysis based on the 16S rRNA gene sequence showed similarity with V. harveyi strain BAA-1116. We sequenced the pathogenic strain V. harveyi E385 and compared the genome with that of the nonpathogenic strain V. harveyi BAA-1116.