Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from  Pseudomonas aeruginosa, there is an urgent requirement for novel approaches to the treatment of bacterial infections. Lectin-like bacteriocins are highly potent protein antibiotics that display an unusual ability to kill a select group of bacteria within a specific genus. In this work, we show how the lectin-like protein antibiotic, pyocin L1, can kill Pseudomonas aeruginosa with extraordinary potency through specific binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide. The CPA is predominantly a homopolymer of the sugar d-rhamnose that although generally rare in nature is found frequently as a component of the lipopolysaccharide of members of the genus Pseudomonas. The targeting of d-rhamnose containing polysaccharides by pyocin L1 and a related lectin-like protein antibiotic, putidacin L1, explains the unusual genus- specific killing activity of the lectin-like bacteriocins. As we learn more about the link between changes to the microbiome and a range of chronic diseases there is a growing realisation that the ability to target specific bacterial pathogens while maintaining the normal gut flora is a desirable property for next generation antibiotics.Due to rapidly increasing rates of antibiotic resistance observed among Gram-negative pathogens, such as  Clostridium difficile infection The ability to target a subgroup of pathogenic bacteria in a complex bacterial community has potential applications in medicine and agriculture where the maintenance of a ‘normal’ microbiome is beneficial. For example, the use of broad spectrum antibiotics to treat bacterial infections is known to cause a range of complications associated with collateral damage to the microbiome, including antibiotic associated diarrhea and P. aeruginosa and the closely related colicins of E. coliP. aeruginosa, whereas colicins show activity against E. coli and some strains of closely related bacteria such as Salmonella spp. 12 uptake P. aeruginosa there is an urgent requirement for the development of novel therapeutic options since its ability to form drug-resistant biofilms in combination with the presence of an outer membrane that is highly impermeable to many classes of antibiotics can make this pathogen essentially untreatable in some groups of patients. This is exemplified in cystic fibrosis patients where chronic lung infection with P. aeruginosa is the leading cause of mortality In contrast to the broad spectrum antibiotics that are widely used in medicine and agriculture, protein antibiotics known as bacteriocins often target a specific bacterial species or a group of closely related bacterial species P. putida (putidacin L1 or LlpABW) P. syringae (LlpAPss642) and P. fluorescens (LlpA1Pf-5) have been characterised and have the unprecedented ability to kill strains of a broad range of bacterial species within the genus Pseudomonas, but are not active outside this genus Xcm761 from Xanthomonas citri pv. malvacearum LMG 761 has the ability to kill various species within the genus XanthomonasAn interesting addition to this group of protein antibiotics is the recently discovered lectin-like bacteriocins that contain two carbohydrate-binding domains of the monocot mannose-binding lectin (MMBL) family Galanthus nivalis agglutinin (GNA) was the first to be characterised G. nivalis or Allium sativum, it is now recognised that proteins of this class are distributed widely throughout prokaryotes and eukaryotes, where they have evolved to mediate diverse functions Lectins are a structurally and evolutionarily diverse class of proteins produced widely by prokaryotes and eukaryotes and are defined by their ability to recognise and bind carbohydrates. This binding is generally highly specific and mediates a range of diverse functions, including cell-cell interaction, immune recognition and cytotoxicity Pseudomonas spp. show complete conservation of two sugar binding motifs on the C-terminal domain and partial conservation of two sites on the N-terminal domain et ald-mannose or a range of mannose containing oligosaccharides. However, Kds for these protein-carbohydrate complexes were reported in the range from 46 mM for methyl-α-d-mannoside to 2 mM for a mannose containing pentasaccharide The lectin-like bacteriocins are not proteolytically processed and thus consist of a single peptide chain, containing tandem β-prism domains. Sequence alignments of members of this class from P. aeruginosa, and show that it utilises lipopolysaccharide (LPS) as a surface receptor, specifically targeting the common polysaccharide antigen (CPA) that is a conserved homopolymer of d-rhamnose. Structural and biophysical analysis shows that the C-terminal carbohydrate binding motifs are responsible for d-rhamnose recognition and that these sites are specific for this sugar over d-mannose. Further to this, we show that the previously described putidacin L1 also selectively binds LPS from susceptible, but not from resistant, P. syringae isolates and shows selectivity for d-rhamnose over d-mannose. This work shows that the physiologically relevant ligand for the QxDxNxVxY carbohydrate binding site of the lectin-like bacteriocins is indeed d-rhamnose and not d-mannose as previously thought. As such, the genus-specific activity of lectin-like bacteriocins from Pseudomonas spp. can be attributed to the widespread inclusion of the rare d-rhamnose in the LPS of members of the genus Pseudomonas.Despite progress in our understanding of the structure and host range of MMBL-like bacteriocins, the mechanism by which these bacteriocins target susceptible strains and exert their antimicrobial effects is unknown. Here we report on the discovery of a novel member of this family, pyocin L1 from P. aeruginosa infections, we searched the genomes of 10 recently sequenced clinical and environmental isolates of P. aeruginosa for genes with homology to known bacteriocins. One putative bacteriocin gene identified in strain C1433, an isolate from a patient with cystic fibrosis, encodes a protein with 31% identity to the lectin-like bacteriocin LlpA1Pf-5, from P. fluorescens. This protein, designated pyocin L1, contained 256-amino acids with a predicted molecular mass of 28413 Da. Alignment of the pyocin L1 protein sequence with other lectin-like bacteriocins, LlpA1Pf-5, LlpAPss642, putidacin L1 (LlpABW), LlpAAu1504 from Burkholderia cenocepacia and LlpAXcm761 from Xanthomonas citri pv. malvacearum shows that pyocin L1 contains tandem MMBL domains with three conserved QxDxNxVxY MMBL sugar-binding motifs  also known as A-band LPS P. aeruginosa produce two distinct LPS-types that differ in their O-antigen, but share the same core oligosaccharide. The CPA is predominantly a homopolymer of d-rhamnose and the O-specific antigen contains a heteropolymeric repeating unit that varies widely among strains wbpZ, we found that production of CPA, as determined by immunoblotting with a CPA-specific monoclonal antibody P. aeruginosa PAO1, which is sensitive to pyocin L1, with insertions in the genes responsible for the transport of CPA to the periplasm wzt and wzm, encode the ATP-binding component and membrane component of a CPA dedicated ABC transporter wzm and wzt  and analysed the pyocin-CPA interaction by isothermal titration calorimetry (ITC). Titration of pyocin L1 into isolated LPS-derived polysaccharides (a mixture of CPA and the O-specific antigen containing polysaccharides) from PAO1 gave rise to strong saturable exothermic heats of binding  was performed. Titration of pyocin L1 into d-rhamnose gave rise to weakly saturable heats of binding that are significantly larger than the heats observed on titration of pyocin L1 into an identical concentration of d-mannose  spectra on addition of d-rhamnose or d-mannose. In the absence of added monosaccharide 15N-HSQC spectra of pyocin L1, which should contain one crosspeak for each non-proline amide NH as well as peaks for the NH groups in various side chains, were well resolved and dispersed, indicative of a folded protein. Chemical shift perturbation monitored by 15N-HSQC allows the mapping of changes to a protein that occur on ligand binding. Addition of either d-rhamnose or d-mannose up to a concentration of 100 mM did not give rise to large or global changes in chemical shifts  and the structural basis of the d-rhamnose specificity of pyocin L1 we determined the X-ray structures of pyocin L1 with bound d-mannose, d-rhamnose and in the unbound form . The DALI server searches the protein data bank (PDB) to identify proteins structurally related to the query structure In an attempt to determine the location of the pyocin L1 und form . Pyocin und form . This inund form . The oveund form . The higd-mannose and d-rhamnose soaked crystals show clear density for sugar moieties in both sites, C1 and C2  and D180A (C2) variants were performed by titrating protein at a concentration of 100 µM into a solution of LPS-derived polysaccharide (1 mg ml−1) from strain PAO1 (d of 0.15 (±0.07) µM for the wild-type pyocin L1-CPA complex. For both the D150A (C1) and D180A (C2) variants, affinity for CPA was reduced. For the pyocin L1 D150A-CPA complex a Kd of 1.52 (±0.51) µM was determined, a 10-fold increase in Kd relative to the wild-type pyocin L1-CPA complex. However, CPA binding to the D180A variant was severely weakened and although heats of binding were still observed the Kd for this complex, which could not be accurately determined, is likely >500 µM. We also produced a double mutant in which both D150A and D180A mutations were present. For this double mutant, no binding to CPA was observed by ITC. These data show that both the C1 and C2 sugar binding motifs are required for full CPA binding, but that the C2 binding site is the major CPA binding determinant. The killing activity of these sugar binding motif variants showed a good correlation with their ability to bind the CPA. Both the D150A and D180A variants showed reduced cytotoxicity against PAO1 relative to pyocin L1, with the D150A showing a greater reduction in activity and for the D150A/D180A variant very low levels of cytotoxicity were observed . Binding of putidacin L1 to the isolated LPS-derived polysaccharides of the above mentioned strains was tested by ITC. Large saturable heats of binding were observed for putidacin L1 and the LPS-derived polysaccharides from LMG 5456 and LMG 2222, while no binding was observed between putidacin L1 and the LPS-derived polysaccharides from LMG 1247, 2563 or DC3000  of pyocin L1 and putidacin L1 is identical. This observation combined with the specificity of putidacin L1 for d-rhamnose, strongly suggests that it also binds to a d-rhamnose containing O-antigen. Indeed branched d-rhamnose O-antigens are common in P. syringaeAlthough -mannose . Interesobserved . It is id-rhamnose containing O-antigens are utilised as surface receptors for lectin-like bacteriocins from Pseudomonas spp. This is an attractive hypothesis since the inclusion of d-rhamnose in the lipopolysaccharides from members of this genus is widespread and could form an important component of the genus specific activity of this group of bacteriocins.Our data for both pyocin L1 and putidacin L1 indicate that d-rhamnose at the conserved QxDxNxVxY sugar binding motifs of the C-terminal lectin domain. The ability of both pyocin L1 and putidacin L1 to recognise d-rhamnose containing carbohydrates is an important component of their ability to target sensitive strains of Pseudomonas spp. The use of the O-antigen as a primary receptor differentiates the lectin-like bacteriocins from other multidomain bacteriocins such as colicins and S-type pyocins (colicin-like bacteriocins) which utilise outer membrane proteins as their primary cell surface receptors In this work we have shown that pyocin L1 targets susceptible cells through binding to the CPA component of LPS and that primary recognition of CPA occurs through binding of et al. has shown through domain swapping experiments that for putidacin L1 (LlpABW) and the homologous lectin-like bacteriocin LlpA1Pf-5 from Pseudomonas fluorescens, species specificity is governed by the identity of the N-terminal lectin domain d-rhamnose containing O-antigens, with the N-terminal domain interacting with species-specific factors and thus determining the precise species and strain specificity of these bacteriocins. Although there are few clues as to how the lectin-like bacteriocins ultimately kill susceptible cells, we have established a clear role for the C-terminal MMBL domain of these proteins. The roles of the N-terminal MMBL domain and the C-terminal extension remain to be discovered et al, it is clear that all three of these regions are required for killing of susceptible cells.In addition to O-antigen recognition, additional factors, as yet to be determined, are clearly also important in strain and species specificity among the lectin-like bacteriocins. Indeed, recent work from Ghequire P. aeruginosal-form of this sugar that is found in nature. Although otherwise rare, d-rhamnose is found frequently as a component of the LPS of plant pathogens and plant associated bacteria such as P. syringaeP. putidaXanthomonas campestrisBurkholderia spp. E. coli, Salmonella and Klebsiella. It is interesting to speculate that since d-rhamnose is a common component of the LPS of bacterial plant pathogens, that some of the many lectins produced by plants may have evolved to target d-rhamnose as part of plant defence to bacterial pathogens.Interestingly, although rhamnose is frequently a component of plant and bacterial glycoconjugates, such as the rhamnolipids of d-rhamnose containing polymers, indicates that the lectin-like bacteriocins would not interact with either plant or animal cells, since these lack d-rhamnose containing glycoconjugates. In addition, these narrow spectrum antibiotics would leave the majority of the soil microbiome and the gut microbiome of plant-eating animals intact and so would be likely to have minimal environmental impact and minimal impact on animal health. This latter property and the potency of these protein antibiotics could also make the use of lectin-like bacteriocins in the treatment of chronic multidrug-resistant P. aeruginosa infections in humans an attractive proposition.The specificity of lectin-like bacteriocins suggests that these protein antibiotics may be useful in combating plant pathogenic bacteria, either through the use of bacteriocin expressing biocontrol strains or by the production of transgenic plants engineered to express these proteins. The specific targeting mechanism described here, binding of P. aeruginosa were grown in LB at 37°C, P. syringae were grown in King's B Media (KB)  at 28°C.Strains and plasmids utilised in this study are presented in Supplementary P. aeruginosa C1433 pyoL1 gene (ACA GAT CAT ATG AAG TCT CCA AAC AAA AGG AGG) and an XhoI site at the end of the gene (ACA GAT CTC GAG GAC CAC GGC GCG CCG TCG TGG ATA GTC GTG GGG CCA A). The PCR product was ligated into the corresponding sites of the E. coli expression vector pET21a to give pETPyoL1 which encodes pyocin L1 with a C-terminal His6 tag separated from the C-terminus of pyocin L1 by a 6 amino acid linker (RRRAVV). Pyocin L1 was overexpressed from E. coli BL21(DE3)pLysS carrying the plasmid pETPyoL1. Five litres of LB broth was inoculated (1∶100) from an overnight culture and cells were grown at 37°C in a shaking incubator to an OD600 = 0.6. Protein production was induced by the addition of 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), the cells were grown at 22°C for a further 20 hand harvested by centrifugation. Cells were resuspended in 20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole (pH 7.5) and lysed using an MSE Soniprep 150 (Wolf Laboratories) and the cell debris was separated by centrifugation. The cell-free lysate was applied to a 5-ml His Trap HP column  equilibrated in 20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole (pH 7.5) and pyocin L1 was eluted over a 5–500 mM imidazole gradient. Pyocin L1 containing fractions were identified by SDS PAGE, pooled and dialyzed overnight into 50 mM Tris-HCl, 200 mM NaCl, pH 7.5 and remaining contaminants were removed by gel filtration chromatography on a Superdex S75 26/600 column  equilibrated in the same buffer. The protein was concentrated using a centrifugal concentrator (Vivaspin 20) with a molecular weight cut off of 5 kDa and stored at −80°C until required. The putidacin L1 open reading frame was synthesised (DNA 2.0) and cloned into pET21a via 5′ NdeI and 3′ XhoI restriction sites. The stop codon was removed in order to utilise the pET21a C-terminal His6 tag. Purification of putidacin L1 was performed as for pyocin L1. Constructs to express the pyocin L1 mutants D31A, D97A, D150A and D180A were created using the QuikChange Site Directed Mutagenesis Kit (Stratagene) utilising pETPyoL1 as a template. The primers used were CAA ATT GGT CAT GCA AGC GGC TGG CAA CTT GGT CCT TTA CG and CGT AAA GGA CCA AGT TGC CAG CCG CTT GCA TGA CCA ATT TG for D31A, GCG TAC CTG AAT CTT CAA GAT GCT GGG GAC TTC GGT ATA TTT TC and GAA AAT ATA CCG AAG TCC CCA GCA TCT TGA AGA TTC AGG TAC GC for D97A, CGC CTA GCG TTT CAG GGA GCT GGC AAC CTA GTG ATC TAT C and GAT AGA TCA CTA GGT TGC CAG CTC CCT GAA ACG CTA GGC G for D150A and GAT AGA GCA GTA GTG CAA GAG GCT GGA AAT TTT GTT ATC TAC AAA G and CTT TGT AGA TAA CAA AAT TTC CAG CCT CTT GCA CTA CTG CTC TAT C for D180A. Mutant proteins were purified as described above for wild-type pyocin L1.Pyocin L1 was amplified from the genomic DNA of the producing strain 600 = 0.6 was added to 6 ml of 0.8% soft agar and poured over an LB or KB agar plate. 5 µl of bacteriocin at varying concentrations was spotted onto the plates and incubated for 20 h at 37 or 28°C.Soft agar overlay spot plates were performed using the method of P. aeruginosa E2 (OD600 = 0.6) was centrifuged and resuspended in 100 µl of LB, to which 100 µl (8 mg ml−1) of purified pyocin L1 was added. The culture was grown for 1 h, plated onto a LB agar plate and incubated for 20 h at 37°C. Isolated colonies were identified as P. aeruginosa using 16S PCR as described previously 1.5 ml of a culture of P. aeruginosa E2 and derived pyocin L1 tolerant mutants were sequenced at the Glasgow Polyomics Facility, generating paired-end reads on an Illumina MiSeq Personal Sequencer. Reads were mapped to the previously sequenced parent genomes of P. aeruginosa E2 using the CLC genomics workbench, MAUVE and RAST to create an ordered annotated genome. The CLC genomics workbench was used for genome comparisons and the identification of SNPs/INDELs.The genomes of P. aeruginosa and P. syringae strains as described previously, with modifications including the omission of the final trifluoroacetic acid hydrolysis and chromatography steps P. aeruginosa and P. syringae respectively, pelleted by centrifugation at 6000 g for 20 min, and resuspended in 50 mM Tris, pH 7.5 containing lysozyme (2 mg ml−1) and DNase I (0.5 mg ml−1). Cells were lysed by sonication and the cell lysate was incubated at 20°C for 30 min before EDTA was added to a final concentration of 2 mM. An equal volume of aqueous phenol was added and the solution was heated at 70°C for 20 min, with vigorous mixing. The solution was then cooled on ice for 30 min, centrifuged at 7000 g for 20 min and the aqueous phase extracted. Proteinase K was added to a final concentration of 0.05 mg ml−1 and dialysed for 12 h against 2×5 L H2O. LPS was pelleted by ultracentrifugation at 100,000 g for 1 h, resuspended in H2O and heated to 60°C for 30 min to remove residual proteinase K activity. LPS-derived carbohydrates were isolated by heating LPS in 2% acetic acid for 1.5 h at 96°C. Lipid A was removed by centrifugation at 13,500 g for 3 min followed by extraction with an equal volume of chloroform. The aqueous phase was then lyophilised.LPS was purified from 1 litre cultures of Purified LPS from wild-type and mutant samples were resolved by electrophoresis on 12% SDS-polyacrylamide gels. The LPS banding patterns were visualised by the Invitrogen ultrafast silver staining method. For immunoblotting LPS was transferred onto nitrocellulose membranes and western immunoblotting was performed as previously described using the CPA-specific monoclonal antibody N1F10 and alkaline phosphatase-conjugated goat anti-mouse Fab2 as the secondary antibody d-rhamnose, d-mannose or l-rhamnose were used as titrants and reactions were performed in 0.2 M sodium phosphate buffer, pH 7.5. d-rhamnose (>97%) was obtained from Carbosynth Limited (UK) and d-mannose and l-rhamnose (>99%) from Sigma-Aldrich (UK). For O-antigen-pyocin L1 binding reactions, pyocin L1 or pyocin L1 variants were used as titrant at 100 or 150 µM with cleaved O-antigen sugars dissolved at 1 mg ml−1 in the chamber. For curve fitting we estimated the molar concentration of LPS-derived CPA containing carbohydrate chains at 20 µM based on an estimated average molecular weight of 10 kDa for CPA containing polysaccharides and estimating the percentage of total LPS represented by CPA containing carbohydrates as 20% of the total by weight d. For O-antigen-putidacin L1 binding reactions, O-antigen was used as the titrant at 3 mg ml−1 with 60 µM putidacin L1 in the chamber. Reactions were performed in 20 mM HEPES buffer pH 7.5. All samples were degassed extensively prior to the experiments. Calorimetric data were calculated by integrating the area under each peak and fitted with a single-site binding model with Microcal LLC Origin software. The heats of dilution for each titration were obtained and subtracted from the raw data.ITC experiments were performed on a VP-ITC microcalorimeter . For monosaccharide binding, titrations were carried out at 299 K with regular 15 µl injections of ligands into 60–100 µM pyocin L1 or putidacin L1 at 300 s intervals. 50 mM 15N labelled proteins (0.1–0.2 mM) and unlabelled ligands, d-rhamnose and d-mannose (100 mM), on a Bruker AVANCE 600 MHz spectrometer. Protein samples were prepared with and without the sugars present and volumes were exchanged at fixed ratios, making sure the protein concentration remained unchanged. The spectra were processed with Topspin and analysed with CCPNmr analysis NMR chemical shift perturbation analysis of sugar binding by pyocin L1 and putidacin L1 was carried out at 305 K and 300 K respectively. Fast-HSQC spectra −1 was screened for crystallisation conditions using the Morpheus and PGA crystallisation screens (Molecular Dimensions) 2, 0.03 M MgCl2, 0.1 M Tris/Bicine, pH 8.5) and condition 2  from the Morpheus screen were selected for optimisation by vapour diffusion in 24 well plates . Clusters of needles from these trays grew after 3–7 days and were mechanically separated. The un-soaked crystals were from condition 1, while soaked crystals were from condition 2. Un-soaked crystals were looped and directly cryo-cooled to 110 K in liquid nitrogen; D-mannose and D-rhamnose soaked crystals were soaked for 2–12 min in artificial mother liquor containing 4 M d-mannose or 2 M d-rhamnose, before cryo-cooling to 110 K. X-ray diffraction data were collected at the Diamond Light Source, Oxfordshire, UK at beam lines I04, I04-1 and I24. Automatic data processing was performed with Xia2 within the EDNA package Purified pyocin L1 at a concentration of 15 mg mlGalanthus nivalis agglutinin (PDB ID: 1MSA). Initial phases from Balbes were improved via density modification and an initial model was built using Phase and Build from the Phenix package Fo-2Fc and Fo-Fc maps d-mannose (PDB ID: BMA) corresponded best to the density of bound d-mannose. The density in the d-rhamnose complex best corresponded to α-d-rhamnose, for which no PDB ligand exists; a model for α-d-rhamnose was prepared by removing the oxygen from carbon 6 of α-d-mannose and submitting these PDB coordinates to the Prodrg server, which generated the model and modeling restraints d-rhamnose was designated with the PDB ID: XXR.A dataset from an un-soaked pyocin L1 crystal was submitted to the Balbes pipeline along with the amino acid sequence for pyocin L1 −1. Buffer was read before and after each sample and an average of the buffer scattering was subtracted from the sample scattering. The data obtained for each sample were analysed using PRIMUS 2) was used to calculate the molecular weight at I(0) and radius of gyration, Rg, of PyoL1. Ab initio models of the protein in solution were built using DAMMIF SAXS was carried out on the X33 beamline at the Deutsches Elektronen Synchrotron . Data were collected on samples of Pyocin L1 in the range of 0.5–5 mg mlFigure S1Sequence alignment of pyocin L1 and previously reported MMBL-like bacteriocins. Dark blue shading designates sequence identity, light blue designates chemically conserved residues. The three conserved MMBL sugar-binding motifs  and the partially conserved motif (N2) are boxed in red.(JPG)Click here for additional data file.Figure S2Genetics of CPA biosynthesis in P. aeruginosa. (A) CPA operon, annotated with location of P. aeruginosa E2 tolerant mutant (M4 and M11) deletion and PAO1 transposon insertion mutants. (B) Summary of CPA biosynthetic pathway, showing function performed by genes, shown to induce pyocin L1 tolerance or resistance.(TIF)Click here for additional data file.Figure S31H-15N HSQC spectra of 15N-labelled pyocin L1 in presence (red) and absence (black) of 100 mM (A) d-rhamnose and (B) d-mannose, showing distinctive chemical shifts upon addition of associating sugars. Chemical shift changes specific to a small number of cross-peaks illustrates association of the sugars with a small subset of amino acids, which likely correspond to the residues within the binding sites. Analogous changes are observed for d-rhamnose and d-mannose titrations indicative that the same sites are binding both ligands. Greater shift magnitude is observed for d-rhamnose, indicative of a greater affinity towards this monosaccharide. Boxed regions include cross-peaks used for chemical shift perturbation analysis as shown in (TIF)Click here for additional data file.Figure S4Small angle X-ray scattering of pyocin L1. (A) Ab initio model of pyocin L1 computed with DAMMIF overlaid with the crystal structure. (B) Guinier plot of scattering data indicates that the protein is monomeric in solution (I(0) gives a molecular mass of 29.53 kDa) by extrapolation of scattering intensity to zero scattering angle. Radius of gyration is 2.72 nm, indicative of a folded, globular monomeric particle in solution.(TIF)Click here for additional data file.Figure S5d-rhamnose in C1, C2 and N2 binding sites of pyocin L1.Coordination of  (A) Stereo view of d-rhamnose coordination by binding site C1 (A), C2 (C) and N1 (E), from d-rhamnose soak data. Core binding motif residues (blue) and additional residues contributing to the pocket (white) are shown. Omit map density for d-rhamnose in binding site C1 (B), C2 (D), N1 (F) calculated by refinement of data from d-rhamnose soaked crystal with model built from unsoaked crystal. Density for all sites contoured to 0.15e/Å3.(TIF)Click here for additional data file.Figure S6d-rhamnose, compared with d-mannose.Putidacin L1 shows specificity for  (A) ITC isotherm of d-rhamnose (50 mM) titrated into putidacin L1 (0.1 mM). Weakly saturable heats are indicative of binding with modest affinity (Kd ∼5–10 mM). (B) ITC isotherm of d-mannose (50 mM) titrated into putidacin L1 (0.1 mM). Binding is undetectable under reaction conditions.(TIF)Click here for additional data file.Figure S7l-rhamnose.Putidacin L1 and pyocin L1 do not bind  ITC isotherms of l-rhamnose (50 mM) titrated into putidacin L1 (A) and pyocin L1 (B) both at (0.1 mM). Binding is undetectable under these conditions.(TIF)Click here for additional data file.Table S1Strains and plasmids used in this work.(PDF)Click here for additional data file.Text S1References for supplementary information.(DOCX)Click here for additional data file.


Alu comprise 11% of the human genome Gypsy, occupy nearly half of the maize genome Fossilised mobile genetic elements, including Long Interspersed Element-1 (LINE-1 or L1) retrotransposons, comprise at least two-thirds of the human genome Dicer (DCR) and then assembled with Argonaute (AGO) and other proteins into the RNA-induced silencing complex (RISC) that, in turn, produces RNAi against retrotransposon transcripts Retrotransposition requires transcription of an RNA template for DNA-primed reverse transcription. Several cellular defence mechanisms have evolved to hinder this process, including: 1) promoter methylation and heterochromatinisation, 2) degradation of retrotransposon transcripts via RNA interference (RNAi), and 3) host factor prevention or destabilisation of reverse transcription. To describe in detail just one of a myriad of specific inhibitory pathways, repeat associated small interfering RNAs (rasiRNAs) are present in plant, worm, fly, fish, and mouse gametes and, therefore, represent a highly conserved defence against germ line retrotransposition PLOS Genetics, Ciaudo et al. Dicer markedly decreases L1-Tf promoter methylation and increases L1-Tf transcription, translation, and copy number in cultured mESCs. In particular, DCR−/− mESCs accumulate a remarkable 860 L1-Tf copies (greater than five megabases of genomic DNA) per cell over 20 passages, versus 255 copies per cell in DCRFlx/Flx controls, based on SYBR-Green qPCR targeting the L1-Tf 5′UTR. High-throughput small RNA sequencing then confirmed that DCR−/− mESCs were depleted of approximately 22 nt molecules found in wild-type mESCs, immunoprecipitated with AGO2 and aligned to L1-Tf, and therefore resembling rasiRNAs. Hence, LINE-1 activation in DCR−/− mESCs coincides with rasiRNA depletion and is also possibly influenced by ablation of Dicer-mediated LINE-1 promoter methylation.In this issue of Dicer- and AGO2-independent small RNAs were found to “paint” the L1-Tf 5′UTR. Again, assessing L1-Tf transcription and copy number, Ciaudo et al. found that deletion of XRN2 and DGCR8, respective members of the RNA surveillance and Drosha-DGCR8 Microprocessor pathways, led to increased L1-Tf transcription but not copy number amplification. These observations agree with other recent reports of small RNAs immunoprecipitated with DGCR8 and enriched for LINE-1 sequences −/− mESCs −/− mESCs with human Dicer and found that these cells recapitulated wild-type mESC LINE-1 suppression and differentiated normally, unlike DCR−/− mESCs.Intriguingly, a second class of Dicer and obscure rasiRNA detection (−/− mESCs Dicer or the Microprocessor increases LINE-1 mobilisation in cancer cells, with the latter result at odds with data generated here from mESCs. Future advances in high-throughput sequencing and single cell genomics should enable characterisation of endogenous LINE-1 mobilisation events in stem cells and further delineate the multifaceted roles of Dicer and other factors in LINE-1 inhibition.Evidence for a reciprocal relationship between rasiRNA depletion and LINE-1 activation significantly advances our understanding of RNAi-mediated control of retrotransposition during mammalian embryogenesis. These data are also important because they address a longstanding question of why rasiRNAs cannot be consistently detected in mammalian somatic cells: small RNAs generated by RNA surveillance and the Microprocessor may cleave the same pool of precursor LINE-1 mRNAs processed by etection . As Ciau


Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease. Microbial ecology used to be a small and specialized field that struggled to identify more than a tiny proportion of the Earth’s microbial biodiversity. Part of the problem was due to the prevalence of pure-culture methods, in which microorganisms had to be removed from their natural environments (which included communities of other organisms) and cultured in laboratories. Recent advances in molecular techniques, sequencing technologies and computational methods have enabled researchers to explore the microbial world at unprecedented levels, with a focus on the natural habitats of microorganisms. The combination of these advances has so far produced remarkable insight into the role of microorganisms in human health and their powerful effects on the natural world, while at the same time developing novel evidence about the evolution and diversification of life on Earth. In this article, we discuss how these advances have allowed researchers to create new lines of inquiry, we summarize important biological and philosophical results from recent publications, and we discuss how our improved understanding of microbial ecology may affect our lives in the coming years.The last decade has seen a transformation and democratization of DNA sequencing (Shendure and Ji These transformations in sequencing technology have correspondingly changed what it means to undertake a sequencing project. When sequences were very expensive (in the late 1970s and early 1980s), it was a substantial accomplishment to sequence even one gene from one species. Correspondingly, the focus was on identifying genes that acted as the best phylogenetic markers. These were short fragments of sequences from which inferences about the patterns of evolution were likely to match the inferred patterns of evolution of the corresponding species. These markers therefore provided efficient readouts of evolutionary history while minimizing sequencing costs. For example, ribosomal RNA genes, which play essential structural and catalytic roles in the ribosome and are thought to be almost exclusively vertically transmitted Lawrence , have beunknown species via bulk DNA extraction directly from the environment. This technique, pioneered by the Pace lab   from the genomes of well-studied organisms can often be assigned at least to the family level, and sometimes at the genus or species level, many sequences cannot confidently be assigned to known named taxonomic groups. The limitation here is primarily the amount of information present in short reads of marker genes for differentiating closely related taxa. Figure One shows that when working with the most informative region of the 16S rRNA gene for broad analyses of bacterial and archaeal communities, the fraction of reads that can be assigned to taxonomic groups increases as expected with the length of the sequence. In real-world experiments  typically find it at less than 1 % abundance in the gut of healthy adults , the abundance threshold that may be set deliberately or may be intrinsically limited by technology or study design , and the fraction of individuals that a taxon must appear into be considered “core” . Additionally, the cost of DNA sequencing posed a barrier until recently to collecting the detailed time-series and spatial datasets that are necessary for ecological modeling in microorganisms. However, some aspects of microbial ecology are substantially easier than in large-organism ecology. For example, the reliance on DNA sequence data means that with advances in technology, even a deep sampling of the population  can be performed rapidly, and observation biases are likely to be less profound than when attempting to glimpse rare and elusive insects or mammals. The ability to collect large-scale information about microbial populations is likely to allow classical ecological models to be applied to the microbial world far more effectively than has been possible in macroecology, because more types of microbes can be simultaneously observed with large population sizes and with replicated sampling.Ecological principles offer more than just ways to stratify the human population . At infancy, our microbial populations go through remarkable changes in structure prior to reaching a resemblance to most adult communities. Inoculation is not necessarily from our mothers, and is substantially influenced by delivery mode. Microbial communities of children delivered vaginally initially tend to resemble their mother’s vaginal communities, while the microbial communities of children delivered by C-section initially tend to resemble human skin communities. Skin inoculations may be obtained from the mother, the medical staff involved in the delivery, or hospital surroundings (many of which harbor communities resembling human skin) (Biasucci et al. A revelatory aspect to studies of the microbiome is that classical ecological models and datasets previously only obtainable for a few economically important systems, such as fisheries, are now testable on the microbial scale because of the ability to assess simultaneously the relative abundance of thousands of species in thousands of samples (Gonzalez et al. The analysis of time-series in microbial ecology has also been limited because the performance of standard signal processing methods is degraded with uneven sampling periods and small numbers of data points (Moller-Levet et al. Overall, the ability to collect far larger amounts of sequence data has led to much broader and deeper characterizations of the human microbiome and microbial communities in other habitats, especially when linked to rich contextual information about the provenance and status of each sample (Knight et al. Some of the solutions to these problems are being sought in large-scale projects such as the Earth Microbiome Project (Gilbert et al.


Saccharomyces cerevisiae (S. cerevisiae) are characterized by low structural stability, and that this is associated with reduced antigenicity and immunogenicity. However, little attention has been devoted to methods of improving the quality of the VLPs. Here, we investigated the effect of carbon source concentration in the medium on the antigenicity and immunogenicity of human papillomavirus (HPV) type 16 L1 VLPs expressed in S. cerevisiae from the galactose promoter. Media containing 2, 4, 6, and 8% carbon source, composed of both glucose and galactose in equal proportion, were used. VLP antigenicity was enhanced in cultures grown on media with 6 or 8% carbon source, compared to those from cultures with less than 6% carbon source. Moreover, the VLPs obtained from these cultures induced higher anti-HPV16 L1 IgG titers and neutralizing antibody titers in immunized mice than those purified from cultures with less than 6% carbon source. Our results indicate that the concentration of the carbon source in the medium plays a crucial role in determining the antigenicity and immunogenicity of HPV type16 L1 VLPs.There is accumulating evidence that virus-like particles (VLPs) recombinantly produced in  The ongoing technological advances in genetic engineering and in the production of recombinant proteins have enabled the development of subunit vaccines that utilize as antigens monomeric proteins derived from pathogens Virus-like particles (VLPs) are multimeric protein complexes similar in shape to naturally occurring virions Saccharomyces cerevisiae (S. cerevisiae) is one of the preferred expression systems used for producing VLPs S. cerevisiae possess the ability to self-assemble into VLPs. Another advantage of the yeast expression system is its low production cost. However, VLPs produced in this system are reported to have low structural stability The recently developed strategy for producing VLPs has provided new insights into mechanisms protecting against pathogens, and yielded an innovative platform for developing high-efficacy vaccines. However, obtaining high-quality VLPs presents a challenge because of their structural complexity. in vitro strategies for increasing VLP antigenicity and immunogenicity, such as redox refolding, VLP maturation, and salt treatment, have been tried with some success in vivo relies on intracellular assembly. In this study, we compared the antigenicity and immunogenicity of HPV type 16 L1 protein (HPV16 L1) VLPs produced in the S. cerevisiae expression system with different concentrations of carbon source. Previously, we found that the concentration and composition of the carbon source used for S. cerevisiae producing HPV16 L1 protein significantly affect the yield of the HPV16 L1 protein Many All animal experiments were treated in accordance with the guideline of Institutional Animal Care and Use Committee, Chung-Ang University IACUC, and the protocol was approved by the IACUC. The conditions of mice were monitored twice a day. Mice were anesthetized intraperitoneally with 10 μl of 4∶1 mixture of Zoletil 50  and Rompun  prior to blood collection.S. cerevisiae Y2805 was transformed with the resulting plasmid (YEGα-MCS-HP16 L1 gene-opt). Transformants were selected on SD-ura medium, a synthetic medium without uracil, and inoculated into 150 mL of YPDG medium. The YPDG medium contained 1% yeast extract, 2% peptone, and various concentrations of carbon source. For the purpose of this study, the selected concentrations were 2, 4, 6 and 8%, and the glucose to galactose ratio was set at 1∶1. Therefore, the percentages of glucose and galactose in the media were 1%, 2%, 3% and 4% each. All components used to prepare media were purchased from Duchefa (Netherlands). Cells were cultured for 6 days at 30°C with shaking at 230 rpm.The codon-optimized HPV16 L1 gene (HPV16 L1 gene-opt), designed to reduce the secondary structure of the mRNA g for 10 min. Thereafter L1 protein was recovered from the cell lysate as pellet by precipitation with 40% saturated ammonium sulfate. The pellet was resuspended in the buffer , and dialyzed against the same buffer for 4 h at room temperature (RT). After dialysis precipitation of contaminating proteins was induced in 10 mM sodium phosphate buffer  for 16 h at 4°C, and the precipitates were removed by centrifugation at 12000×g for 10 min. The supernatant (fraction containing L1 protein) was dialyzed against binding buffer for cation-exchange chromatography  for 3 h at RT and loaded onto a column packed with P-11 phosphocellulose cation-exchange resin . The column was washed with five column volumes of the binding buffer, and bound proteins were eluted by successive addition of buffer containing 0.6, 0.7, 0.8, 0.9 and 1 M NaCl. L1 protein fractions were collected and concentrated using an Amicon Ultra-4  and dialyzed against storage buffer .HPV16 L1 protein was purified as previously described Protein concentrations were measured by the Bradford protein assay  with bovine serum albumin  as a standard. The purity and quantity of L1 were determined by SDS-PAGE and Western blot analysis.http://rsbweb.nih.gov/ij/).SDS-PAGE and Western blotting were performed as described Electron microscopy was performed on a TEM200CX at a final magnification of 234000×. The purified HPV16 L1 VLPs were absorbed onto carbon-coated grids, stained with 2% phosphotungstic acid and analyzed under the microscope o-phenylenediamine , and optical density was measured at an absorbance of 492 nm.The ELISA was carried out as described DLS was performed using an ELSZ-2 system  as described Six-week-old female BALB/c mice  were immunized three times subcutaneously with purified HPV16 L1 VLPs at two-weekly intervals. The mice were divided into five groups of 8, 9, or 10 mice each. The control group received PBS, and the remaining groups received HPV16 L1 VLPs purified from cultures containing 2, 4, 6, or 8% carbon source. The mice received 10 or 1000 ng purified HPV16 L1 VLPs per dose in combination with aluminum hydroxide (200 μg per dose). The doses were as recommended in a previous report The anti-HPV16 L1 IgG titers of mouse sera were measured as described HPV16 PsV was prepared according to a published method The neutralizing antibody titer of a mouse serum was defined as the reciprocal of the highest dilution that caused a reduction of at least 50% in secreted embryonic alkaline phosphatase (SEAP) activity Production of HPV16 L1 protein was evaluated as a function of carbon source concentration , and theWe confirmed that the L1 protein finally recovered was able to form VLPs (data not shown). The concentration of L1 in each preparation was estimated by SDS-PAGE and Western blotting  prior toTo investigate the neutralizing epitopes in each VLP preparation, ELISAs were performed using neutralizing Mabs, H16.V5 and H16.E70 . It is sS. cerevisiae contain 2 – 4% carbon source due to the fact that higher concentrations can cause osmotic stress to the yeast and lead to higher alcohol production, which is potentially detrimental to cultures Typically, cultures designed to produce heterologous proteins in in vitroIn contrast with conventional fermentation strategies, in this study we obtained superior-quality HPV16 L1 VLPs from cultures containing 6 and 8% carbon source and after 6 days of culture. As shown in Previously we found that the levels of conformational neutralizing epitopes on the surface of HPV16 VLPs increased with increasing culture duration when the culture contained more than 6% carbon source in vitro treatment of redox reagent affects VLP conformation and maturation S. cerevisiae producing HPV L1  Representative plot of all VLPs: numbers in parentheses indicate the hydrodynamic diameters of the HPV16 L1 VLPs. (B) The mean ± SEM of four independent experiments.(TIF)Click here for additional data file.Figure S2GSSG and GSH level of S. cerevisiae producing HPV16 L1 protein. Cells were culture in YPDG medium containing 7% glucose and 1% galactose for 144 h at 30°C. This culture condition showed the highest production yield of the L1 protein (see reference (TIF)Click here for additional data file.


A 54-year-old woman with a past history of silicone augmentation mammoplasty was admitted with fever and dyspnea with diffuse interstitial shadows on computed tomography (CT). Although radiological findings were atypical, we diagnosed sarcoidosis by laboratory, microbiological, and bronchoalveolar lavage fluid analysis. Corticosteroids ameliorated the condition, but she had recurrent of fever and CT revealed miliary nodules while interstitial shadows disappeared. Liver biopsy showed that noncaseating granuloma and Ziehl-Neelsen stain was positive. We diagnosed miliary tuberculosis which developed during corticosteroid therapy. Antituberculotic therapy resulted in favorable outcome. Possibility exists that onset of sarcoidosis was induced by mammoplasty, namely, human adjuvant disease. Sarcoidosis is a systemic disease of unknown etiology that primarily affects the lungs and is characterized by noncaseous granuloma. On the other hand, tuberculosis is an infectious disease characterized by caseous granuloma. Differentiation of sarcoidosis from tuberculosis is important, as the treatment of sarcoidosis is opposite to that of tuberculosis. However, differentiation can be difficult because clinical and histological findings are similar. Given these similarities, etiological relationships between sarcoidosis and tuberculosis have been documented. The prevalence of sarcoidosis is high in patients who have previously suffered from tuberculosis –3, and tHuman adjuvant disease (HAD) is characterized by autoimmune-like symptoms following cosmetic mammoplasty . SarcoidHerein, we report the case of a patient with sarcoidosis following mammoplasty complicated by miliary tuberculosis during treatment with corticosteroids. Relationships between sarcoidosis, tuberculosis, and HAD are discussed.A 54-year-old woman was admitted to our hospital with fever, general malaise, and dyspnea. She had undergone bilateral breast augmentation surgery with silicone bags 5 years previously, with resurgery on the right side due to bag rupture 7 months before presentation. Seven days before admission, she became febrile and noticed dyspnea. She visited her family doctor and was diagnosed with pneumonia, but treatment with antibiotic therapy had no noticeable effect. She was then referred to our hospital for further examination and treatment.The patient was 158 cm tall, weighed 46 kg, and blood pressure was 100/50 mmHg, heart rate was 74 beats/min, body temperature was 37.0°C, and respiratory rate was 30 breaths/min on admission. Inspiratory rales were heard at both lung bases. The examination of the blood revealed severe thrombocytopenia with prolonged prothrombin time and high levels of fibrinogen degradation product (FDP) . ChemicaMycobacterium tuberculosis. Fever recurred on day 20 and liver biopsy was performed to identify the cause of fever. We considered the possibility of sarcoidosis based on elevated levels of angiotensin-converting enzyme (ACE) (63.0 IU/L) and lysozyme (11.8 mg/mL). On day 23, Liver biopsy confirmed Langhans' giant cell and noncaseating granuloma , likely caused by rifampicin. On day 59, the WBC count was fully recovered, and rifampicin was restarted at a low dose (150 mg). Fever was again seen on day 64, and CT was performed on day 68. Although miliary nodules in both lung fields had almost disappeared  and initiated methylprednisolone pulse therapy (1 g/day for 3 consecutive days) for interstitial pneumonia and gabexate mesilate and antithrombin III for DIC. Antibiotic  was administrated to cover the possibility of concomitant bacterial pneumonia. On day 4, respiratory status and interstitial opacity on CT were improved . Howeverranuloma . On day ranuloma . On day specimen , but negappeared , sickly appeared . These nappeared . On day appeared .Mycobacterium tuberculosis as proven by Ziehl-Neelsen staining and noncaseous granuloma in the liver. Since noncaseous granuloma is not specific for sarcoidosis and can also occur in tuberculosis, the series of events in the present case may be attributable to miliary tuberculosis from the beginning. However, this possibility is considered unlikely for the following reasons.The most characteristic feature of the present case was the coexistence of Mycobacterium tuberculosis on Ziehl-Neelsen-stained tissue or sputum culture. Although the yield of acid-fast smears from sputum is usually low (46.4–50%) [First, interstitial shadowing on CT and the respiratory status completely ameliorated after first steroid pulse without the use of antituberculotic therapy. The steroid therapy has brought down fever for 7 days. If she was affected by tuberculosis from the beginning, we could not control her fever only by steroid therapy. Second, bronchoscopy on day 18 failed to identify 6.4–50%) , tissue 6.4–50%) . Third, The classic findings of sarcoidosis on lung CT are widespread small nodules with bronchovascular and subpleural distributions, thickened interlobular septa, architectural distortion, and conglomerate masses . RadiogrThe etiology of sarcoidosis remains unclear. However, onset of sarcoidosis in some cases is causally related to mammoplasty –9. In thIn summary, we reported the case of a patient with sarcoidosis following mammoplasty complicated by miliary tuberculosis during treatment with corticosteroids. HAD may have induced or triggered the onset of sarcoidosis. Sarcoidosis needs to be distinguished from miliary tuberculosis because the therapeutic strategies for these two pathologies are in opposition.


Thus, anti-PD-L1 antibody might have a therapeutic potential in attenuating liver injury in sepsis.Liver plays a major role in hypermetabolism and produces acute phase proteins during systemic inflammatory response syndrome and it is of vital importance in host defense and bacteria clearance. Our previous studies indicated that programmed death-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1) are crucial modulators of host immune responses during sepsis. Our current study was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture (CLP) model. Our results indicated that there was a significant increase of PD-L1 expression in liver after CLP challenge compared to sham-operated controls, in terms of levels of mRNA transcription and immunohistochemistry. Anti-PD-L1 antibody significantly alleviated the morphology of liver injury in CLP mice. Anti-PD-L1 antibody administration decreased ALT and AST release in CLP mice, decreased the levels of tumor necrosis factor (TNF)- Sepsis remains a major clinical challenge in intensive care units (ICU), characterized by the systemic inflammatory response and multiple organ failure . Liver iIt has been proved that the high rates of morbidity and mortality associated with sepsis may be contributed by immunosuppression caused by impaired pathogen clearance after infection –7. It isProgrammed death-1 (PD-1) is a newly identified coinhibitory receptor, which has been proved to be an important modulator of immune responses , 12. PD-+ T cells, CD8+ T cells, and CD19+ B cells. Expression of PD-1 on T cells and of PD-L1 on monocytes was dramatically upregulated in septic shock patients. PD-1/PD-L1 pathway blockade in vitro with anti-PD-L1 antibody decreased apoptosis of T cells induced by TNFα or T-cell receptor ligation. Meanwhile, this blockade potentiated the lipopolysaccharide-induced TNFα and IL-6 production and decreased IL-10 production by monocytes in vitro. In short, the PD-1/PD-L1 pathway might play an essential role in sepsis-induced immunosuppression. Our animal research found that PD-L1 blockade improved the survival of septic mice [α and interleukin (IL)-6 productions, decreased IL-10 production, and enhanced bacterial clearance. Anti-PD-L1 antibody administration may be a promising therapeutic strategy for sepsis-induced immunosuppression.Our previous work showed that the expression of PD-1 and PD-L1 was evaluated in septic patients . We examtic mice . The expLiver played a key role in the immune tolerance of a variety of diseases. However, the effect of PD-L1 blockade with antibodies on sepsis-induced liver injury and its molecular mechanism remains unclear. Thus, our current research was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture model. We want to determine the expression of PD-L1 in liver during sepsis and provide a preliminary result of the role of PD-L1 in sepsis-induced liver injury.Male 8- to 10-week-old C57BL/6 mice, weighing 22 g to 30 g each, were purchased from the Animals Experimentation Center of Second Military Medical University. All mice were housed in air-filtered, temperature controlled units with 12-hour light-dark cycles and had free access to food and water. All experiments were approved by the Institutional Animal Care and Use Committee of our university.μg anti-PD-L1 antibody (Anti-Mice CD274 (B7-H1) PE, Clone: MIH5, purchased from R&D Systems) in 200 μL of PBS was injected intraperitoneally 1 hour after the operation per mouse. Mouse from the isotype group was injected with a dose of 50 μg (200 μL) control antibody  accordingly.CLP-induced polymicrobial sepsis was performed as described previously . BrieflyMice were euthanized 24 hours after CLP or sham-operated surgery. Blood samples were taken from the heart. After separation from whole blood by centrifugation, plasma were used for the determination of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activities by commercially available ELISA kits  according to the manufacturer's instructions. μm tissue sections were cut from the paraffin blocks and stained with hematoxylin and eosin. The tissue sections were examined under light microscopy by a senior independent pathologist.The right lobe of liver was rapidly harvested after blood obtaining. The liver was then fixed in 10% formalin and then paraffin embedded. 5 μg/mL. Secondary antibodies were used in the morning to bind with primary antibodies after PBS washing and sections were incubated in room temperature for 20 min. The secondary antibodies were detected with DAB according to the manufacturer's instructions, and sections were counterstained with hematoxylin. Negative controls without primary antibody and positive control tissue were included in all experiments to ensure the staining quality.The left lobe of liver was also cut off to be frozen by microtome-cryostat. Consecutive 4-micron sections of the specimens were air-dried for 10 min and then fixed in acetone for 10 min. Endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxidase in PBS for 30 min at room temperature. Sections were then washed three times in PBS. After blocking nonspecific binding with normal solcoseryl for 20 min at room temperature, sections were incubated in a humidified chamber at 4°C overnight with primary antibodies, Anti-PD-L1 antibody, which were purchased from eBioscience  and used at the final concentrations of 20 α were measured by real-time polymerase chain reaction (RT-PCR). Small cubes of liver were obtained immediately after the death of mice. Total RNA in the cube was extracted using RNeasy Mini kit . 100 ng RNA was used for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative RT-PCR was performed using SYBR Green (TaKaRa) on an ABI PRISM 7900 Sequence Detector  with SDS 2.1 software. Each reaction was performed in quadruplicate, with final calculations resulting from means of quadruplicate wells. The ΔΔCq method was used to determine the difference of the mean expression levels of PD-L1, IL-6, IL-10, and TNF-α between study subjects with different genotypes of rs4755453. For each individual, the relative expression level ΔCq (Cq T − Cq E) of PD-L1, IL-6, IL-10, and TNF-α was normalized with GAPDH and then transformed into relative quantity using the RQ formula . The primers for PD-L1 were forward 5′-tgctgcataatcagctacgg-3′ and reverse 5′-gctggtcacattgagaagca-3′. The primers for IL-6 were forward 5′-atggatgctaccaaactggat-3′ and reverse 5′-tgaaggactctggctttgtct-3′. The primers for IL-10 were 5′-ccagttttacctggtagaagtgatg-3′ and reverse 3′-tgtctaggtcctggagtccagcagactc-5′. The primers for TNF-α were 5′-catcttctcaaaattcgagtgacaa-3′ and reverse 5′-tgggagtagacaaggtacaaccc-3′.To evaluate the gene expression of mediators in the inflammatory responses, the mRNA levels of PD-L1, IL-6, IL-10, and TNF-t tests were done when 2 groups were compared. Graphs are displayed as mean with error bars representing the standard error. A P value < 0.05 (two-tailed) was considered statistically significant.All data were analyzed using GraphPad Prism software 5.0.1 . Means and standard errors of the means were calculated in experiments. Paired To examine the localization of PD-L1 expressions in liver tissues, we performed immunohistochemical staining . Among aP < 0.01). In order to clarify the expression of PD-L1 in liver quantitatively, we performed real-time PCR. Our results indicated that there is a ~5.7-fold significant increase of PD-L1 mRNA expression in liver of mice 24 hours after CLP challenge than that in sham group  . In contrast, anti-PD-L1 antibody decreased levels of ALT and AST  (P < 0.01). Thus, anti-PD-L1 antibody could reduce the level of ALT and AST levels , indicating liver damage, compared with sham group  . There is no significant difference between CLP group and Isotype group in terms of each cytokine (P > 0.05)  . These rThe main finding of this study is that PD-L1 expression was regulated in the liver of mice with sepsis, based on levels of mRNA transcription and Immunohistochemistry of liver. PD-L1 blockade with antibodies could decrease the level of CLP-induced septic liver injury.CLP-induced sepsis caused a significant inflammatory response and damage in a liver characterized both biochemically (ALT and AST activity) and histologically (hematoxylin-eosin-stained section). Our findings are in accordance with the report by Koskinas et al. . They shα were involved in sepsis, which contributes to tissue and organ damage. Our study found that anti-PD-L1 antibody suppressed not only proinflammatory cytokines (IL-6 and TNF-α) but also anti-inflammatory cytokine (IL-10). These results are partially in accordance with our previous findings. Zhang et al. [α and Il-6 and decreased the level of IL-10 in CLP murine plasma. Collectively, these data indicated that the protective effects of anti-PD-L1 antibody on septic liver injury might be partially achieved by inhibition of proinflammatory cytokines.Sepsis was associated with overproductions of proinflammatory cytokines, which can lead to the recruitment of leukocytes and tissue damage , 21. A ng et al.  found thWith the use of CLP-induced sepsis, a well-established animal model for sepsis, our findings indicated that PD-L1 expression was regulated in the liver of mice with sepsis. Mice with sepsis had a significant elevation of PD-L1 expression in liver, based on the levels of mRNA and transcription immunohistochemistry staining of liver. It was indicated that PD-L1 was expressed on Kupffer cells (KCs) and liver sinusoidal epithelial cells in murine liver tissues (LSECs) . HepatocSeveral limitations in our study should be noted. We did not explore more specific mechanisms underlying the protective role of anti-PD-L1 antibody in sepsis-induced liver injury. It is reported that liver damage during sepsis is manifested by functional change rather than structure damage. A time course study of the role of PD-L1 in sepsis-induced liver injury is warranted. Our current research chose a single dose of anti-PD-L1 antibody to use on the mice, which was based on our previous findings. The effects of anti-PD-L1 antibody at different doses in the model also need to be clarified. Furthermore, whether the blockade of PD-L1 plays a vital role in other organ damages, that is, kidney, intestinal, and heart, during sepsis also needs to be clarified.To conclude, we found elevated PD-L1 expression in liver of mice with experimental sepsis. PD-L1 blockade with antibodies could exert protective roles against sepsis-induced liver injury, characterized by alleviating liver pathological injury, reducing ALT and AST release, and attenuating liver inflammatory responses. Thus, anti-PD-L1 antibody might have a therapeutic potential in reducing liver injury in sepsis.


We aimed to describe haemodynamic changes when haemodialysis is instituted in the critically ill. Three hypotheses are tested: (1) the initial session is associated with cardiovascular instability; (2) the initial session is associated with more cardiovascular instability compared to subsequent sessions; and (3) looking at unstable sessions alone, there will be a greater proportion of potentially harmful changes in the initial sessions compared to subsequent ones.Data were collected for 209 patients, identifying 1,605 dialysis sessions. Analysis was performed on hourly records, classifying sessions as stable/unstable by a cut-off >±20% change in baseline physiology (HR/MAP). Data from 3 hours prior to and 4 hours after dialysis were included, and average and minimum values derived. Three time comparisons were made . Initial sessions were analysed separately from subsequent sessions to derive two groups. If a session was identified as being unstable, then the nature of instability was examined by recording whether changes crossed defined physiological ranges. The changes seen in unstable sessions could be described as to their effects: being harmful/potentially harmful, or beneficial/potentially beneficial.P = 0.68, reinforcing the lack of significant variance.Discarding incomplete data, 181 initial and 1,382 subsequent sessions were analysed. A session was deemed to be stable if there was no significant change (>±20%) in the time-averaged or minimum MAP/HR across time comparisons. By this definition 85/181 initial sessions were unstable . Therefore Hypothesis 1 is accepted. This compares to 44% of subsequent sessions (95% CI 41.1 to 46.3). Comparing these proportions and their respective CI gives a 95% CI for the standard error of the difference of -4% to 10%. Therefore Hypothesis 2 is rejected. In initial sessions there were 92/1,020 harmful changes. This gives a proportion of 9.0% (95% CI SEM 7.4 to 10.9). In the subsequent sessions there were 712/7,248 harmful changes. This gives a proportion of 9.8% (95% CI SEM 9.1 to 10.5). Comparing the two unpaired proportions gives a difference of -0.08% with a 95% CI of the SE of the difference of -2.5 to +1.2. Hypothesis 3 is rejected. Fisher's exact test gives a result of Our results reject the claims that using haemodialysis is an inherently unstable choice of therapy. Although proportionally more of the initial sessions are classed as unstable, the majority of MAP and HR changes are beneficial in nature.


The excitation and absorption properties of grating coupled graphene surface plasmons were studied. It was found that whether a mode can be excited is mainly determined by the frequency of incident light and the duty ratio of gratings. In the structure consisting graphene bilayer, a blueshift of the excitation frequency existed when the distance between neighbor graphene layer were decreased gradually. In graphene-grating multilayer structures, a strong absorption (approximately 90% at maximum) was found in near-THz range. Recently, a lot of work has been done based on graphene due to its unique properties in electric, magnetic, thermal, etc.-3. Graphσ, which consists of both intraband and interband from Kubo formula[ω/μc was small[Up to date, the graphene is modeled usually to be an extremely thin film with a conductivity  formula-9. The ias small. Both tras small-12. In oIn the previous works, many numerical approaches were used to study the structured graphene, for example the finite element method (FEM), finite In this paper, we studied the binary grating bounded by graphene on both sides. The rigorous coupled wave analysis (RCWA),17 was uω ≾ μc (see[Previous research has shown that the conductivity of graphene came from the contribution of intraband and interband-22. The  μc (see). Then t μc  was positive, no solution of Equation 3 was found in real, which meant the TE mode GSP could not be excited.For TM mode, put Equation 2 into Equation 4, we foundneff = β/k0 = βc/ω as the effective index of GSP. After making a transformation of  → , the dispersion relations were obtained and plotted in Figure kΛ0 = 2π/λ0, λ0 = 1 μm. As a local mode, GSP modes were same as the surface plasmon polaritons (SPPs). They cannot be excited directly from the air. And in our work, gratings were used to provide an external wave vector to match the phase condition.Here, we defined h to be the depth of grating (thickness of gratings). The h was also the distance between two graphene layers. In multilayer structures of Figure h was the longitudinal period. The structures were designed to only contain two kinds of interfaces.In Figure J = σE, while graphene was included.In common, the conventional RCWA based on the Floquet's theorem was unabIn RCWA, the field was expanded into the form ofJ can also be expended to the sum of spatial harmonics with different wave vector components. To obtain the reflection, transmission, absorption, field distribution, and other optical properties of such structures as shown in Figure Hy field considering the induced current,So the current density Hy will also be continuous at the interface if each spatial harmonics subcomponent satisfied the boundary conditions independently,According to the principle of superposition, n was the order, ± in subscripts represented approaching to y0 from two different directions. After the modification on the RCWA program, we can utilize it to deal with the graphene-containing structures.in which For a structure with a binary grating bounded by graphene layers on two sides shown in Figure ε1-graphene-ε1 and ε1-graphene-ε2. When GSP is propagating along the interfaces, the phase shifts were φ1 and φ2 for the two kinds of interfaces, respectively. δ was the total phase loss considering two abrupt phase changes when GSP propagates across the joints between the two kinds of interfaces in a grating period. At the excitation frequency, the phase change in a grating period should satisfy the relationIn the structure shown in Figure N is the integer and can be rewritten aswhich was known to be the phase matching conditions,28. In Ef2 = L/Λ and f1 = 1 - f2, β1 and β2 were the wave vectors of GSP on two kinds of interfaces, respectively. When N was a nonnegative integer, the GSP mode could be excited, and N can be defined as the order of surface modes. The resonant frequencies can be obtained both from absorption spectrum in Figure δ = 0). They were given in Table where The field distributions of orders 1 and 2 of the structure in Figure f, where f = φ1/(φ1 + φ2), φ1, φ2 had the same meaning as Equation 9. From the discussion above, when the phase match conditions were satisfied, GSPs could be coupled and absorption peaks should appear. But some break points appeared as shown in Figure f of controlling the excitation of GSP.By using the modified RCWA, the absorption spectrum was obtained in Figure From the field distributions in Figure x0 is the distance of A and B in the form of light path (k0x0 = L1β1 = φ1 = (φ1 +φ2)f = 2πNf). Because in real space, different interfaces (ε1/ε1 and ε1/ε2) had different propagating constants, the expression might be complex. Here, the light path of x was used. It is found that scatting points A and B had a phase difference of π. This was caused by the different geometric symmetries. From Equation 11, when sin(k0x0/2) = 0, i.e., f = m/N , field amplitude F would always be 0, which meant that the field cannot be excited. It was a cancelation process of two sets of standing waves that are coherent. So, for GSP mode of N, N + 1 of none absorption points appeared.First two terms were GSP excited by one set of points  was the coupling coefficient and n, h, and ∆θ were order of GSP mode, thickness of grating, and phase difference of GSPs on two graphene layers, respectively. Essentially, the GSPs were surface waves so they interact with each other via evanescent interactions, and the coupling intensity decayed exponentially with h increasing. For fixed h, the lower order modes had larger skin depth (stronger coupling intensity) than the higher orders; then, the stronger coupling resulted in a large spectra shift. The phase difference of ∆θ also had affection to the absorption frequencies. However, in our case, the wavelength (15 meV ~ 82.8 μm) was much larger than the thickness of grating layer (h = 10 μm), it is reasonable to assume ∆θ is approximately 0. This can also be obtained clearly from the field distribution in Figure κ ∝ e-hq(n), whereIn Equation 12, κ(xup = xdown = x0eiωt- (no phase difference between GSP on neighbor layers), it is found that the resonant frequency becameSuppose the solution of having the form of h was small (h < 4 μm), the larger κ ∝ e-h was the larger shift of resonant frequency would be. And obviously, κ was approaching 0 rapidly when h was large enough, which meant that the resonant frequency became a stable value ofκ was also related to the order of GSP. The high order mode had a small skin deep with weak coupling intensity and less blueshift. When h tends to be 0, the grating became too thin to excite the surface mode. This was why the absorption disappeared when h = 0 in Figure When Moreover, the behavior of multilayer structures shown in Figure The field distributions of Figure On conclusion, the rigorous coupled wave analysis was modified to compute the excitation of graphene surface plasmon in graphene-containing binary gratings structures. Under the phase matching conditions, the excitation of the graphene surface plasmonics was determined by the distance between graphene layers and duty ratio of gratings, and the mode suppression can be realized by modifying the grating constant and duty ratio. A blueshift of the excitation frequency was obtained for enhanced coupling between GSP of neighbor graphene layers. Increasing the number of graphene layers had almost no effect on the excitation frequency of GSP but would lead to a high absorption with negligible reflection in near-THz range. Finally, the resonant frequency and absorptions can be easily modified by manipulating the structure parameter, including grating constant, duty ratio, and distance between the graphene layers and number of grating, and graphene-containing grating might become potential applications of THz region, such as optical absorption devices, optical nonlinear, optical enhancement, and so on.The authors declare that they have no competing interests.HYU, XJ, and XS conceived the idea. HYU, PL, HYA, and XS wrote the codes, calculated the results, and made the conclusions. HYU, XS, and PL contributed to the preparation and revision of the manuscript. All the authors read and approved the final manuscript.