Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N₂-fixing root nodules on diverse and globally distributed angiosperms in the “actinorhizal” symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%–98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses.     

Isolation,structure elucidation and biological activity of angucycline antibiotics from an epiphytic yew streptomycete

In the course of study of epiphytic microorganisms occurring on the surface of roots of Taxus baccata L. a new strain Streptomyces sp. AC113 was isolated. According to 16S ribosomal DNA‐based identification the new strain is 99% identical with Streptomyces flavidofuscus. This strain cultivated in an arginine glycerol medium produced three major metabolites identified as (–)‐8‐O ‐methyltetrangomycin ( 1 ), 8‐O ‐methyltetrangulol ( 2 ) and 8‐O ‐methyl‐7‐deoxo‐7‐hydroxytetrangomycin ( 3 ). The chemical structures of these angucyclines were elucidated by 1D and 2D NMR as well as by mass spectrometry. Isolated angucycline metabolites showed significant antimicrobial activity against Bacillus cereus and Listeria mocytogenes. Cytotoxic activities of compounds 1 , 2 and 3 against four cell lines (B16, HT‐29 and non – tumor V79, L929) were evaluated. Compound 3 was the most potent anticancer agents with IC₅₀ 0.054 μg/ml against cell line B16. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Co‐production of γ ‐glutamylcysteine and glutathione by mutant strain Saccharomyces cerevisiae FC‐3 and its kinetic analysis

Co‐production of γ ‐glutamylcysteine (γ ‐GC) and glutathione (GSH) by a novel mutant strain Saccharomyces cerevisiae FC‐3 and its kinetic analysis were investigated. The strain could produce γ ‐GC and GSH with high yields (4.22 and 14.3 mg/g‐DCW, respectively) in batch submerged cultures. Effects of medium components and cultivation conditions on cell growth and the contents of intracellular γ ‐GC and GSH were examined. Results show that 2% (w/v) sucrose and 2.5% (w/v) yeast extract are the best carbon and nitrogen sources, respectively, and supplement of three amino acids (glycine, cysteine and glutamate), each at 0.08% (w/v), in the medium could enhance γ ‐GC and GSH production. In addition, optimal operating conditions are at the initial pH value of 7.0, 30 °C and 200 rev/min. Moreover, results obtained from kinetic analyses reveal that γ ‐GC production is mixed‐type growth associated, but GSH production is growth‐associated. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Semi‐Interpenetrating Hydrogel Networks of Poly(2‐hydroxyethyl methacrylate) with Poly[(D,L‐lactic acid)‐co‐(ε‐caprolactam)]

Semi‐interpenetrating hydrogel networks (IPNs) composed of poly(2‐hydroxyethyl methacrylate), PHEMA, and poly[(D ,L ‐lactic acid)‐co‐(ε‐caprolactam)], copoly(D,L ‐LA/ε‐CLM), were synthesized. Linear copoly(D,L ‐LA/ε‐CLM) chains were interpenetrated into the crosslinked three‐dimensional networks of PHEMA. Physical properties of IPN samples such as stress‐strain behavior, swelling, extraction and glass transition (T_g) were examined. Glass transition temperature of the PHEMA was shifted to lower temperatures, indicating interpenetration of PHEMA and copoly(D,L ‐LA/ε‐CLM) chains. The extraction results showed that the entrapping level between two components of PHEMA/copoly(D,L ‐LA/ε‐CLM) in simultaneous semi‐IPN is very high. These results confirm that the decrease in swelling ratios with increase in copoly(D,L ‐LA/ε‐CLM) contents is probably due to the entanglement effect. Semi‐IPNs showed higher ultimate strength and modulus but lower elongation with respect to pure PHEMA hydrogel. The semi‐IPN samples exhibited brittle fracture morphology which is consistent with tensile properties.     

Biofilm formation in moderately halophilic bacteria is influenced by varying salinity levels

Bacteria in a biofilm have a co‐dependent lifestyle resulting in a harmonized and complex coordination of the bacterial cells within an exopolysaccharide (EPS) matrix. We hypothesized that biofilm formation and EPS production in salt‐tolerant bacteria are helpful for plant growth improvement in saline soil, but that they are influenced differently. To investigate this hypothesis, we tested the effect of different salinity levels on the biofilm formation of the bacterial strains PAa6 (Halomonas meridiana), HT2 (Kushneria indalinina) and ST2 (Halomonas aquamarina) on different abiotic and biotic surfaces. Maximum biofilm formation was established at 1 M salt concentration. However, EPS production was maximal at 0–1 M NaCl stress. We also studied the effect of salt stress on EPS produced by the bacterial strains and confirmed the presence of EPS on Cicer arietinum var. CM 98 roots and in soil at different salinity levels, using Alcian blue staining. Overall, the strain PAa6 was more effective in biofilm formation and EPS production. Under saline and non‐saline conditions, this strain also colonized the plant roots more efficiently as compared to the other two strains. We conclude that the strain PAa6 has the potential of biofilm formation and EPS production at different salinity levels. The presence of EPS in the biofilm helped the bacterial strains to better colonize the roots. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Synthesis and Radical Polymerization Behavior of Methacrylamides Having L‐Leucyl‐L‐alanine Oligopeptide Moieties. Effect of the Peptide Chain Length on the Radical Polymerizability

Synthesis and radical polymerization of methacrylamides having L ‐leucyl‐L ‐alanine oligopeptide moieties, N‐methacryloyl‐(L ‐leucyl‐L ‐alanine)_n methyl ester (MA‐(LA)_n‐M, n = 2 ˜ 4), were carried out. The monomers were synthesized by the condensation of (L ‐leucyl‐L ‐alanine)_1˜3 methyl ester·trifluoroacetate with N‐methacryloyl‐L ‐leucyl‐L ‐alanine using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride or N,N ′‐dicyclohexyl‐carbodiimide as coupling reagents. The radical polymerization was carried out in 1,1,1,3,3,3‐hexafluoro‐2‐propanol using 2,2′‐azoisobutyronitrile as an initiator at 60°C. The monomer with a longer peptide chain showed a smaller conversion and degree of polymerization.     

Alnus incana response on treatment with fluorescent Pseudomonas sp. strain 267

Fluorescent Pseudomonas sp. strain 267 was evaluated for its ability to stimulate the growth of A. incana in vitro under gnotobiotic and nongnotobiotic conditions, as well as in the presence and absence of FeCl₃ as iron source. Bacterization of grey alder roots with Pseudomonas sp. strain 267 promoted plant growth under each of the studied conditions. The dry weight of leaves and roots increased significantly when compared with the uninoculated plants. Chlorophyll concentration in the leaves of grey alder, treated with fluorescent pseudomonad, was positively correlated with plant growth stimulation. Its concentration showed 50% increase over the control uninoculated plants. Moreover, it is supposed, that A. incana growth promotion by Pseudomonas sp. strain 267 may result from secretion of vitamins by this bacterium, which can be utilized by plant.     

Regulation of alveolar gas conductance by NO in man,as based on studies with NO donors and inhibitors of NO production

Aim: Nitric oxide (NO) is a mediator of the pulmonary vessel tone and permeability. We hypothesized that it may also regulate the alveolar‐capillary membrane gas conductance and lung diffusion capacity. Methods: In 20 healthy subjects (age = 23 ± 3 years) we measured lung diffusion capacity for carbon monoxide (DLco), its determinants (membrane conductance, D_m, and pulmonary capillary blood volume, V_c), systolic pulmonary artery pressure (PAPs) and pulmonary vascular resistance (PVR). Measurements were performed before and after administration of N^g ‐monomethyl‐l ‐arginine (l ‐NMMA, 0.5 mg kg^?1 min^?1), as a NO production inhibitor, and l ‐arginine (l ‐Arg, 0.5 mg kg^?1 min¹) as a NO pathway activator. The effects of l ‐NMMA were also tested in combination with active l ‐Arg and inactive stereoisomer d ‐Arg vehicled by 150 mL of 5%d ‐glucose solution. For l ‐Arg and l ‐NMMA, saline (150 mL) was also tested as a vehicle. Results: l ‐NMMA reduced D_m (?41%P < 0.01), DLco (?20%, P < 0.01) and cardiac output (CO), and increased PAPs and PVR. In 10 additional subjects, a dose of l ‐NMMA of 0.03 mg kg^?1 min¹ infused in the main stem of the pulmonary artery was able to lower D_m (?32%, P < 0.01) despite no effect on PVR and CO. D_m depression was significantly greater when l ‐NMMA was vehicled by saline than by glucose. l ‐Arg but not d ‐Arg abolished the effects of l ‐NMMA. l ‐Arg alone increased D_m (+14%, P < 0.01). Conclusion: The findings indicate that NO mediates the respiratory effects of l ‐NMMA and l ‐Arg, and is involved in the physiology of the alveolar‐capillary membrane gas conductance in humans. NO deficiency may cause an excessive endothelial sodium exchange/water conduction and fluid leakage in alveolar interstitial space, lengthening the air–blood path and depressing diffusion capacity.     

Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP‐1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH‐20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP‐1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1–10 μg ml^–1 and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 μg ml^–1 under greenhouse conditions. The efficacy of FP‐1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp ‐tagged strain of KPE62302H confirmed its ability to colonize tomato plants. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll‐like receptor 2 (TLR2), which leads to the c‐Jun N‐terminal kinase (JNK)‐mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild‐type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of d ‐alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, d ‐alanine‐bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d ‐alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.     

Poly(L‐lactide) Nanoparticles via Ring‐Opening Polymerization in Non‐aqueous Emulsion

The preparation of poly(L ‐lactide) nanoparticles via ring‐opening polymerization (ROP) of L ‐lactide is conducted in non‐aqueous emulsion. In this process, acetonitrile is dispersed in either cyclohexane or n‐hexane as the continuous phase and stabilized by a PI‐b‐PEO, respectively, a PI‐b‐PS copolymer as emulsifier. The air and moisture sensitive N‐heterocyclic carbene 1,3‐bis(2,4,6‐trimethylphenyl)‐2‐ididazolidinylidene (SIMes) catalyzes the polymerization of L ‐lactide at ambient temperatures. Spherical poly(L ‐lactide) nanoparticles with an average diameter of 70 nm and a tunable molecular weight are generated. Hence, the non‐aqueous emulsion technique demonstrates its good applicability toward the generation of well‐defined poly(L ‐lactide) nanoparticles under very mild conditions.     

Polylactones, 45. Homo‐ and Copolymerizations of 3‐Methylmorpholine‐2,5‐dione Initiated With a Cyclic Tin Alkoxide

Morpholine‐2,5‐dione and D ,L ‐3‐methylmorpholine‐2,5‐dione were polymerized with 2,2‐dibutyl‐2‐stanna‐1,3‐dioxepane (DSDOP) as initiator with variation of time and monomer/initiator (M/I)‐ratio. For comparison a few polymerizations were initiated with Sn(II) 2‐ethylhexanoate. The DSDOP initiated polymerizations gave slightly higher molecular weights, but the molecular weights were rather low in all cases and did not parallel the M/I‐ratio. Furthermore, D ,L ‐3‐methylmorpholine‐2,5‐dione was copolymerized with ε‐caprolactone (ε‐CL) or with L ‐lactide. ¹³C NMR spectra proved the formation of nearly random sequences. Therefore, the 1 : 1 copolymers were amorphous. Higher feed ratios of ε‐CL or L ‐lactide yielded higher molecular weights (at constant M/I‐ratio) and they yielded crystalline copolyesters as evidenced by DSC‐measurements.     

Protection and Polymerization of Functional Monomers, 31. Living Anionic Polymerization of Styrene Derivatives m,m′‐Disubstituted with Acetal‐Protected Monosaccharide Residues

Two new styrene derivatives m,m′‐disubstituted with acetal‐protected monosaccharide residues, 3,5‐bis(1,2:5,6‐di‐O‐isopropylidene‐α‐D ‐glucofuranose‐3‐oxymethyl)styrene ( 1 ) and 3,5‐bis(1,2:3,4‐di‐O‐isopropylidene‐α‐D ‐galactopyranose‐6‐oxymethyl)styrene ( 2 ) were synthesized viahigh yielding eight reaction steps starting from isophthalic acid. Their anionic polymerizations were carried out with sec‐BuLi in THF at –78°C for 30 min. Both monomers, 1 and 2 , were found to undergo living anionic polymerization to afford quantitatively the polymers of predictable molecular weights and narrow molecular weight distributions (M̄_w/M̄_n < 1.08). Novel AB and BA diblock copolymers of 1 and styrene were also successfully synthesized. Complete deprotection of the acetal protective groups by treatment with trifluoroacetic acid was achieved to quantitatively regenerate D ‐glucose and D ‐galactose. The resulting polymers were highly water‐soluble polymers as expected.     

Occurrence of diversified N‐acyl homoserine lactone mediated biofilm‐forming bacteria in rice rhizoplane

Quorum sensing (QS)‐mediated biofilm‐forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N‐acyl homoserine lactone (AHL) as the QS signal in the roots of a rice plant grown under field conditions. The AHL‐producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS‐positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX‐PCR fingerprinting showed high variability among the isolates. Most of the best biofilm‐forming isolates produced a N‐butyryl dl ‐homoserine lactone (a C4‐AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7‐4; QS36‐2), A. enteropelongenes (QS20‐8), A. veronii (QS36‐3), Enterobacter sp. (QS20‐11), Klebsiella pneumoniae (QS24‐6), Kosakonia cowanii (QS24‐21), Providentia rettigeri (QS24‐2), Sphingomonas aquatilis (QS24‐17), and Pseudomonas sihuiensis (QS24‐20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.     

In vivo detection of d‐amino acid oxidase with hyperpolarized d‐[1‐13C]alanine

d ‐amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d ‐amino acids to their corresponding α‐keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d ‐amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N‐methyl‐d ‐aspartate (NMDA) receptor hypo/hyper‐function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d ‐[1‐¹³C]alanine. Following a bolus of hyperpolarized d ‐alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate‐to‐d ‐alanine ratio was comparable to the values measured when the l ‐enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d ‐[1‐¹³C]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate‐to‐d ‐alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1‐¹³C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d ‐[1‐¹³C]alanine as a probe of d ‐amino acid metabolism.     

The effect of gender and genetic polymorphisms on matrix metalloprotease (MMP) and tissue inhibitor (TIMP) plasma levels in different infectious and non‐infectious conditions

Matrix metalloproteases (MMPs) are increased in different infections due to their role in controlling immune responses and are regulated by tissue inhibitors (TIMPs). Different MMP promoter single nucleotide polymorphisms (SNPs) induce changes in MMP genes, mRNA and protein expression. Gender might also modify MMP plasma levels. In order to determine the weight of these variables on MMP secretion we studied MMP‐1, ‐2, ‐3, ‐8, ‐9, ‐10, ‐13 and TIMP‐1, ‐2, ‐4 plasma levels in 90 patients with severe bacterial sepsis, 102 with anti‐retroviral (ARV)‐treated HIV monoinfection, 111 with ARV‐treated HIV–hepatitis C virus (HCV) co‐infection and 86 non‐infected controls (45 stroke and 41 trauma patients). MMP‐1(‐1607 1G/2G), MMP‐3(‐1612 5A/6A), MMP‐8(‐799C/T), MMP‐9(‐1562 C/T) and MMP‐13(‐77A/G) SNPs were genotyped. MMP‐3 plasma levels were significantly higher in men than in women in each diagnostic group, and MMP‐3 SNP allele 6A carriers also had higher levels than allele 5A carriers, an effect that was magnified by sepsis. Independent predictors of higher MMP‐3 levels were male gender (P = 0·0001), MMP‐3(‐1612 5A/6A) SNP (P = 0·001), higher levels of TIMP‐4 (P = 0·004) and MMP‐8 (P = 0·006) and lower levels of MMP‐1 (P = 0·03) by multivariate analysis. No strong associations with gender or SNPs were observed for other MMPs or TIMPs. In conclusion, male gender and MMP‐3(‐1612 5A/6A) 6A allele carriage increased MMP‐3 plasma levels significantly, especially in patients with severe bacterial sepsis. This confounding gender effect needs to be addressed when evaluating MMP‐3 plasma levels in any infectious or non‐infectious condition.     

Relationship of polymorphisms located in tumor necrosis factor region and HLA loci among Croatians

Polymorphism of TNFa, TNFb, and TNFd microsatellites, linkage disequilibrium (LD) within TNF region as well as relationship of TNF microsatellites with alleles at HLA‐A, ‐B, and ‐DRB1 loci was investigated on a sample of 160 Croatians, previously typed for HLA‐A, ‐B, and ‐DRB1 loci. Analysis of the relationship of TNF alleles and HLA specificities revealed that very strong associations exist between TNFa2, TNFb3, and TNFd2 alleles and HLA‐A*01, ‐B*08, and ‐DRB1*03 specificities, therefore placing them in the 8.1 ancestral haplotype. Similar findings were observed for TNFa11, TNFb4, and TNFd4 alleles and HLA specificities, which are a part of the 7.1 ancestral haplotype. Finally, multiple associations with significant P‐values were also observed between TNFa10, TNFb4, and TNFd4 microsatellite alleles and HLA‐A*02, ‐B*18, ‐DRB1*11 specificities which form the 18.3 ancestral hapolotype. Am. J. Hum. Biol. 2009. © 2008 Wiley‐Liss, Inc.     

Saccharide polymers, 4. Synthesis and polymerisation of 1,2‐unsaturated fructopyranoid derivatives

Unsaturated fructopyranose derivatives like 2,6‐anhydro‐3,4,5‐tri‐O‐benzoyl‐1‐desoxy‐β‐D ‐arabino‐ hex‐1‐enopyranose ( 3 ) and 2,6‐anhydro‐3,4,5‐tri‐O‐acetyl‐1‐desoxy‐β‐D ‐arabino‐hex‐1‐enopyranose ( 6 ), briefly called “Bz‐exo‐fructal” ( 3 ) and “Ac‐exo‐fructal” ( 6 ), were synthesised. These sugar monomers, which are exo‐cyclic vinyl ethers, were investigated in polymerisation reactions. The corresponding “saccharide polymers”, homo‐ and copolymers, were synthesised under free radical conditions. The structure and composition of the “saccharide polymers” were determined by elemental analysis, ¹H and ¹³C NMR, and FT‐IR spectroscopy. Characterisation and properties of the various polymers in terms of molecular weight, optical rotation, and glass transition temperature are reported.     

HIF‐1α triggers long‐lasting glutamate excitotoxicity via system xc− in cerebral ischaemia–reperfusion

Hypoxia‐inducible factor 1α (HIF ‐1α) controls many genes involved in physiological and pathological processes. However, its roles in glutamatergic transmission and excitotoxicity are unclear. Here, we proposed that HIF ‐1α might contribute to glutamate‐mediated excitotoxicity during cerebral ischaemia–reperfusion (CIR ) and investigated its molecular mechanism. We showed that an HIF ‐1α conditional knockout mouse displayed an inhibition in CIR ‐induced elevation of extracellular glutamate and N ‐methyl‐d ‐aspartate receptor (NMDAR ) activation. By gene screening for glutamate transporters in cortical cells, we found that HIF ‐1α mainly regulates the cystine–glutamate transporter (system x_c^?) subunit xCT by directly binding to its promoter; xCT and its function are up‐regulated in the ischaemic brains of rodents and humans, and the effects lasted for several days. Genetic deletion of xCT in cortical cells of mice inhibits either oxygen glucose deprivation/reoxygenation (OGDR ) or CIR ‐mediated glutamate excitotoxicity in vitro and in vivo . Pharmaceutical inhibition of system x_c^? by a clinically approved anti‐cancer drug, sorafenib, improves infarct volume and functional outcome in rodents with CIR and its therapeutic window is at least 3 days. Taken together, these findings reveal that HIF ‐1α plays a role in CIR ‐induced glutamate excitotoxicity via the long‐lasting activation of system x_c^?‐dependent glutamate outflow and suggest that system x_c^? is a promising therapeutic target with an extended therapeutic window in stroke. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prediction and characterization of helper T‐cell epitopes from pneumococcal surface adhesin A

Pneumococcal surface adhesin A (PsaA) is a multifunctional lipoprotein known to bind nasopharyngeal epithelial cells, and is significantly involved in bacterial adherence and virulence. Identification of PsaA peptides that optimally bind human leucocyte antigen (HLA) and elicit a potent immune response would be of great importance to vaccine development. However, this is hindered by the multitude of HLA polymorphisms in humans. To identify the conserved immunodominant epitopes, we used an experimental dataset of 28 PsaA synthetic peptides and in silico methods to predict specific peptide‐binding to HLA and murine MHC class II molecules. We also characterized spleen and cervical lymph node (CLN) ‐derived T helper (Th) lymphocyte cytokine responses to these peptides after Streptococcus pneumoniae strain EF3030 challenge in mice. Individual, yet overlapping, peptides 15 amino acids in length revealed residues of PsaA that consistently caused the highest interferon‐γ, interleukin‐2 (IL‐2), IL‐5 and IL‐17 responses and proliferation as well as moderate IL‐10 and IL‐4 responses by ex vivo re‐stimulated splenic and CLN CD4⁺ T cells isolated from S. pneumoniae strain EF3030‐challenged F₁ (B6 ×  BALB/c) mice. In silico analysis revealed that peptides from PsaA may interact with a broad range of HLA‐DP, ‐DQ and ‐DR alleles, due in part to regions lacking β‐turns and asparagine endopeptidase sites. These data suggest that Th cell peptides (7, 19, 20, 22, 23 and 24) screened for secondary structures and MHC class II peptide‐binding affinities can elicit T helper cytokine and proliferative responses to PsaA peptides.