Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

Correction to: Cell Death and Differentiation (2015) 22, 921­934; doi:10.1038/cdd.2015.5; published online 20 February 2015. Since the publication of this paper, the authors have noticed the y-axis label of Figure 7e was incorrect. It should be % of the fESP slope. This has now been rectified and the corrected article appears in this issue together with this corrigendum.

Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

The accumulation of amyloid-ß protein (Aß) is an early event associated with synaptic and mitochondrial damage in Alzheimer's disease (AD). Recent studies have implicated the filamentous actin (F-actin) severing protein, Cofilin, in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of the AD pathogenic process and how Aß impinges its signals to Cofilin from the neuronal surface are unknown. In this study, we found that Aß42 oligomers (Aß42O, amyloid-ß protein 1-42 oligomers) bind with high affinity to low or intermediate activation conformers of ß1-integrin, resulting in the loss of surface ß1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of ß1-integrin prevented Aß42O-induced Cofilin activation, and allosteric modulation or activation of ß1-integrin significantly reduced Aß42O binding to neurons while blocking Aß42O-induced reactive oxygen species (ROS) production, mitochondrial dysfunction, depletion of F-actin/focal Vinculin, and apoptosis. Cofilin, in turn, was required for Aß42O-induced loss of cell surface ß1-integrin, disruption of F-actin/focal Talin-Vinculin, and depletion of F-actin-associated postsynaptic proteins. SSH1 reduction, which mitigated Cofilin activation, prevented Aß42O-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, AD mouse model (APP (amyloid precursor protein)/PS1) brains contained increased SSH1/Cofilin and decreased SSH1/14-3-3 complexes, indicative of SSH1-Cofilin activation via release of SSH1 from 14-3-3. Finally, genetic reduction in Cofilin rescued APP/Aß-induced synaptic protein loss and gliosis in vivo as well as deficits in long-term potentiation (LTP) and contextual memory in APP/PS1 mice. These novel findings therefore implicate the essential involvement of the ß1-integrin-SSH1-Cofilin pathway in mitochondrial and synaptic dysfunction in AD.

Phytoextraction of heavy metals from contaminated soil by co-cropping with chelator application and assessment of associated leaching risk.

Phytoextraction using hyperaccumulating plants is generally time-consuming and requires the cessation of agriculture. We coupled chelators and a co-cropping system to enhance phytoextraction rates, while allowing for agricultural production. An experiment on I m3 lysimeter beds was conducted with a co-cropping system consisting of the hyperaccumulator Sedum alfredii and low-accumulating corn (Zea Mays, cv. Huidan-4), with addition ofa mixture of chelators (MC), to assess the efficiency of chelator enhanced co-crop phytoextraction and the leaching risk caused by the chelator. The results showed that the addition of MC promoted the growth of S. alfredii in the first crop (spring-summer season) and significantly increased the metal phytoextraction. The DTPA-extractable and total metal concentrations in the topsoil were also reduced more significantly with the addition of MC compared with the control treatments. However, mono-cropped S. alfredii without MC was more suitable for maximizing S. alfredii growth and therefore phytoextraction of Zn and Cd during the autumn-winter seasons. No adverse impact to groundwater due to MC application was observed during the experiments with three crops and three MC applications. But elevated total Cd and Pb concentrations among subsoils compared to the initial subsoil concentrations were found for the co-crop + MC treatment after the third crop.

Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and NarP during anaerobic growth of Escherichia coli: new insights into microbial physiology.

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

Surface runoff losses of phosphorus from Virginia soils amended with turkey manure using phytase and high available phosphorus corn diets.

Many states have passed legislation that regulates agricultural P applications based on soil P levels and crop P uptake in an attempt to protect surface waters from nonpoint P inputs. Phytase enzyme and high available phosphorus (HAP) corn supplements to poultry feed are considered potential remedies to this problem because they can reduce total P concentrations in manure. However, less is known about their water solubility of P and potential nonpoint-source P losses when land-applied. This study was conducted to determine the effects of phytase enzyme and HAP corn supplemented diets on runoff P concentrations from pasture soils receiving surface applications of turkey manure. Manure from five poultry diets consisting of various combinations of phytase enzyme, HAP corn, and normal phytic acid (NPA) corn were surface-applied at 60 kg P ha(-1) to runoff boxes containing tall fescue (Festuca arundinacea Schreb.) and placed under a rainfall simulator for runoff collection. The alternative diets caused a decrease in manure total P and water soluble phosphorus (WSP) compared with the standard diet. Runoff dissolved reactive phosphorus (DRP) concentrations were significantly higher from HAP manure-amended soils while DRP losses from other manure treatments were not significantly different from each other. The DRP concentrations in runoff were not directly related to manure WSP. Instead, because the mass of manure applied varied for each treatment causing different amounts of manure particles lost in runoff, the runoff DRP concentrations were influenced by a combination of runoff sediment concentrations and manure WSP.

Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level.

The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pylori regulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 microM NiCl(2) resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptional ureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pylori urease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences.

Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.

Roles of rpoN, fliA, and flgR in expression of flagella in Campylobacter jejuni.

Three potential regulators of flagellar expression present in the genome sequence of Campylobacter jejuni NCTC 11168, the genes rpoN, flgR, and fliA, which encode the alternative sigma factor sigma(54), the sigma(54)-associated transcriptional activator FlgR, and the flagellar sigma factor sigma(28), respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated in C. jejuni strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that the rpoN and flgR mutants were nonflagellate and that the fliA mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of rpoN, flgR, and fliA in the expression of flagellin.

The iron-induced ferredoxin FdxA of Campylobacter jejuni is involved in aerotolerance.

A gene encoding a putative 2[4Fe--4S] ferredoxin (FdxA) was identified upstream of, and divergent to the peroxide stress defense gene ahpC of the microaerophilic pathogen Campylobacter jejuni. The transcription start site of fdxA was located 27 and 28 bp upstream of the fdxA start codon. Transcriptional fusions of the fdxA promoter to a lacZ reporter gene demonstrated that expression of fdxA is iron-induced, and thus oppositely regulated to the iron-repressed ahpC gene. Insertional mutagenesis of the fdxA gene did not affect microaerobic growth of C. jejuni, but significantly reduced aerotolerance of C. jejuni. The fdxA gene is the first reported iron-induced gene of C. jejuni, and encodes a novel component of its oxidative stress defense.

Zanamivir: a rational approach to influenza B.

Influenza B viruses have co-circulated with the HIN1 and H3N2 subtypes of influenza A since 1977. Influenza A viruses are found in various animals, whereas influenza B viruses are probably restricted to humans. The lack of an animal reservoir means that the virus has no potential for genetic reassortment across species. In addition, influenza B viruses are more serologically homogeneous than influenza A viruses. Thus, the chance of influenza B causing a pandemic is much lower than that of influenza A. However, influenza B viruses are still a frequent cause of local disease outbreaks and epidemics as a result of antigenic drift. Any prophylactic or therapeutic measure must, therefore, be effective against both influenza A and B viruses. Zanamivir is the first widely approved neuraminidase inhibitor for the treatment of influenza. It is delivered directly to the primary site of viral replication, the respiratory tract, and is well tolerated and effective in the treatment of both influenza A and B. Data in prophylaxis are also encouraging. Zanamivir is the only drug proven to be clinically effective against both influenza A and B virus infections.

Fungal and parasitic infections of the eye.

The unique structure of the human eye as well as exposure of the eye directly to the environment renders it vulnerable to a number of uncommon infectious diseases caused by fungi and parasites. Host defenses directed against these microorganisms, once anatomical barriers are breached, are often insufficient to prevent loss of vision. Therefore, the timely identification and treatment of the involved microorganisms are paramount. The anatomy of the eye and its surrounding structures is presented with an emphasis upon the association of the anatomy with specific infection of fungi and parasites. For example, filamentous fungal infections of the eye are usually due to penetrating trauma by objects contaminated by vegetable matter of the cornea or globe or, by extension, of infection from adjacent paranasal sinuses. Fungal endophthalmitis and chorioretinitis, on the other hand, are usually the result of antecedent fungemia seeding the ocular tissue. Candida spp. are the most common cause of endogenous endophthalmitis, although initial infection with the dimorphic fungi may lead to infection and scarring of the chorioretina. Contact lens wear is associated with keratitis caused by yeasts, filamentous fungi, and Acanthamoebae spp. Most parasitic infections of the eye, however, arise following bloodborne carriage of the microorganism to the eye or adjacent structures.

A comparison of prestorage WBC-reduced whole-blood-derived platelets and bedside-filtered whole-blood-derived platelets in autologous progenitor cell transplant.

BACKGROUND: Prestorage WBC-reduced platelet concentrates (PCs) can be manufactured from platelet-rich plasma (PRP) by in-line filtration of PRP. There are few published data on the clinical use of these products, as compared to bedside-filtered pools of standard PCs (S-PCs) manufactured from PRP. STUDY DESIGN AND METHODS: A prospective, randomized trial was conducted in autologous progenitor cell transplant patients requiring platelet transfusions with each patient as his or her own control who was given a pool of 5 units of WBC-reduced PCs and a pool of 6 units of S-PCs within a 3-hour period. The pools were characterized before transfusion for platelet and WBC content, P-selectin expression, and IL-8. The patients were monitored with platelet counts and vital signs and observed for reactions. Data were analyzed using Mann-Whitney U tests. RESULTS: Thirty-three transfusions were administered to 13 patients. Median platelet content in the WBC-reduced PC pools was lower than that in the S-PC pools (3.3 vs. 4.0 x 10(11), p<0.01). Median WBC content was 4 to 5 log less in the WBC-reduced PC pools (2.5 x 10(4) vs. 4.6 x 10(8), p<0.01). Median IL-8 levels (pg/mL) were lower in the WBC-reduced PC pools (2 vs. 36, p<0.01). No differences were observed in CCI, but the median absolute increase after transfusion of the S-PC pools was higher (25 vs. 19 x 10(9)/L, p<0.01), which reflected the larger size of the S-PC pools. No overall differences in vital signs were recorded. Two reactions were observed, both in temporal association with the transfusion of pools of S-PCs. CONCLUSIONS: A pool consisting of 5 units of WBC-reduced PCs gave a median platelet increment of 19 x 10(9) per L in these thrombocytopenic patients and has a median WBC content 1 to 2 log below the accepted threshold for primary alloimmunization or CMV transmission.

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor.

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.

Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence.

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.

Pragmatic assessment and therapy for persons with brain damage: what have clinicians gleaned in two decades?

Pragmatic competence comprises a number of interrelated skills which manifest in real-time in a range of adaptive behaviors and which are driven by underlying cognitive processes that appear to be variably compromised in brain damage. Differential neurological profiles reflect different pragmatic outcomes. The essence of pragmatic assessment and therapy for clinicians is therefore to capture accurately, measure, and, where possible, enhance the ability of the individual to adapt to a changing communicative environment. Assessment measures differ along a number of dimensions. A distinction is drawn between testing and assessment and the argument proposed that for clinical purposes, it is helpful to keep the notions of functional and pragmatic distinct.

An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni.

Microaerophiles like Campylobacter jejuni must resist oxidative stresses during transmission or infection. Growth of C. jejuni 81116 under iron limitation greatly increased the expression of two polypeptides of 26 and 55 kDa. The identification of these proteins by N-terminal amino acid sequencing showed both to be involved in the defense against oxidative stress. The 55-kDa polypeptide was identical to C. jejuni catalase (KatA), whereas the N terminus of the 26-kDa polypeptide was homologous to a 26-kDa Helicobacter pylori protein. The gene encoding the C. jejuni 26-kDa protein was cloned, and the encoded protein showed significant homology to the small subunit of alkyl hydroperoxide reductase (AhpC). The upstream region of ahpC encoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences contained flhB and motB homologs, which are involved in flagellar motility. There was no evidence for an adjacent homolog of ahpF, encoding the large subunit of alkyl hydroperoxide reductase. Reporter gene studies showed that iron regulation of ahpC and katA is achieved at the transcriptional level. Insertional mutagenesis of the ahpC gene resulted in an increased sensitivity to oxidative stresses caused by cumene hydroperoxide and exposure to atmospheric oxygen, while resistance to hydrogen peroxide was not affected. The C. jejuni AhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.