Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Identification and functional characterization of a cis-acting positive DNA element regulating CYP 2B1/B2 gene transcription in rat liver

A positive cis-acting DNA element in the near 5'-upstream region of the CYP2B1/B2 genes in rat liver was found to play an important role in the transcription of these genes. An oligonucleotide covering -69 to -98 nt mimicked the gel mobility shift pattern given by the fragment -179 to +29 nt, which was earlier found adequate to confer the regulatory features of this gene. Two major complexes were seen, of which the slower and faster moving complexes became intense under uninduced and Phenobarbitone-induced conditions respectively. Minigene cloned DNA plasmid covering -179 to +181 nt in pUC 19 and Bal 31 mutants derived from this parent were transcribed in whole nuclei and cell free transcription extracts and mutants containing only upto -75 nt of the upstream were poorly transcribed. Transcription extracts from phenobarbitone-injected rat liver nuclei were significantly more active than extracts from uninduced rats in transcribing the minigene constructs. Addition of the oligonucleotide (-69 to -98nt) specifically inhibited the transcription of the minigene construct (-179 to +181 nt) in the cell free transcription system. It is therefore, concluded that the region -69 to -98 nt acts as a positive cis-acting element in the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbitone.

Negative sequence compensation within fundamental positive sequence reference frame for a stiff micro-grid generation in a wind power system using slip ring induction machine

An isolated wind power generation scheme using slip ring induction machine (SRIM) is proposed. The proposed scheme maintains constant load voltage and frequency irrespective of the wind speed or load variation. The power circuit consists of two back-to-back connected inverters with a common dc link, where one inverter is directly connected to the rotor side of SRIM and the other inverter is connected to the stator side of the SRIM through LC filter. Developing a negative sequence compensation method to ensure that, even under the presence of unbalanced load, the generator experiences almost balanced three-phase current and most of the unbalanced current is directed through the stator side converter is the focus here. The SRIM controller varies the speed of the generator with variation in the wind speed to extract maximum power. The difference of the generated power and the load power is either stored in or extracted from a battery bank, which is interfaced to the common dc link through a multiphase bidirectional fly-back dc-dc converter. The SRIM control scheme, maximum power point extraction algorithm and the fly-back converter topology are incorporated from available literature. The proposed scheme is both simulated and experimentally verified.

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial

Background The irreversible ErbB family blocker afatinib and the reversible EGFR tyrosine kinase inhibitor gefitinib are approved for first-line treatment of EGFR mutation-positive non-small-cell lung cancer (NSCLC). We aimed to compare the efficacy and safety of afatinib and gefitinib in this setting. Methods This multicentre, international, open-label, exploratory, randomised controlled phase 2B trial (LUX-Lung 7) was done at 64 centres in 13 countries. Treatment-naive patients with stage IIIB or IV NSCLC and a common EGFR mutation (exon 19 deletion or Leu858Arg) were randomly assigned (1:1) to receive afatinib (40 mg per day) or gefitinib (250 mg per day) until disease progression, or beyond if deemed beneficial by the investigator. Randomisation, stratified by EGFR mutation type and status of brain metastases, was done centrally using a validated number generating system implemented via an interactive voice or web-based response system with a block size of four. Clinicians and patients were not masked to treatment allocation; independent review of tumour response was done in a blinded manner. Coprimary endpoints were progression-free survival by independent central review, time-to-treatment failure, and overall survival. Efficacy analyses were done in the intention-to-treat population and safety analyses were done in patients who received at least one dose of study drug. This ongoing study is registered with ClinicalTrials.gov, number NCT01466660. Findings Between Dec 13, 2011, and Aug 8, 2013, 319 patients were randomly assigned (160 to afatinib and 159 to gefitinib). Median follow-up was 27·3 months (IQR 15·3–33·9). Progression-free survival (median 11·0 months [95% CI 10·6–12·9] with afatinib vs 10·9 months [9·1–11·5] with gefitinib; hazard ratio [HR] 0·73 [95% CI 0·57–0·95], p=0·017) and time-to-treatment failure (median 13·7 months [95% CI 11·9–15·0] with afatinib vs 11·5 months [10·1–13·1] with gefitinib; HR 0·73 [95% CI 0·58–0·92], p=0·0073) were significantly longer with afatinib than with gefitinib. Overall survival data are not mature. The most common treatment-related grade 3 or 4 adverse events were diarrhoea (20 [13%] of 160 patients given afatinib vs two [1%] of 159 given gefitinib) and rash or acne (15 [9%] patients given afatinib vs five [3%] of those given gefitinib) and liver enzyme elevations (no patients given afatinib vs 14 [9%] of those given gefitinib). Serious treatment-related adverse events occurred in 17 (11%) patients in the afatinib group and seven (4%) in the gefitinib group. Ten (6%) patients in each group discontinued treatment due to drug-related adverse events. 15 (9%) fatal adverse events occurred in the afatinib group and ten (6%) in the gefitinib group. All but one of these deaths were considered unrelated to treatment; one patient in the gefitinib group died from drug-related hepatic and renal failure. Interpretation Afatinib significantly improved outcomes in treatment-naive patients with EGFR-mutated NSCLC compared with gefitinib, with a manageable tolerability profile. These data are potentially important for clinical decision making in this patient population.

Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials

Background We aimed to assess the effect of afatinib on overall survival of patients with EGFR mutation-positive lung adenocarcinoma through an analysis of data from two open-label, randomised, phase 3 trials. Methods Previously untreated patients with EGFR mutation-positive stage IIIB or IV lung adenocarcinoma were enrolled in LUX-Lung 3 (n=345) and LUX-Lung 6 (n=364). These patients were randomly assigned in a 2:1 ratio to receive afatinib or chemotherapy (pemetrexed-cisplatin [LUX-Lung 3] or gemcitabine-cisplatin [LUX-Lung 6]), stratified by EGFR mutation (exon 19 deletion [del19], Leu858Arg, or other) and ethnic origin (LUX-Lung 3 only). We planned analyses of mature overall survival data in the intention-to-treat population after 209 (LUX-Lung 3) and 237 (LUX-Lung 6) deaths. These ongoing studies are registered with ClinicalTrials.gov, numbers NCT00949650 and NCT01121393. Findings Median follow-up in LUX-Lung 3 was 41 months (IQR 35–44); 213 (62%) of 345 patients had died. Median follow-up in LUX-Lung 6 was 33 months (IQR 31–37); 246 (68%) of 364 patients had died. In LUX-Lung 3, median overall survival was 28·2 months (95% CI 24·6–33·6) in the afatinib group and 28·2 months (20·7–33·2) in the pemetrexed-cisplatin group (HR 0·88, 95% CI 0·66–1·17, p=0·39). In LUX-Lung 6, median overall survival was 23·1 months (95% CI 20·4–27·3) in the afatinib group and 23·5 months (18·0–25·6) in the gemcitabine-cisplatin group (HR 0·93, 95% CI 0·72–1·22, p=0·61). However, in preplanned analyses, overall survival was significantly longer for patients with del19-positive tumours in the afatinib group than in the chemotherapy group in both trials: in LUX-Lung 3, median overall survival was 33·3 months (95% CI 26·8–41·5) in the afatinib group versus 21·1 months (16·3–30·7) in the chemotherapy group (HR 0·54, 95% CI 0·36–0·79, p=0·0015); in LUX-Lung 6, it was 31·4 months (95% CI 24·2–35·3) versus 18·4 months (14·6–25·6), respectively (HR 0·64, 95% CI 0·44–0·94, p=0·023). By contrast, there were no significant differences by treatment group for patients with EGFR Leu858Arg-positive tumours in either trial: in LUX-Lung 3, median overall survival was 27·6 months (19·8–41·7) in the afatinib group versus 40·3 months (24·3–not estimable) in the chemotherapy group (HR 1·30, 95% CI 0·80–2·11, p=0·29); in LUX-Lung 6, it was 19·6 months (95% CI 17·0–22·1) versus 24·3 months (19·0–27·0), respectively (HR 1·22, 95% CI 0·81–1·83, p=0·34). In both trials, the most common afatinib-related grade 3–4 adverse events were rash or acne (37 [16%] of 229 patients in LUX-Lung 3 and 35 [15%] of 239 patients in LUX-Lung 6), diarrhoea (33 [14%] and 13 [5%]), paronychia (26 [11%] in LUX-Lung 3 only), and stomatitis or mucositis (13 [5%] in LUX-Lung 6 only). In LUX-Lung 3, neutropenia (20 [18%] of 111 patients), fatigue (14 [13%]) and leucopenia (nine [8%]) were the most common chemotherapy-related grade 3–4 adverse events, while in LUX-Lung 6, the most common chemotherapy-related grade 3–4 adverse events were neutropenia (30 [27%] of 113 patients), vomiting (22 [19%]), and leucopenia (17 [15%]). Interpretation Although afatinib did not improve overall survival in the whole population of either trial, overall survival was improved with the drug for patients with del19 EGFR mutations. The absence of an effect in patients with Leu858Arg EGFR mutations suggests that EGFR del19-positive disease might be distinct from Leu858Arg-positive disease and that these subgroups should be analysed separately in future trials.

HER2 positive gastric and gastroesophageal adenocarcinoma; An Irish tertiary center experience

Background: Trastuzumab has been approved for patients with human epidermal growth factor receptor 2 (HER2) over expression and gene amplification metastatic gastric cancer. Here we present the prevalence of HER2 positive gastric cancer in an Irish population, the use of Trastuzumab in first line and beyond progression. Methods: The study was conducted in St James's Hospital, Dublin. A retrospective analysis of the date of patients with HER2 positive gastric cancer over a period of 3 years was carried out. Her2 positive was defined as immunohistochemistry (IHC) score of +3, of IHC score of +2 and increased gene copy number by fluorescence in situ hybridization (FISH). Overall survival was calculated from the day of initiation of treatment with Trastuzumab until death. Results: During the study period 140 patients with gastric and gastro-esophageal junction adenocarcinoma were treated. Out of those, 30 (21.4%) had HER2 positive disease. Among HER2 positive disease patients 18 (12.8%) were treated with first line Trastuzumab containing regimen with a median overall survival of 13 months. Nine (50%) developed progressive disease while on Trastuzumab and of those, 4 (22.2%) patients continued on Trastuzumab beyond progression, two (11.1%) of whom achieved stable disease and a prolonged survival. Conclusion: HER2 positivity rate in an Irish population with advanced gastric and gastro-esophageal junction adenocarcinoma is 21.4%. Treatment with Trastuzumab in the first line in combination with chemotherapy is a reasonable approach. Continuation of Trastuzumab beyond progression is a feasible strategy that requires further exploration.

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motio

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

Existence of multiple positive solutions for N-laplacian in a bounded domain in R-N

Let Ohm be a bounded domain in IRN, N greater than or equal to 2, lambda > 0, q is an element of (0, N - 1) and alpha is an element of (1, N/N-1 In this article we show the existence of at least two positive solutions for the following quasilinear elliptic problem with an exponential type nonlinearity:

Two-way converter logic for positive and negative binary numbers

This paper describes a hardware implementation of a two-way converter logic by which conversion between numbers from positive to negative binary representation is possible. Index terms: (i) Negative radix, (ii) Positive radix, (iii) Two-way conversion.

Heme is a positive regulator of cytochrome P-450 gene transcription

Inhibitors of heme biosynthesis such as CoCl2, 3-amino-1,2,4-triazole, and thioacetamide block the 3-methylcholanthrene-mediated induction of cytochrome P-450 (c + d) messenger RNAs and their transcription in rat liver. This effect is specific, since the messenger RNA levels for albumin and glutathione transferase (Ya + Yc) and their transcription are not significantly influenced under conditions of heme depletion. Exogenous administration of heme at very low doses (50 μg/100 g body wt) is able to completely counteract the effects of the heme biosynthetic inhibitors on cytochrome P-450 (c + d) messenger RNA levels and their transcription. This constitutes a direct proof for the role of heme as a positive regulator of cytochrome P-450 gene transcription.

Wire-cylinder corona discharge with the wire at positive potential studies of iron oxides

A wire-cylinder corona discharge was studied in nitrogen and dry air in crossed electric and magnetic fields for values of magnetic field ranging from 0 to 3000 G with the wire at positive potential. In the absence of a magnetic field pre-onset streamers and pulses were observed in nitrogen. In both nitrogen and dry air breakdown streamers were observed just before spark breakdown of the gap. Furthermore, experiments in dry air at atmospheric pressure in an electric field indicate regular pre-onset streamers appearing at time intervals of 19.5 µs. The appearance of regular pre-onset streamers suggests that it is not possible for negative ions to form a sheath close to the anode as postulated by Hermstein (1960) for the formation of steady or glow corona in a point-plane gap.

A new method for modulating the activity of parvalbumin-positive neurons and cerebellar Purkinje cells in vivo

Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.

Potentiodynamic behaviour of β-lead dioxide in neutral media at positive potentials

The behaviour of the PbO2 electrode in NaNO3, Na2SO4 NaClO4 and NaCl in the pH range 3.0–10.5 has been studied by cyclic voltammetry. When the electrode is cycled between 0.30 and 1.90 V, a large cathodic current peak appears in the negative scan; in the subsequent cycle, two anodic peaks appear. The addition of H2O2 at low concentrations to the electrolyte also results in two anodic peaks at the same potentials. A number of possible explanations for the appearance of the cathodic peak, and a mechanism for the oxidation of PbO to PbO2 through Pb3O4 corresponding to the two anodic peaks, are proposed.