Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Comprehensive Evaluation of the Effects of Enalapril on Matrix Metalloproteinases Levels in Hypertension

Angiotensin-converting enzyme inhibitors (ACEi) may downregulate matrix metalloproteinases (MMPs). We examined whether enalapril affects MMP-2, MMP-8, and MMP-9 levels and activity, and their endogenous inhibitors (tissue inhibitors of MMPs, TIMP-1 and TIMP-2) levels in hypertensive patients. Moreover, we assessed the effects of enalaprilat on MMP-9 and TIMP-1 secretion by human endothelial cells (HUVECs). Thirty-eight hypertensive patients received enalapril for 8 weeks and were compared with thirty-eight normotensive controls. Blood samples were collected at baseline and after treatment. Plasma ACE activity was determined by a fluorimetric assay. Plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA and gelatin zymography. A fluorogenic peptide cleavage assay was used to measure MMP activity. HUVECs cells were stimulated by phorbol-12-myristate-13-acetate (PMA) and the effects of enalaprilat (10(-10) to 10(-6) M) on MMP-9 and TIMP-1 levels were determined. Enalapril decreased blood pressure and ACE activity in hypertensive patients (P < 0.05), but had no effects on plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 levels, or MMP activity. Enalaprilat had no effects on PMA-induced increases in MMP-9 and TIMP-1 secretion by HUVECs or on MMP activity. We show consistent evidence, both in vivo and in vitro, that enalapril does not affect MMPs and TIMPs levels in hypertensive patients.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

N-4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N-4-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene) hydrazinecarbothioamide) and its N-4-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N-4-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N-4-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N-4-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC50: MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5) M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization. (C) 2012 Elsevier Ltd. All rights reserved.

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.

Pain in photodynamic therapy: mechanism of action and management strategies

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by irradiation with a light source at wavelengths matching the absorption spectrum of the photosensitizer. In many countries around the world, topical photodynamic therapy has been approved for treatment of cutaneous oncologic conditions such as actinic keratosis, Bowen's disease, and superficial basal cell carcinoma. Multicenter, randomized, controlled studies have confirmed its efficacy and superior cosmetic outcomes compared to conventional therapies. Nevertheless, this therapeutic method presents some adverse effects, such as erythema, edema, pigmentation, pustules, and pain. There is no doubt that pain is the most severe of the adverse effects, being sometimes responsible for definitive treatment interruption. The pain mechanism has not yet been fully understood, which makes complete pain control a challenge to be conquered. In spite of that, this literature review presents some useful pain management strategies as well as the most important pain-related factors in photodynamic therapy.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

Background The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation. Results Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. Conclusions Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

Dynamical speckles patterns of action potential transmission effects in squid giant axon membrane.

Undoubtedly the most important result of the investigations in physiology and biophysics was the discovery of the electrochemical mechanism of propagation of the action potential in nerves that was made by Hodgkin and Huxley during the first half of the past century. Since some decades ago diverse experiments about the electro optical properties of the axon membrane there was published using the most diverse optical experimental ‘procedures POT 6-10’. In this paper some results of a dynamical speckle technique applied for obtaining microscopic images of a section of a squid giant axon membrane during the activation by electrical impulses and his digital process are presented.