Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires'' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm⁺, showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm⁺ background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.     

Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments

Background

Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not.

Methods

We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures.

Results

3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures.

General significance

This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.     

Determination of culturable indoor airborne fungi during normal and dust event days in Ahvaz, Iran

The first purpose of our study was to determine culturable indoor airborne fungi in different places in Ahvaz during normal and dust event days. Ahvaz is the biggest city in southwest of Iran which has suffered from dust storms in recent decade. To minimize personal exposure to polluted air, governmental and private sectors excluding critical places such as hospitals, fire department and security organization are closed during dust event days. Therefore, the second objective of this survey was to see whether this plan (forced shutdown) is in place. Samples were collected from July 2010 to March 2011 by using quick take 30 equipped with a single-stage sampler. Fungal population was measured in school, university and hospital during normal and dust event days. Five dominant fungi genera in three sampling stations were Cladosporium, Alternaria, Aspergillus, Penicillium and Rhizopus the concentrations (CFU/m³) of which were higher during dust events than normal days. Airborne fungi such as Aspergillus, Mucor and Rhizopus are opportunistic fungi which can cause aspergillusis and mucormycosis infections in immunodeficiency individuals, vulnerable groups as well as healthy people. The results of this study confirmed the measure of forced shutdown in Ahvaz city due to increased concentration of fungi during dust event days.     

Monitoring the biofertilizing potential and establishment of inoculated cyanobacteria in soil using physiological and molecular markers

This paper aims to develop methods for quantifying their establishment; using physiological activity (chlorophyll as a growth index and nitrogen-fixing potential as a measure of their biofertilizing capacity), along with evaluation based on DNA fingerprints generated using repeat sequences/palindromes. Time course studies were undertaken in liquid and soil microcosm experiments inoculated with a set of four rhizosphere cyanobacterial strains (BF1 Anabaena sp., BF2 Nostoc sp., BF3 Nostoc sp., BF4 Anabaena sp.). Observations revealed the synergistic effect of three-membered combinations (especially the i.e. BF1 + 2 + 3, 1 + 2 + 4, 1 + 3 + 4) in terms of enhancing chlorophyll and acetylene reducing activity. PCR-based amplification profiles (using short tandemly repetitive repeat (STRR) 1A, STRR_mod, and HIP_AT sequences) proved discriminative in monitoring the presence of the inoculated cyanobacteria in soil microcosm. Future work is in progress to assess the utility of the selected markers/primers in pot experiments, followed by field-level experiments with crop.     

Delineating Novel Therapeutic Drug and Vaccine Targets for Staphylococcus cornubiensis NW1T Through Computational Analysis

International Journal of Peptide Research and Therapeutics - Staphylococcal multidrug resistance is an emerging future threat. Among staphylococci species, Staphylococcus intermedius group (SIG)...     

Towards a Methodology for Validation of Centrality Measures in Complex Networks

Background

Living systems are associated with Social networks — networks made up of nodes, some of which may be more important in various aspects as compared to others. While different quantitative measures labeled as “centralities” have previously been used in the network analysis community to find out influential nodes in a network, it is debatable how valid the centrality measures actually are. In other words, the research question that remains unanswered is: how exactly do these measures perform in the real world? So, as an example, if a centrality of a particular node identifies it to be important, is the node actually important?

Purpose

The goal of this paper is not just to perform a traditional social network analysis but rather to evaluate different centrality measures by conducting an empirical study analyzing exactly how do network centralities correlate with data from published multidisciplinary network data sets.

Method

We take standard published network data sets while using a random network to establish a baseline. These data sets included the Zachary''s Karate Club network, dolphin social network and a neural network of nematode Caenorhabditis elegans. Each of the data sets was analyzed in terms of different centrality measures and compared with existing knowledge from associated published articles to review the role of each centrality measure in the determination of influential nodes.

Results

Our empirical analysis demonstrates that in the chosen network data sets, nodes which had a high Closeness Centrality also had a high Eccentricity Centrality. Likewise high Degree Centrality also correlated closely with a high Eigenvector Centrality. Whereas Betweenness Centrality varied according to network topology and did not demonstrate any noticeable pattern. In terms of identification of key nodes, we discovered that as compared with other centrality measures, Eigenvector and Eccentricity Centralities were better able to identify important nodes.     

A Convenient Method for the Synthesis of (Prop-2-Ynyloxy)Benzene Derivatives via Reaction with Propargyl Bromide,Their Optimization,Scope and Biological Evaluation

A highly convenient method has been developed for the synthesis of (prop-2-ynyloxy) benzene and its derivatives. Differently substituted phenol and aniline derivatives were allowed to react with propargyl bromide in the presence of K₂CO₃ base and acetone as solvent. The compounds were synthesized in good yields (53–85%). Low cost, high yields and easy availability of compounds helped in the synthesis. Electron withdrawing groups favor the formation of stable phenoxide ion thus in turn favors the formation of product while electron donating groups do not favor the reaction. Phenol derivatives gave good yields as compared to that of aniline. As aprotic polar solvents favor S_N2 type reactions so acetone provided best solvation for the reactions. K₂CO₃ was proved to be good for the synthesis. Antibacterial, Antiurease and NO scavenging activity of synthesized compounds were also examined. 4-bromo-2-chloro-1-(prop-2-ynyloxy)benzene 2a was found most active compound against urease enzyme with a percentage inhibition of 82.00±0.09 at 100 µg/mL with IC₅₀ value of 60.2. 2-bromo-4-methyl-1-(prop-2-ynyloxy)benzene 2d was found potent antibacterial against Bacillus subtillus showing excellent inhibitory action with percentage inhibition of 55.67±0.26 at 100 µg/ml wih IC₅₀ value of 79.9. Based on results, it can be concluded that some of the synthesized compounds may have potential antiurease and antibacterial effects against several harmful substances.     

Enemies make you stronger: Coevolution between fruit fly host and bacterial pathogen increases postinfection survivorship in the host

Multiple laboratory studies have evolved hosts against a nonevolving pathogen to address questions about evolution of immune responses. However, an ecologically more relevant scenario is one where hosts and pathogens can coevolve. Such coevolution between the antagonists, depending on the mutual selection pressure and additive variance in the respective populations, can potentially lead to a different pattern of evolution in the hosts compared to a situation where the host evolves against a nonevolving pathogen. In the present study, we used Drosophila melanogaster as the host and Pseudomonas entomophila as the pathogen. We let the host populations either evolve against a nonevolving pathogen or coevolve with the same pathogen. We found that the coevolving hosts on average evolved higher survivorship against the coevolving pathogen and ancestral (nonevolving) pathogen relative to the hosts evolving against a nonevolving pathogen. The coevolving pathogens evolved greater ability to induce host mortality even in nonlocal (novel) hosts compared to infection by an ancestral (nonevolving) pathogen. Thus, our results clearly show that the evolved traits in the host and the pathogen under coevolution can be different from one‐sided adaptation. In addition, our results also show that the coevolving host–pathogen interactions can involve certain general mechanisms in the pathogen, leading to increased mortality induction in nonlocal or novel hosts.     

Lack of functional selectin ligand interactions compromises long term tumor protection by CD8+ T cells

Central memory CD8⁺ T cells expressing the adhesion molecule CD62L (L-selectin) are potent mediators of anti-cancer immunity due to their ability to proliferate extensively upon antigen re-stimulation. The interaction of selectin with its ligands mediates leukocyte rolling along high endothelial venules. Mice deficient in α(1,3) Fucosyltransferase IV and VII (FtDKO) lack functional L, P and E selectin ligands. Thus, we addressed whether the lack of selectin ligand interactions alters tumor protection by CD8⁺ T cells in FtDKO mice. Listeria monocytogenes-OVA (LM-OVA) infection evoked potent OVA-specific CD8⁺ T cells that proliferated and contracted at similar kinetics and phenotype in FtDKO and wild-type mice. Additionally, OVA-specific CD8⁺ T cells in both mouse strains exhibited similar phenotypic differentiation, in vivo cytolytic activity and IFN-γ expression. However, FtDKO mice succumbed to B16-OVA tumors significantly earlier than wild-type mice. In contrast, FtDKO mice evoked strong recall memory CD8⁺ T cell responses and protection to systemic LM-OVA re-challenge. The diminished tumor protection in FtDKO mice was not related to defective antigen presentation by dendritic cells or reduced proliferation of antigen-specific CD8⁺ T cells. However, WT or FtDKO OVA-specific CD8⁺ T cells showed significantly reduced ability to traffic to lymph nodes upon adoptive transfer into naïve FtDKO recipients. Furthermore, FtDKO OVA-specific CD8⁺ T cells displayed poor ability to infiltrate tumors growing in WT mice. These results reveal that selectin ligand expression on host endothelium as well CD8⁺ T cells may be important for their efficient and continued extravasation into peripheral tumors.