Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

AbstractRapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

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Social role of the media: Control of social reality

Politea ◽

10.5937/pol1204229d ◽

2012 ◽

Vol 2 (4) ◽

pp. 229-246 ◽

Cited By ~ 1

Author(s):

Nemanja Djukic

Keyword(s):

Social Role ◽

Social Reality ◽

Media Control ◽

The Media

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The relationship between intracellular calcium levels and limb bud chondrogenesis in vitro

Development ◽

10.1242/dev.100.1.73 ◽

1987 ◽

Vol 100 (1) ◽

pp. 73-81

Author(s):

J.A. Bee ◽

R. Jeffries

Keyword(s):

Phenotypic Expression ◽

Culture Conditions ◽

Limb Bud ◽

Concentration Effects ◽

Cartilage Differentiation ◽

Standard Culture ◽

The Relationship ◽

Plateau Level

Under standard culture conditions, chondrogenic expression by stage-21 embryonic chick limb bud mesenchyme is dependent upon high cell plating densities. Alternatively, when cultured in suspension aggregating limb bud cells differentiate exclusively as cartilage. We have previously demonstrated that the aggregation of prechondrogenic limb bud cells is specifically mediated by a Ca2+ -dependent mechanism. In the present paper, we examine the involvement of calcium cations in chondrogenic expression in vitro. During cartilage differentiation, we demonstrate that limb bud cells elevate their intracellular Ca2+ levels to achieve a conserved plateau level. This increase in intracellular Ca2+ levels does not occur in sparse cell cultures, which also fail to demonstrate cartilage differentiation. Although elevation of extracellular Ca2+ concentration effects precocious chondrogenesis, ultimately this is substantially lower than in control cultures. In contrast, elevation of intracellular Ca2+ levels by the addition of 0á1 μm-A23187 readily stimulates precocious and extensive cartilage differentiation. 0á1μm-A23187 initially elevates intracellular Ca2+ levels to that required for cartilage differentiation but this then continues to increase concomitant with a reduction in cartilage nodule size. 10μm-retinoic acid completely inhibits chondrogenesis in vitro and elevates intracellular Ca2+ to particularly high levels. Our data indicate the central role of controlled intracellular Ca2+ levels to normal chondrogenic expression. Deviation from this level by cells that either fail to achieve or that exceed it inhibits subsequent cartilage development, and can cause a loss of phenotypic expression by differentiated cartilage.

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Plasmalogens: targets for oxidants and major lipophilic antioxidants

Biochemical Society Transactions ◽

10.1042/bst0320147 ◽

2004 ◽

Vol 32 (1) ◽

pp. 147-150 ◽

Cited By ~ 108

Author(s):

B. Engelmann

Keyword(s):

Fatty Acids ◽

Polyunsaturated Fatty Acids ◽

Lipid Oxidation ◽

De Novo ◽

Peroxyl Radicals ◽

Plasma Lipoproteins ◽

Future Work

Cellular membranes and plasma lipoproteins are less efficiently protected against oxidative stress than the various aqueous compartments of mammalian organisms. Here, previous results on the role of plasmalogens in lipid oxidation are evaluated on the basis of criteria required for an antioxidant. The plasmalogen-specific enol ether double bond is targeted by a vast variety of oxidants, including peroxyl radicals, metal ions, singlet oxygen and halogenating species. Oxidation of the vinyl ether markedly prevents the oxidation of highly polyunsaturated fatty acids, and products of plasmalogen degradation do not propagate lipid oxidation. This protection is also demonstrated intramolecularly, thus ascertaining the function of plasmalogens as a major storage pool for polyunsaturated fatty acids. Although cells rapidly incorporate and synthesize plasmalogens de novo, their plasmalogen contents can be deliberately increased by supplementation with precursors. Thus plasmalogens terminate lipid-oxidation processes, are present in adequate locations at sufficient concentrations, and are rapidly regenerated, classifying them as efficient antioxidants in vitro. Future work should address the in vivo role of plasmalogens in lipid oxidation and the biological function of plasmalogen interactions with oxidants.

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Ectoine from Bacterial and Algal Origin Is a Compatible Solute in Microalgae

Marine Drugs ◽

10.3390/md18010042 ◽

2020 ◽

Vol 18 (1) ◽

pp. 42 ◽

Cited By ~ 9

Author(s):

Simona Fenizia ◽

Kathleen Thume ◽

Marino Wirgenings ◽

Georg Pohnert

Keyword(s):

Phaeodactylum Tricornutum ◽

Culture Conditions ◽

Compatible Solute ◽

Associated Bacteria ◽

The Family ◽

Polar Metabolites ◽

Standard Culture ◽

Polar Low ◽

Dual Origin

Osmoregulation in phytoplankton is attributed to several highly polar low-molecular-weight metabolites. A widely accepted model considers dimethylsulfoniopropionate (DMSP) as the most important and abundant osmotically active metabolite. Using an optimized procedure for the extraction and detection of highly polar metabolites, we expand the group of phytoplankton osmolytes by identifying ectoine in several microalgae. Ectoine is known as a bacterial compatible solute, but, to the best of our knowledge, was never considered as a phytoplankton-derived product. Given the ability of microalgae to take up zwitterions, such as DMSP, we tested the hypothesis that the algal ectoine is derived from associated bacteria. We therefore analyzed methanol extracts of xenic and axenic cultures of two different species of microalgae and could detect elevated concentrations of ectoine in those that harbor associated bacteria. However, also microalgae without an associated microbiome contain ectoine in smaller amounts, pointing towards a dual origin of this metabolite in the algae from their own biosynthesis as well as from uptake. We also tested the role of ectoine in the osmoadaptation of microalgae. In the model diatoms Thalassiosira weissflogii and Phaeodactylum tricornutum, elevated amounts of ectoine were found when cultivated in seawater with salinities of 50 PSU compared to the standard culture conditions of 35 PSU. Therefore, we add ectoine to the family of osmoadaptive metabolites in phytoplankton and prove a new, potentially synergistic metabolic interplay of bacteria and algae.

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Russian Media in the Digital Age: Propaganda Rewired

Russian Politics ◽

10.1163/2451-8921-00104004 ◽

2016 ◽

Vol 1 (4) ◽

pp. 398-417 ◽

Cited By ~ 8

Author(s):

Sarah Oates

Keyword(s):

Case Studies ◽

Political Communication ◽

Digital Age ◽

Information Control ◽

Media Environment ◽

Media Control ◽

Russian Media ◽

The Media ◽

The Russian Federation

This article reflects on the role of media in the Russian Federation through the concept of “rewired propaganda.” The approach highlights how the Russian regime copes with challenges to its information hegemony in the digital age. The study employs two critical case studies to examine the Russian political communication sphere: the 2011–12 election protests and the downing of Malaysia Airlines Flight 17 by a Russian missile in 2014. The article argues that a key vector of analysis is understanding strategic narrative as the critical measurement of media control. The findings suggests that it is not so much who owns or controls the media that is key to understanding information control; rather, it is knowing who is constructing and disseminating the most compelling national narrative that holds the key to power in Russia. This focus on rewired propaganda and recasting of the debate will permit an analysis of the role of the media in the post-Soviet state even as the overall media environment has shifted with the advent of the digital age. On balance, the two case studies demonstrate that Russian elites have continued to adapt to growing challenges, showing an ability to use many facets of communication to consolidate an information dominance over citizens.

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Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

10.1101/200477 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alexander Harms ◽

Cinzia Fino ◽

Michael A. Sørensen ◽

Szabolcs Semsey ◽

Kenn Gerdes

Keyword(s):

Antibiotic Treatment ◽

Bacterial Growth ◽

Culture Conditions ◽

Growth Stages ◽

Persister Cells ◽

E Coli ◽

Common Basis ◽

Mutant Strains ◽

Future Work

AbstractBacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drivesEscherichia colipersister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage φ80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules inE. colipersister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.ImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

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Protocol Interactions among User Agents, Application Servers, and Media Servers

Digital Arts and Entertainment ◽

10.4018/978-1-4666-6114-1.ch024 ◽

2014 ◽

pp. 527-542

Author(s):

Alessandro Amirante ◽

Tobia Castaldi ◽

Lorenzo Miniero ◽

Simon Pietro Romano

Keyword(s):

The Other ◽

The Internet ◽

Application Server ◽

Current Standard ◽

Media Control ◽

Complex Interactions ◽

The Media ◽

Media Server ◽

Open Issues

In this chapter, the authors focus on the complex interactions involving the various actors participating in a multimedia session over the Internet. More precisely, bearing in mind the current standard proposals coming from both the 3GPP and the IETF, they investigate some of the issues that have to be faced when separation of responsibilities comes to the fore. The scenario the authors analyze is one in which one or more user agents are put into communication with a media server through the mediation of an application server. In such scenario, the application server does play the role of a middlebox for all that concerns signaling, since it is responsible for the transparent negotiation of a session among the entities (the user agents on one side and the media server on the other) that will be exchanging media during the communication phase. In this chapter, the authors highlight that protocol interactions become really complex under the depicted circumstances. They provide a survey of the current standardization efforts related to media control, together with a discussion of open issues and potential solutions.

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