Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.     

A Facile and Specific Assay for Quantifying MicroRNA by an Optimized RT-qPCR Approach

Background

The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.

Results

We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.

Conclusions

The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.     

A Competitive Enzyme-Linked Immunosorbent Assay to Quantify Acetaldehyde-Protein Adducts That Accumulate in Dry Seeds during Aging

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify endogenous acetaldehyde-protein adducts (APAs) produced in plant seeds at low acetaldehyde concentrations without exogenous reducing agents. The key point of this technique is the use of a gelatin-acetaldehyde adduct, which is synthesized under 1 mM acetaldehyde and 10 mM NaCNBH3, to pre-coat plate wells to obtain the proper binding parameters for the quantification of APA in seed proteins. Compared with the traditional, direct ELISA method, the competitive one has higher sensitivity and less background. Using competitive ELISA, we determined the accumulation of endogenous APAs in seeds in relation to the loss of seed viability. Lettuce seeds were exposed to 2 mM gaseous acetaldehyde during storage for 30 or 45 d; the relative humidity and temperature of storage were studied independently. Viability decreased only in acetaldehyde-treated seeds, as either the temperature or the relative humidity increased. A loss in viability was accompanied by an increase in the accumulation of APA. The APA content also increased as viability decreased in five species of seeds, which were aged naturally without exposure to acetaldehyde. It is suggested that the modification of functional seed proteins with endogenously evolved acetaldehyde may be an important cause of seed aging.     

Environmental Indication: A Field Test of an Ecosystem Approach to Quantify Biological Self-Organization

In this study, we take an ecosystem approach to examine the degree of biological self-organization at the ecosystem level. An integrated set of indicators is derived from a theoretical framework and tested by field data from an ecosystem research project focusing on the Bornhöved Lake district in northern Germany. This field test is based on a comparison of the self-organized phenomena that comprise the carbon, water, and energy budgets of two adjacent edaphically and climatically similar ecosystems, that have vastly different levels of human interference—a crop field and a beech forest. In terms of biomass storage, biologically incorporated nitrogen and phosphorus, species number, total ecosystem respiration per total biomass (qCO₂), total ecosystem assimilation per available nutrients, and transpiration per total evapotranspiration, we found clear differences between the systems. Ecosystem surface temperature and R_n/K* were found to be of limited utility in characterizing the two systems. The study is rooted in the concept of ecological integrity, an influential idea at the interface of ecological and environmental debate that has acquired a number of different meanings. Among other interpretations, it can be viewed as a guiding principle for sustainable land use that aims at long-term protection of ecological life-support systems. Effective use of any interpretation of this concept requires a theoretically consistent and applicable set of indicators. Therefore, we also discuss the integration of the indicator set and its potential use in monitoring programs.     

Synapse formation: let's stick together

Synapse formation requires the precise alignment and attachment of presynaptic and postsynaptic cells. Homophilic cell adhesion molecules have now been found to have a role in these processes on both sides of the synaptic cleft.     

Quantifying the Number of Pregnancies at Risk of Malaria in 2007: A Demographic Study

Background

Comprehensive and contemporary estimates of the number of pregnancies at risk of malaria are not currently available, particularly for endemic areas outside of Africa. We derived global estimates of the number of women who became pregnant in 2007 in areas with Plasmodium falciparum and P. vivax transmission.

Methods and Findings

A recently published map of the global limits of P. falciparum transmission and an updated map of the limits of P. vivax transmission were combined with gridded population data and growth rates to estimate total populations at risk of malaria in 2007. Country-specific demographic data from the United Nations on age, sex, and total fertility rates were used to estimate the number of women of child-bearing age and the annual rate of live births. Subregional estimates of the number of induced abortions and country-specific stillbirths rates were obtained from recently published reviews. The number of miscarriages was estimated from the number of live births and corrected for induced abortion rates. The number of clinically recognised pregnancies at risk was then calculated as the sum of the number of live births, induced abortions, spontaneous miscarriages, and stillbirths among the population at risk in 2007. In 2007, 125.2 million pregnancies occurred in areas with P. falciparum and/or P. vivax transmission resulting in 82.6 million live births. This included 77.4, 30.3, 13.1, and 4.3 million pregnancies in the countries falling under the World Health Organization (WHO) regional offices for South-East-Asia (SEARO) and the Western-Pacific (WPRO) combined, Africa (AFRO), Europe and the Eastern Mediterranean (EURO/EMRO), and the Americas (AMRO), respectively. Of 85.3 million pregnancies in areas with P. falciparum transmission, 54.7 million occurred in areas with stable transmission and 30.6 million in areas with unstable transmission (clinical incidence <1 per 10,000 population/year); 92.9 million occurred in areas with P. vivax transmission, 53.0 million of which occurred in areas in which P. falciparum and P. vivax co-exist and 39.9 million in temperate regions with P. vivax transmission only.

Conclusions

In 2007, 54.7 million pregnancies occurred in areas with stable P. falciparum malaria and a further 70.5 million in areas with exceptionally low malaria transmission or with P. vivax only. These represent the first contemporary estimates of the global distribution of the number of pregnancies at risk of P. falciparum and P. vivax malaria and provide a first step towards a more informed estimate of the geographical distribution of infection rates and the corresponding disease burden of malaria in pregnancy. Please see later in the article for the Editors'' Summary     

From Tango to Quadrilla: Current Views of the Immunological Synapse

All T cell functions require establishing contacts with other cells. In the last ten years, the immunological synapse, the contact-site between T cells and their partners, has been the object of numerous investigations and recent advances in imaging technologies have provided significant insights into the mechanism of immunological synapse formation and its functional outcomes. Considering all the available data, the immunological synapse can be defined as a dynamic structure, formed between a T cell and one or more antigen-presenting cells, showing lipid and protein segregation, signaling compartmentalization, and bidirectional information exchange though soluble and membrane-bound transmitters. In this review, we present the current views on the immunological synapse and discuss about some interesting unresolved questions.Key words: T lymphocyte, immunological synapse, signaling, microcluster, TCR, lipid raft, costimulation     

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.     

Under Construction: Building the Macromolecular Superstructure and Signaling Components of an Electrical Synapse

A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36-containing electrical synapses in brain--specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.     

SomatiCA: Identifying,Characterizing and Quantifying Somatic Copy Number Aberrations from Cancer Genome Sequencing Data

Whole genome sequencing of matched tumor-normal sample pairs is becoming routine in cancer research. However, analysis of somatic copy-number changes from sequencing data is still challenging because of insufficient sequencing coverage, unknown tumor sample purity and subclonal heterogeneity. Here we describe a computational framework, named SomatiCA, which explicitly accounts for tumor purity and subclonality in the analysis of somatic copy-number profiles. Taking read depths (RD) and lesser allele frequencies (LAF) as input, SomatiCA will output 1) admixture rate for each tumor sample, 2) somatic allelic copy-number for each genomic segment, 3) fraction of tumor cells with subclonal change in each somatic copy number aberration (SCNA), and 4) a list of substantial genomic aberration events including gain, loss and LOH. SomatiCA is available as a Bioconductor R package at http://www.bioconductor.org/packages/2.13/bioc/html/SomatiCA.html.     

Coordinated Feeding Behavior in Trichoplax,an Animal without Synapses

Trichoplax is a small disk-shaped marine metazoan that adheres to substrates and locomotes by ciliary gliding. Despite having only six cell types and lacking synapses Trichoplax coordinates a complex sequence of behaviors culminating in external digestion of algae. We combine live cell imaging with electron microscopy to show how this is accomplished. When Trichoplax glides over a patch of algae, its cilia stop beating so it ceases moving. A subset of one of the cell types, lipophils, simultaneously secretes granules whose content rapidly lyses algae. This secretion is accurately targeted, as only lipophils located near algae release granules. The animal pauses while the algal content is ingested, and then resumes gliding. Global control of gliding is coordinated with precise local control of lipophil secretion suggesting the presence of mechanisms for cellular communication and integration.     

Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKγ, are major unrecognized components of this synapse type. We demonstrate that MRCKγ can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.     

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.To this end, the standard β-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.     

Synapse scaffolding: intersection of endocytosis and growth

The Drosophila dynamin-associated protein Dap160, homolog of the vertebrate Intersectins, is thought likely to act as a molecular scaffold in the synaptic periactive zone. New mutant analyses have revealed separable roles for Dap160 in the regulation of vesicular endocytosis and synaptic growth.     

A Co-Culture System with an Organotypic Lung Slice and an Immortal Alveolar Macrophage Cell Line to Quantify Silica-Induced Inflammation

There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages) of wild type and P2rx7^−/− mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7^−/− mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7^−/− mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7^−/− mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.