22 research outputs found
Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells
Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion
Evaluation of NMR-based strategies to differentiate fresh from frozen-thawed fish supported by multivariate data analysis
The differentiation of fresh and frozen-thawed fish is a relevant authenticity aspect as in the European Union fish holds a high statistical risk of being adulterated. Here, nuclear magnetic resonance spectroscopy (NMR) in combination with principal components analysis followed by linear discriminant analysis (PCA-LDA) was used for a non-targeted based differentiation of fresh from frozen-thawed fish. To identify the most promising NMR approach(es), six different approaches were applied to 96 fish samples (mackerel, trout, cod). These approaches included different sample preparation procedures and different NMR methods to investigate both the lipid fraction and the polar fraction of the fish samples. After cross-validation embedded in a Monte Carlo resampling design, six independent classification models were obtained. Evaluation of the multivariate data analysis revealed that the most promising approaches were the 1H NMR analysis of the lipid fraction (correct prediction of about 90.0%) and the H NMR based screening of minor components of the lipid fraction with a correct prediction of about 91.9%. H NMR analysis of the water extract of the fish samples showed a correct prediction of about 82.6%. Hence, a general differentiation of fresh from frozen-thawed fish via non-targeted NMR is feasible, even though the underlying sample batch contained different fish species. Additional fish samples need to be analyzed with the three most promising NMR approaches to further improve the developed classification models
Loop-Mediated Isothermal Amplification (LAMP)-Based Method for Rapid Mushroom Species Identification
Toxic mushroom species, such as the death cap (Amanita
phalloides), are responsible for most mushroom poisonings.
In the present work, novel loop-mediated isothermal amplification
(LAMP) assays were used for the differentiation of even closely related
edible and toxic mushroom species. The applicability of these methods
was tested by cross-reaction studies and analysis of spiked mushroom
samples (raw and fried material). Contaminations at the level of 2%
(w/w) could be detected in different mushroom blends. Three detection
methods were used: agarose gel analysis, fluorimetric real-time detection,
and visual detection by lateral flow dipsticks (LFD). The LAMP assay
combined with LFD detection allows the identification of A. phalloides in about 2 h (including DNA extraction)
at a very low level of technical equipment (micropestle, water bath,
and mobile centrifuge), which makes this technique perfectly suited
for on-site applications
Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments
8 páginas, 3 figuras, 1 tablaConventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU lawThis work was funded by the European Union INTERREG Atlantic Area Program (“LabelFish”, project 2011-1/163)Peer reviewe
Disseminated Emergomycosis in a Person with HIV Infection, Uganda
We describe emergomycosis in a patient in Uganda with HIV infection. We tested a formalin-fixed, paraffin-embedded skin biopsy to identify Emergomyces pasteurianus or a closely related pathogen by sequencing broad-range fungal PCR amplicons. Results suggest that emergomycosis is more widespread and genetically diverse than previously documented. PCR on tissue blocks may help clarify emergomycosis epidemiology.Peer Reviewe
Emergomycosis: Case report of a disseminated Emergomyces pasteurianus infection and review of an emerging fungal infection
Real-Time PCR Assays for the Quantitation of rDNA from Apricot and Other Plant Species in Marzipan
Marzipan
or marzipan raw paste is a typical German sweet which
is consumed directly or is used as an ingredient in the bakery industry/confectionery
(e.g., in stollen) and as filling for chocolate candies. Almonds (blanched
and pealed) and sugar are the only ingredients for marzipan production
according to German food guidelines. Especially for the confectionery
industry, the use of persipan, which contains apricot or peach kernels
instead of almonds, is preferred due to its stronger aroma. In most
of the companies, both raw pastes are produced, in most cases on the
same production line, running the risk of an unintended cross contamination.
Additionally, due to high almond market values, dilutions of marzipan
with cheaper seeds may occur. Especially in the case of apricot and
almond, the close relationship of both species is a challenge for
the analysis. DNA based methods for the qualitative detection of apricot,
peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in
marzipan have recently been published. In this study, different quantitation
strategies on the basis of real-time PCR have been evaluated and a
relative quantitation method with a reference amplification product
was shown to give the best results. As the real-time PCR is based
on the high copy rDNA-cluster, even contaminations <1% can be reliably
quantitated
Marzipan: Polymerase Chain Reaction-Driven Methods for Authenticity Control
According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected
Development of a Multiplex Real-Time PCR for Determination of Apricot in Marzipan Using the Plexor System
Marzipan is a confectionary which
is mostly offered in form of
filled chocolate, pralines, or pure. According to the German guidelines
for oil seeds only almonds, sugar and water are admitted ingredients
of marzipan. A product very similar in taste is persipan which is
used in the confectionary industry because of its stronger flavor.
For persipan production almonds are replaced by debittered apricot
or peach kernels. To guarantee high quality products for consumers,
German raw paste producers have agreed a limit of apricot kernels
in marzipan raw paste of 0.5%. Different DNA-based methods for quantitation
of persipan contaminations in marzipan are already published. To increase
the detection specificity compared to published intercalation dye-based
assays, the present work demonstrate the utilization of a multiplex
real-time PCR based on the Plexor technology. Thus, the present work
enables the detection of at least 0.1% apricot DNA in almond DNA or
less. By analyzing DNA mixtures, the theoretical limit of quantification
of the duplex PCR for the quantitation of persipan raw paste DNA in
marzipan raw paste DNA was determined as 0.05%