A Raman-spectroscopy-based approach for detection and discrimination of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> phages at low titer in raw milk

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~?10⁷ pfu/mL) and have lower titers (10²–10³ pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (10² pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (R²) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).     

Comparative sequence and structure analysis of eIF1A and eIF1AD

Background

Eukaryotic translation initiation factor 1A (eIF1A) is universally conserved in all organisms. It has multiple functions in translation initiation, including assembly of the ribosomal pre-initiation complexes, mRNA binding, scanning, and ribosomal subunit joining. eIF1A binds directly to the small ribosomal subunit, as well as to several other translation initiation factors. The structure of an eIF1A homolog, the eIF1A domain-containing protein (eIF1AD) was recently determined but its biological functions are unknown. Since eIF1AD has a known structure, as well as a homolog, whose structure and functions have been extensively studied, it is a very attractive target for sequence and structure analysis.

Results

Structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A, including a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We identified conserved surfaces and sequence motifs in the folded domain as well as the C-terminal tail of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. We have also identified a set of trypanosomatid-specific surface determinants in eIF1A that could be a promising target for development of treatments against these parasites.

Conclusions

The results described here identify regions in eIF1A and eIF1AD that are likely to play major functional roles and are promising therapeutic targets. Our findings and hypotheses will promote new research and help elucidate the functions of eIF1AD.

     

CITRIC: cold-inducible translational readthrough in the chloroplast of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> using a novel temperature-sensitive transfer RNA

Background

The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnW_UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.

Results

A mutated version of our trnW_UCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.

Conclusions

CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.

     

Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

Parental food provisioning is related to nestling stress response in wild great tit nestlings: implications for the development of personality

Background

Variation in early nutrition is known to play an important role in shaping the behavioural development of individuals. Parental prey selection may have long-lasting behavioural influences. In birds foraging on arthropods, for instance, the specific prey types, e.g. spiders and caterpillars, matter as they have different levels of taurine which may have an effect on personality development. Here we investigated how naturally occurring variation in the amounts of spiders and caterpillars, provisioned to nestlings at day 4 and 8 after hatching, is related to the response to handling stress in a wild passerine, the great tit (Parus major). Broods were cross-fostered in a split-brood design allowing us to separate maternal and genetic effects from early rearing effects. Adult provisioning behaviour was monitored on day four and day eight after hatching using video recordings. Individual nestlings were subjected to a handling stress test at an age of 14 days, which is a validated proxy for exploratory behaviour as an adult.

Results

Variation in handling stress was mainly determined by the rearing environment. We show that, contrary to our predictions, not the amount of spider biomass, but the amount of caterpillar biomass delivered per nestling significantly affected individual performance in the stress test. Chicks provisioned with lower amounts of caterpillars exhibited a stronger stress response, reflecting faster exploratory behaviour later on in life, than individuals who received larger amounts of caterpillars.

Conclusions

These results suggest that natural variation in parental behaviour in wild birds modulates the developmental trajectories of their offspring's personality via food provisioning. Since parental provisioning behaviour might also reflect the local environmental conditions, provisioning behaviour may influence how nestlings respond to these local environmental conditions.

     

Physiological and biochemical mechanisms of the ornamental <Emphasis Type="Italic">Eugenia myrtifolia</Emphasis> L. plants for coping with NaCl stress and recovery

Main Conclusion

We studied the response of Eugenia myrtifolia L. plants, an ornamental shrub native to tropical and subtropical areas, to salt stress in order to facilitate the use of these plants in Mediterranean areas for landscaping. E. myrtifolia plants implement a series of adaptations to acclimate to salinity, including morphological, physiological and biochemical changes. Furthermore, the post-recovery period seems to be detected by Eugenia plants as a new stress situation. Different physiological and biochemical changes in Eugenia myrtifolia L. plants after being subjected to NaCl stress for up to 30 days (Phase I) and after recovery from salinity (Phase II) were studied. Eugenia plants proved to be tolerant to NaCl concentrations between 44 and 88 mM, displaying a series of adaptative mechanisms to cope with salt-stress, including the accumulation of toxic ions in roots. Plants increased their root/shoot ratio and decreased their leaf area, leaf water potential and stomatal conductance in order to limit water loss. In addition, they displayed different strategies to protect the photosynthetic machinery, including the limited accumulation of toxic ions in leaves, increase in chlorophyll content, changes in chlorophyll fluorescence parameters, leaf anatomy and antioxidant defence mechanisms. Anatomical modifications in leaves, including an increase in palisade parenchyma and intercellular spaces and decrease in spongy parenchyma, served to facilitate CO₂ diffusion in a situation of reduced stomatal aperture. Salinity produced oxidative stress in Eugenia plants as evidenced by oxidative stress parameters values and a reduction in APX and ASC levels. Nevertheless, SOD and GSH contents increased. The post-recovery period is detected as a new stress situation, as observed through effects on plant growth and alterations in chlorophyll fluorescence and oxidative stress parameters.

     

Large-scale production of foreign proteins via the novel plant transient expression system in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factor-single chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agro-mediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.     

Survey of <Emphasis Type="Italic">Alternaria</Emphasis> toxin contamination in food from the German market,using a rapid HPLC-MS/MS approach

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA₁ and TA₂. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1 % of all analyzed samples (n?=?96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.