Cellular components of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: immunohistochemical (MHC class II) and electron-microscopic observations

This report deals with the distribution, morphology and specific topical relationships of bone-marrow-derived cells (free cells) in the spinal meninges and dorsal root ganglia of the normal rat. The morphology of these cells has been studied by transmission and scanning electron microscopy. Cells expressing the major histocompatibility complex (MHC) class II gene product have been recognized by immunofluorescence. At the level of the transmission electron microscope, free cells are found in all layers of the meninges. Many of them display characteristic ultrastructural features of macrophages, whereas others show a highly vacuolated cytoplasm and are endowed with many processes. These elements lack a conspicuous lysosomal system and might represent dendritic cells. Scanning electron microscopy has revealed that free cells contact the cerebrospinal fluid via abundant cytoplasmic processes that cross the cell layers of the pia mater and of the arachnoid. Cells expressing the MHC class II antigen are also found in all layers of the meninges. They are particularly abundant in the layers immediately adjacent to the subarachnoid space, in the neighbourhood of dural vessels, along the spinal roots and in the dural funnels. In addition to the meninges, strong immunoreactivity for MHC class II antigen is observed in the dorsal root ganglia. The ultrastructural and immunohistochemical findings of this study suggest the existence of a well-developed system of immunological surveillance of the subarachnoid space and of the dorsal root ganglia.     

Is there hybridization between diploid and tetraploid Euphrasia in a secondary contact zone?

Premise

Strong postzygotic reproductive isolating barriers are usually expected to limit the extent of natural hybridization between species with contrasting ploidy. However, genomic sequencing has revealed previously overlooked examples of natural cross-ploidy hybridization in some flowering plant genera, suggesting that the phenomenon may be more common than once thought. We investigated potential cross-ploidy hybridization in British eyebrights (Euphrasia, Orobanchaceae), a group from which 13 putative cross-ploidy hybrid combinations have been reported based on morphology.

Methods

We analyzed a contact zone between diploid Euphrasia rostkoviana and tetraploid E. arctica in Wales. We sequenced part of the internal transcribed spacer (ITS) of nuclear ribosomal DNA and used genotyping by sequencing (GBS) to look for evidence of cross-ploidy hybridization and introgression.

Results

Common variant sites in the ITS region were fixed between diploids and tetraploids, indicating a strong barrier to hybridization. Clustering analyses of 356 single-nucleotide polymorphisms (SNPs) generated using GBS clearly separated samples by ploidy and revealed strong genetic structure (F_ST = 0.44). However, the F_ST distribution across all SNPs was bimodal, indicating potential differential selection on loci between diploids and tetraploids. Demographic inference suggested potential gene flow, limited to around one or fewer migrants per generation.

Conclusions

Our results suggest that recent cross-ploidy hybridization is rare or absent in a site of secondary contact in Euphrasia. While a strong ploidy barrier prevents hybridization over ecological timescales, such hybrids may form in stable populations over evolutionary timescales, potentially allowing cross-ploidy introgression to take place.     

Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.     

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Computer simulation of antibody binding specificity

A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives⁸) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form. © 1993 Wiley-Liss, Inc.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.