Application of the imidate procedure to α-d-galactosyltion

Using the imidate procedure, 2,3,4,6-tetra-O-benzyl-1-O-(N-methylacetimidoyl)-β-d-galactopyranose was condensed with various monosaccharides to provide, in good yield and with high stereoselectivity, α-linked disaccharides.     

Using Stable Isotopes to Differentiate Trophic Feeding Channels within Soil Food Webs

The soil is probably the most diverse habitat there is, with organisms ranging in sizes from less than 1 μm to several metres in length. However, it is increasingly evident that we know little about the interactions occurring between these organisms, the functions that they perform as individual species, or together within their different feeding guilds. These interactions between groups of organisms and physical and chemical processes shape the soil as a habitat and influence the nature of the soil food web with consequences for the above‐ground vegetation and food web. Protists are known as one of the most abundant groups of bacterivores within the soil; however, they are also consumers of a number of other food sources. Even though they are responsible for a large proportion of the mineralisation of bacterial biomass and have a large impact on the C and N cycles within the soil they are regularly overlooked when investigating the complete soil food web. Recently, stable isotopes have been used to determine trophic interactions and here we describe how this technique has been used to highlight linkages between protists and the soil food web.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Effects of Stress Modifier Biostimulants on Vegetative Growth,Nutrients, and Antioxidants Contents of Garden Thyme (Thymus vulgaris L.) Under Water Deficit Conditions

The aim of this study is to investigate the effect of stress modulators on vegetative growth, antioxidants, and nutrient content of Thymus vulgaris L. under water deficit stress conditions. A factorial experiment was performed in the form of a randomized complete block design with 10 treatments and 3 replications in the 2019–2020 growing season. The factors were stress modulators at 5 levels (ZN: zinc nano-fertilizer, AA: amino acid, SW: seaweed, HA: humic acid and C: control) and irrigation regime at 2 levels [FIrr: full irrigation (100% field capacity) and DIrr: deficit irrigation (50% field capacity)]. The highest plant height, number of branches, and total dry weight of the garden thyme plant were observed in the foliar application of HA and SW under full irrigation conditions. Relative water content, chlorophyll a and b, and uptake of nutrients (N, P, and K) were reduced under water deficit stress, but the foliar application of stress modulators increased relative water content, chlorophyll content, and nutrient uptake of the garden thyme plant significantly compared with control. The water deficit increased proline content, total flavonoid, and phenol content in the garden thyme plant. So, the highest total flavonoid and phenol content was obtained from plants treated with HA, whereas proline content was higher in the control plants. Soluble sugars and essential oil increased significantly under water deficit stress conditions. The foliar application of HA compared to the control plant increased soluble sugars and essential oil in garden thymes. The activities of catalase, superoxide dismutase, and ascorbate peroxidase enzymes were improved in stress modulator treatments such as HA and SW compared to control plants under water deficit stress conditions. The plants of garden thymes showed a good response to stress modulator treatments under water stress conditions, and HA and SW treatments were found to be more effective.

     

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.     

Analysis of the Binding Moiety Mediating the Interaction between Monocarboxylate Transporters and Carbonic Anhydrase II

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster ⁴³¹EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster ⁴⁸⁹EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.