Rapid removal of magnetic particles from large volumes of suspensions

Summary Two simple and rapid procedures for removal of fine magnetic particles from large volumes of suspensions are described. One of them is based on the flow of magnetic suspension through the modified glass pipette placed on a flat magnet, in the second one the magnetic suspension is poured on a plastic film covering the magnet.     

Predicting species distribution and abundance responses to climate change: why it is essential to include biotic interactions across trophic levels

Current predictions on species responses to climate change strongly rely on projecting altered environmental conditions on species distributions. However, it is increasingly acknowledged that climate change also influences species interactions. We review and synthesize literature information on biotic interactions and use it to argue that the abundance of species and the direction of selection during climate change vary depending on how their trophic interactions become disrupted. Plant abundance can be controlled by aboveground and belowground multitrophic level interactions with herbivores, pathogens, symbionts and their enemies. We discuss how these interactions may alter during climate change and the resulting species range shifts. We suggest conceptual analogies between species responses to climate warming and exotic species introduced in new ranges. There are also important differences: the herbivores, pathogens and mutualistic symbionts of range-expanding species and their enemies may co-migrate, and the continuous gene flow under climate warming can make adaptation in the expansion zone of range expanders different from that of cross-continental exotic species. We conclude that under climate change, results of altered species interactions may vary, ranging from species becoming rare to disproportionately abundant. Taking these possibilities into account will provide a new perspective on predicting species distribution under climate change.     

In Vitro Analysis of the Staphylococcus aureus Lipoteichoic Acid Synthase Enzyme Using Fluorescently Labeled Lipids

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane-embedded lipoteichoic acid synthase enzyme, LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as the substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin-layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of the fluorescently labeled PG lipid substrate, in contrast to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds other parts of the lipid substrate, besides the glycerolphosphate head group. Furthermore, eLtaS activity was Mn²⁺ ion dependent; Mg²⁺ and Ca²⁺ supported only weak enzyme activity. Addition of Zn²⁺ or EDTA inhibited enzyme activity even in the presence of Mn²⁺. The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activities of other members of the lipoteichoic acid synthase enzyme family.Lipoteichoic acid (LTA) is a crucial component of the cell wall envelope in Gram-positive bacteria. Diverse functions have been ascribed to LTA, including regulation of the activity of hydrolytic enzymes (4), an essential role in divalent cation homeostasis (2, 26, 37), and retention of noncovalently attached proteins within the cell wall envelope (20, 41). In addition, functions of LTA in host-pathogen interactions have been reported (44). d-Alanine modifications on LTA protect bacteria from killing by cationic antimicrobial peptides (36, 43) and are critical during the infection and colonization processes (1, 5, 10). On the other hand, LTA may also play a positive role for the host in wound healing, by preventing excessive inflammation (25).In the Gram-positive bacterial pathogen Staphylococcus aureus and in many other bacteria belonging to the Firmicutes, including Bacillus, Listeria, Streptococcus, Enterococcus, and Lactococcus spp., LTA is composed of a linear 1,3-linked polyglycerolphosphate backbone chain that is tethered via a glycolipid anchor to the bacterial membrane (6, 9). Recently, the staphylococcal protein LtaS was identified and shown to be responsible for polyglycerolphosphate LTA synthesis in vivo (14). An S. aureus strain depleted of LtaS is unable to synthesize LTA and shows severe growth and morphological defects (14); an S. aureus ltaS deletion strain is viable at 30°C only in a growth medium containing at least 1% NaCl or at higher temperatures at high salt (7.5%) or high sucrose (40%) concentrations (35). Taken together, these findings provide further evidence for the importance of this abundant cell envelope component for normal cell morphology and physiology.Pulse-chase experiments have provided strong biochemical evidence that the glycerolphosphate subunits of LTA are derived from the head group of the membrane lipid phosphatidylglycerol (PG) (7, 8, 12). A rapid and almost complete turnover of the nonacylated glycerolphosphate group of PG into LTA is observed in S. aureus and other Gram-positive bacteria that synthesize polyglycerolphosphate LTA (23, 24). It is assumed that the LtaS enzyme cleaves the head group of PG and uses this glycerolphosphate subunit to polymerize the LTA backbone chain.One or more LtaS-like enzymes are encoded in the genomes of Gram-positive bacteria that synthesize polyglycerolphosphate LTA (14). S. aureus LtaS and all other members of this enzyme family are predicted to contain five N-terminal transmembrane helices followed by an extracellular C-terminal enzymatic domain (eLtaS) (14, 29). The LtaS enzyme is processed in S. aureus, and the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall envelope (11, 29, 45). The crystal structure of the S. aureus eLtaS domain, alone and in a complex with soluble glycerolphosphate and the soluble domain of the Bacillus subtilis LtaS (LtaS_Bs) enzyme (YflE), identified a threonine as the catalytic residue. This is based on the location of the glycerolphosphate head group in the active site for S. aureus LtaS and on threonine phosphorylation in the B. subtilis enzyme structure (29, 37). Replacement of this threonine residue with an alanine renders the S. aureus enzyme inactive and unable to synthesize LTA in vivo (29). In addition, a Mn²⁺ ion was detected in the active center of the S. aureus LtaS structure, while the B. subtilis enzyme contained a Mg²⁺ ion.To provide insight into the enzymatic activity of the S. aureus lipoteichoic acid synthase enzyme, we developed an in vitro assay for this enzyme using purified recombinant eLtaS and fluorescently labeled PG as a substrate. Using thin-layer chromatography (TLC) and mass spectrometry analysis of the lipid reaction products, we show that eLtaS protein is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol (NBD-DAG). Furthermore, we provide experimental evidence that LtaS requires Mn²⁺ for enzyme activity, while Zn²⁺ inhibits enzyme function. Our results suggest that LtaS has a narrow substrate specificity, with PG serving as a substrate while phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS) do not. Lastly, we show that this in vitro assay can be used to study the enzyme functions of other members of this protein family, such as the Listeria monocytogenes LTA synthase (LtaS_Lm) and LTA primase (LtaP_Lm) enzymes. This study is the first in vitro characterization of lipoteichoic acid synthase enzymes and an important first step towards the development of an assay to screen and identify enzyme-specific inhibitors for this new and important class of bacterial enzymes.     

Estimation of fungal biomass in forest litter and soil

The contents of fungal biomass markers were analysed in the fruit bodies of dominant basidiomycetes from an ectomycorrhiza-dominated coniferous forest, and used to estimate the fungal biomass content in the litter and soil. The content of ergosterol (3.8 ± 2.0 mg g^?1 dry fungal biomass) and the phospholipid fatty acid 18:2ω6,9 (11.6 ± 4.3 mg g^?1) showed less variation than the internal transcribed spacer (ITS) copy numbers (375 ± 294 × 10⁹ copies g^?1). A high level of variation in the ITS copy numbers (per ng DNA) was also found among fungal taxa. The content of fungal biomass in the litter and soil, calculated using the mean contents, varied between 0.66 and 6.24 mg g^?1 fungal biomass in the litter, and 0.22 and 0.68 mg g^?1 in the soil. The ratio of fungal biomass in the litter to that in the soil varied greatly among the markers. The estimates of fungal biomass obtained with different biomarkers are not exactly comparable, and caution should be used when analysing taxon abundance using PCR amplification of fungal rDNA.     

Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase

Lipoteichoic acid (LTA) is a crucial cell envelope component in Gram-positive bacteria. In Staphylococcus aureus, the polyglycerolphosphate LTA molecule is synthesized by LtaS, a membrane-embedded enzyme with five N-terminal transmembrane helices (5TM domain) that are connected via a linker region to the C-terminal extracellular enzymatic domain (eLtaS). The LtaS enzyme is processed during bacterial growth, and the eLtaS domain is released from the bacterial membrane. Here we provide experimental evidence that the proteolytic cleavage following residues 215Ala-Leu-Ala217 is performed by the essential S. aureus signal peptidase SpsB, as depletion of spsB results in reduced LtaS processing. In addition, the introduction of a proline residue at the +1 position with respect to the cleavage site, a substitution known to inhibit signal peptidase-dependent cleavage, abolished LtaS processing at this site. It was further shown that the 5TM domain is crucial for enzyme function. The observation that the construction of hybrid proteins between two functional LtaS-type enzymes resulted in the production of proteins unable to synthesize LTA suggests that specific interactions between the 5TM and eLtaS domains are required for function. No enzyme activity was detected upon expression of the 5TM and eLtaS domains as separate fragments, indicating that the two domains cannot assemble postsynthesis to form a functional enzyme. Taken together, our data suggest that only the full-length LtaS enzyme is active in the LTA synthesis pathway and that the proteolytic cleavage step is used as a mechanism to irreversibly inactivate the enzyme.     

Cytoplasmic domain of NCAM140 interacts with ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1)

The neural cell adhesion molecule NCAM is implicated in different neurodevelopmental processes and in synaptic plasticity in adult brain. The cytoplasmic domain of NCAM interacts with several cytoskeletal proteins and signaling molecules. To identify novel interaction partners of the cytosolic domain of NCAM a protein macroarray has been performed. We identified the ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1) as an interaction partner of NCAM140. Ufc1 is one of the enzymes involved in modification of proteins with the ubiquitin-like molecule ubiquitin-fold modifier-1 (Ufm1). We also observed a partial co-localization of NCAM140 with Ufc1 and Ufm1 and increased endocytosis of NCAM140 in the presence of Ufm1 suggesting a possible ufmylation of NCAM140 and a potential novel function of Ufm1 for cell surface proteins.     

Novel chemistry of invasive plants: exotic species have more unique metabolomic profiles than native congeners

It is often assumed that exotic plants can become invasive when they possess novel secondary chemistry compared with native plants in the introduced range. Using untargeted metabolomic fingerprinting, we compared a broad range of metabolites of six successful exotic plant species and their native congeners of the family Asteraceae. Our results showed that plant chemistry is highly species‐specific and diverse among both exotic and native species. Nonetheless, the exotic species had on average a higher total number of metabolites and more species‐unique metabolites compared with their native congeners. Herbivory led to an overall increase in metabolites in all plant species. Generalist herbivore performance was lower on most of the exotic species compared with the native species. We conclude that high chemical diversity and large phytochemical uniqueness of the exotic species could be indicative of biological invasion potential.     

Influence of knee angle and individual flexibility on the flexion-relaxation response of the low back musculature.

In many occupational settings (e.g. agriculture and construction) workers are asked to maintain static flexed postures of the low back for extended periods of time. Recent research indicates that the resulting strain in the viscoelastic, ligamentous tissues may have a deleterious effect on the stability of the spine and the normal reflex response of spinal tissues. The purpose of this study was to evaluate the previously described flexion-relaxation response in terms of the interactive effect of trunk flexion angle (30 degrees, 50 degrees, 70 degrees, 90 degrees ), knee flexion angle (0 degrees (straight knees), 20 degrees, 40 degrees ) and individual flexibiliteky (low, medium, and high). These conditions were tested under two levels of loading: no load (just supporting the weight of the torso) and trunk extension moment equal to 50% of the subject's posture-specific maximum voluntary trunk extension capacity. Surface electromyographic (EMG) data were collected from the multifidus, the longissimus, the iliocostalis, the vastus medialis, the rectus femoris, the vastus lateralis, the biceps femoris, and the gastrocnemius-soleus group from a sample of eight male participants as they performed isometric weight holding tasks in the postures defined by the combinations of trunk angle and knee angle. The results of this study showed that knee angle did have a significant effect on the lumbar extensor muscle activity but only consistently at the 90 degrees trunk angle. Participant flexibility showed a consistent trend of decreasing lumbar extensor muscle activity with decreased flexibility across all trunk angle values. Most interesting was the interactive response of flexibility and knee angle, wherein the flexibility of the participant influenced the trunk angles at which the knee flexion angle affected the flexion-relaxation response. Highly flexible subjects showed an effect of knee angle on the flexion-relaxation response only at the 90 degrees trunk angle; subjects in the medium flexibility category showed a similar response in both the 70 degrees and 90 degrees trunk angles; subject in the low flexibility group showed no knee angle effect on the flexion-relaxation response. Overall the results confirm previous results with regard to the contribution of the passive tissues to the overall trunk extension moment but also show that the tension in the bi-articular biceps femoris, which was influenced by knee flexion angle and flexibility, affects the ratio of active extensor moment contributions of the lumbar extensor musculature to passive extensor moment contributions from the muscular and ligamentous tissues. The results of this study provide empirical data describing this complicated, interactive response.