An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

NITROGEN LIMITATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) LEADS TO INCREASED SUSCEPTIBILITY TO DAMAGE BY ULTRAVIOLET‐B RADIATION BUT ALSO INCREASED REPAIR CAPACITY1

Chl fluorescence was used to measure the photosynthetic capacity of the green alga Dunaliella tertiolecta in order to investigate interactions between susceptibility to acute UV‐B radiation (UVBR, 280–320 nm) exposure and decreased nitrogen availability. Under UVBR exposure the decline in the fluorescence parameters F_v/F_m (the maximum effective quantum yield Φ_PSIIe‐max) and F_v′/F_m′ (the operational quantum yield of PSII, Φ_PSIIe) were enhanced with higher UVBR fluxes, with the data well described by the Kok model, inferring that a dynamic balance existed between damage and repair with the repair proportional to the pool size of inactivated targets. When UVBR exposure was coupled with nitrogen limitation, the inhibition of photosynthesis was intensified. Under the more severely N‐limited conditions, the damage rate increased. Unexpectedly, repair rates were also stimulated under N‐limited conditions, although this was insufficient to counteract the increase in damage, so the overall effect of N limitation was an enhancement of UVBR‐induced inhibition of photosynthesis.     

Dominance of a clonal green sulfur bacterial population in a stratified lake

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA ]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline ( c . 8 × 10⁶ GSB cells mL⁻¹) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA , and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.     

Single‐cell genomics based on Raman sorting reveals novel carotenoid‐containing bacteria in the Red Sea

Cell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.     

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

Phagocytosis of erythrocytes by the proximal tubule of the rat kidney

Summary Morphological examination of kidney biopsies from patients with glomerulonephritis and hematuria has revealed the presence of erythrocytes within epithelial cells of the proximal tubule. This observation suggested that the proximal tubule might be capable of phagocytizing morphologically intact erythrocytes. To examine this possibility small quantities of heparinized autologous blood were injected into surface convolutions of proximal tubules of the rat kidney using standard micropuncture techniques. At time intervals ranging from 10 min to 120 h after injection, the kidneys were preserved for light and transmission electron microscopy by drip-fixation with a half-strength Karnovsky's glutaraldehyde-formaldehyde fixative.During the initial 6 h there was a flattening of the brush border and accumulation of electron-dense material representing hemoglobin in apical vacuoles and in lysosome-like structures. From 6 to 15 h after micropuncture, there was progressive loss of the brush border and the simultaneous formation of pseudopodia-like evaginations that extended from the apical plasma membrane and surrounded the individual erythrocytes. By 18 and 24 h, erythrocytes were observed in the proximal tubule cells. At later time intervals, edema, lymphocytic infiltration, and fibrosis were observed in the interstitium. In addition, crystalline structures were present in the lumen and the cells of both proximal and distal tubules. These findings suggest that in addition to their well-established ability to pinocytize hemoglobin and other proteins, the cells of the proximal tubule are capable of phagocytizing morphologically intact autologous erythrocytes. It is possible that phagocytosis by the proximal tubule cells may play a role in the disposal of erythrocytes from the tubular fluid in hematuric conditions.     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.     

Fish Oil Supplementation Alters the Plasma Lipidomic Profile and Increases Long-Chain PUFAs of Phospholipids and Triglycerides in Healthy Subjects

Background

While beneficial health effects of fish and fish oil consumption are well documented, the incorporation of n-3 polyunsaturated fatty acids in plasma lipid classes is not completely understood. The aim of this study was to investigate the effect of fish oil supplementation on the plasma lipidomic profile in healthy subjects.

Methodology/Principal Findings

In a double-blinded randomized controlled parallel-group study, healthy subjects received capsules containing either 8 g/d of fish oil (FO) (1.6 g/d EPA+DHA) (n = 16) or 8 g/d of high oleic sunflower oil (HOSO) (n = 17) for seven weeks. During the first three weeks of intervention, the subjects completed a fully controlled diet period. BMI and total serum triglycerides, total-, LDL- and HDL-cholesterol were unchanged during the intervention period. Lipidomic analyses were performed using Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (QTOFMS), where 568 lipids were detected and 260 identified. Both t-tests and Multi-Block Partial Least Square Regression (MBPLSR) analysis were performed for analysing differences between the intervention groups. The intervention groups were well separated by the lipidomic data after three weeks of intervention. Several lipid classes such as phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylglycerol, and triglycerides contributed strongly to this separation. Twenty-three lipids were significantly decreased (FDR<0.05) in the FO group after three weeks compared with the HOSO group, whereas fifty-one were increased including selected phospholipids and triglycerides of long-chain polyunsaturated fatty acids. After seven weeks of intervention the two intervention groups showed similar grouping.

Conclusions/Significance

In healthy subjects, fish oil supplementation alters lipid metabolism and increases the proportion of phospholipids and triglycerides containing long-chain polyunsaturated fatty acids. Whether the beneficial effects of fish oil supplementation may be explained by a remodeling of the plasma lipids into phospholipids and triglycerides of long-chain polyunsaturated fatty acids needs to be further investigated.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01034423","term_id":"NCT01034423"}}NCT01034423     

Widely distributed native and alien plant species differ in arbuscular mycorrhizal associations and related functional trait interactions

It is debated whether alien plants in new environments benefit from being mycorrhizal and whether widely distributed natives and aliens differ in their associations with mycorrhizal fungi. Here, we compared whether species differing in their origin status, i.e. natives, archaeophytes (alien species introduced before the year 1500) and neophytes (introduced after the year 1500), and arbuscular mycorrhizal (AM) status (obligate, facultative, non‐mycorrhizal) differ in their area of occupancy in Germany (i.e. number of occupied grid cells, each ~130 km²). We used generalized linear models, incorporating main effects and up to three‐way interactions combining AM status, origin status and plant functional traits. The latter were chosen to describe the possible trade‐off in carbon allocation either towards the symbiosis or to other plant structures, such as storage organs (significant interactions involving traits were assumed to indicate the existence of such trade‐offs). AM status significantly explained the area of occupancy of natives and neophytes – with facultative mycorrhizal species occupying the largest area in both groups – but was less pronounced among archaeophytes. Archaeophytes may have reduced dependency on AM fungi, as they are generally agricultural weeds and the symbiosis potentially becomes obsolete for plants growing in habitats providing a steady provision of nutrients. Trait interactions between AM status and other functional traits were almost exclusively detected for neophytes. While facultative mycorrhizal neophytes benefit from trade‐offs with other traits related to high C cost in terms of area of occupancy, such trade‐offs were almost absent among natives. This indicates that natives and neophytes benefit differently from the symbiosis and suggests that native AM fungal partners might be less important for neophytic than for native plant species or that more time is required to establish similar relationships between neophytes and native fungal symbionts.     

Factors controlling nitrification and denitrification: A laboratory study with gel-stabilized liquid cattle manure

Nitrification and denitrification were studied in a millimeterscale microenvironment using a two-phase system with a liquid manure-saturated layer. Samples consisted of liquid cattle manure and air-dried soil stabilized with silica gel, placed between two aerobic soil phases with a water content near field capacity. A high potential for NH₄ ⁺ oxidation developed within 0–2 mm distance from the interface, and NH₄ ⁺ diffused only 10–20 mm into the soil. Some NH₄ ⁺ was probably immobilized by microorganisms in the soil between 0 and 4 days, after which nitrification was the only sink for NH₄ ⁺. A potential for denitrification developed within the manure-saturated zone. Maximum rates of both potential and actual denitrification were recorded by Day 4, but denitrification continued for at least 2–3 weeks. The potential for nitrification peaked after 14 days. When the pH of the manure was adjusted to 5.5, nitrification was reduced close to the interface, and NH₄ ⁺ penetrated further into the soil before it was oxidized. The pH adjustment had an inhibitory effect on denitrification: Both potential and actual rates of denitrification were almost eliminated for several days. The size of the manure-saturated layer strongly affected denitrification losses. With layers of 8 and 16 mm thickness, losses equivalent to 33 and 40% of the original NH₄ ⁺ pool, respectively, were estimated. When manure corresponding to a 12 mm layer was homogeneously mixed with the soil, only 0.3% was lost.Offprint requests to: S. O. Petersen.