Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

The Fascinating Coat Surrounding Mycobacteria

The mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane.

OMA and OPA—Software-Supported Mass Spectra Analysis of Native and Modified Nucleic Acids

The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated doublestranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Hylan versus hyaluronic acid for osteoarthritis of the knee: a systematic review and meta-analysis

OBJECTIVE: To compare the effectiveness and safety of intraarticular high-molecular hylan with standard preparations of hyaluronic acids in osteoarthritis of the knee. METHODS: We performed a systematic review and meta-analysis of randomized controlled trials comparing hylan with a hyaluronic acid in patients with knee osteoarthritis. Trials were identified by systematic searches of Central, Medline, EMBase, Cinahl, the Food and Drug Administration, and Science Citation Index supplemented by hand searches of conference proceedings and reference lists (last update November 2006). Literature screening and data extraction were performed in duplicate. Effect sizes were calculated from differences in means of pain-related outcomes between treatment and control groups at the end of the trial, divided by the pooled standard deviation. Trials were combined using random-effects meta-analysis. RESULTS: Thirteen trials with a pooled total of 2,085 patients contributed to the meta-analysis. The pooled effect size was -0.27 (95% confidence interval [95% CI] -0.55, 0.01), favoring hylan, but between-trial heterogeneity was high (I(2) = 88%). Trials with blinded patients, adequate concealment of allocation, and an intent-to-treat analysis had pooled effect sizes near null. The meta-analyses on safety revealed an increased risk associated with hylan for any local adverse events (relative risk [RR] 1.91; 95% CI 1.04, 3.49; I(2) = 28%) and for flares (RR 2.04; 95% CI 1.18, 3.53; I(2) = 0%). CONCLUSION: Given the likely lack of a superior effectiveness of hylan over hyaluronic acids and the increased risk of local adverse events associated with hylan, we discourage the use of intraarticular hylan in patients with knee osteoarthritis in clinical research or practice.

Omega-3 poly-unsaturated fatty acids for the prevention of severe neutropenic enterocolitis in patients with acute myeloid leukemia

Neutropenic enterocolitis is a potentially fatal complication of myeloablative chemotherapy in patients with acute myeloid leukemia. Omega-3 polyunsaturated fatty acids (PUFA) are precursors of potent anti-inflammatory prostaglandins. Our aim was to explore the safety and effectiveness of omega-3 PUFA added to parenteral nutrition in protecting leukemia patients from severe enterocolitis. Fourteen patients with acute myeloid leukemia who received omega-3 PUFA in a Phase II trial were compared with 66 consecutive control patients not getting this intervention. We performed crude and adjusted comparisons, using inverse probability of treatment weighting for adjusted analysis, and blind outcome assessment to minimize assessor bias. Primary outcome was severe enterocolitis (≥Grade 3). The crude odds ratio of Grade 3 colitis or higher was 1.36 (95% CI 0.37 to 4.96, P = 0.64), and the adjusted odds ratio was 0.79 (95% CI 0.35 to 1.78, P = 0.57). There was little evidence to suggest differences between groups in serious adverse events and overall mortality. Our results provide little evidence that addition of omega-3 PUFA is beneficial in this condition. Routine treatment with omega-3 PUFA is currently not warranted.

Fish or n3-PUFA intake and body composition: a systematic review and meta-analysis.

Obesity is a major public health issue and an important contributor to the global burden of chronic disease and disability. Studies indicate that fish and omega-3 polyunsaturated fatty acids (n3-PUFA) supplements may help prevent cardiovascular and metabolic diseases. However, the effect of fish oil on body composition is still uncertain, so we performed a systematic review of randomized controlled trials and the first meta-analysis on the association between fish or fish oil intake and body composition measures. We found evidence that participants taking fish or fish oil lost 0.59 kg more body weight than controls (95% confidence interval [CI]: -0.96 to -0.21). Treatment groups lost 0.24 kg m(-2) (body mass index) more than controls (-0.40 to -0.08), and 0.49 % more body fat than controls (-0.97 to -0.01). Fish or fish oil reduced waist circumference by 0.81 cm (-1.34 to -0.28) compared with control. There was no difference for fat mass and lean body mass. Further research is needed to confirm or refute our findings and to reveal possible mechanisms by which n3-PUFAs might reduce weight.

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

Milk fatty acids as possible biomarkers to early diagnose elevated concentrations of blood plasma nonesterified fatty acids in dairy cows

Most cows encounter a state of negative energy balance during the periparturient period, which may lead to metabolic disorders and impaired fertility. The aim of this study was to assess the potential of milk fatty acids as diagnostic tools of detrimental levels of blood plasma nonesterified fatty acids (NEFA), defined as NEFA concentrations beyond 0.6 mmol/L, in a data set of 92 early lactating cows fed a glucogenic or lipogenic diet and subjected to 0-, 30-, or 60-d dry period before parturition. Milk was collected in wk 2, 3, 4, and 8 (n = 368) and blood was sampled weekly from wk 2 to 8 after parturition. Milk was analyzed for milk fatty acids and blood plasma for NEFA. Data were classified as "at risk of detrimental blood plasma NEFA" (NEFA ≥ 0.6 mmol/L) and "not at risk of detrimental blood plasma NEFA" (NEFA <0.6 mmol/L). Concentrations of 45 milk fatty acids and milk fat C18:1 cis-9-to-C15:0 ratio were subjected to a discriminant analysis. Milk fat C18:1 cis-9 revealed the most discriminating variable to identify detrimental blood plasma NEFA. A false positive rate of 10% allowed us to diagnose 46% of the detrimental blood plasma NEFA cases based on a milk fat C18:1 cis-9 concentration of at least 230 g/kg of milk fatty acids. Additionally, it was assessed whether the milk fat C18:1 cis-9 concentrations of wk 2 could be used as an early warning for detrimental blood plasma NEFA risk during the first 8 wk in lactation. Cows with at least 240 g/kg of C18:1 cis-9 in milk fat had about 50% chance to encounter blood plasma NEFA values of 0.6 mmol/L or more during the first 8 wk of lactation, with a false positive rate of 11.4%. Profit simulations were based on costs for cows suffering from detrimental blood plasma NEFA, and costs for preventive treatment based on daily dosing of propylene glycol for 3 wk. Given the relatively low incidence rate (8% of all observations), continuous monitoring of milk fatty acids during the first 8 wk of lactation to diagnose detrimental blood plasma NEFA does not seem cost effective. On the contrary, milk fat C18:1 cis-9 of the second lactation week could be an early warning of cows at risk of detrimental blood NEFA. In this case, selective treatment may be cost effective.

Porcine cathelicidins efficiently complex and deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α responses.

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine-rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide-DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid-complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.