Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).     

The haemoxisome: a haem-iron containing structure in the Rhodnius prolixus midgut cells

Rhodnius prolixus midgut was analysed using transmission electron microscopy and electron spectroscopic imaging in order to localize the cellular structures involved in haem metabolism. In the posterior midgut, special cellular electron-dense structures were observed. These structures are here designated haemoxisomes. Haemoxisomes are present in the epithelial cells at various time points after a blood meal. Several days after the blood meal, some of them become less electron-dense. By electron spectroscopic imaging, large amounts of iron and oxygen were detected in these cellular structures. The iron is probably bound to the porphyrin ring as an iron-protoporphyrin IX complex, as detected using the diaminobenzidine technique. An interesting observation was the presence of endoplasmic reticulum surrounding the haemoxisomes during some special periods. Iron content was monitored in the posterior midgut epithelium and was found to be constant at the initial days after a blood meal, but slightly higher at the end of the digestive process (from 13th up to 20th day). These results are in agreement with the observation that the appearance of the haemoxisomes changes at the end of the digestive process. The ability to degrade haem seems to depend on the presence of endoplasmic reticulum as observed using a haem degradation assay in the presence of an endoplasmic reticulum-enriched fraction. Taken together these results suggest that haemoxisomes may play a role in intracellular haem detoxification.     

The N-terminal 12 residue long peptide of HIV gp41 is the minimal peptide sufficient to induce significant T-cell-like membrane destabilization in vitro

Here, we predicted the minimal N-terminal fragment of gp41 required to induce significant membrane destabilization using IMPALA. This algorithm is dedicated to predict peptide interaction with a membrane. We based our prediction of the minimal fusion peptide on the tilted peptide theory. This theory proposes that some protein fragments having a peculiar distribution of hydrophobicity adopt a tilted orientation at a hydrophobic/hydrophilic interface. As a result of this orientation, tilted peptides should disrupt the interface. We analysed in silico the membrane-interacting properties of gp41 N-terminal peptides of different length derived from the isolate BRU and from an alignment of 710 HIV strains available on the Los Alamos National Laboratory. Molecular modelling results indicated that the 12 residue long peptide should be the minimal fusion peptide. We then assayed lipid-mixing and leakage of T-cell-like liposomes with N-terminal peptides of different length as first challenge of our predictions. Experimental results confirmed that the 12 residue long peptide is necessary and sufficient to induce membrane destabilization to the same extent as the 23 residue long fusion peptide. In silico analysis of some fusion-incompetent mutants presented in the literature further revealed that they cannot insert into a modelled membrane correctly tilted. According to this work, the tilted peptide model appears to explain at least partly the membrane destabilization properties of HIV fusion peptide.     

Determining ecosystem functioning in Brazilian biomes through foliar carbon and nitrogen concentrations and stable isotope ratios

By analyzing 6,480 tree leaf samples from 57 sites within Brazilian biomes, we considered whether vegetation types in terrestrial ecosystems reflect biogeochemical diversity and whether they fit into a leaf economics spectrum (LES). To achieve this, we investigated the relations among leaf carbon (C) and nitrogen (N) concentrations, their isotope natural abundance and C:N ratio. In addition, we tested their correlations with mean annual temperature (MAT) and precipitation (MAP), as climatic factors. We found consistent differences in the C and N concentrations and their isotopic composition among the vegetation types. MAP is the main climatic driver of changes in N, C:N ratio, δ¹⁵N, and δ¹³C, correlating negatively with N and positively with C:N ratio. These relations show that these biomes follow an LES. The Caatinga had the highest δ¹⁵N values, suggesting that N residence time in soil is longer due to low leaching and plant uptake. We observed that MAP is not the only factor influencing δ¹³C values in different biomes; instead canopy effect probably explains the highest values observed in the Cerrado. Our results reinforce earlier findings that life diversity in the tropics reflects biogeochemistry diversity and leaf δ¹⁵N opens the possibility for investigating plant trade-offs dictated by the LES. Finally, we expect our findings to contribute to a better understanding of the tropics in global climate models.

     

Effects of ezetimibe on markers of synthesis and absorption of cholesterol in high-risk patients with elevated C-reactive protein

AimsHigh-risk subjects with elevated C-reactive protein (CRP) are at high risk for cardiovascular events and frequently require potent statins or combined lipid-lowering therapy to achieve lipid targets and decrease inflammation. Our study aimed at evaluating the effects of three lipid-modifying therapies on LDL-cholesterol, CRP levels and markers of cholesterol absorption and synthesis.Main methodsA prospective intervention study was performed in high cardiovascular risk individuals receiving atorvastatin 10 mg daily for four weeks. Those with CRP ≥ 2.0 mg/L were randomized to another four-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or the combination of atorvastatin 40 mg / ezetimibe 10 mg. Lipids, markers of cholesterol absorption (campesterol and β-sitosterol), and synthesis (desmosterol), as well as CRP were quantified at baseline and end of study.Key findingsOne hundred and twenty two individuals were included. Atorvastatin alone or combined with ezetimibe reduced both LDL-cholesterol and CRP (P < 0.002 vs. baseline; Wilcoxon); ezetimibe did not modify CRP. Ezetimibe-based therapies reduced absorption markers and their ratios to cholesterol (P < 0.0001 vs. baseline, for all; Wilcoxon), whereas atorvastatin alone increased campesterol/cholesterol and β-sitosterol/cholesterol ratios (P < 0.05 vs. baseline; Wilcoxon). In addition, ezetimibe also increased desmosterol and desmosterol/cholesterol ratio (P < 0.0001 vs. baseline; Wilcoxon).SignificanceThese results contribute to understanding the link between cellular cholesterol homeostasis, inflammation and lipid-modifying therapies. Our findings highlight the broader benefit of combined therapy with a potent statin and ezetimibe decreasing inflammation, and preventing increase in cholesterol biosynthesis, an effect not observed with ezetimibe alone.     

Incidence of toxoplasma retinochoroiditis in patients with ankylosing spondylitis after using TNF-α blockers

TNF-α blockers are associated with reactivation of latent granulomatous infections and almost 6% of the world population has some chorioretinitis (CR) caused by Toxoplasma gondii. Thus, the blockade of TNF-α could reactivate a latent toxoplasmosis infection (LTxI). This study was conducted to evaluate the prevalence and incidence of chronic and active CR related to T. gondii in patients with ankylosing spondylitis (AS). A total of 74 eyes from 37 active AS outpatients starting TNFα blockers were compared with 35 AS patients, matched to age and sex, under conventional therapy in a prospective and controlled trial. All patients underwent serological tests for T. gondii, as well as periodic ophthalmologic examination during 12 months. Active CR was defined if a white, focal retinochoroidal lesion with overlying vitreous inflammation had been found. Retinochoroidal lesions with sharp edges, hyperpigmented borders and atrophic center were defined as CR scars. At baseline, no patient had active CR. From the 144 eyes examined, almost 6% had CR scars and only 2.1% had a typical toxoplasmic CR scar and all of them were negative for HLA-B27. During 12 months of follow-up, no recurrence or new CR were observed. AS patients using TNF-α blockers do not have a higher risk of acute or chronic CR caused by T. gondii.     

Monophyletic origin of magnetotaxis and the first magnetosomes

Horizontal gene transfer (HGT), the transfer of genetic material other than by descent, is thought to have played significant roles in the evolution and distribution of genes in prokaryotes. These include those responsible for the ability of motile, aquatic magnetotactic bacteria (MTB) to align and swim along magnetic field lines and the biomineralization of magnetosomes that are responsible for this behaviour. There is some genomic evidence that HGT might be responsible for the distribution of magnetosome genes in different phylogenetic groups of bacteria. For example, in the genomes of a number of MTB, magnetosome genes are present as clusters within a larger structure known as the magnetosome genomic island surrounded by mobile elements such as insertion sequences and transposases as well as tRNA genes. Despite this, there is no strong direct proof of HGT between these organisms. Here we show that a phylogenetic tree based on magnetosome protein amino acid sequences from a number of MTB was congruent with the tree based on the organisms' 16S rRNA gene sequences. This shows that evolution and divergence of these proteins and the 16S rRNA gene occurred similarly. This suggests that magnetotaxis originated monophyletically in the Proteobacteria phylum and implies that the common ancestor of all Proteobacteria was magnetotactic.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

The association of mid-regional pro-adrenomedullin and mid-regional pro-atrial natriuretic peptide with mortality in an incident dialysis cohort

High levels of the plasma peptides mid-regional pro-adrenomedullin (MR-proADM) and mid-regional pro-atrial natriuretic peptide (MR-proANP) are associated with clinical outcomes in the general population. Data in patients with chronic kidney disease are sparse. We therefore investigated the association of MR-proANP and MR-proADM levels with all-cause and cardiovascular (CV) mortality, CV events and peripheral arterial disease in 201 incident dialysis patients of the INVOR-Study prospectively followed for a period of up to more than 7 years. The overall mortality rate was 43%, thereof 43% due to CV events. Both baseline MR-proANP and MR-proADM were associated with higher risk of all-cause (HR = 1.44, p = 0.001 and HR = 1.32, p = 0.002, respectively) and CV mortality (HR = 1.75, p<0.001 and HR = 1.41, p = 0.007, respectively) after adjustment for age, sex, previous CV events, diabetes mellitus and time-dependent type of renal replacement therapy. We then stratified patients in high risk (both peptides in the upper tertile), intermediate risk (only one of the two peptides in the upper tertile) and low risk (none in the upper tertile). Although demographic, clinical and laboratory variables were similar among the intermediate and high risk group, to be with both parameters in the upper tertile was associated with a 3-fold higher risk for all-cause (HR = 2.87, p<0.001) and CV mortality (HR = 3.58, p = 0.001). In summary, among incident dialysis patients MR-proANP and MR-proADM were shown to be associated with all-cause and CV mortality, with the highest risk when both parameters were in the upper tertiles.