Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

Background  

Since bacteria embedded in biofilms are far more difficult to eradicate than planktonic infections, it would be useful to know whether certain Staphylococcus aureus lineages are especially involved in strong biofilm formation. For this reason, in vitro biofilm formation of 228 clinical S. aureus isolates of distinct clonal lineages was investigated.     

Integrative analysis of epigenetics data identifies gene-specific regulatory elements

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.     

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.     

In vitro degradation behavior of microspheres based on cross-linked dextran

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.     

Biventricular pacing in chronic heart failure acutely facilitates the arterial baroreflex

Metabolic and mechanical stress in the failing heart activates the cardiac sympathetic afferent reflex (CSAR). It has been demonstrated that cardiac resynchronization therapy (CRT) acutely reduces MSNA in clinical responders. Mechanistically, this beneficial effect might be explained by acute deactivation of the CSAR. In addition to sympathoexcitation, CSAR inhibits the arterial baroreflex at the level of the nucleus tractus solitarii. Hence, in responders, CRT is likely to remove/reduce this inhibition. Therefore, we hypothesized that CRT acutely facilitates the arterial baroreflex. One day after implantation of a CRT device in 32 patients with chronic heart failure (LVEF; 27 +/- 6%), we measured noninvasive baroreflex sensitivity (BRS) and heart rate variability (HRV) in two conditions: CRT device switched on and switched off (on/off order randomized). BRS changes were correlated with the difference in unpaced/paced LVEF, a measure of acute mechanical response to CRT. CRT increased BRS by 35% from 2.96 to 3.79 ms/mmHg (P < 0.02) and increased HRV (standard deviation of the intervals between normal beats) from 18.5 to 24.0 ms (P < 0.01). The CRT-induced relative change in BRS correlated with the change in LVEF (r = 0.44; P < 0.01). In conclusion, CRT acutely increases BRS and HRV. This favorable response of the autonomic nervous system might be caused by CRT-induced CSAR deactivation. Follow-up studies should verify the mechanism of the acute response and the possible predictive value of an acute positive BRS response.     

Passive transverse mechanical properties as a function of temperature of rat skeletal muscle in vitro

The objective of the current study was to determine the in vitro passive transverse mechanical properties of skeletal muscle with Dynamic Mechanical Thermal Analysis (DMTA) tests. The starting hypotheses was that the time-temperature-superposition principle could be used to expand the DMTA results to a 1 kHz frequency range. Experiments were performed with rat hind leg skeletal muscle tissue samples on a rotational rheometer using a parallel plate geometry. Because of the small size and low modulus of the samples, the standard test geometry was altered and the samples were shifted from the center to the edge of the plates. From strain sweep tests it became clear that for strains smaller than 0.003 the muscle tissue behaves linearly. In the linear region storage moduli ranged between 24 kPa (omega = 1 rad/s) and 42 kPa (omega = 100 rad/s) at T = 4 degrees C and 22 kPa and 33 kPa at 29 degrees C within the experimental frequency range. The loss modulus decreased with increasing frequency and ranged between 7 and 4 kPa at 4 degrees C and 4.5 and 3.5 kPa at 29 degrees C. Although the properties are clearly temperature dependent, a temperature shift in phase angle delta could not be detected, thus Time Temperature Superposition is not allowed for skeletal muscle in vitro.     

Pyrococcus furiosus 4Fe-ferredoxin, chemisorbed on gold, exhibits gated reduction and ionic strength dependent dimerization

Pyrococcus furiosus ferredoxin is a small metalloprotein that shuttles electrons between redox enzymes. In its native 4Fe-4S form the protein is highly thermostable. In addition to three cluster-ligating cysteines, two surface cysteine residues (C21 and C48) are present. We used the reactivity of these surface thiols to directly immobilize ferredoxin on a bare gold electrode, with an orientation in which the cluster is exposed to solution. Voltammetry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) studies established the immobilization of the 4Fe form. Native and recombinant wild-type ferredoxins were compared with the C48S, C21S, and C21S/C48S mutants. The variants with one and two surface cysteines can be directly chemisorbed on bare gold. Cyclic voltammetry demonstrated that the reduction potentials are similar to those in solution. The interfacial electron transfer kinetics revealed that the reduction is gated by the interconversion between two oxidized species. AFM images showed that dimers are chemisorbed at low ionic strength, while monomers are present at high ionic strength. XPS spectra revealed the presence of S, Fe, C, N, and O at the surface, which are assigned to the corresponding atoms in the peptide and the cofactor. Analysis of the sulfur spectrum corroborates that both C21 and C48 form gold-thiolate bonds. Moreover, two inorganic sulfide and two iron species were identified, suggesting an inhomogeneous charge distribution in the 4Fe-4S cluster. In conclusion, P. furiosus ferredoxin can be directly and vectorially chemisorbed on gold with retention of its properties. This may provide a biocompatible electrode surface with docking sites for redox enzymes.     

Haemostatic defect in non-immune patients with falciparum malaria: no evidence of diffuse intravascular coagulation.

Nine non-immune patients with imported falciparum malaria were examined for signs of diffuse intravascular coagulation (DIC). Although all had thrombocytopenia initially and some later had a decline in plasma fibrinogen concentrations, DIC was never detected, even in severely affected patients with coma and kidney damage. None of the patients were given heparin and all recovered without residual symptoms. Heparin administration should probably be considered only when clear-cut DIC, which possibly never occurs in falciparum malaria, has been demonstrated.     

Versatile polymer microspheres for injection therapy: aspects of fluoroscopic traceability and biofunctionalization

Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles' surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl-2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive space-occupying objects to particles that cross-talk with surrounding tissues.