Quantitative proteomic analysis of sokotrasterol sulfate-stimulated primary human endothelial cells

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Average cell viability levels of human dental pulp stem cells: an accurate combinatorial index for quality control in tissue engineering

Background aimsOne of the most important issues in tissue engineering (TE) is the search for a suitable stem cell reservoir with optimal cell viability levels for the development of new tissues relevant for therapeutic needs. The aim of this study was to evaluate the cell viability levels of 10 sequential cell passages of human dental pulp stem cells (hDPSC) to determine their potential for TE techniques.MethodsTo assess the average cell viability levels of hDPSC, four cell viability assays were used in a combinatorial approach: trypan blue exclusion test, water-soluble tetrazolium 1 assay, live/dead assay and electron probe x-ray microanalysis.ResultsThe results showed that cell viability as determined by trypan blue staining and live/dead assays was greater than 85%, with a significant decrease at the second passage (P < 0.05) and a significant increase at the ninth passage (P < 0.05). Electron probe x-ray microanalysis showed that the highest cell viability corresponded to the ninth passage, with the lowest K/Na values found at the third passage. No statistical differences were found among the different passages for the water-soluble tetrazolium 1 assay (P = 0.219).ConclusionsAssessment of average cell viability levels showed that the highest viability of hDPSC was reached after nine passages, suggesting that this passage would be the most adequate for use in TE protocols.     

Response of cylindrospermopsin production and release in Aphanizomenon flos-aquae (Cyanobacteria) to varying light and temperature conditions

The influence of light and temperature on the cylindrospermopsin (CYN) production of two Aphanizomenon flos-aquae strains, isolated from North-eastern German lakes, was investigated with semi-continuously growing cultures. A light gradient from 10 to 60 μE m⁻² s⁻¹ in combination with temperatures of 16, 20, and 25 °C was tested.CYN concentrations varied by a maximum factor of 2.7 in strain 10E9 with a significant decrease with increasing temperature. Strain 22D11 showed less pronounced changes, i.e. by a factor of 1.6, and without clear relationship to temperature.Reaction patterns of CYN production to changing light intensities are different at different temperatures. In both strains CYN concentrations increase significantly at 20 °C between 10 and 60 μE m⁻² s⁻¹, whereas they decrease significantly at 25 °C in the same light gradient. The amount of synthesised CYN is not reflected by growth rates of the strains in a uniform manner. Nonetheless several temperature–light combinations which constitute physiological stress seem to trigger CYN production and particularly CYN release from cells. The lowest growth rate observed at 16 °C and 60 μE m⁻² s⁻¹ of strain 22D11 may reflect photoinhibition due to the lower temperature and related limited CO₂-fixation. Under these conditions, extracellular CYN concentrations increased to 58% of total CYN, while the share of extracellular CYN of all other light and temperature regimes was 11–26%. From the results and the experimental design we conclude an active release of the toxin into medium to be more likely than mere leakage from cells.     

Therapeutic Hypothermia Modifies Perinatal Asphyxia-Induced Changes of the Corpus Callosum and Outcome in Neonates

What Is Known about this Subject?

Diffusion-weighted MRI has demonstrated changes in the corpus callosum of term neonates with perinatal asphyxia. The severity of cerebral changes demonstrated using diffusion-weighted MRI is difficult to assess without measuring values of the Apparent Diffusion Coefficient (ADC).

What Is New?

ADC values of the anterior part of the corpus callosum are slightly higher than of the posterior part in full term infants with perinatal asphyxia. Low ADC values of the corpus callosum were associated with an adverse outcome in infants with perinatal asphyxia. In infants treated with hypothermia lower ADC values than with normothermia were associated with a poor outcome, supporting neuroprotective effects of hypothermia

Background

Using MRI, changes can be detected in the corpus callosum (CC) following perinatal asphyxia which are associated with later neurodevelopmental outcome.

Aim

To study the association between the apparent diffusion coefficient of water (ADC) in the CC on MRI in neonates with perinatal asphyxia and neurodevelopmental outcome at 18 months of age.

Subjects, Methods

Of 121 infants 32 (26%) died and 13 (11%) survived with an adverse neurological outcome. Sixty-five (54%) received therapeutic hypothermia. MRI was performed within 7 days after birth using a 1.5 T or 3.0 T system, and ADC values were measured in the anterior and posterior CC. The association between ADC and composite outcome (death or abnormal neurodevelopment) was analyzed for both normothermia and hypothermia cases using receiver operating characteristics.

Results

ADC values of the posterior CC were lower than of the anterior part (mean difference 0.050 x 10^-3 mm²/s, p<0.001). Field strength did not affect ADC values. ADC values of the posterior part of the CC were significantly lower in infants with basal ganglia/thalamus or near total brain injury (p<0.001). Lower ADC values were associated with an adverse outcome, but cut-off levels were lower after hypothermia (1.024 x 10^-3 mm²/s vs 0.969 x 10^-3 mm²/s)

Conclusion

Low ADC values of the posterior part of the corpus callosum are associated with an adverse outcome in term or near term neonates with perinatal asphyxia. Therapeutic hypothermia slightly modifies this association, showing that lower values were needed for an adverse outcome.     

Periconceptional health and lifestyle factors of both parents affect the risk of live-born children with orofacial clefts

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (CL/P) or cleft palate only (CPO) are orofacial clefts and have a multifactorial etiology. The identification of amendable parental risk factors may contribute to a reduced occurrence of these malformations in the future. METHODS: Standardized demographic and periconceptional exposure data from 284 parents of a child with CL/P, 66 parents of a child with a CPO and 222 parents of a child without congenital malformations were collected at approximately 24 months after the periconceptional period of the index child. Univariate and multivariate logistic regression analyses were used to estimate relative risks by odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: Univariate results suggest that low parental education, periconceptional maternal medication use and illnesses, paternal smoking, and first-trimester maternal common cold increased CL/P risk. Pregnancy planning and periconceptional folic acid supplementation, however, reduced CL/P risk by approximately 50% (OR, 0.5; 95% CI, 0.3-0.8) and 40% (OR, 0.6; 95% CI, 0.4-0.9), respectively. Mostly comparable results were obtained for CPO. Being a boy (OR, 2.0; 95% CI, 1.4-3.0), folic acid supplementation (OR, 0.6; 95% CI, 0.4-0.9), and low paternal education (OR, 1.6; 95% CI, 1.0-2.3) mainly determined CL/P in the multivariate analyses, compared to low paternal (OR, 4.5; 95% CI, 2.1-9.4) and maternal medication use (OR, 2.0; 95% CI, 1.0-4.0) for CPO. CONCLUSIONS: Preconceptional counseling for orofacial cleft risk assessment should pay attention to maternal medication use, periconceptional folic acid supplementation, and exposures of the father. These determinants can be amended, thereby modifying orofacial cleft risk.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

The F gene of rodent brain-adapted mumps virus is a major determinant of neurovirulence

Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuV(KH)) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuV(JL5)) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.