Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.     

Electrocardiographic monitoring in the Göttingen minipig

The purpose of the study reported here was to determine conditions for electrocardiographic monitoring in the G?ttingen minipig in view of its use as a second non-rodent species in toxicology studies. Electrocardiograms were recorded from conscious minipigs (6/sex) maintained in a sling. The three standard bipolar limb leads (I, II, III), the three augmented unipolar limb leads (aVR, aVL, aVF), the triangular Nehb-Sp?ri leads (dorsal, axial, ventral) and their corresponding unipolar leads were recorded, and automated analysis of amplitudes and intervals was made. Major QRS patterns were not observed for any of the bipolar and unipolar leads. For triangular leads, the amplitude of waves was higher than that for limb leads, and the rS pattern dominated for dorsal, axial ventral and aV(F)-Ventral leads. The qR pattern dominated in the aV(R)-dorsal lead, whereas consistency and dominant patterns were not observed for the aV(L)-axial lead. For limb leads, the position of the electrode affected the ECG. Electrodes placed on the cubital and stifle joints were the preferred positions since the P- and R-waves were clearly identifiable with amplitudes > 0.2 mV. Also, the T-wave amplitude was (positive or negative) > 0.2 mV in at least two leads, making the determination of the QT-interval accurate. For the triangular leads, the position of the electrode had less influence on the amplitude of deflections. However, if the axial lead is to be used for calculation of intervals and amplitudes, the xyphoid process is the preferred position. In conclusion, the triangular lead system is recommended for recording ECGs in minipigs. Limb leads could be used in connection. The cubital and stifle joints for standard limb leads and the neck, sacrum, and xyphoid process for triangular leads are the preferred positions for electrodes.     

Antipredator responses to skin extract of redbelly dace, <Emphasis Type="Italic">Phoxinus eos, </Emphasis>by free-ranging populations of redbelly dace and fathead minnows,<Emphasis Type="Italic">Pimephales promelas</Emphasis>

Synopsis Fishes in the superorder Ostariophysi possess specialized epidermal cells that contain a chemical alarm cue. The alarm cue is released when the skin is damaged during a predatory attack. Therefore, the cue serves as a reliable indicator of predation risk to nearby conspecifics and ecologically similar heterospecifics with which it shares predators. Antipredator behavior in response to these alarm cues has been demonstrated in numerous studies in confined spaces (laboratory aquaria, field traps, fluvarium). When tested on a natural field population however, behavioral response has been inconsistent. Here, we expose free-ranging redbelly dace and fathead minnows to skin extract of redbelly dace and record their behavioral response with an underwater video camera. We observed avoidance of areas in which skin extract was introduced, but no avoidance of areas in which water (control) was introduced. These data confirm the ecological function of skin extract in mediating predator–prey interactions in aquatic habitats, and argue against the hypothesis that alarm reactions are an artifact of confined spaces.     

A Discrete Event Simulation Model for Evaluating the Performances of an M/G/C/C State Dependent Queuing System

M/G/C/C state dependent queuing networks consider service rates as a function of the number of residing entities (e.g., pedestrians, vehicles, and products). However, modeling such dynamic rates is not supported in modern Discrete Simulation System (DES) software. We designed an approach to cater this limitation and used it to construct the M/G/C/C state-dependent queuing model in Arena software. Using the model, we have evaluated and analyzed the impacts of various arrival rates to the throughput, the blocking probability, the expected service time and the expected number of entities in a complex network topology. Results indicated that there is a range of arrival rates for each network where the simulation results fluctuate drastically across replications and this causes the simulation results and analytical results exhibit discrepancies. Detail results that show how tally the simulation results and the analytical results in both abstract and graphical forms and some scientific justifications for these have been documented and discussed.     

Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.     

Polymorphisms of stress-related genes and the risk of nonsyndromic cleft lip with or without cleft palate

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NCL/P) is a common structural malformation with a complex and multifactorial etiology. It has been shown that maternal psychological stress in the periconceptional period can contribute to an increase in the risk of NCL/P affecting pregnancy. METHODS: Twenty‐four single nucleotide polymorphisms of 11 stress‐related genes (COMT, CRHR1, FKBP5, GABRA6, HSD11β2, MAOA, NPY, NR3C1, SERPINA6, SLC6A4, and TPH2) were investigated in 220 healthy mothers of children with facial clefts and 210 matched controls using restriction fragment‐length polymorphism and high‐resolution melting analysis. RESULTS: We found that polymorphisms in SLC6A4, TPH2, and SERPINA6 appear to be maternal factors increasing the risk of having a child with facial clefts. The closest correlations with NCL/P were found for the SLC6A4 rs2020942 and TPH2 rs10879357 gene variants (odds ratio [OR], 1.720; 95% confidence interval [CI], 1.158–2.553; p = 0.0069; p_trend = 0.0036; and OR, 1.837; 95% CI, 1.226–2.753, p = 0.0030, p_trend = 0.0057; respectively). Moreover, haplotype analysis revealed that several combinations of markers in SLC6A4, TPH2, and SERPINA6 might be significantly associated with the risk of NCL/P affected pregnancies. However, these associations were not statistically significant after correction for multiple testing. CONCLUSION: This study suggests that nucleotide variants of genes encoding components of the hypothalamus‐pituitary‐adrenal axis and serotoninergic system have a role in the etiology of NCL/P in the Polish population. SLC6A4, TPH2, and SERPINA6 might be novel candidate genes for this common congenital anomaly. Birth Defects Research (Part A), 2011. © 2011 Wiley‐Liss, Inc.     

Dielectric properties of two diastereoisomers of the arabinose and their equimolar mixture

Dielectric relaxation measurements were performed on two enantiomers, d- and l-arabinose and their equimolar mixture, and compared to dielectric data obtained for d-ribose. d-Arabinose differs from d-ribose by having the opposite configuration at C2. This study reveals that both d- and l- of arabinose exhibit α-relaxation peaks with the same shape for the same α-relaxation time τ_α, and the same steepness index for the T_g-scale T-dependence of τ_α. However, the two isomers have slightly different glass transition temperatures T_g’s, and their secondary γ-relaxation times also differ slightly from the previously observed γ-relaxation in d-ribose at the same temperature. However, when samples of both investigated monosaccharides are annealed at higher temperatures, their glass transition temperatures become nearly identical. This is an effect of the mutarotation process, which leads to the formation of pairs of the enantiomers and accordingly they should have the same physical properties. The width of the α-relaxation of d- and l-arabinose is broader than that of d-ribose, as reflected by the smaller stretch exponent in the Kohlrausch-Williams-Watts function used to fit the data of the former (β_KWW = 0.46 ± 0.01) than the latter (β_KWW = 0.55 ± 0.01). The width of the α-relaxation of racemic mixture of the d- and l-arabinose is slightly broader than that of the pure isomers. While the dielectric loss data of d-ribose in the glassy state at ambient and elevated pressures show an inflexion indicating the presence of the JG β-relaxation, the data of d- and l-arabinose show no such feature for identification of the supposedly universal JG β-relaxation. Nevertheless, on comparing the loss spectra of d-arabinose with that of d-ribose, the presence of the JG β-relaxation in d-arabinose has been rationalized.     

The seeds of success: release from fungal attack on seeds may influence the invasiveness of alien <Emphasis Type="Italic">Impatiens</Emphasis>

Although closely related, Impatiens glandulifera and Impatiens balfourii differ in their invasiveness in Europe; only the former is highly invasive there. Following the assumptions of the enemy release hypothesis (ERH), we tested whether these differences may be explained by the levels of seed infestation by pathogenic fungi. Using seeds collected along the Swiss-Italian border, we recorded four true pathogens of seeds: Fusarium culmorum, F. oxysporum, F. sporotrichoides, and Giberella avenacea. In Italy the seeds of I. balfourii were infected by fungal pathogens more often than those of I. glandulifera, while in Switzerland both species were under the same level of pressure. However, the overall differences in pathogen abundance were consistent with the ERH: seeds of the more invasive species were attacked less. This could be a result of differences between the communities of fungal pathogens attacking the seeds of both species in each country. The number of colonies of secondary pathogens (Cladosporium cladosporioides, Alternaria alternata) correlated negatively with the number of colonies of true pathogens; we suggest that the secondary pathogens may have prevented the occurrence of the true pathogens. The reason for the between-country differences in the fungal pathogen communities is unclear. A possible explanation is that Italy and Switzerland differ in their road and green-area maintenance work schemes, which may have influenced pathogen pressure on seeds. This study is one of the few that offers results indicating that release from enemies may be crucial to the invasion success of plants as early as the seed stage.