Efficient side-chain and backbone assignment in large proteins: Application to tGCN5

In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast ¹³C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a ¹³ C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOCSY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.     

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation

Background

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn''s disease.

Methods and Findings

To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.

Conclusion

These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.     

Effects of Radiofrequency Catheter Ablation of Atrial Fibrillation on Soluble P-Selectin,Von Willebrand Factor and IL-6 in the Peripheral and Cardiac Circulation

Background

Catheter ablation (CA) of atrial fibrillation (AF) is associated with inflammatory response, endothelial damage and with increased risk of thrombosis. However, whether these processes differ in peripheral and cardiac circulation is unknown.

Methods

Plasma markers (von Willebrand factor (vWf), soluble P-selectin (sPsel) and interleukin-6 (IL-6)) were measured by ELISA at three time points in 80 patients (62±10 years, 63% males, 41% paroxysmal AF) undergoing CA. These were at baseline – from femoral vein (FV) and left atrium (LA) before ablation; directly after ablation – from the pulmonary vein (PV), LA and FV; and 24 hours after procedure – from a cubital vein (CV).

Results

The levels of vWF and IL6 – but not sP-sel – increased significantly 24h after procedure (p<0.001). Baseline vWF was significantly associated with persistent AF (Beta = .303, p = 0.006 and Beta = .300, p = 0.006 for peripheral and cardiac levels, respectively), while persistent AF (Beta = .250, p = 0.031) and LAA flow pattern (Beta = .386, p<0.001) remained associated with vWF in cardiac blood after ablation. Advanced age was significantly associated with IL6 levels at baseline and after ablation in peripheral and cardiac blood. There were no clinical, procedural or anti-coagulation characteristics associated with sP-sel levels in cardiac blood, while peripheral sP-sel levels were associated with hypertension before (Beta = −.307, p = 0.007) and with persistent AF after ablation (Beta = −.262, p = 0.020).

Conclusions

vWF levels are higher in persistent AF and are associated with LAA rheological pattern after AF ablation. Increase of peripheral vWF and IL6 levels after procedure supports current AF ablation management with careful control of post-procedural anticoagulation to avoid ablation-related thromboembolism.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

The systematic distribution of vascular epiphytes – a critical update

Vascular epiphyte species exclusively, or at least primarily, germinate and grow on other plants without contact with the soil and, in contrast to mistletoes, without parasitizing their hosts. The last review of the systematic distribution of this diverse group of plants dates back more than two decades. The present study pursues three major goals: (1) it critically discusses conceptual problems arising from the distinction of epiphytes from non‐epiphytes; (2) it presents a compilation of epiphytic diversity derived from a vast number of sources; and (3) it arranges epiphyte diversity in an up‐to‐date taxonomic framework. The resulting compilation, which identifies 27 614 species of vascular epiphytes (including primary hemiepiphytes) representing 913 genera in 73 families, or approximately 9% of extant vascular plant diversity, is meant to be an important tool for studies on the ecology and evolution of epiphytes, but also for comparative studies with a focus on other life forms. © 2013 The Linnean Society of London     

Genotype-phenotype correlation in neurofibromatosis type-1: NF1 whole gene deletions lead to high tumor-burden and increased tumor-growth

Neurofibromatosis type-1 (NF1) patients suffer from cutaneous and subcutaneous neurofibromas (CNF) and large plexiform neurofibromas (PNF). Whole gene deletions of the NF1 gene can cause a more severe phenotype compared to smaller intragenic changes. Two distinct groups of NF1 whole gene deletions are type-1 deletions and atypical deletions. Our aim was to assess volumes and averaged annual growth-rates of CNF and PNF in patients with NF1 whole gene deletions and to compare these with NF1 patients without large deletions of the NF1 gene.We retrospectively evaluated 140 whole-body MR examinations of 38 patients with NF1 whole gene deletions (type-1 group: n = 27/atypical group n = 11) and an age- and sex matched collective of 38 NF1-patients. Age-dependent subgroups were created (0–18 vs >18 years). Sixty-four patients received follow-up MRI examinations (NF1whole gene deletion n = 32/control group n = 32). Whole-body tumor-volumes were semi-automatically assessed (MedX, V3.42). Tumor volumes and averaged annual growth-rates were compared.Median tumor-burden was significantly higher in the type-1 group (418ml; IQR 77 – 950ml, p = 0.012) but not in the atypical group (356ml;IQR 140–1190ml, p = 0.099) when compared to the controls (49ml; IQR 11–691ml). Averaged annual growth rates were significantly higher in both the type-1 group (14%/year; IQR 45–36%/year, p = 0.004) and atypical group (11%/year; IQR 5–23%/year, p = 0.014) compared to the controls (4%/year; IQR1–8%/year). Averaged annual growth rates were significantly higher in pediatric patients with type-1 deletions (21%/year) compared with adult patients (8%/year, p = 0.014) and also compared with pediatric patients without large deletions of the NF1 gene (3.3%/year, p = 0.0015).NF1 whole gene deletions cause a more severe phenotype of NF1 with higher tumor burden and higher growth-rates compared to NF1 patients without large deletions of the NF1 gene. In particular, pediatric patients with type-1 deletions display a pronounced tumor growth.     

The effect of temperature on the development times of eggs,naupliar and copepodite stages of five species of cyclopoid copepods

The duration times of eggs, combined naupliar instars and of the different copepodite stages of five species of cyclopoid copepods — Acanthocyclops robustus, Cyclops vicinus, Diacyclops bicuspidatus, Mesocyclops leuckarti, and Thermocyclops crassus — were investigated at five different temperatures. The five species can be divided in two groups: two species, C. vicinus and D. bicuspidatus, adapted to cold water conditions and three species, A. robustus, M. leuckarti and T. crassus adapted to warm water conditions. The cold water species showed a faster egg development than M. leuckarti and T. crassus at 5–15 °C. The eggs and instars of the warm water species M. leuckarti tend to develop faster than those of the former two species at higher temperatures. A. robustus showed the shortest egg and instar development at 10–25 °C. The warm water species T. crassus produced no eggs at 10 °C and temperatures below. At higher temperatures (20, 25 °C) the egg and instar duration times were similar or longer than those of the other species. When cultured in total darkness a great part of the C_IV respectively C_V copepodites of the summer forms entered arrest and the percentage of copepodites that showed an arrest of development was highest at lowest temperatures. The present results are compared with data from literature and differences are discussed.     

DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.